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Sample records for broccoli coq5 methyltransferase

  1. Involvement of a broccoli COQ5 methyltransferase in the production of volatile selenium compounds.

    Science.gov (United States)

    Zhou, Xin; Yuan, Youxi; Yang, Yong; Rutzke, Michael; Thannhauser, Theodore W; Kochian, Leon V; Li, Li

    2009-10-01

    Selenium (Se) is an essential micronutrient for animals and humans but becomes toxic at high dosage. Biologically based Se volatilization, which converts Se into volatile compounds, provides an important means for cleanup of Se-polluted environments. To identify novel genes whose products are involved in Se volatilization from plants, a broccoli (Brassica oleracea var italica) cDNA encoding COQ5 methyltransferase (BoCOQ5-2) in the ubiquinone biosynthetic pathway was isolated. Its function was authenticated by complementing a yeast coq5 mutant and by detecting increased cellular ubiquinone levels in the BoCOQ5-2-transformed bacteria. BoCOQ5-2 was found to promote Se volatilization in both bacteria and transgenic Arabidopsis (Arabidopsis thaliana) plants. Bacteria expressing BoCOQ5-2 produced an over 160-fold increase in volatile Se compounds when they were exposed to selenate. Consequently, the BoCOQ5-2-transformed bacteria had dramatically enhanced tolerance to selenate and a reduced level of Se accumulation. Transgenic Arabidopsis expressing BoCOQ5-2 volatilized three times more Se than the vector-only control plants when treated with selenite and exhibited an increased tolerance to Se. In addition, the BoCOQ5-2 transgenic plants suppressed the generation of reactive oxygen species induced by selenite. BoCOQ5-2 represents, to our knowledge, the first plant enzyme that is not known to be directly involved in sulfur/Se metabolism yet was found to mediate Se volatilization. This discovery opens up new prospects regarding our understanding of the complete metabolism of Se and may lead to ways to modify Se-accumulator plants with increased efficiency for phytoremediation of Se-contaminated environments.

  2. Involvement of a Broccoli COQ5 Methyltransferase in the Production of Volatile Selenium Compounds[C][OA

    Science.gov (United States)

    Zhou, Xin; Yuan, Youxi; Yang, Yong; Rutzke, Michael; Thannhauser, Theodore W.; Kochian, Leon V.; Li, Li

    2009-01-01

    Selenium (Se) is an essential micronutrient for animals and humans but becomes toxic at high dosage. Biologically based Se volatilization, which converts Se into volatile compounds, provides an important means for cleanup of Se-polluted environments. To identify novel genes whose products are involved in Se volatilization from plants, a broccoli (Brassica oleracea var italica) cDNA encoding COQ5 methyltransferase (BoCOQ5-2) in the ubiquinone biosynthetic pathway was isolated. Its function was authenticated by complementing a yeast coq5 mutant and by detecting increased cellular ubiquinone levels in the BoCOQ5-2-transformed bacteria. BoCOQ5-2 was found to promote Se volatilization in both bacteria and transgenic Arabidopsis (Arabidopsis thaliana) plants. Bacteria expressing BoCOQ5-2 produced an over 160-fold increase in volatile Se compounds when they were exposed to selenate. Consequently, the BoCOQ5-2-transformed bacteria had dramatically enhanced tolerance to selenate and a reduced level of Se accumulation. Transgenic Arabidopsis expressing BoCOQ5-2 volatilized three times more Se than the vector-only control plants when treated with selenite and exhibited an increased tolerance to Se. In addition, the BoCOQ5-2 transgenic plants suppressed the generation of reactive oxygen species induced by selenite. BoCOQ5-2 represents, to our knowledge, the first plant enzyme that is not known to be directly involved in sulfur/Se metabolism yet was found to mediate Se volatilization. This discovery opens up new prospects regarding our understanding of the complete metabolism of Se and may lead to ways to modify Se-accumulator plants with increased efficiency for phytoremediation of Se-contaminated environments. PMID:19656903

  3. Molecular and biochemical characterization of the selenocysteine Se-methyltransferase gene and Se-methylselenocysteine synthesis in broccoli.

    Science.gov (United States)

    Lyi, Sangbom M; Heller, Laurence I; Rutzke, Michael; Welch, Ross M; Kochian, Leon V; Li, Li

    2005-05-01

    Selenium (Se) plays an indispensable role in human nutrition and has been implicated to have important health benefits, including being a cancer preventative agent. While different forms of Se vary in their anticarcinogenic efficacy, Se-methylselenocysteine (SeMSC) has been demonstrated to be one of the most effective chemopreventative compounds. Broccoli (Brassica oleracea var. italica) is known for its ability to accumulate high levels of Se with the majority of the selenoamino acids in the form of Se-methylselenocysteine. Therefore, it serves as a good model to study the regulation of SeMSC accumulation in plants. A cDNA encoding selenocysteine Se-methyltransferase, the key enzyme responsible for SeMSC formation, was cloned from broccoli using a homocysteine S-methyltransferase gene probe from Arabidopsis (Arabidopsis thaliana). This clone, designated as BoSMT, was functionally expressed in Escherichia coli, and its identity was confirmed by its substrate specificity in the methylation of selenocysteine. The BoSMT gene represents a single copy sequence in the broccoli genome. Examination of BoSMT gene expression and SeMSC accumulation in response to selenate, selenite, and sulfate treatments showed that the BoSMT transcript and SeMSC synthesis were significantly up-regulated in plants exposed to selenate but were low in plants supplied with selenite. Simultaneous treatment of selenate with selenite significantly reduced SeMSC production. In addition, high levels of sulfate suppressed selenate uptake, resulting in a dramatic reduction of BoSMT mRNA level and SeMSC accumulation. Our results reveal that SeMSC accumulation closely correlated with the BoSMT gene expression and the total Se status in tissues and provide important information for maximizing the SeMSC production in this beneficial vegetable plant.

  4. Identification of Resistance to Blackrot in Broccoli

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The screening for Black rot(Xanthomonas campestris PV.campestris)resistance in broccoli (Brassica oleracea L.Var.italica Planch) lines plays an important role in breeding for Black rot resistance and is of great value to practical utilization.The broccoli in bred line"91-9318-4"was used as the experimental material in the trial.X0-2 strain of Xanthomonas campestris PV.campestris was and purified from the infected broccoli plants in the field.The spraying method was adopted in the inoculation.The trial was designed as the randomized blocks of two factors with three replications.Factor A was the foliar age and factor B was the concetrations of inoculum.Factor A had three levels A1,A2 and A3 (the 2nd 4th and 6th true-leaf stage),factor B had three levels B1,B2 and B3(106 counts/mL,107 counts/mL and 108 counts/mL).26 breeding materials of broccoli were identified for resistance to Black rot by means of the acquired method.One resistant breeding material was obtained,the identifying results showed that this screening method is practical.

  5. Agronomy of strip intercropping broccoli with alyssum for biological control of aphids

    Science.gov (United States)

    Organic broccoli growers in California typically control aphids by intercropping broccoli with strips of alyssum (Lobularia maritima (L.) Desv.) which attracts hoverflies (Diptera: Syrphidae) that are important predators of aphids. A three year study with transplanted organic broccoli in Salinas, ...

  6. Machine vision for a selective broccoli harvesting robot

    NARCIS (Netherlands)

    Blok, Pieter M.; Barth, Ruud; Berg, Van Den Wim

    2016-01-01

    The selective hand-harvest of fresh market broccoli is labor-intensive and comprises about 35% of the total production costs. This research was conducted to determine whether machine vision can be used to detect broccoli heads, as a first step in the development of a fully autonomous selective harve

  7. Persistence and performance of esfenvalerate residues on broccoli.

    Science.gov (United States)

    Antonious, George F

    2002-01-01

    The efficacy of esfenvalerate (84 g litre-1 EC; Asana XL) at 7.0 g AI ha-1 on broccoli was tested against the flea beetle, Phyllotreta cruciferae Goeze (Chrysomelidae: Coleoptera) and the imported cabbage worm, Pieris rapae L (Pieridae: Lepidoptera) under field conditions. Insect populations were monitored before and after treatment. Periodic sweep-net collections and examination of the leaves in treated and untreated broccoli plots revealed mean reductions of P cruciferae levels of nearly 98% 1 week post-application compared with untreated plots. The residual toxicity of esfenvalerate was also effective for 2 weeks in reducing population density of P rapae by 69% on broccoli leaves. The impact of esfenvalerate on feeding damage to broccoli leaves was established by counting the number of feeding holes made by P cruciferae on spring broccoli and P rapae on fall broccoli. As the leaf area ingested increased, a linear relationship was seen between the number of holes and number of insects. Results indicated that forage destruction by the two pests was significantly reduced by esfenvalerate application. Esfenvalerate was extracted from broccoli at 1 h and 1, 3, 7, 10, and 14 days post-application for residue analysis. Residues on spring broccoli were 12.2, 5.2 and 2.9 micrograms cm-2 on the leaves and 0.13, 0.05 and 0.02 microgram g-1 on the heads at 1 h, 1 and 3 days, respectively. Only trace levels (0.001 microgram g-1) were detected in/on the heads 14 days after spraying. On the basis of half-life (T1/2) values, persistence of esfenvalerate on spring broccoli leaves (T1/2 = 1 day) was shorter than on fall broccoli (T1/2 = 1.6 days). T1/2 values were 2.1 and 3.6 days on spring and fall broccoli heads, respectively. The implications of these residue levels on re-entry times into treated fields are discussed.

  8. Secondary Broccoli Production Depending On Sowing And Planting Dates

    OpenAIRE

    Todorova, Desislava

    2014-01-01

    The investigation was carried out during the period of 2009-2011 at the Institute of Agriculture – Kyustendil, located in the Southwest Bulgaria. Four broccoli (Brassica oleracea var. italica Plenck) hybrids were grown by the technology of late field production with different sowing and planting dates. The aim of the research was to establish a relationship between planting time and secondary yield of broccoli. Some morphological characteristics and production traits of an additional yield of...

  9. Plant characters of broccoli determinants of head production

    OpenAIRE

    Fernanda Daniela Brandelero; Betania Brum; Lindolfo Storck; Jessica Cardoso; Talita Slota Kutz; Thiago de Oliveira Vargas

    2016-01-01

    ABSTRACT: The increasing consumption of single-head broccoli is due to several factors, among them there are food production in minimally processing form and the existence of hybrids that adapt to various climates, in addition to the simple harvesting of this typical architecture.This study aimed to identify the most relevant plant characters of broccoli, represented by growth characters, which are determinant in the production and canopy area. The study was conducted in an experimental area ...

  10. Production and postharvest quality maintenance of single unit and bunching broccoli in Virginia

    OpenAIRE

    Jett, Lewis W.

    1990-01-01

    Broccoli (Brassica oleracea L. var. italica) has become an increasingly popular vegetable with American consumers. Much of the attractiveness of fresh broccoli is derived from this vegetable's high nutrition and excellent organoleptic properties. In a consumer response survey, Virginia Master Gardeners indicated a preference for broccoli that has less stalk and more florets by weight. The objectives of this research were to produce single unit broccoli, and to examine vac...

  11. Histone methyltransferases in cancer

    DEFF Research Database (Denmark)

    Albert, Mareike; Helin, Kristian

    2009-01-01

    Cancer is perceived as a heterogeneous group of diseases that is characterized by aberrant patterns of gene expression. In the last decade, an increasing amount of data has pointed to a key role for epigenetic alterations in human cancer. In this review, we focus on a subclass of epigenetic...... regulators, namely histone methyltransferases (HMTs). Several HMTs have been linked to different types of cancer; however, in most cases we only have limited knowledge regarding the molecular mechanisms by which the HMTs contribute to disease development. We summarize the current knowledge regarding some...

  12. Food safety evaluation of broccoli and radish sprouts.

    Science.gov (United States)

    Martínez-Villaluenga, Cristina; Frías, Juana; Gulewicz, Piotr; Gulewicz, Krzysztof; Vidal-Valverde, Concepción

    2008-05-01

    Three cultivars of broccoli seeds (Brassica oleracea var. italica), cv. Tiburon, cv. Belstar and cv. Lucky, and two cultivars of radish seeds (Raphanus sativus), cv. Rebel and cv. Bolide, were germinated for three and five days and safety aspects such as microbiological counts and biogenic amines were investigated. Cytotoxicity evaluation was also carried out. Broccoli and radish sprouts contained numbers of mesophilic, psychrotrophic, total and faecal coliform bacteria which are the usual counts for minimally processed germinated seeds. Putrescine, cadaverine, histamine, tyramine, spermidine and spermine increased during sprout production although these levels were below those permitted by legislation (5 mg/100 g of edible food). Broccoli and radish sprouts demonstrated no toxic effects on proliferation and viability of HL-60 cells and should be included in our diets as healthy and safe fresh foods.

  13. Detection of the Diversity of Cytoplasmic Male Sterility Sources in Broccoli (Brassica Oleracea var. Italica) Using Mitochondrial Markers

    OpenAIRE

    Shu, Jinshuai; Liu, Yumei; Li, Zhansheng; Zhang, Lili; Fang, Zhiyuan; Yang, Limei; Zhuang, Mu; Zhang, Yangyong; Lv, Honghao

    2016-01-01

    Broccoli (Brassica oleracea var. italica) is an important commercial vegetable crop. As part of an efficient pollination system, cytoplasmic male sterility (CMS) has been widely used for broccoli hybrid production. Identifying the original sources of CMS in broccoli accessions has become an important part of broccoli breeding. In this study, the diversity of the CMS sources of 39 broccoli accessions, including 19 CMS lines and 20 hybrids, were analyzed using mitochondrial markers. All CMS acc...

  14. Dietary Broccoli Alters Rat Cecal Microbiota to Improve Glucoraphanin Hydrolysis to Bioactive Isothiocyanates

    Science.gov (United States)

    Liu, Xiaoji; Wang, Yanling; Hoeflinger, Jennifer L.; Neme, Bárbara P.; Jeffery, Elizabeth H.; Miller, Michael J.

    2017-01-01

    Broccoli consumption brings many health benefits, including reducing the risk of cancer and inflammatory diseases. The objectives of this study were to identify global alterations in the cecal microbiota composition using 16S rRNA sequencing analysis and glucoraphanin (GRP) hydrolysis to isothiocyanates ex vivo by the cecal microbiota, following different broccoli diets. Rats were randomized to consume AIN93G (control) or different broccoli diets; AIN93G plus cooked broccoli, a GRP-rich powder, raw broccoli, or myrosinase-treated cooked broccoli. Feeding raw or cooked broccoli for four days or longer both changed the cecal microbiota composition and caused a greater production of isothiocyanates ex vivo. A more than two-fold increase in NAD(P)H: quinone oxidoreductase 1 activity of the host colon mucosa after feeding cooked broccoli for seven days confirmed the positive health benefits. Further studies revealed that dietary GRP was specifically responsible for the increased microbial GRP hydrolysis ex vivo, whereas changes in the cecal microbial communities were attributed to other broccoli components. Interestingly, a three-day withdrawal from a raw broccoli diet reversed the increased microbial GRP hydrolysis ex vivo. Findings suggest that enhanced conversion of GRP to bioactive isothiocyanates by the cecal microbiota requires four or more days of broccoli consumption and is reversible. PMID:28287418

  15. Genetics Home Reference: guanidinoacetate methyltransferase deficiency

    Science.gov (United States)

    ... Facebook Share on Twitter Your Guide to Understanding Genetic Conditions Search MENU Toggle navigation Home Page Search ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions guanidinoacetate methyltransferase deficiency guanidinoacetate methyltransferase ...

  16. A Market-Window Analysis for Crown-Cut Broccoli Produced in Southwest Virginia

    OpenAIRE

    Dillard, John; Kmak, Heather; Russ, M.J.; Coale, Charles W., Jr.; Bratsch, Tony; Reaves, Dixie Watts

    2006-01-01

    This research determines the profitability for farmers of marketing film-wrapped Crown-Cut broccoli in Southwest Virginia. Historical price data was collected in three eastern terminal markets (Atlanta, Baltimore, and Philadelphia) for Crown-Cut and bunched broccoli from October 1998 to January 2005. Cost-of-production budgets were estimated for Crown-Cut broccoli. The economic data was entered into a market window analysis and the profitability of each market was evaluated. The profitability...

  17. Broccoli, PTEN deletion and prostate cancer: where is the link?

    Directory of Open Access Journals (Sweden)

    Bardelli Alberto

    2010-12-01

    Full Text Available Abstract The concept that vegetables and fruits are relevant sources of cancer-preventive substances is strongly supported by population studies. Among others, cruciferous vegetables like broccoli, cabbage, cauliflower and Brussels sprouts are thought to affect the development of various types of cancers and especially prostate tumors. Yet, the identification of the molecular mechanisms by which the 'active' compounds contained in these vegetables mediate their anticancer activity has historically lagged behind. Accordingly, direct laboratory evidence of how individual nutrients affect cancer genes and the pathways they control remains the major obstacle to progress in this research field. Here we review a recent report investigating the interaction between sulforaphane, a dietary isothiocyanate derived from broccoli, and expression of the PTEN tumor suppressor gene in pre malignant prostate tissue.

  18. Effect of Se treatment on the volatile compounds in broccoli.

    Science.gov (United States)

    Lv, Jiayu; Wu, Jie; Zuo, Jinhua; Fan, Linlin; Shi, Junyan; Gao, Lipu; Li, Miao; Wang, Qing

    2017-02-01

    Broccoli contains high levels of bioactive compounds but deteriorates and senesces easily. In the present study, freshly harvested broccoli was treated with selenite and stored at two different temperatures. The effect of selenite treatment on sensory quality and postharvest physiology were analyzed. Volatile components were assessed by HS-SPME combined with GC-MS and EN. The metabolism of Se and S was also examined. Results indicated that Se treatment had a significant effect on maintaining the sensory quality, suppressing the respiration intensity and ethylene production, as well as increasing the content of Se and decreasing the content of S. In particular, significant differences in the composition of volatile compounds were present between control and Se-treated. The differences were mainly due to differences in alcohols and sulfide compounds. These results demonstrate that Se treatment can have a positive effect on maintaining quality and enhancing its sensory quality through the release of volatile compounds.

  19. THE INFLUENCE OF NUTRITION ON THE MAIN PRODUCTION OF BROCCOLI

    Directory of Open Access Journals (Sweden)

    Mădălina Doltu

    2013-07-01

    Full Text Available The research was realized in the unheated greenhouse of Horting Institute Bucharest, in 2012 year. It have aimed the influence of one phase of fertilization with ammonium nitrate (NH4NO3 on inflorescences of broccoli, at a hybrid F1 - Ironman. The fertilizer was used before the formation of main inflorescences, in different doses (100 kg/ha, 150 kg/ha, without fertilizer. Harvesting was done in 4 stages. A phase of fertilization with ammonium nitrate has influenced the main production of broccoli: the variant with fertilization 150 kg/ha has obtained the highest percentage of inflorescences formed, the average weight/inflorescence largest and superior production compared with other variants. Were obtained direct linear correlations between the doses of ammonium nitrate (NH4NO3 and the production aspects followed (percentage of main inflorescences harvested, average weight/inflorescence, total production, significances of the correlations very high.

  20. Studies on statistical optimization of sulforaphane production from broccoli seed

    OpenAIRE

    Wu, Yuanfeng; Mao, Jianwei; Mei,Lehe; Liu, Shiwang

    2013-01-01

    Background: Natural sulforaphane (SF) has been of increasing interest for nutraceutical and pharmaceutical industries due to its anti-cancer effect. The main objective of the present work was to optimize the production of SF from broccoli seed using response surface methodology. Results: Three major factors (hydrolysis time, water volume and ethyl acetate volume) were screened out through Plackett-Burman (PB) factorial design. The methods of steepest ascent combined with central composite des...

  1. Plant characters of broccoli determinants of head production

    Directory of Open Access Journals (Sweden)

    Fernanda Daniela Brandelero

    2016-06-01

    Full Text Available ABSTRACT: The increasing consumption of single-head broccoli is due to several factors, among them there are food production in minimally processing form and the existence of hybrids that adapt to various climates, in addition to the simple harvesting of this typical architecture.This study aimed to identify the most relevant plant characters of broccoli, represented by growth characters, which are determinant in the production and canopy area. The study was conducted in an experimental area in Pato Branco city, PR. The 11 characters were evaluated for 365 plants, spaced with 0.8x0.5m, on a blank experiment. The characters of group 1 (height, number of leaves, stem height, stem diameter were evaluated on the 21 and 58 day after transplanting (DAT, and the leaf area was evaluated on the 17 and 32 DAT. The characters of group 2 were quantity of fresh head mass and canopy area. At the initial stage of cultivation, on the 17 and 21 DAT, variations in the plants characters did not lead toany variation in production. The higher number of leaves and the larger stem diameter on the 58 DAT determined the greater mass of the broccoli heads.

  2. Controlling rheology and structure of sweet potato starch noodles with high broccoli powder content by hydrocolloids

    NARCIS (Netherlands)

    Silva, E.; Birkenhake, M.; Scholten, E.; Sagis, L.M.C.; Linden, van der E.

    2013-01-01

    Incorporating high volume fractions of broccoli powder in starch noodle dough has a major effect on its shear modulus, as a result of significant swelling of the broccoli particles. Several hydrocolloids with distinct water binding capacity (locust bean gum (LBG), guar gum, konjac glucomannan (KG),

  3. High amounts of broccoli in pasta-like products: nutritional evaluation and sensory acceptability

    NARCIS (Netherlands)

    Silva, E.; Gerritsen, L.; Dekker, M.; Linden, van der E.; Scholten, E.

    2013-01-01

    Pasta and noodles were enriched with concentrations of broccoli powder (BP) up to 30% (v/v). To ensure the benefits from the broccoli nutrients, their leakage during cooking should be prevented. Such leakage is determined by the microstructure. In a previous study we have shown that the microstructu

  4. Differences in tolerance of broccoli and cabbage cultivars to clomazone herbicide

    Science.gov (United States)

    Clomazone herbicide is registered for cabbage in the U.S., but not for other cultivar groups within Brassica oleracea. Greenhouse and field experiments were designed to compare the tolerance of broccoli and cabbage cultivars to clomazone and assess its potential for weed management in broccoli. Fo...

  5. A research approach for quality based design of healthy foods: Dried broccoli as a case study

    NARCIS (Netherlands)

    Oliviero, T.; Verkerk, R.; Dekker, M.

    2013-01-01

    An approach for process design based on optimization of product quality attributes is presented. Adsorption drying of broccoli with retention of its health benefits is taken as an example to illustrate the approach. Related to its content of glucosinolates, broccoli has a high potential to reduce th

  6. Effect of Hot Water Treatment on Postharvest Shelf Life and Quality of Broccoli

    Institute of Scientific and Technical Information of China (English)

    WU Ping; LI Wu

    2003-01-01

    Broccoli was stored at 0, 10, or 20℃ after immersion in hot water (38 -52℃ ) for 10 or 30min. Yellowing of broccoli was significantly slowed and shelf life significantly increased when broccoli wastreated with hot water at 42 -46℃ and then stored at 10 or 20℃. Heat injury occurred when treatment washigher than 46℃ in some varieties. Broccoli lasted 2 -3 days longer when stored at 10℃ and 1 -2 days longerwhen stored at 20℃ after hot water treatment at 46℃. There was no significant effect of treatment on shelflife after long time storage at 0℃. Weight loss was reduced by hot water treatment and the respiration behav-ior of the broccoli was also changed.

  7. Selenium enrichment of broccoli: interactions between selenium and secondary plant compounds.

    Science.gov (United States)

    Finley, John W; Sigrid-Keck, Anna; Robbins, Rebecca J; Hintze, Korry J

    2005-05-01

    Multiple components of broccoli, such as sulforaphane (Sf) and phenolic acids, may inhibit cancer. Additionally, broccoli can accumulate selenium (Se), and Se has been demonstrated to reduce the risk of cancer. Studies were conducted to determine whether enhancement of broccoli with Se would produce a plant with superior health benefits. Although increasing the concentration of Se in broccoli from 800 microg/g resulted in inhibition of colon cancer in rats, it also decreased the Sf content by >80% and inhibited production of most phenolic acids. The inclusion of Se-enriched broccoli in the diet of rats induced the activity of the selenoprotein thioredoxin reductase beyond the maximum activity induced by Se alone. These results emphasize the complex interactions of bioactive chemicals in a food; attempts to maximize one component may affect accumulation of another, and consumption of high amounts of multiple bioactive compounds may result in unexpected metabolic interactions within the body.

  8. ADAPTABILITY, FERTILIZER EFFECT AND NUTRITION CONTENT OF VARIOUS BROCCOLI CULTIVAR (Brassica oleracea L. var. italica INTRODUCED IN KOPANG, CENTRAL LOMBOK

    Directory of Open Access Journals (Sweden)

    Zaenul Gafari

    2015-09-01

    Full Text Available This research was aimed to determine adaptability of eight introduced broccoli cultivars in the tropical climate, the effect of fertilizers, as well as determine the content of vitamin A, C and fiber owned by the cultivar. This research was using randomized complete block design ( RCBD with two factors. The first factorsare different broccoli cultivars namely Broccoli 'Lucky', Broccoli 'B - 106', Broccoli 'B-116', Broccoli 'B-112', Broccoli 'Green 2109' F1, Broccoli 'Green calabrase' and Broccoli 'Green Magic 'and Broccoli' Mujur'as a control. The second factors fertilizer of ZA, manure and urea. This research was performed in Kopang, Central Lombok, West Nusa Tenggara, for seven months from December 2014 to Juni 2015.Analysis of the nutrient content was done at the Laboratory of Analytical Chemistry, University of Mataram, Lombok. Parameters observed in this research were plant height, number and weight of leaves, and quality of curd. Data were analyzed by analysis of variance (ANOVA if significantly different then tested further by Duncan's Multiple Range Test (DMRT at 5% level. The results showed all the cultivars able to adapt. Broccoli 'B-106' is a cultivar that has the quality and quantity of curd most excellent and able to adapt to high temperatures. ZA fertilizer application will produce curd weight and diameter greater than that ofmanure or urea. The research also showed that cultivar B-112 has the highest content of vitamin A, B-116 has the highest vitamin C and Green 2109F1 has the highest fibers.

  9. THE INFLUENCE OF NUTRITION ON THE MAIN PRODUCTION OF BROCCOLI

    OpenAIRE

    Mădălina Doltu; Marian Bogoescu; Dorin Sora

    2013-01-01

    The research was realized in the unheated greenhouse of Horting Institute Bucharest, in 2012 year. It have aimed the influence of one phase of fertilization with ammonium nitrate (NH4NO3) on inflorescences of broccoli, at a hybrid F1 - Ironman. The fertilizer was used before the formation of main inflorescences, in different doses (100 kg/ha, 150 kg/ha, without fertilizer). Harvesting was done in 4 stages. A phase of fertilization with ammonium nitrate has influenced the main production of br...

  10. Broccoli consumption and chronic atrophic gastritis among Japanese males: an epidemiological investigation.

    Directory of Open Access Journals (Sweden)

    Sato K

    2004-06-01

    Full Text Available Previous in vitro and animal experiments have shown that sulforaphane, which is abundant in broccoli, inhibits Helicobacter pylori (H. pylori infection and blocks gastric tumor formation. This suggests that broccoli consumption prevents chronic atrophic gastritis (CAG introduced by H. pylori infection and, therefore, gastric cancer. For an epidemiological investigation of the relationship between the broccoli consumption and CAG, a cross-sectional study of 438 male employees, aged 39 to 60 years, of a Japanese steel company was conducted. CAG was serologically determined with serum cut-off values set at pepsinogen I < or = 70 ng/ml and a ratio of serum pepsinogen I/pepsinogen II < or = 3.0. Broccoli consumption (weekly frequency and diet were monitored by using a 31-item food frequency questionnaire. The prevalence of CAG among men who ate broccoli once or more weekly was twice as high as that among men who consumed a negligible amount (P < 0.05. Multiple logistic regression analysis indicated that broccoli consumption once or more weekly significantly increased the risk for CAG (odds ratio, 3.06; 95% confidence interval, 1.12-8.38; P < 0.05, after controlling for age, education, cigarette smoking, and alcohol consumption. The present study failed to show an expected association between frequent broccoli consumption and a low prevalence of CAG.

  11. Effect of NaCl treatments on glucosinolate metabolism in broccoli sprouts

    Institute of Scientific and Technical Information of China (English)

    Rong-fang GUO; Gao-feng YUAN; Qiao-mei WANG

    2013-01-01

    To understand the regulation mechanism of NaCl on glucosinolate metabolism in broccoli sprouts,the germination rate,fresh weight,contents of glucosinolates and sulforaphane,as well as myrosinase activity of broccoli sprouts germinated under 0,20,40,60,80,and 100 mmol/L of NaCl were investigated in our experiment.The results showed that glucoerucin,glucobrassicin,and 4-hydroxy glucobrassicin in 7-d-old broccoli sprouts were significantly enhanced and the activity of myrosinase was inhibited by 100 mmol/L of NaCl.However,the total glucosinolate content in 7-d-old broccoli sprouts was markedly decreased although the fresh weight was significantly increased after treatment with NaCl at relatively low concentrations(20,40,and 60 mmol/L).NaCl treatment at the concentration of 60 mmol/L for 5 d maintained higher biomass and comparatively higher content of glucosinolates in sprouts of broccoli with decreased myrosinase activity.A relatively high level of NaCl treatment(100 mmol/L)significantly increased the content of sulforaphane in 7-d-old broccoli sprouts compared with the control.These results indicate that broccoli sprouts grown under a suitable concentration of NaCl could be desirable for human nutrition.

  12. Monitoring the microbial populations and temperatures of fresh broccoli from harvest to retail display.

    Science.gov (United States)

    Dallaire, R; LeBlanc, D I; Tranchant, C C; Vasseur, L; Delaquis, P; Beaulieu, C

    2006-05-01

    Microbial populations and the temperature of fresh broccoli were monitored at several steps of a supply chain by sampling 33 distinct lots of locally grown produce over two seasons during harvest, storage, wholesale handling, and retail display. Imported broccoli was also sampled, but only at retail display. Microbiological analyses were conducted on the florets of 201 local and 60 imported broccoli samples to determine populations of total aerobic bacteria (aerobic colony count), fecal coliforms, Escherichia coli, and Listeria monocytogenes. All the samples had mean aerobic colony counts ranging between 4 and 6 log CFU/g, but L. monocytogenes was not detected (limit of detection =100 CFU/g). Fecal coliforms and E. coli (limit of detection =20 most probable number per 100 g) were found in 22 of 126 samples of local broccoli collected at various steps of the production and distribution system during the first season. None was found in 75 samples collected in the second season. Fecal coliforms and E. coli were found in 2 of 60 imported broccoli samples. Broccoli temperatures were relatively well controlled throughout the production and distribution system. No clear change in produce microbial populations was evident between harvest and retail display, during both sampling seasons. However, a large experimental variability was found, possibly associated with the high variability of the initial levels of microbial populations on broccoli at harvest.

  13. Polymer coating, germination and vigor of broccoli seeds

    Directory of Open Access Journals (Sweden)

    Almeida Celina de

    2005-01-01

    Full Text Available Brassica oleracea var italica occupies a special place in the internal and external market of vegetables seeds. Vegetables producers demand seeds with high degree of purity, germination and vigor, since seeds' quality is the basis for the success of the production. In this work, broccoli seeds were coated in a spouted bed, by an aqueous suspension of hidroxy-ethyl-cellulose. Effects of the operating variables: spouting air temperature, atomizing air pressure and coating suspension flow rate over the dependent variables: seeds germination, seeds accelerated aging and the speed of seeds germination in soil, were investigated in a factorial scheme trial. The maximum processing time was 120 min. A totally randomized experiment evaluated and compared seeds germination and vigor of the coated and non-coated seeds. There was no identifiable, pronounced difference on germination of coated and non-coated seeds, accelerated aging of seeds, and speed of seeds germination in the soil. Coating with hidroxy-ethyl-cellulose in the spouted bed did not alter broccoli seeds physiologic quality. The surface of coated seeds presented satisfactory distribution and spreading of the polymer film, uniform and individual coating and homogeneous aspect.

  14. Heat Transfer during Blanching and Hydrocooling of Broccoli Florets.

    Science.gov (United States)

    Iribe-Salazar, Rosalina; Caro-Corrales, José; Hernández-Calderón, Óscar; Zazueta-Niebla, Jorge; Gutiérrez-Dorado, Roberto; Carrazco-Escalante, Marco; Vázquez-López, Yessica

    2015-12-01

    The objective of this work was to simulate heat transfer during blanching (90 °C) and hydrocooling (5 °C) of broccoli florets (Brassica oleracea L. Italica) and to evaluate the impact of these processes on the physicochemical and nutrimental quality properties. Thermophysical properties (thermal conductivity [line heat source], specific heat capacity [differential scanning calorimetry], and bulk density [volume displacement]) of stem and inflorescence were measured as a function of temperature (5, 10, 20, 40, 60, and 80 °C). The activation energy and the frequency factor (Arrhenius model) of these thermophysical properties were calculated. A 3-dimensional finite element model was developed to predict the temperature history at different points inside the product. Comparison of the theoretical and experimental temperature histories was carried out. Quality parameters (firmness, total color difference, and vitamin C content) and peroxidase activity were measured. The satisfactory validation of the finite element model allows the prediction of temperature histories and profiles under different process conditions, which could lead to an eventual optimization aimed to minimize the nutritional and sensorial losses in broccoli florets.

  15. Response of broccoli to soil water tension under drip irrigation

    Directory of Open Access Journals (Sweden)

    Bartolomeu Felix Tangune

    2016-02-01

    Full Text Available We evaluated the effect of different soil water tensions on the production of broccoli cultivated in a protected environment under drip irrigation in order to establish criteria for the adequate management of irrigation. A completely randomized block design was used, comprising six treatments and four replicates. The treatments included six soil water tensions (15, 30, 45, 60, 75 and 90 kPa. Soil water tension was monitored with granular matrix sensors installed at depths of 0.2 m (decision sensors and 0.4 m (seepage control sensors. Total and marketable fresh weight of broccoli heads, average diameter of marketable heads, height of marketable heads, and total and marketable yield were greatest when the soil water tension at a depth of 0.2 m was 15 kPa, at which the mean values of the evaluated variables were 0.84 kg, 0.76 kg, 20.5 cm, 11.7 cm; 26.5 t ha?1, and 23.7 t ha?1, respectively. Treatments did not significantly affect efficiency of water use or height of marketable heads.

  16. Enzymology of Mammalian DNA Methyltransferases.

    Science.gov (United States)

    Jurkowska, Renata Z; Jeltsch, Albert

    2016-01-01

    DNA methylation is currently one of the hottest topics in basic and biomedical research. Despite tremendous progress in understanding the structures and biochemical properties of the mammalian DNA nucleotide methyltransferases (DNMTs), principles of their regulation in cells have only begun to be uncovered. In mammals, DNA methylation is introduced by the DNMT1, DNMT3A, and DNMT3B enzymes, which are all large multi-domain proteins. These enzymes contain a catalytic C-terminal domain with a characteristic cytosine-C5 methyltransferase fold and an N-terminal part with different domains that interacts with other proteins and chromatin and is involved in targeting and regulation of the DNMTs. The subnuclear localization of the DNMT enzymes plays an important role in their biological function: DNMT1 is localized to replicating DNA via interaction with PCNA and UHRF1. DNMT3 enzymes bind to heterochromatin via protein multimerization and are targeted to chromatin by their ADD and PWWP domains. Recently, a novel regulatory mechanism has been discovered in DNMTs, as latest structural and functional data demonstrated that the catalytic activities of all three enzymes are under tight allosteric control of their N-terminal domains having autoinhibitory functions. This mechanism provides numerous possibilities for the precise regulation of the methyltransferases via controlling the binding and release of autoinhibitory domains by protein factors, noncoding RNAs, or by posttranslational modifications of the DNMTs. In this chapter, we summarize key enzymatic properties of DNMTs, including their specificity and processivity, and afterward we focus on the regulation of their activity and targeting via allosteric processes, protein interactors, and posttranslational modifications.

  17. The use of legume cover crops in no-tillage broccoli and cabbage production

    OpenAIRE

    Seward, David L.

    1985-01-01

    Field experiments were conducted in 1983 and 1984 to compare conventional tillage (CT) versus no-tillage (NT) production of broccoli and cabbage. The tillage treatments were applied in combination with four rates of applied nitrogen fertilizer and three cover crops: hairy vetch (Vigia villosa Roth), Austrian winter pea (Fisum arvenu L.), and cereal rye (Secale cereale L.). Transplants of 'Premium Crop' broccoli (Brassica oleracea var. italica Plenck) and 'Market Prize'...

  18. Broccoli production depending on the seed production system and organic and mineral fertilizer

    OpenAIRE

    LOPES, José Carlos; Mauri,Janaína; Ferreira,Adésio; Alexandre,Rodrigo S.; Freitas,Allan R de

    2012-01-01

    The experiment was conducted in a greenhouse at the Universidade Federal do Espirito Santo, in Alegre, Espirito Santo state, Brazil, to evaluate the broccoli production depending on the seed production system and organic and mineral fertilization. Seeds of the broccoli cultivar Ramoso Piracicaba were originated from lots of seed obtained in an organic and conventional production system. The experimental design was of randomized blocks, with four replications in a factorial arrangement combina...

  19. Cultivation conditions and selenium fertilization alter the phenolic profile, glucosinolate, and sulforaphane content of broccoli.

    Science.gov (United States)

    Robbins, Rebecca J; Keck, Anna-Sigrid; Banuelos, Gary; Finley, John W

    2005-01-01

    Broccoli is a food often consumed for its potential health-promoting properties. The health benefits of broccoli are partly associated with secondary plant compounds that have bioactivity; glucosinolates and phenolic acids are two of the most abundant and important in broccoli. In an effort to determine how variety, stress, and production conditions affect the production of these bioactive components broccoli was grown in the greenhouse with and without selenium (Se) fertilization, and in the field under conventional or organic farming procedures and with or without water stress. High-performance liquid chromatography/mass spectrometry was used to separate and identify 12 primary phenolic compounds. Variety had a major effect: There was a preponderance of flavonoids in the Majestic variety, but hydroxycinnamic esters were relatively more abundant in the Legacy variety. Organic farming and water stress decreased the overall production of phenolics. Se fertilization increased glucosinolates in general, and sulforaphane in particular, up to a point; above that Se fertilization decreased glucosinolate production. Organic farming and water stress also decreased glucosinolate production. These data show environmental and genetic variation in phenolics and glucosinolates in broccoli, and warn that not all broccoli may contain all health-promoting bioactive components. They further show that selection for one bioactive component (Se) may decrease the content of other bioactive components such as phenolics and glucosinolates.

  20. Consumer acceptability and sensory profile of cooked broccoli with mustard seeds added to improve chemoprotective properties.

    Science.gov (United States)

    Ghawi, Sameer Khalil; Shen, Yuchi; Niranjan, Keshavan; Methven, Lisa

    2014-09-01

    Broccoli, a rich source of glucosinolates, is a commonly consumed vegetable of the Brassica family. Hydrolysis products of glucosinolates, isothiocyanates, have been associated with health benefits and contribute to the flavor of Brassica. However, boiling broccoli causes the myrosinase enzyme needed for hydrolysis to denature. In order to ensure hydrolysis, broccoli must either be mildly cooked or active sources of myrosinase, such as mustard seed powder, can be added postcooking. In this study, samples of broccoli were prepared in 6 different ways; standard boiling, standard boiling followed by the addition of mustard seeds, sous vide cooking at low temperature (70 °C) and sous vide cooking at higher temperature (100 °C) and sous vide cooking at higher temperature followed by the addition of mustard seeds at 2 different concentrations. The majority of consumers disliked the mildly cooked broccoli samples (70 °C, 12 min, sous vide) which had a hard and stringy texture. The highest mean consumer liking was for standard boiled samples (100 °C, 7 min). Addition of 1% mustard seed powder developed sensory attributes, such as pungency, burning sensation, mustard odor, and flavor. One cluster of consumers (32%) found mustard seeds to be a good complement to cooked broccoli; however, the majority disliked the mustard-derived sensory attributes. Where the mustard seeds were partially processed, doubling the addition to 2% led to only the same level of mustard and pungent flavors as 1% unprocessed seeds, and mean consumer liking remained unaltered. This suggests that optimization of the addition level of partially processed mustard seeds may be a route to enhance bioactivity of cooked broccoli without compromising consumer acceptability.

  1. Incidence of Bacterial Disease and Yield of Broccoli as Influenced by Different Rain Protectors and Varieties during the Rainy Season in Southern Thailand

    OpenAIRE

    Karistsapol Nooprom; Quanchit Santipracha

    2014-01-01

    This study is mainly focused on evaluating the effects of different rain protectors and broccoli varieties to find out whether rain protector and variety is suitable or not for broccoli production during rainy season. Broccoli was experimented at Prince of Songkla University, Hat Yai, Songkhla, southern Thailand. Study revealed that broccoli growing under plastic sheet and green shade net had lower incidence of soft rot disease (1.62 and 3.75%, respectively) than those grown in open field (13...

  2. Microbiological quality and production of botulinal toxin in film-packaged broccoli, carrots, and green beans.

    Science.gov (United States)

    Hao, Y Y; Brackett, R E; Beuchat, L R; Doyle, M P

    1999-05-01

    The production of toxin by a 10-strain mixture of proteolytic Clostridium botulinum in fresh produce packaged in polyethylene films with different oxygen permeability was determined. Broccoli florets, shredded carrots, and green beans inoculated with approximately 10(2) C. botulinum spores per g were placed in bags (1.4 kg per bag) composed of four films with different oxygen transmission rates (OTRs). Broccoli was packaged in bags with OTRs of 3 (7,000 cm3/m2/24 h) and 4 (16,000 cm3/m2/24 h), and green beans were packaged in bags with OTRs of 2 (6,000 cm3/m2/24 h) and 4. Broccoli and green beans in bags were compressed and heat-sealed. Shredded carrots were packaged in bags with OTRs of 1 (3,000 cm3/m2/24 h) and 3 and vacuum-sealed. Produce was stored at 4, 13, and 21 degrees C for up to 27 (broccoli) or 28 (carrots and green bean) days and analyzed periodically. At each sampling time, gas composition within the bags, pH of the produce microbial population (total aerobic and anaerobic microorganisms, lactic acid bacteria, psychrotrophic bacteria, yeasts, and molds), and the presence or absence of botulinal toxin were determined. Packaging material affected the quality of vegetables, especially broccoli stored at 4 and 13 degrees C. For example, broccoli was scored as "good" after 22 days at 4 degrees C when it was packaged in film with higher gas permeability (OTR of 4), whereas broccoli appeared to be in "poor" condition when packaged in film with lower gas permeability (OTR of 3). With the exception of lactic acid bacteria, packaging material did not noticeably influence the growth of microorganisms. Lactic acid bacteria grew better in broccoli packaged in bags with an OTR of 3 than in those with an OTR of 4 at all temperatures. Botulinal toxin was detected in broccoli packaged in bags with an OTR of 3 and stored at 13 degrees C for 21 days and in those with an OTR of 4 and 3 and stored at 21 degrees C for 10 days. All toxic samples were visibly spoiled. Toxin

  3. Caffeine synthase and related methyltransferases in plants.

    Science.gov (United States)

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region.

  4. Glucosinolate biosynthesis in hairy root cultures of broccoli (Brassica oleracea var. italica).

    Science.gov (United States)

    Kim, Sun-Ju; Park, Woo Tae; Uddin, Md Romij; Kim, Yeon Bok; Nam, Sang-Yong; Jho, Kwang Hyun; Park, Sang Un

    2013-02-01

    Here we present previously unreported glucosinolate production by hairy root cultures of broccoli (B. oleracea var. italica). Growth media greatly influenced the growth and glucosinolate content of hairy root cultures of broccoli. Seven glucosinolates, glucoraphanin, gluconapin, glucoerucin, glucobrassicin, 4-methoxyglucobrassicin, gluconasturtiin, and neoglucobrassicin, were identified by analysis of the broccoli hairy root cultures. Both half and full strength B5 and SH media enabled the highest accumulation of glucosinolates. In most cases, the levels of glucosinolates were higher in SH and BS media. Among the 7 glucosinolates, the accumulation of neoglucobrassicin was very high, irrespective of growth medium. The neoglucobrassicin content was 7.4-fold higher in SH medium than 1/2 MS, in which its level was the lowest. The 1/2 B5 medium supported the production of the highest amounts of glucobrassicin and 4-methoxyglucobrassicin, the levels for which were 36.2- and 7.9- fold higher, respectively, than their lowest content in 1/2 MS medium. The 1/2 SH medium enabled the highest accumulation of glucoraphanin and gluconapin in the broccoli hairy root cultures, whose levels were 1.8- and 4.6-fold higher, respectively, than their lowest content in 1/2 MS medium. Our results suggest that hairy root cultures of broccoli could be a valuable alternative approach for the production of glucosinolate compounds.

  5. Growth and yield of broccoli fertilized with doses of velvet bean in greenhouse

    Directory of Open Access Journals (Sweden)

    Ellen Rúbia Diniz

    2015-06-01

    Full Text Available The dose effects of green manure in vegetable crops production are still poorly understood. There are few scientific studies indicate that increasing the dose may influence plants characteristics. The objectives were to evaluate the effect of doses of velvet bean green manure on growth and yield, the partitioning of dry matter and nitrogen (N in plants of broccoli, as well as determine the apparent recovery and physiological efficiency of use N. We established four treatments plus a control. The treatments consisted of four levels of green manure: 0, 3, 6, 9 t ha-1 on a dry matter basis, with the addition of 12 t ha-1 of compost. Doses of green manure applied influenced the growth and yield of broccoli. The distribution of dry matter between plant parts of broccoli is not influenced by the doses of green manure. Doses of green manure influenced the amount of N accumulated in the inflorescence and whole plant of broccoli. The apparent recovery of N from green manure and efficiency of N use by plants of broccoli are positively associated with the applied doses.

  6. Effect of matrix and particle type on rheological, textural and structural properties of broccoli pasta and noodles

    NARCIS (Netherlands)

    Silva, E.; Sagis, L.M.C.; Linden, van der E.; Scholten, E.

    2013-01-01

    Durum wheat semolina (DWS) pasta and sweet potato starch (SPS) noodles were incorporated with dif ferent volume fractions and types of broccoli powder (up to 20% v/v). The incorporation of high volume fractions of broccoli powder produced in-house in SPS noodles increases the modulus of the dough an

  7. Separation and Purification of Sulforaphane from Broccoli by Solid Phase Extraction

    Directory of Open Access Journals (Sweden)

    Kyung Ho Row

    2011-03-01

    Full Text Available A simple solid-phase extraction (SPE method for the determination of sulforaphane in broccoli has been developed. The optimal conditions were found to be use of a silica SPE cartridge, and ethyl acetate and dichloromethane as washing and eluting solvents, respectively, which could eliminate interferences originating from the broccoli matrix. The extracts were sufficiently clean to be directly injected into high-performance liquid chromatography (HPLC for further chromatographic analysis. Good linearity was obtained from 0.05 to 200 μg/mL (r = 0.998 for sulforaphane with the relative standard deviations less than 3.6%. The mean recoveries of sulforaphane from broccoli were more than 90.8% and the detection limit (S/N = 3:1 was 0.02 μg/mL. The SPE method provides a higher yield of sulforaphane from crude extracts compared to conventional liquid-liquid extraction.

  8. Effect of Different Growth State of Broccoli (Brassica oleracea var. italica) on Low Temperature Induction

    Institute of Scientific and Technical Information of China (English)

    JIANG Xin-mei; LI Yue-fang; YU Xi-hong

    2005-01-01

    In order to study the responding of different growth state on low temperature in Broccoli (Brassica oleracea var. italica), we took prematurity broccoli hybrid as the objects. It was found that growth state was varied under different sowing time , moreover, stem became wider with the increasing of light density at the same leaf age.Seedling age of responding to low temperature vernalization only when they grew five leaves above and with over 3.03± 0.07 centimeter stalk width in "Qingfeng Broccoli 103". The older leaf age was and the stronger plant was, the more sensitive and the shorter demanded duration time on low temperature was, and the shorter time required when the plants entered into critical period of floral bud differentiation.

  9. The potential to intensify sulforaphane formation in cooked broccoli (Brassica oleracea var. italica) using mustard seeds (Sinapis alba).

    Science.gov (United States)

    Ghawi, Sameer Khalil; Methven, Lisa; Niranjan, Keshavan

    2013-06-01

    Sulforaphane, a naturally occurring cancer chemopreventive, is the hydrolysis product of glucoraphanin, the main glucosinolate in broccoli. The hydrolysis requires myrosinase isoenzyme to be present in sufficient activity; however, processing leads to its denaturation and hence reduced hydrolysis. In this study, the effect of adding mustard seeds, which contain a more resilient isoform of myrosinase, to processed broccoli was investigated with a view to intensify the formation of sulforaphane. Thermal inactivation of myrosinase from both broccoli and mustard seeds was studied. Thermal degradation of broccoli glucoraphanin was investigated in addition to the effects of thermal processing on the formation of sulforaphane and sulforaphane nitrile. Limited thermal degradation of glucoraphanin (less than 12%) was observed when broccoli was placed in vacuum sealed bag (sous vide) and cooked in a water bath at 100°C for 8 and 12 min. Boiling broccoli in water prevented the formation of any significant levels of sulforaphane due to inactivated myrosinase. However, addition of powdered mustard seeds to the heat processed broccoli significantly increased the formation of sulforaphane.

  10. Modelling the change in colour in broccoli and green beans during blanching

    NARCIS (Netherlands)

    Tijskens, L.M.M.; Schijvens, E.P.H.M.; Biekman, E.S.A.

    2001-01-01

    The green colour of vegetables changes considerably during heat treatments like blanching. Green beans from two different countries and growing seasons, and the stems and florets of broccoli were heat-treated from 40 up to 96 °C. The colour was monitored with the CIE-Lab system. Expressing the green

  11. Identification of the phenolic components of collard greens, kale, and Chinese broccoli

    Science.gov (United States)

    An LC-MS profiling method was used to comprehensively study the phenolic components of collard greens, kale, and Chinese broccoli. This study led to the identification of 45 flavonoids and 13 hydroxycinnamic acid derivatives. All three plant materials contained kaempferol glycosides and acylgentio...

  12. Growth and yield responses of broccoli cultivars to different rates of nitrogen at western Chitwan, Nepal

    DEFF Research Database (Denmark)

    Giri, Raj Kumar; Sharma, Moha Datta; Shakya, Santa Man;

    2013-01-01

    A field experiment was conducted with the objective to determine the optimum rate of nitrogen (N) fertilizer for effective growth and yield of two varieties of broccoli in southern plain of Nepal. The experiment was laid out with two-factorial completely random block design (RCBD) comprising two ...

  13. Moisture Sorption Isotherms of Broccoli Interpreted with the Flory-Huggins Free Volume Theory

    NARCIS (Netherlands)

    Jin, X.; Sman, R.G.M. van der; Maanen, J.F.C. van; Deventer, H.C. van; Straten, G. van; Boom, R.M.; Boxtel, A.J.B. van

    2014-01-01

    In this work, the Flory Huggins Free Volume theory is used to interpret the sorption isotherms of broccoli from its composition and using physical properties of the components. This theory considers the mixing properties of water, biopolymers and solutes and has the potential to describe the sorptio

  14. Purification and characterization of broccoli (Brassica oleracea var. italica) myrosinase (β-thioglucosidase glucohydrolase).

    Science.gov (United States)

    Mahn, Andrea; Angulo, Alejandro; Cabañas, Fernanda

    2014-12-03

    Myrosinase (β-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.

  15. Generation of Se-fortified broccoli as functional food: impact of Se fertilization on S metabolism.

    Science.gov (United States)

    Hsu, Fu-Chen; Wirtz, Markus; Heppel, Simon C; Bogs, Jochen; Krämer, Ute; Khan, Muhammad Sayyar; Bub, Achim; Hell, Rüdiger; Rausch, Thomas

    2011-02-01

    Selenium (Se)-fortified broccoli (Brassica oleracea var. italica) has been proposed as a functional food for cancer prevention, based on its high glucosinolate (GSL) content and capacity for Se accumulation. However, as selenate and sulphate share the initial assimilation route, Se fertilization could interfere with sulphur metabolism and plant growth. Consequently, GSL accumulation could be compromised. To evaluate these potentially adverse effects of Se fertilization, we performed a comprehensive study on sand-grown young broccoli plants (weekly selenate applications of 0.8 µmol plant(-1) via the root) and field-grown adult broccoli plants during head formation (single foliar selenate application: 25.3 or 253 µmol plant(-1) ). The results show that under these conditions, Se application does not affect plant growth, contents of cysteine, glutathione, total GSL, glucoraphanin (major aliphatic GSL) or the expression of BoMYB28 (encoding a functionally confirmed master regulator for aliphatic GSL biosynthesis). Conversely, due to the changed expression of sulphate transporters (BoSULTR1;1, 1;2, 2;1, and 2;2), sulphate and total S contents increased in the shoot of young plants while decreasing in the root. We conclude that broccoli can be fertilized with Se without reduction in GSL content, even with Se accumulation exceeding the level recommended for human consumption.

  16. Ingestion of broccoli sprouts does not improve endothelial function in humans with hypertension

    DEFF Research Database (Denmark)

    Christiansen, Buris; Bellostas Muguerza, Natalia; Petersen, Atheline Major;

    2010-01-01

    be the case in humans suffering from essential hypertension. METHODS: 40 hypertensive individuals without diabetes and with normal levels of cholesterol were examined. The participants were randomized either to ingest 10 g dried broccoli sprouts, a natural donor of glucosinolates with high in vitro...

  17. Dielectric properties and heating rate of broccoli powder as related to radio-frequency heating

    Science.gov (United States)

    Recently, Salmonella contamination was identified in low-moisture foods including dried vegetable powder. Radio Frequency (RF) dielectric heating is a potential alternative pasteurization method with short heating time. Dielectric properties of broccoli powder with 6.9, 9.1, 12.2, and 14.9%, w. b....

  18. Broccoli Cultivar Performance under Organic and Conventional Management Systems and Implications for Crop Improvement

    NARCIS (Netherlands)

    Renaud, E.N.C.; Lammerts Van Bueren, E.; Caldas Paulo, M.J.; Eeuwijk, van F.A.; Juvik, J.A.; Hutton, M.G.; Myers, J.R.

    2014-01-01

    To determine if present commercial broccoli cultivars meet the diverse needs of organic management systems, such as adaptation to low N input, mechanical weed management, and no chemical pesticide use, and to propose the selection environments for crop improvement for organic production, we compared

  19. Impact of thermal processing on sulforaphane yield from broccoli ( Brassica oleracea L. ssp. italica).

    Science.gov (United States)

    Wang, Grace C; Farnham, Mark; Jeffery, Elizabeth H

    2012-07-11

    In broccoli, sulforaphane forms when the glucosinolate glucoraphanin is hydrolyzed by the endogenous plant thiohydrolase myrosinase. A myrosinase cofactor directs hydrolysis away from the formation of bioactive sulforaphane and toward an inactive product, sulforaphane nitrile. The cofactor is more heat sensitive than myrosinase, presenting an opportunity to preferentially direct hydrolysis toward sulforaphane formation through regulation of thermal processing. Four broccoli cultivars were microwave heated, boiled, or steamed for various lengths of time. Production of nitrile during hydrolysis of unheated broccoli varied among cultivars from 91 to 52% of hydrolysis products (Pinnacle > Marathon > Patriot > Brigadier). Boiling and microwave heating caused an initial loss of nitrile, with a concomitant increase in sulforaphane, followed by loss of sulforaphane, all within 1 min. In contrast, steaming enhanced sulforaphane yield between 1.0 and 3.0 min in all but Brigadier. These data are proof of concept that steaming for 1.0-3.0 min provides less nitrile and more sulforaphane yield from a broccoli meal.

  20. Fumigation with Phosphine for Postharvest Insect Control on Lettuce, Broccoli, and Strawberries

    Science.gov (United States)

    U.S. exported lettuce, broccoli, and strawberries often harbor western flower thrips (Frankliniella occidentalis), a quarantined pest in Taiwan, and therefore require quarantine treatment. Pure phosphine fumigation at a low temperature of 2°C was studied as an alternative to methyl bromide to contro...

  1. Broccoli consumption interacts with GSTM1 to perturb oncogenic signalling pathways in the prostate.

    Directory of Open Access Journals (Sweden)

    Maria Traka

    Full Text Available BACKGROUND: Epidemiological studies suggest that people who consume more than one portion of cruciferous vegetables per week are at lower risk of both the incidence of prostate cancer and of developing aggressive prostate cancer but there is little understanding of the underlying mechanisms. In this study, we quantify and interpret changes in global gene expression patterns in the human prostate gland before, during and after a 12 month broccoli-rich diet. METHODS AND FINDINGS: Volunteers were randomly assigned to either a broccoli-rich or a pea-rich diet. After six months there were no differences in gene expression between glutathione S-transferase mu 1 (GSTM1 positive and null individuals on the pea-rich diet but significant differences between GSTM1 genotypes on the broccoli-rich diet, associated with transforming growth factor beta 1 (TGFbeta1 and epidermal growth factor (EGF signalling pathways. Comparison of biopsies obtained pre and post intervention revealed more changes in gene expression occurred in individuals on a broccoli-rich diet than in those on a pea-rich diet. While there were changes in androgen signalling, regardless of diet, men on the broccoli diet had additional changes to mRNA processing, and TGFbeta1, EGF and insulin signalling. We also provide evidence that sulforaphane (the isothiocyanate derived from 4-methylsuphinylbutyl glucosinolate that accumulates in broccoli chemically interacts with TGFbeta1, EGF and insulin peptides to form thioureas, and enhances TGFbeta1/Smad-mediated transcription. CONCLUSIONS: These findings suggest that consuming broccoli interacts with GSTM1 genotype to result in complex changes to signalling pathways associated with inflammation and carcinogenesis in the prostate. We propose that these changes may be mediated through the chemical interaction of isothiocyanates with signalling peptides in the plasma. This study provides, for the first time, experimental evidence obtained in humans to

  2. BROCCOLI: Software for Fast fMRI Analysis on Many-Core CPUs and GPUs

    Directory of Open Access Journals (Sweden)

    Anders eEklund

    2014-03-01

    Full Text Available Analysis of functional magnetic resonance imaging (fMRI data is becoming ever more computationally demanding as temporal and spatial resolutions improve, and large, publicly available data sets proliferate. Moreover, methodological improvements in the neuroimaging pipeline, such as non-linear spatial normalization, non-parametric permutation tests and Bayesian Markov Chain Monte Carlo approaches, can dramatically increase the computational burden. Despite these challenges, there do not yet exist any fMRI software packages which leverage inexpensive and powerful graphics processing units (GPUs to perform these analyses. Here, we therefore present BROCCOLI, a free software package written in OpenCL (Open Computing Language that can be used for parallel analysis of fMRI data on a large variety of hardware configurations. BROCCOLI has, for example, been tested with an Intel CPU, an Nvidia GPU and an AMD GPU. These tests show that parallel processing of fMRI data can lead to significantly faster analysis pipelines. This speedup can be achieved on relatively standard hardware, but further, dramatic speed improvements require only a modest investment in GPU hardware. BROCCOLI (running on a GPU can perform non-linear spatial normalization to a 1 mm3 brain template in 4-6 seconds, and run a second level permutation test with 10,000 permutations in about a minute. These non-parametric tests are generally more robust than their parametric counterparts, and can also enable more sophisticated analyses by estimating complicated null distributions. Additionally, BROCCOLI includes support for Bayesian first-level fMRI analysis using a Gibbs sampler. The new software is freely available under GNU GPL3 and can be downloaded from github (https://github.com/wanderine/BROCCOLI/.

  3. Effect of meal composition and cooking duration on the fate of sulforaphane following consumption of broccoli by healthy human subjects.

    Science.gov (United States)

    Rungapamestry, Vanessa; Duncan, Alan J; Fuller, Zoë; Ratcliffe, Brian

    2007-04-01

    The isothiocyanate, sulforaphane, has been implicated in the cancer-protective effects of brassica vegetables. When broccoli is consumed, sulforaphane is released from hydrolysis of glucoraphanin by plant myrosinase and/or colonic microbiota. The influence of meal composition and broccoli-cooking duration on isothiocyanate uptake was investigated in a designed experiment. Volunteers (n 12) were each offered a meal, with or without beef, together with 150 g lightly cooked broccoli (microwaved 2.0 min) or fully cooked broccoli (microwaved 5.5 min), or a broccoli seed extract. They received 3 g mustard containing pre-formed allyl isothiocyanate (AITC) with each meal. Urinary output of allyl (AMA) and sulforaphane (SFMA) mercapturic acids, the biomarkers of production of AITC and sulforaphane respectively, were measured for 24 h after meal consumption. The estimated yield of sulforaphane in vivo was about 3-fold higher after consumption of lightly cooked broccoli than fully cooked broccoli. Absorption of AITC from mustard was about 1.3-fold higher following consumption of the meat-containing meal compared with the non meat-containing alternative. The meal matrix did not significantly influence the hydrolysis of glucoraphanin and its excretion as SFMA from broccoli. Isothiocyanates may interact with the meal matrix to a greater extent if they are ingested pre-formed rather than after their production from hydrolysis of glucosinolates in vivo. The main influence on the production of isothiocyanates in vivo is the way in which brassica vegetables are cooked, rather than the effect of the meal matrix.

  4. Catechol-O-methyltransferase and Parkinson's disease.

    OpenAIRE

    Tai CH; Wu RM

    2002-01-01

    Parkinson's disease (PD) is one of the main causes of neurological disability in the elderly. Levodopa is the gold standard for treating this disease, but chronic levodopa therapy is complicated by motor fluctuation and dyskinesia. The catechol-O-methyltransferase (COMT) inhibitors represent a new class of antiparkinsonian drugs. When coadministered with levodopa/decarboxylase inhibitor, 2 COMT inhibitors, tolcapone and entacapone have been shown to improve the clinical benefit of levodopa. C...

  5. Influence of different blanching methods on colour, ascorbic acid and phenolics content of broccoli.

    Science.gov (United States)

    Severini, C; Giuliani, R; De Filippis, A; Derossi, A; De Pilli, T

    2016-01-01

    Lack of nutrients in cooking water, high energetic costs, high water consumption and recycling are some drawbacks of vegetable blanching. Those disadvantages could be bypassed using microwave blanching. Three blanching methods (microwave, boiling water and steaming) were compared in this study in order to determine their effects on some functional properties of broccoli. In addition, the thermal damage on broccoli colour was evaluated. The effectiveness of each blanching process was performed measuring the lost of peroxidase activity, that results more rapidly in microwaves and steam treatments (50 and 60 s respectively) than in boiling water treatment (120 s). The colour indexes did not allow to discriminate a significant difference among treatments. The increase of treatment time caused a vitamin C decrease in samples blanched by boiling water and steam; this trend was not observed in microwaved samples. The phenols content did not significantly vary depending both on type and on time of treatment.

  6. Response surface optimization and identification of isothiocyanates produced from broccoli sprouts.

    Science.gov (United States)

    Guo, Qianghui; Guo, Liping; Wang, Zhiying; Zhuang, Yan; Gu, Zhenxin

    2013-12-01

    Isothiocyanates (ITCs) are proved as one of natural anticarcinogenic compounds, which are produced from the decomposition of glucosinolates by myrosinase. The present study optimized the enzymolysis conditions (pH, addition of EDTA and ascorbic acid) for ITCs production from glucosinolates in broccoli sprouts using response surface methodology. ITCs production was clearly enhanced by a suitable pH, addition content of EDTA and ascorbic acid. The optimal enzymolysis conditions were determined to be adding EDTA 0.02 mmol and 0.16 mg ascorbic acid to 4 ml of the homogenized phosphate-citrate buffer solution (pH 4.00). ITCs profiles were identified and seven kinds of individual ITCs were detected, among which sulforaphane accounted the most. Four kinds of individual ITCs including isobutyl isothiocyanate, 4-isothiocyanato-1-butene, 1-isothiocyanato-3-methyl-butane and 1-isothiocyanato-butane are firstly reported in broccoli sprouts.

  7. Effects of different cooking methods on health-promoting compounds of broccoli

    Institute of Scientific and Technical Information of China (English)

    Gao-feng YUAN; Bo SUN; Jing YUAN; Qiao-mei WANG

    2009-01-01

    The effects of five domestic cooking methods, including steaming, microwaving, boiling, stir-frying, and stir-frying followed by boiling (stir-frying/boiling), on the nutrients and health-promoting compounds of broccoli were investigated. The results show that all cooking treatments, except steaming, caused significant losses of chlorophyll and vitamin C and significant decreases of total soluble proteins and soluble sugars. Total aliphatic and indole glucosinolates were significantly modified by all cooking treatments but not by steaming. In general, the steaming led to the lowest loss of total glucosinolates, while stir-frying and stir-frying/boiling presented the highest loss. Stir-frying and stir-frying/boiling, the two most popular methods for most homemade dishes in China, cause great losses of chlorophyll, soluble protein, soluble sugar, vitamin C, and glucosinolates, but the steaming method appears the best in retention of the nutrients in cooking broccoli.

  8. Effect of Nitrogen and Boron in Seed Yield and Yield Attributing Characters of Broccoli

    Directory of Open Access Journals (Sweden)

    A. Khanal

    2015-09-01

    Full Text Available Plant nutrient is one of the limiting factors affecting crop production. Nitrogen and boron are major nutrients in case of broccoli. So, an experiment was carried out to evaluate the effect of nitrogen and boron in seed yield and yield attributing characters of broccoli in Rampur, Chitwan during winter season. The experiment was laid out in factorial RCBD design with four levels of nitrogen and two levels of boron. Each plot consists of 25 plants which were separated by 60 * 60 cm spacing. There are altogether eight treatments replicates thrice. Local variety Calabrese was used. Significant effect of different dose of nitrogen and boron on yield attributing characters was found. Also interactive effect of nitrogen and boron in number of pods, pod length, seed yield and number of seeds per pod was found significantly different.

  9. Untargeted Metabolomics Reveals Predominant Alterations in Lipid Metabolism Following Light Exposure in Broccoli Sprouts

    Directory of Open Access Journals (Sweden)

    Mariateresa Maldini

    2015-06-01

    Full Text Available The consumption of vegetables belonging to the family Brassicaceae (e.g., broccoli and cauliflower is linked to a reduced incidence of cancer and cardiovascular diseases. The molecular composition of such plants is strongly affected by growing conditions. Here we developed an unbiased metabolomics approach to investigate the effect of light and dark exposure on the metabolome of broccoli sprouts and we applied such an approach to provide a bird’s-eye view of the overall metabolic response after light exposure. Broccoli seeds were germinated and grown hydroponically for five days in total darkness or with a light/dark photoperiod (16 h light/8 h dark cycle. We used an ultra-performance liquid-chromatography system coupled to an ion-mobility, time-of-flight mass spectrometer to profile the large array of metabolites present in the sprouts. Differences at the metabolite level between groups were analyzed using multivariate statistical analyses, including principal component analysis and correlation analysis. Altered metabolites were identified by searching publicly available and in-house databases. Metabolite pathway analyses were used to support the identification of subtle but significant changes among groups of related metabolites that may have gone unnoticed with conventional approaches. Besides the chlorophyll pathway, light exposure activated the biosynthesis and metabolism of sterol lipids, prenol lipids, and polyunsaturated lipids, which are essential for the photosynthetic machinery. Our results also revealed that light exposure increased the levels of polyketides, including flavonoids, and oxylipins, which play essential roles in the plant’s developmental processes and defense mechanism against herbivores. This study highlights the significant contribution of light exposure to the ultimate metabolic phenotype, which might affect the cellular physiology and nutritional value of broccoli sprouts. Furthermore, this study highlights the

  10. Ozone effects on yield quality of spring oilseed rape and broccoli

    Science.gov (United States)

    Vandermeiren, Karine; De Bock, Maarten; Horemans, Nele; Guisez, Yves; Ceulemans, Reinhart; De Temmerman, Ludwig

    2012-02-01

    The impact of elevated tropospheric ozone (O 3) on the quality of spring oilseed rape ( Brassica napus cv Ability) and broccoli ( Brassica oleracea L. cv Italic cv Monaco) was assessed during a three year Open - Top Chamber (OTC) experiment. Current ambient O 3 levels were compared to an increase of 20 and 40 ppb during 8 h per day over the entire growing season. The qualitative responses were expressed as a function of the accumulated hourly O 3 concentrations over a threshold of 40 ppb (AOT40) and the phytotoxic O 3 dose above a threshold of 6 nmol s -1 m -2 projected leaf area (POD 6). Our results provide clear evidence that O 3 has an influence on the qualitative attributes of the harvested products of these Brassica species. The responses were comparable whether they were expressed as a function of the accumulated O 3 concentrations or of the modelled O 3 uptake. The protein concentration of oilseed rape seeds and broccoli heads was significantly increased in response to O 3. There was also a shift in the fatty acid composition of the vegetable oil derived from seeds of oilseed rape. Oleic acid (18:1) declined significantly ( p broccoli an important shift occurred from indolic to aliphatic GSLs although the total GSL concentration was not changed. The increase in the aliphatic/indolic GSL ratio ( p broccoli were not influenced by O 3; glutathione (GSH) was slightly increased in response to a higher O 3 uptake ( p < 0.05). The consequences of these changes with regard to food and feed quality and human health are discussed.

  11. Computer Vision Based Analysis of Broccoli for Application in a Selective Autonomous Harvester

    OpenAIRE

    Ramirez, Rachael Angela

    2006-01-01

    As technology advances in all areas of society and industry, the technology used to produce one of life's essentials - food - is also improving. The majority of agriculture production in developed countries has gone from family farms to industrial operations. With the advent of large-scale farming, the automation of basic farming operations has increasingly made practical and economic sense. Broccoli, which is still harvested by hand, is one of the most expensive crops to produce. ...

  12. Broccoli yield in response to top-dressing fertilization with green manure and biofertilizer

    Directory of Open Access Journals (Sweden)

    Gilberto Bernardo de Freitas

    2011-10-01

    Full Text Available The objective of this work was to evaluate the productive performance of broccoli under different top-dressing organic fertilizations. The experiment was conducted under protected cultivation, in a completely randomized design with four replications, with two plants per experimental unit. Broccoli seedlings were produced in a commercial substrate in styrofoam trays. The seedlings were transplanted to plastic pots containing 10.0 L of substrate made up of subsoil and organic compost at the ratio of 3:1 (v/v, respectively, which is equivalent to about 20.0 t ha-1 of organic compost at planting. After seedling establishment, the top-dressing fertilization treatments were applied: gliricidia biomass associated or not with liquid biofertilizer of cattle manure to the soil and bokashi. Two control treatments were established: one with mineral fertilization recommended for the crop and the other without top-dressing fertilization. The broccoli production was evaluated (commercial standard. Plants that received mineral fertilizer were more productive, however, they were not significantly different (p>0.05, by Dunnet test, from the plants fertilized with 2.5 t ha-1 gliricidiabiomass (dry mass associated with liquid biofertilizer (2.0 L m-2 applied to soil. Top-dressing fertilizations with only gliricidia, at 2.5 and 5.0 t ha-1 of biomass (dry mass, resulted in no significant increase in production of broccoli inflorescence. The use of bokashi in addition to gliricidia biomass and liquid biofertilizer reduced the efficiency of the fertilization compared with plants that received only gliricidia and liquid biofertilizer.

  13. Growth and yield of broccoli fertilized with doses of velvet bean in greenhouse

    OpenAIRE

    Ellen Rúbia Diniz; Thiago de Oliveira Vargas; Ricardo Henrique Silva Santos; Alysson Roberto de Almeida; Urias Batista Martins de Mattos

    2015-01-01

    The dose effects of green manure in vegetable crops production are still poorly understood. There are few scientific studies indicate that increasing the dose may influence plants characteristics. The objectives were to evaluate the effect of doses of velvet bean green manure on growth and yield, the partitioning of dry matter and nitrogen (N) in plants of broccoli, as well as determine the apparent recovery and physiological efficiency of use N. We established four treatments plus a control....

  14. Organic broccoli production on transition soils: Comparing cover crops, tillage and sidedress N

    OpenAIRE

    Schellenberg, D.L.; Morse, R.D.; Welbaum, G.E.

    2009-01-01

    Metadata only record Little information is available about how farmers in transition to organic practices should manage short- and long-term N fertility. The objectives of this research were (1) to evaluate the leguminous cover crops lablab (Dolichos lablab L.), soybean (Glycine max L.), sunn hemp (Crotalaria juncea L.) and a mixture of sunn hemp and cowpea (Vigna sinensis Endl.) as N sources; (2) to compare N availability and broccoli yield when cover crops were incorporated with conventi...

  15. Integration of High Residue/No-till and Farmscaping Systems in Organic Production of Broccoli

    OpenAIRE

    Benson, Gordon Brinkley, 1964-

    2006-01-01

    High-biomass cover cropping enhances marketable yields in organic production of vegetables, linked to the improvement of soil quality and weed control. Although, during transition from chemical to organic cover-cropping production, especially with no-till systems, reduction of nitrogen availability to the main crop and increase in weed and pest pressure may occur. In 2004-2005, summer and fall broccoli (Brassica oleraceae L. Botrytis Group) crops were grown in twin rows on permanent (contro...

  16. Utility of the broccoli sign in the distinction of prolapsed uterine tumor from cervical tumor

    Energy Technology Data Exchange (ETDEWEB)

    Jha, Priyanka; Chang, Stephanie T. [Department of Radiology, University of California San Francisco, 505 Parnassus Avenue, San Francisco, CA 94143-0628 (United States); Rabban, Joseph T. [Department of Anatomic Pathology, University of California San Francisco, 505 Parnassus Avenue, San Francisco, CA 94143-0628 (United States); Chen, Lee-may [Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, 505 Parnassus Avenue, San Francisco, CA 94143-0628 (United States); Yeh, Benjamin M. [Department of Radiology, University of California San Francisco, 505 Parnassus Avenue, San Francisco, CA 94143-0628 (United States); Coakley, Fergus V., E-mail: Fergus.Coakley@radiology.ucsf.edu [Department of Radiology, University of California San Francisco, 505 Parnassus Avenue, San Francisco, CA 94143-0628 (United States)

    2012-08-15

    Objective: To describe the utility, histopathological basis, and clinical correlates of the broccoli sign. Methods: The committee on human research approved this HIPAA compliant study and waived written informed consent. Based on the records of the senior author and our multidisciplinary Gynecologic Oncology Tumor Board, we retrospectively identified thirteen women (mean age of 48.8 years; range, 34-74) with a cervical mass seen at MR imaging (n = 13) or CT (n = 5) that demonstrated the previously reported broccoli sign (i.e., a soft tissue stalk connecting the cervical mass to the uterine cavity) on one or other modality. All available clinical, imaging, and histopathological records were reviewed, with particular emphasis on initially suspected diagnosis, final proven diagnosis, and outcome. Results: Cervical cancer was the initial clinically suspected diagnosis in 6 of 13 patients. Surgical resection demonstrated prolapsed uterine tumor in all patients, consisting of endometrioid adenocarcinoma (n = 7), carcinosarcoma (n = 2), adenosarcoma (n = 1), and leiomyoma (n = 3). Excluding the three patients with leiomyomas, currently, 7 patients with malignant tumors are disease free after a mean interval of 15 months (range, 3-45) and 3 patients have been lost to follow-up. Conclusion: A stalk connecting an apparent cervical mass seen at CT or MR imaging to the endometrial cavity ('broccoli sign') favors the diagnosis of a prolapsed uterine tumor; these prolapsed uterine tumors can often be malignant but appear to have a good prognosis.

  17. Bioactive glucosinolates and antioxidant properties of broccoli seeds cultivated in Thailand

    Directory of Open Access Journals (Sweden)

    Sarunya Chuanphongpanich

    2006-03-01

    Full Text Available One of the most significant health concerns of cruciferous vegetables is the presence of biologically active compounds, glucosinolates. Broccoli (Brassica oleracea var. italica is a nutritionally important crop grown all over the world. Glucosinolates have been found to have anti-cancer properties. The primary purpose of this study was to evaluate glucosinolate content and antioxidant property in the seeds of broccoli cultivars widely grown in Thailand. Glucosinolates were analyzed with high performance liquid chromatography (HPLC. Total and individual glucosinolate levels varied significantly among cultivars. In all broccoli seeds, 4-methylsulfinylbutylglucosinolate (glucoraphanin was the predominant glucosinolate. The highest total glucosinolates was 65.5 µmol/g DW in ‘Top Green #067’ cultivar, followed by ‘Packman’ (58.6, ‘Green Queen’ (51.2, ‘Pak Ging’ (25.5 and ‘Rod Fai’ (20.3. The antioxidant capacities, including ABTS radical scavenging activity and ferrous ion chelating ability in the methanol and water extracts, were found to be high.

  18. Validation of the Analytical Method for the Determination of Flavonoids in Broccoli

    Directory of Open Access Journals (Sweden)

    Tuszyńska Magdalena

    2014-09-01

    Full Text Available A simple, accurate and selective HPLC method was developed and validated for determination of quercetin and kaempferol, which are the main flavonols in broccoli. The separation was achieved on a reversed-phase C18 column using a mobile phase composed of methanol/water (60/40 and phosphoric acid 0.2% at a flow rate of 1.0 ml min-1. The detection was carried out on a DAD detector at 370 nm. This method was validated according to the requirements for new methods, which include selectivity, linearity, precision, accuracy, limit of detection and limit of quantitation. The current method demonstrates good linearity, with R2 > 0.99. The recovery is within 98.07-102.15% and 97.92-101.83% for quercetin and kaempferol, respectively. The method is selective, in that quercetin and kaempferol are well separated from other compounds of broccoli with good resolution. The low limit of detection and limit of quantitation of quercetin and kaempferol enable the detection and quantitation of these flavonoids in broccoli at low con–centrations.

  19. Dietary chemoprevention of PhIP induced carcinogenesis in male Fischer 344 rats with tomato and broccoli.

    Directory of Open Access Journals (Sweden)

    Kirstie Canene-Adams

    Full Text Available The heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-B]pyridine (PhIP, found in meats cooked at high temperatures, has been implicated in epidemiological and rodent studies for causing breast, prostate, and colorectal cancers. A previous animal study using a xenograft model has shown that whole tomato and broccoli, when eaten in combination, exhibit a marked effect on tumor reduction compared to when eaten alone. Our aim was to determine if PhIP-induced carcinogenesis can be prevented by dietary consumption of whole tomato + broccoli powders. Male Fischer 344 rats (n = 45 were randomized into the following treatment groups: control (AIN93G diet, PhIP (200 ppm in AIN93G diet for the first 20 weeks of the study, or tomato + broccoli + PhIP (mixed in AIN93G diet at 10% each and fed with PhIP for 20 weeks, and then without PhIP for 32 weeks. Study animals were monitored for 52 weeks and were euthanized as necessary based on a set of criteria for health status and tumor burden. Although there appeared to be some hepatic and intestinal toxicity due to the combination of PhIP and tomato + broccoli, these rodents had improved survival and reduced incidence and/or severity of PhIP-induced neoplastic lesions compared to the PhIP-alone treated group. Rats eating tomato + broccoli exhibited a marked decrease in the number and size of cribiform prostatic intraepitheilial neoplasia/carcinoma in situ (cribiform PIN/CIS lesions and in the incidence of invasive intestinal adenocarcinomas and skin carcinomas. Although the apparent toxic effects of combined PhIP and tomato + broccoli need additional study, the results of this study support the hypothesis that a diet rich in tomato and broccoli can reduce or prevent dietary carcinogen-induced cancers.

  20. Detection of the Diversity of Cytoplasmic Male Sterility Sources in Broccoli (Brassica Oleracea var. Italica) Using Mitochondrial Markers.

    Science.gov (United States)

    Shu, Jinshuai; Liu, Yumei; Li, Zhansheng; Zhang, Lili; Fang, Zhiyuan; Yang, Limei; Zhuang, Mu; Zhang, Yangyong; Lv, Honghao

    2016-01-01

    Broccoli (Brassica oleracea var. italica) is an important commercial vegetable crop. As part of an efficient pollination system, cytoplasmic male sterility (CMS) has been widely used for broccoli hybrid production. Identifying the original sources of CMS in broccoli accessions has become an important part of broccoli breeding. In this study, the diversity of the CMS sources of 39 broccoli accessions, including 19 CMS lines and 20 hybrids, were analyzed using mitochondrial markers. All CMS accessions contained the ogu orf138-related DNA fragment and the key genes of nap CMS, pol CMS, and tour CMS were not detected. The 39 CMS accessions were divided into five groups using six orf138-related and two simple sequence repeat markers. We observed that ogu CMS R3 constituted 79.49% of the CMS sources. CMS6 and CMS26 were differentiated from the other accessions using a specific primer. CMS32 was distinguished from the other accessions based on a 78-nucleotide deletion at the same locus as the orf138-related sequence. When the coefficient was about 0.90, five CMS accessions (13CMS6, 13CMS23, 13CMS24, 13CMS37, and 13CMS39) exhibiting abnormal floral organs with poor seed setting were grouped together. The polymerase chain reaction amplification profiles for these five accessions differed from those of the other accessions. We identified eight useful molecular markers that can be used to detect CMS types during broccoli breeding. Our data also provide important information relevant to future studies on the possible origins and molecular mechanisms of CMS in broccoli.

  1. Improving cancer immunotherapy with DNA methyltransferase inhibitors.

    Science.gov (United States)

    Saleh, Mohammad H; Wang, Lei; Goldberg, Michael S

    2016-07-01

    Immunotherapy confers durable clinical benefit to melanoma, lung, and kidney cancer patients. Challengingly, most other solid tumors, including ovarian carcinoma, are not particularly responsive to immunotherapy, so combination with a complementary therapy may be beneficial. Recent findings suggest that epigenetic modifying drugs can prime antitumor immunity by increasing expression of tumor-associated antigens, chemokines, and activating ligands by cancer cells as well as cytokines by immune cells. This review, drawing from both preclinical and clinical data, describes some of the mechanisms of action that enable DNA methyltransferase inhibitors to facilitate the establishment of antitumor immunity.

  2. EFFECTS OF PLANTING DATES AND VARIETIES ON GROWTH AND YIELD OF BROCCOLI DURING RAINY SEASON

    Directory of Open Access Journals (Sweden)

    Karistsapol Nooprom

    2013-01-01

    Full Text Available Broccoli (Brassica oleracea var. italica is one of the most popular vegetable crops in many countries of the world as its nutritional importance. It has the most exacting climatic and cultural requirements, which limit its commercial production to a few favored locations. In Thailand, broccoli has been expending in recent years and amount of increasing production. It is best planted in the North where the weather is cool or in the Northeast during winter season. The South has tropical climates and raining all year round, but farmers can produce broccoli during early summer by using the heat-tolerant varieties. Although the broccoli is highly priced and still wanted by consumers in the rainy season, the farmers might not choose to grow as the problems with low productivity. Thus, the selection of suitable planting dates and varieties is crucial for successful cultivation during rainy season in the South. The experiment was performed to evaluate the effects of planting dates and broccoli varieties on their growth and yield responses during rainy season. Four broccoli varieties: ‘Top Green’, ‘Green Queen’, ‘Yok Kheo’ and ‘Special’ were plated for six planting dates: July, August, September, October, November and December at Prince of Songkla University, Thailand, between July, 2011 and January, 2012. Split-plot in a randomized complete block design was used in this experiment with four replications. This study showed that four broccoli varieties had highly seedling survival rates of 70.31-99.22% when planting in July-December except ‘Special’ of 54.68% when planting in August. ‘Yok Kheo’ had plant diameter of 67.54-75.24 cm when planting in July and December, not significantly different from ‘Top Green’ planting in July, August and November of 69.15-71.24 cm and ‘Green Queen’ in July and September of 68.69-68.88 cm. The highest total yield was obtained by ‘Yok Kheo

  3. Characteristic of phenolic compound and antioxidant activity of fermented broccoli (Brassica oleracea L. ssp.) beverage by lactic acid bacteria (LAB)

    Science.gov (United States)

    Maryati, Yati; Susilowati, Agustine; Melanie, Hakiki; Lotulung, Puspa D.

    2017-01-01

    Broccoli (Brassica oleracea L. ssp.) has a relatively high nutrient content, especially as a source of vitamins, minerals and fiber and contain bioactive compounds that act as antioxidants. In order to increase the nutritional value and innovate new products, fermentation process involving rich-antioxidants lactic acid bacteria (LAB) was done. The aim of this study is to determine the content of bioactive components, such as total polyphenols, total acid and antioxidant activity of the mixed culture of LAB (L. bulgaricus, S. thermophulus, L. acidophilus, Bd. bifidum)-fermented broccoli extracts. Ratio of fermented broccoli extract and concentration of starter cultureLAB was varied in the range of 5, 10, 15 and 20% (v/v), and the alterations of characteristics of the fermented broccoli extract, before and after fermentation (0 and 24 hours), were evaluated. The results showed that fermentation functional beverage broccoli with different concentrations of LAB cultures affect the antioxidant activity, total polyphenols, total acid and total cell of LAB generated. The optimum conditions obtained for the highest antioxidant activity of 6.74%, at aculture concentration of 20% during fermentation time of 24 h with a pH value of 4.29, total sugar of 10.89%, total acids of 0.97%, total polyphenols of 0.076%, and total LAB of 13.02 + 0.05 log cfu /ml.

  4. Novel non-specific DNA adenine methyltransferases

    Science.gov (United States)

    Drozdz, Marek; Piekarowicz, Andrzej; Bujnicki, Janusz M.; Radlinska, Monika

    2012-01-01

    The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N6-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N6-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N6-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential ‘sequence specificity’ could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation. PMID:22102579

  5. Effects of industrial pre-freezing processing and freezing handling on glucosinolates and antioxidant attributes in broccoli florets.

    Science.gov (United States)

    Cai, Congxi; Miao, Huiying; Qian, Hongmei; Yao, Leishuan; Wang, Bingliang; Wang, Qiaomei

    2016-11-01

    The effects of industrial pre-freezing processing and freezing handling on the contents of glucosinolates and antioxidants (vitamin C, polyphenols, carotenoid and chlorophyll), as well as the antioxidant capacity in broccoli (Brassica oleracea L. var. italica) florets were investigated in the present study. Our results showed that the glucosinolate accumulations were significantly decreased after pre-freezing processing, whereas elevated levels of phenols, carotenoids, chlorophyll, and also antioxidant capacity were observed in frozen broccoli florets. The contents of vitamin C remained constant during above mentioned processing. In conclusion, the current industrial freezing processing method is a good practice for the preservation of main antioxidant nutrients in broccoli florets, although some improvements in pre-freezing processing, such as steam blanching and ice-water cooling, are needed to attenuate the decrease in glucosinolate content.

  6. Evolution of novel O-methyltransferases from the Vanilla planifolia caffeic acid O-methyltransferase.

    Science.gov (United States)

    Li, Huaijun Michael; Rotter, David; Hartman, Thomas G; Pak, Fulya E; Havkin-Frenkel, Daphna; Belanger, Faith C

    2006-06-01

    The biosynthesis of many plant secondary compounds involves the methylation of one or more hydroxyl groups, catalyzed by O-methyltransferases (OMTs). Here, we report the characterization of two OMTs, Van OMT-2 and Van OMT-3, from the orchid Vanilla planifolia Andrews. These enzymes catalyze the methylation of a single outer hydroxyl group in substrates possessing a 1,2,3-trihydroxybenzene moiety, such as methyl gallate and myricetin. This is a substrate requirement not previously reported for any OMTs. Based on sequence analysis these enzymes are most similar to caffeic acid O-methyltransferases (COMTs), but they have negligible activity with typical COMT substrates. Seven of 12 conserved substrate-binding residues in COMTs are altered in Van OMT-2 and Van OMT-3. Phylogenetic analysis of the sequences suggests that Van OMT-2 and Van OMT-3 evolved from the V. planifolia COMT. These V. planifolia OMTs are new instances of COMT-like enzymes with novel substrate preferences.

  7. Distinction between the Cfr Methyltransferase Conferring Antibiotic Resistance and the Housekeeping RlmN Methyltransferase

    DEFF Research Database (Denmark)

    Atkinson, Gemma C; Hansen, Lykke H; Tenson, Tanel

    2013-01-01

    The cfr gene encodes the Cfr methyltransferase that primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. The rlmN gene encodes the Rlm......N methyltransferase that methylates C-2 in A2503 in 23S rRNA and A37 in tRNA, but RlmN does not significantly influence antibiotic resistance. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine....... The differentiation between the two classes is supported by previous and new experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. Finally, evolutionary aspects of the distribution of Cfr- and RlmN-like enzymes are discussed....

  8. Combination treatments for killing Escherichia coli O157:H7 on alfalfa, radish, broccoli, and mung bean seeds.

    Science.gov (United States)

    Bari, M L; Nei, D; Enomoto, K; Todoriki, S; Kawamoto, S

    2009-03-01

    In this study, the effectiveness of prolonged dry-heat treatment (50 degrees C) alone or in combination with chemical treatments (1% oxalic acid, 0.03% phytic acid, 50% ethanol, electrolyzed acidic water, and electrolyzed alkaline water) in eliminating Escherichia coli O157:H7 on laboratory-inoculated alfalfa, radish, broccoli, and mung bean seeds was compared with that of dry-heat treatment in combination with irradiation treatment. Dry-heat treatment for 17 or 24 h alone could reduce E. coli O157:H7 numbers to below detectable levels in radish, broccoli, and alfalfa seeds, but was unable to reduce the pathogen numbers to below the detectable level in mung bean seeds. In addition, dry-heat treatment for 17 h plus sanitizer treatments were effective in greatly reducing pathogen populations on radish, broccoli, and alfalfa seeds, without compromising the quality of the sprouts, but these treatments did not eliminate the pathogen from radish and alfalfa seeds. Seventeen hours of dry heat followed by a 1.0-kGy dose of irradiation completely eliminated E. coli O157:H7 from radish and mung bean seeds, whereas only a minimum radiation dose of 0.25 kGy was required to completely eliminate the pathogen from broccoli and alfalfa seeds. Dry heat in combination with radiation doses of up to 1.0 kGy did not negatively impact the seed germination rate or length of alfalfa, broccoli, and radish seeds or the length of alfalfa, broccoli, and radish sprouts, but did decrease the length of mung bean sprouts.

  9. Effects of different substrates and salinity on growth and yield of broccoli (Brassica oleracea var. italica in soilless culture

    Directory of Open Access Journals (Sweden)

    2015-11-01

    Full Text Available Salinity is a very serious problem for agricultural development, especially in arid and semi-arid regions. On the other hand, many benefits of soilless culture have caused the expansion of these systems in these areas. In this research, in order to investigate the effects of different substrates and salinity stress on physiological characteristics and yield of broccoli, an experiment was conducted as factorial, in which the main factor was substrate (cocopeat, perlite, sand, 25% cocopeat+75% perlite, 25% perlite+75% cocopeat and 50% peat+50% pumice and the sub factor was salinity stress (0, 100 and 150 mM NaCl with 4 replications. Results indicated that substrate, different levels of salinity and their interaction had significant effects on morphological characteristics, days to flowering, fresh and dry weight of leaves, nutrients concentration and weight of the broccoli head. Maximum values of the aforementioned factors were observed in the zero salinity level and 50% peat+50% pumice substrate. This means that substrate can be effective on the effect of salinity on plants. High levels of salinity caused a reduction in growth and yield of broccoli and this growth reduction was accompanied by the reduction of chlorophyll and content of Ca, K, and Fe and increased Na and Cl concentration in leaf tissue. The best growing medium, either in saline or non-saline conditions, was 50% peat+50% pumice. Although broccoli growth was not suitable in perlite medium, this substrate caused its early maturing. In this experiment, salinity treatment reduced the head weight of broccoli, in addition to decreasing the quality of yield. According to the results, 50% peat+50 pumice is recommended as a proper substrate for production of broccoli.

  10. A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase.

    Science.gov (United States)

    Ticak, Tomislav; Kountz, Duncan J; Girosky, Kimberly E; Krzycki, Joseph A; Ferguson, Donald J

    2014-10-28

    COG5598 comprises a large number of proteins related to MttB, the trimethylamine:corrinoid methyltransferase. MttB has a genetically encoded pyrrolysine residue proposed essential for catalysis. MttB is the only known trimethylamine methyltransferase, yet the great majority of members of COG5598 lack pyrrolysine, leaving the activity of these proteins an open question. Here, we describe the function of one of the nonpyrrolysine members of this large protein family. Three nonpyrrolysine MttB homologs are encoded in Desulfitobacterium hafniense, a Gram-positive strict anaerobe present in both the environment and human intestine. D. hafniense was found capable of growth on glycine betaine with electron acceptors such as nitrate or fumarate, producing dimethylglycine and CO2 as products. Examination of the genome revealed genes for tetrahydrofolate-linked oxidation of a methyl group originating from a methylated corrinoid protein, but no obvious means to carry out corrinoid methylation with glycine betaine. DSY3156, encoding one of the nonpyrrolysine MttB homologs, was up-regulated during growth on glycine betaine. The recombinant DSY3156 protein converts glycine betaine and cob(I)alamin to dimethylglycine and methylcobalamin. To our knowledge, DSY3156 is the first glycine betaine:corrinoid methyltransferase described, and a designation of MtgB is proposed. In addition, DSY3157, an adjacently encoded protein, was shown to be a methylcobalamin:tetrahydrofolate methyltransferase and is designated MtgA. Homologs of MtgB are widely distributed, especially in marine bacterioplankton and nitrogen-fixing plant symbionts. They are also found in multiple members of the human microbiome, and may play a beneficial role in trimethylamine homeostasis, which in recent years has been directly tied to human cardiovascular health.

  11. Structures of NS5 Methyltransferase from Zika Virus

    Directory of Open Access Journals (Sweden)

    Javier Coloma

    2016-09-01

    Full Text Available The Zika virus (ZIKV poses a major public health emergency. To aid in the development of antivirals, we present two high-resolution crystal structures of the ZIKV NS5 methyltransferase: one bound to S-adenosylmethionine (SAM and the other bound to SAM and 7-methyl guanosine diphosphate (7-MeGpp. We identify features of ZIKV NS5 methyltransferase that lend to structure-based antiviral drug discovery. Specifically, SAM analogs with functionalities on the Cβ atom of the methionine portion of the molecules that occupy the RNA binding tunnel may provide better specificity relative to human RNA methyltransferases.

  12. IDENTIFICATION OF ANTAGONISTS OF Xanthomonas campestris ISOLATED FROM RHIZOSPHERE ZONE OF BROCCOLI FARM AT KEMBANG MERTA VILLAGE, TABANAN, BALI

    Directory of Open Access Journals (Sweden)

    Nadya Treesna Wulansari

    2015-03-01

    Full Text Available The main objectives of this research were to isolate and identify antagonists of Xanthomonas campestris from rhizosphere zone of broccoli plants. Soil samples were collected from broccoli farm located at Kembang Merta village, Tabanan, Bali. Isolation and identification of the antagonists were conducted at the Laboratory of Microbiology, Udayana University. Two fungal (Trichoderma harzianum and Trichoderma viride and two bacterial (Bacillus sp. and Pseudomonas sp. antagonists potentially to be developed as biocontrol agents of Xanthomonas campestris were successfully identified in this research

  13. Broccoli Microgreens: A Mineral-Rich Crop That Can Diversify Food Systems

    Science.gov (United States)

    Weber, Carolyn F.

    2017-01-01

    Current malnourishment statistics are high and are exacerbated by contemporary agricultural practices that damage the very environments on which the production of nutritious food depends. As the World’s population grows at an unprecedented rate, food systems must be revised to provide adequate nutrition while minimizing environmental impacts. One specific nutritional problem that needs attention is mineral (e.g., Fe and Zn) malnutrition, which impacts over two-thirds of the World’s people living in countries of every economic status. Microgreens, the edible cotyledons of many vegetables, herbs, and flowers, is a newly emerging crop that may be a dense source of nutrition and has the potential to be produced in just about any locale. This study examined the mineral concentration of broccoli microgreens produced using compost-based and hydroponic growing methods that are easily implemented in one’s own home. The nutritional value of the resulting microgreens was quantitatively compared to published nutritional data for the mature vegetable. Nutritional data were also considered in the context of the resource demands (i.e., water, fertilizer, and energy) of producing microgreens in order to gain insights into the potential for local microgreen production to diversify food systems, particularly for urban areas, while minimizing the overall environmental impacts of broccoli farming. Regardless of how they were grown, microgreens had larger quantities of Mg, Mn, Cu, and Zn than the vegetable. However, compost-grown (C) microgreens had higher P, K, Mg, Mn, Zn, Fe, Ca, Na, and Cu concentrations than the vegetable. For eight nutritionally important minerals (P, K, Ca, Mg, Mn, Fe, Zn, and Na), the average C microgreen:vegetable nutrient ratio was 1.73. Extrapolation from experimental data presented here indicates that broccoli microgreens would require 158–236 times less water than it does to grow a nutritionally equivalent amount of mature vegetable in the fields of

  14. Economic Efficiency of Broccoli Production Depending on Sowing and Planting Dates

    OpenAIRE

    KRISHKOVA, Iliana; Todorova, Desislava

    2014-01-01

    An experiment was set up to study the economic effect of different sowing and planting dates. The field trial was conducted in the experimental garden of the Institute of Agriculture - Kyustendil over the period 20082011 via the block method in four replications. Three sowing and six planting dates were examined for the four broccoli hybrids used: Fiesta F1, Coronado F1, Marathon F1 and Parthenon F1. The plants were grown according to the adopted methodology for late field production. The flo...

  15. Interactions within the mammalian DNA methyltransferase family

    Directory of Open Access Journals (Sweden)

    Ehrenhofer-Murray Ann E

    2003-05-01

    Full Text Available Abstract Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase.

  16. Monolignol 4-O-methyltransferases and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Incidence of Bacterial Disease and Yield of Broccoli as Influenced by Different Rain Protectors and Varieties during the Rainy Season in Southern Thailand

    Directory of Open Access Journals (Sweden)

    Karistsapol Nooprom

    2014-04-01

    Full Text Available This study is mainly focused on evaluating the effects of different rain protectors and broccoli varieties to find out whether rain protector and variety is suitable or not for broccoli production during rainy season. Broccoli was experimented at Prince of Songkla University, Hat Yai, Songkhla, southern Thailand. Study revealed that broccoli growing under plastic sheet and green shade net had lower incidence of soft rot disease (1.62 and 3.75%, respectively than those grown in open field (13.33% while their growing under plastic sheet (1.50% had lower incidence of black rot disease than those grown under green shade net and open field (18.75 and 32.88%, respectively. All broccoli varieties were found to be statistically different in their response towards soft rot and black rot diseases. However, the Top Green had the highest diseases incidence (8.33 and 21.08%, respectively while the Yok Kheo had the lowest diseases incidence (4.62 and 0.00%, respectively. The highest total yield was obtained from the Yok Kheo when those grown under plastic sheet (13.48 t/ha while the Top Green had lowest yield when those grown in open field (3.94 t/ha. Therefore, the most suitable method for broccoli production during rainy season in southern Thailand was to grow under plastic sheet and green shade net by using the three varieties of broccoli.

  18. Optimization of a blanching step to maximize sulforaphane synthesis in broccoli florets.

    Science.gov (United States)

    Pérez, Carmen; Barrientos, Herna; Román, Juan; Mahn, Andrea

    2014-02-15

    A blanching step was designed to favor sulforaphane synthesis in broccoli. Blanching was optimised through a central composite design, and the effects of temperature (50-70 °C) and immersion time in water (5-15 min) on the content of total glucosinolates, glucoraphanin, sulforaphane, and myrosinase activity were determined. Results were analysed by ANOVA and the optimal condition was determined through response surface methodology. Temperature between 50 and 60 °C significantly increased sulforaphane content (pblanching at 70 and 74 °C diminished significantly this content, compared to fresh broccoli. The optimal blanching conditions given by the statistical model were immersion in water at 57 °C for 13 min; coinciding with the minimum glucosinolates and glucoraphanin content, and with the maximum myrosinase activity. In the optimal conditions, the predicted response of 4.0 μmol sulforaphane/g dry matter was confirmed experimentally. This value represents a 237% increase with respect to the fresh vegetable.

  19. Phenotypic and genotypic characterization of Xanthomonas campestris strains isolated from cabbage, kale and broccoli

    Directory of Open Access Journals (Sweden)

    Popović Tatjana

    2013-01-01

    Full Text Available Thirty-six strains of Xanthomonas campestris pv. campestris (Xcc isolated from cabbage, kale and broccoli were identified according to their pathogenicity, phenotypic and genotypic characterization. Pathogenicity was confirmed by the injection method with a hypodermic syringe into the mesophilic tissue of cabbage leaves. All strains were Gramnegative, aerobic, catalase-positive, oxidase-negative, grew at 35°C, produced levan, H2S and indole, did not reduce nitrate, hydrolyzed Tween 80, starch, gelatin and esculin and did not show tolerance to 0.1 and 0.02% TTC. The strains produced acid from d-arabinose, arginine, dulcitol, galactose, d-glucose, maltose, mannose, sorbitol, sucrose and xylose. The genetic characterization was based on the sequence analyses of 16S rDNA and ERIC and BOX PCR. Strains of different pathovars were also used to compare PCR resulting patterns. BOX-PCR of the strains from kale and broccoli, obtained using (GTG5 primer, yielded patterns with a high similarity level to pathovar reference strain Xcc. The strains from cabbage yielded BOX and ERIC product patterns, distinguishing them from the other tested strains and reference strains. 16S rDNA of the representative strains was closely related to Xcc strain ATCC 33913. ERIC PCR and BOX using (GTG5 primer generated different Xcc patterns and were effective in distinguishing strains from different plant hosts. [Projekat Ministarstva nauke Republike Srbije, br. III43010 i br. III46007

  20. Structural characterization of a broccoli polysaccharide and evaluation of anti-cancer cell proliferation effects.

    Science.gov (United States)

    Xu, Lishan; Cao, Jingjing; Chen, Wenrong

    2015-08-01

    Broccoli is a widely consumed vegetable with abundant amount of nutrients, which bring numerous beneficial effects on human health. The structural information of water-soluble polysaccharides in broccoli was eludicated for the first time in this work. A purified polysaccharide fraction (BPCa) was obtained by column chromatography. It comprised of arabinose (Ara), galactose (Gal), rhamnose (Rha) with a molar ratio of 5.3:0.8:1.0. Nuclear magnetic resonnance spectra data revealed that α-L-1,5-Araf and α-L-1,3,5-Araf are present in the backbone, while α-L-Araf terminal was attended in side chain. α-L-1,2-Rhap was found to be linked to α-L-1,5-Araf in heteronuclear multiple bond correlation spectra. The presences of β-D-1,4-Galp and α-D-1,4-GalpA were also detected. Furthermore, BPCa showed significant anti-cancer cell proliferation activities against HepG2, Siha and MDA-MB-231 carcinoma cell lines. The results indicated that BPCa had a good potential to be applied as functional food additives.

  1. Persistence of Indicator and Pathogenic Microorganisms in Broccoli following Manure Spreading and Irrigation with Fecally Contaminated Water: Field Experiment.

    Science.gov (United States)

    Généreux, Mylène; Breton, Marie Jo; Fairbrother, John Morris; Fravalo, Philippe; Côté, Caroline

    2015-10-01

    In 2011 and 2012, trials consisting of experimental plots were carried out to evaluate the presence of pathogenic (Listeria monocytogenes, Salmonella) and prevalence of indicator (Escherichia coli) microorganisms in broccoli fertilized with liquid hog manure or mineral fertilizers and irrigated zero, one, or two times with E. coli-contaminated water. In 2011, results showed that E. coli contamination in broccoli heads was affected by the interval between irrigation and sampling (P = 0.0236), with a significant decrease between the first and third day following irrigation (P = 0.0064). In 2012, irrigation frequency significantly increased E. coli prevalence in broccoli samples (P = 0.0499). In 2012, E. coli counts in the soil were significantly influenced by the type of fertilizer applied, as plots receiving liquid hog manure showed higher bacterial counts (P = 0.0006). L. monocytogenes was recovered in one broccoli sample, but geno-serogrouping differentiated the isolate from those recovered in manure and irrigation water. The L. monocytogenes serogroup IIA, pulsotype 188 strain was found in six soil samples and in irrigation water applied 5 days before soil sampling. This study highlights the link between E. coli levels in irrigation water, irrigation frequency, and interval between irrigation and harvest on produce contamination. It also demonstrates that L. monocytogenes introduced into the soil following irrigation can persist for up to 5 days.

  2. Variation in Broccoli Cultivar Phytochemical Content under Organic and Conventional Management Systems: Implications in Breeding for Nutrition

    NARCIS (Netherlands)

    Renaud, E.N.C.; Lammerts Van Bueren, E.; Myers, J.R.; Caldas Paulo, M.J.; Eeuwijk, van F.A.; Zhu, N.; Juvik, J.A.

    2014-01-01

    Organic agriculture requires cultivars that can adapt to organic crop management systems without the use of synthetic pesticides as well as genotypes with improved nutritional value. The aim of this study encompassing 16 experiments was to compare 23 broccoli cultivars for the content of phytochemic

  3. Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence.

    Science.gov (United States)

    Liu, Mao-Sen; Li, Hui-Chun; Chang, You-Min; Wu, Min-Tze; Chen, Long-Fang Oliver

    2011-09-01

    Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli (Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase (ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days' post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.

  4. Optimisation of enzymatic production of sulforaphane in broccoli sprouts and their total antioxidant activity at different growth and storage days.

    Science.gov (United States)

    Tian, Ming; Xu, Xiaoyun; Hu, Hao; Liu, Yu; Pan, Siyi

    2017-01-01

    Sulforaphane, a type of isothiocyanate hydrolysed from glucosinolate, is a powerful anticancer compound naturally found in food especially in broccoli sprouts. Despite the function of sulforaphane has been extensively studied in recent years, little attention has been given to methods that can maximize the production of this compound in broccoli sprouts. The present study optimised the enzymolysis conditions for sulforaphane production in broccoli sprouts using response surface methodology. The maximum sulforaphane production (246.95 μg/g DW) was achieved using a solid-liquid ratio of 1:30, hydrolysis time of 1.5 h, ascorbic acid content of 3.95 mg/g DW sample, and temperature of 65 °C. The highest sulforaphane content in broccoli sprouts were 233.80 μg/g DW in 5-day-old sprouts and 1555.95 μg/g DW at day 4 of storage. The highest antioxidant activities were 37.22 U/min/g DW in 3-day-old sprouts and 35.08 U/min/g DW on 4th day of storage.

  5. Biotechnological applications in in vitro plant regeneration studies of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

    Science.gov (United States)

    Kumar, Pankaj; Srivastava, Dinesh Kumar

    2016-04-01

    Biotechnology holds promise for genetic improvement of important vegetable crops. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop of the family Brassicaceae. However, various biotic and abiotic stresses cause enormous crop yield losses during commercial cultivation of broccoli. Establishment of a reliable, reproducible and efficient in vitro plant regeneration system with cell and tissue culture is a vital prerequisite for biotechnological application of crop improvement programme. An in vitro plant regeneration technique refers to culturing, cell division, cell multiplication, de-differentiation and differentiation of cells, protoplasts, tissues and organs on defined liquid/solid medium under aseptic and controlled environment. Recent progress in the field of plant tissue culture has made this area one of the most dynamic and promising in experimental biology. There are many published reports on in vitro plant regeneration studies in broccoli including direct organogenesis, indirect organogenesis and somatic embryogenesis. This review summarizes those plant regeneration studies in broccoli that could be helpful in drawing the attention of the researchers and scientists to work on it to produce healthy, biotic and abiotic stress resistant plant material and to carry out genetic transformation studies for the production of transgenic plants.

  6. Pure phosphine fumigation treatment at low temperature for postharvest control of western flower thrips on lettuce, broccoli, asparagus, and strawberries

    Science.gov (United States)

    U.S. exported lettuce, broccoli, asparagus, and strawberries often harbor western flower thrips (Frankliniella occidentalis), a quarantined pest in Taiwan, and therefore require quarantine treatment. Pure phosphine fumigation at a low temperature of 2°C was studied as an alternative fumigant to meth...

  7. Low temperature phosphine fumigation for postharvest control of western flower thrips (Thysanoptera: Thripidae) on lettuce, broccoli, asparagus, and strawberry.

    Science.gov (United States)

    Liu, Yong-Biao

    2008-12-01

    U.S. exported lettuce, broccoli, asparagus, and strawberries often harbor western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), a quarantined pest in Taiwan, and therefore require quarantine treatment. Fumigation with diluted pure phosphine at a low temperature of 2 degrees C was studied to control western flower thrips and to determine effects on the quality of the treated products. Total thrips control was achieved in > or = 18-h fumigation treatments with > or = 250 ppm phosphine. One day fumigation treatment with 1,000 ppm phosphine was tested on lettuce and broccoli. One-day fumigation treatments with 500 ppm and 1,000 ppm phosphine were tested on asparagus and strawberry. Visual quality of lettuce, broccoli, and asparagus was evaluated after 2-wk posttreatment storage. Strawberry quality was evaluated immediately after fumigation and after 1-wk posttreatment storage. For all the products, there were no significant differences between the treatments and the controls in postharvest quality, and there were no injuries caused by the fumigation treatments. Therefore, phosphine fumigation at low temperature was promising for postharvest control of western flower thrips on lettuce, broccoli, asparagus, and strawberry.

  8. The response of broccoli (Brassica oleracea convar. italica) varieties on foliar application of selenium: uptake, translocation, and speciation.

    Science.gov (United States)

    Šindelářová, Kristýna; Száková, Jiřina; Tremlová, Jana; Mestek, Oto; Praus, Lukáš; Kaňa, Antonín; Najmanová, Jana; Tlustoš, Pavel

    2015-01-01

    A model small-scale field experiment was set up to investigate selenium (Se) uptake by four different varieties of broccoli plants, as well as the effect of Se foliar application on the uptake of essential elements for plants calcium (Ca), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), phosphorus (P), sulfur (S), and zinc (Zn). Foliar application of sodium selenate (Na2SeO4) was carried out at two rates (25 and 50 g Se/ha), and an untreated control variant was included. Analyses of individual parts of broccoli were performed, whereby it was found that Se in the plant accumulates mainly in the flower heads and slightly less in the leaves, stems, and roots, regardless of the Se rate and broccoli variety. In most cases, there was a statistically significant increase of Se content in all parts of the plant, while there was no confirmed systematic influence of the addition of Se on the changing intake of other monitored elements. Selenization of broccoli leads to an effective increase in the Se content at a rate of 25 g/ha, whereas the higher rate did not result in a substantial increase of Se content compared to the lower rate in all varieties. Therefore, the rate of 25 g/ha can be recommended as effective to produce broccoli with an increased Se content suitable for consumption. Moreover, Se application resulted in an adequate increase of the main organic compounds of Se, such as selenocystine (SeCys2), selenomethionine (SeMet), and Se-methylselenocysteine (Se-MeSeCys).

  9. Systematic Comparisons of Orthologous Selenocysteine Methyltransferase and Homocysteine Methyltransferase Genes from Seven Monocots Species

    Directory of Open Access Journals (Sweden)

    De-yong ZHAO

    2015-06-01

    Full Text Available Identifying and manipulating genes underlying selenium metabolism could be helpful for increasing selenium content in crop grain, which is an important way to overcome diseases resulted from selenium deficiency. A reciprocal smallest distance algorithm (RSD approach was applied using two experimentally confirmed Homocysteine S-Methyltransferases genes (HMT1 and HMT2 and a putative Selenocysteine Methyltransferase (SMT from dicots plant Arabidopsis thaliana, to explore their orthologs in seven sequenced diploid monocot species: Oryza sativa, Zea mays, Sorghum bicolor, Brachypodium distachyon, Hordeum vulgare, Aegilops tauschii (the D-genome donor of common wheat and Triticum urartu (the A-genome donor of common wheat. HMT1 was apparently diverged from HMT2 and most of SMT orthologs were the same with that of HMT2 in this study, leading to the hypothesis that SMT and HMT originate from one common ancestor gene. Identifying orthologs provide candidates for further experimental confirmation; also it could be helpful in designing primers to clone SMT or HMT orthologs in other crops.

  10. Effect of cooking on the concentration of bioactive compounds in broccoli (Brassica oleracea var. Avenger) and cauliflower (Brassica oleracea var. Alphina F1) grown in an organic system.

    Science.gov (United States)

    Dos Reis, Luzia Caroline Ramos; de Oliveira, Viviani Ruffo; Hagen, Martine Elisabeth Kienzle; Jablonski, André; Flôres, Simone Hickmann; de Oliveira Rios, Alessandro

    2015-04-01

    Brassica vegetables have been shown to have antioxidant capacities due to the presence of carotenoids, flavonoids and vitamins. This study evaluates the influence of different processing conditions (boiling, steaming, microwaving and sous vide) on the stability of flavonoids, carotenoids and vitamin A in broccoli and cauliflower inflorescences grown in an organic system. Results indicated that sous vide processing resulted in greater antioxidant capacity and that all processes contributed in some way to an increased content of antioxidant compounds in both cauliflower and broccoli.

  11. Changes in SeMSC, Glucosinolates and Sulforaphane Levels, and in Proteome Profile in Broccoli (Brassica oleracea var. Italica Fertilized with Sodium Selenate

    Directory of Open Access Journals (Sweden)

    Alejandra Moenne

    2013-05-01

    Full Text Available The aim of this work was to analyze the effect of sodium selenate fortification on the content of selenomethyl selenocysteine (SeMSC, total glucosinolates and sulforaphane, as well as the changes in protein profile of the inflorescences of broccoli (Brassica oleracea var. Italica. Two experimental groups were considered: plants treated with 100 mmol/L sodium selenate (final concentration in the pot and control plants treated with water. Fortification began 2 weeks after transplantation and was repeated once a week during 10 weeks. Broccoli florets were harvested when they reached appropriate size. SeMSC content in broccoli florets increased significantly with sodium selenate fortification; but total glucosinolates and sulforaphane content as well as myrosinase activity were not affected. The protein profile of broccoli florets changed due to fortification with sodium selenate. Some proteins involved in general stress-responses were up-regulated, whereas down-regulated proteins were identified as proteins involved in protection against pathogens. This is the first attempt to evaluate the physiological effect of fortification with sodium selenate on broccoli at protein level. The results of this work will contribute to better understanding the metabolic processes related with selenium uptake and accumulation in broccoli.

  12. Influence of thermal processing on hydrolysis and stability of folate poly-gamma-glutamates in broccoli (Brassica oleracea var. italica), carrot (Daucus carota) and tomato (Lycopersicon esculentum).

    Science.gov (United States)

    Munyaka, Ann Wambui; Verlinde, Philippe; Mukisa, Ivan Muzira; Oey, Indrawati; Van Loey, Ann; Hendrickx, Marc

    2010-04-14

    The folate poly-gamma-glutamate profile, their concentrations, and hydrolysis by endogenous gamma-glutamyl hydrolase (GGH) were evaluated in broccoli, carrot and tomato. Further studies on the effect of time and temperature on folate poly-gamma-glutamate hydrolysis and stability were carried out in broccoli since this vegetable showed the highest long-chain and total folate poly-gamma-glutamate concentration. The evolution of l-ascorbic acid, total phenols and Trolox equivalent antioxidant capacity (TEAC) values was evaluated in parallel. Upon thermal inactivation of GGH prior to crushing, it was observed that broccoli, carrot and tomato contained poly-gamma-glutamates with one to seven glutamate residues but differed in the predominant poly-gamma-glutamates. Crushing of raw broccoli, carrot and tomato resulted in significant poly-gamma-glutamate profile changes in broccoli and carrot (indicating GGH-catalyzed hydrolysis) but not in tomato. In this study, the actual crushing of raw broccoli matrix had a greater effect on folate poly-gamma-glutamate hydrolysis than incubation conditions (0-30 min at 25-55 degrees C). During treatments at 25-140 degrees C, folate retention was higher at 80 and 100 degrees C than at the other temperatures. A similar trend in thermal stability was observed for folates, vitamin C, total phenols and TEAC value, an indication that conditions that result in endogenous antioxidants degradation might also result in folate degradation.

  13. Effect of blanching temperature and dipping time on drying time of broccoli.

    Science.gov (United States)

    Doymaz, Ibrahim

    2014-03-01

    Drying characteristics of broccoli was experimentally studied in cabinet-type convective dryer. Experiments were conducted using constant air velocity of 2 m/s and air temperature of 60 . Drying rate increased with increasing blanching temperature and thus reduced the drying time. The experimental drying data were used to fit twelve drying models and drying rate constants and coefficients of the models were determined by non-linear regression analysis. Estimations by the Midilli et al. model were in good agreement with the experimental data obtained. Fick's second law was used to calculate the effective moisture diffusivity, which varied from 1.987 to 3.577 × 10(-8) m(2)/s for the given blanching temperature range. The rehydration ratio was observed to increase with increasing blanching temperature.

  14. Dietary constituents of broccoli and other cruciferous vegetables: implications for prevention and therapy of cancer.

    Science.gov (United States)

    Herr, Ingrid; Büchler, Markus W

    2010-08-01

    Over the past several decades, research on the action of bioactive constituents of plants has focused predominantly on their cancer-preventive properties. Today it can be explained why the consumption of fruits and vegetables may lead to a reduced frequency of certain cancer entities and why certain foods have therapeutic effects. Secondary plant products and especially glucosinolates from vegetables of the cruciferae family are supposed to have anti-carcinogenic potential. The present article gives an overview about secondary plant products in general and focuses to mechanisms of preventive and therapeutic effects of cruciferae, particular the brassica family and their famous member broccoli. Also, this article summarizes our knowledge of safety, tolerance and metabolism of glucosinolates and their therapeutic active degradation products isothiocyanates in animals and clinical studies.

  15. [Effects of different nitrogen forms and their ratios on broccoli yield, quality, and nutrient absorption].

    Science.gov (United States)

    Liu, Zhao-Fan; Zhang, Guo-Bin; Yu, Ji-Hua; Yang, Hai-Xing; Shi, Gui-Ying; Ma, Yan-Xia; Li, Jie

    2013-07-01

    A field experiment was conducted to study the effects of different nitrogen forms ((NH2)2CO-N, NO(3-)-N, NH(4+)-N, and NO(3-)-NH4+) and their ratios on the yield formation, quality, and nutrient (N, P, and K) absorption of broccoli (Brassica oleracea). Fertilization with NO(3-)-N increased the accumulation of nitrate and soluble sugars in ball flower. When the NO(3-)-N: NH(4+)-N ratio was ranged from 3:7 to 5:5 and from 5:5 to 7:3, respectively, the accumulation of nitrate in the ball flower was reduced, while the soluble sugars content was promoted. Fertilization with (NH2)2CO-N and NH(4+)-N was conducive to the improvement of Vc content in the ball flower. N fertilization increased the accumulation of N, P, and K in plants, with the highest contents of N, P, and K observed at rosette stage. Throughout the entire growth period, NH(4+)-N fertilization improved the plant N content, whereas NO(3-)-N fertilization improved the plant K content. At different growth stages, the effects of different N sources on plant P content varied. As compared with applying single N forms, the NO(3-)-N:NH(4+)-N ratio ranged from 3:7 to 5:5 could improve the yield significantly. It was suggested that a combined application of NO(3-)-N and NH(4+)-N with an appropriate ratio could improve the productivity, quality, and economic return of broccoli.

  16. Ozone dose-response relationships for spring oilseed rape and broccoli

    Science.gov (United States)

    De Bock, Maarten; Op de Beeck, Maarten; De Temmerman, Ludwig; Guisez, Yves; Ceulemans, Reinhart; Vandermeiren, Karine

    2011-03-01

    Tropospheric ozone is an important air pollutant with known detrimental effects for several crops. Ozone effects on seed yield, oil percentage, oil yield and 1000 seed weight were examined for spring oilseed rape ( Brassica napus cv. Ability). For broccoli ( Brassica oleracea L. cv. Italica cv. Monaco) the effects on fresh marketable weight and total dry weight were studied. Current ozone levels were compared with an increase of 20 and 40 ppb during 8 h per day, over the entire growing season. Oilseed rape seed yield was negatively correlated with ozone dose indices calculated from emergence until harvest. This resulted in an R2 of 0.24 and 0.26 ( p crops (UNECE, Mills, 2004), can indeed be applied for spring oilseed rape. The reduction of oilseed rape yield showed the highest correlation with the ozone uptake during the vegetative growth stage: when only the first 47 days after emergence were used to calculate POD 6, R2 values increased up to 0.476 or even 0.545 when the first 23 days were excluded. The highest ozone treatments, corresponding to the future ambient level by 2100 (IPCC, Meehl et al., 2007), led to a reduction of approximately 30% in oilseed rape seed yield in comparison to the current ozone concentrations. Oil percentage was also significantly reduced in response to ozone ( p oil yield was even more severely affected by elevated ozone exposure compared to seed yield: critical levels for oil yield dropped to 3.2 ppm h and 3.9 mmol m -2. For broccoli the applied ozone doses had no effect on yield.

  17. Characterization of a multifunctional methyltransferase from the orchid Vanilla planifolia.

    Science.gov (United States)

    Pak, F E; Gropper, S; Dai, W D; Havkin-Frenkel, D; Belanger, F C

    2004-07-01

    The final enzymatic step in the synthesis of the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) is believed to be methylation of 3,4-dihydroxybenzaldehyde. We have isolated and functionally characterized a cDNA that encodes a multifunctional methyltransferase from Vanilla planifolia tissue cultures that can catalyze the conversion of 3,4-dihydroxybenzaldehyde to vanillin, although 3,4-dihydroxybenzaldehyde is not the preferred substrate. The higher catalytic efficiency of the purified recombinant enzyme with the substrates caffeoyl aldehyde and 5-OH-coniferaldehyde, and its tissue distribution, suggest this methyltransferase may primarily function in lignin biosynthesis. However, since the enzyme characterized here does have 3,4-dihydroxybenzaldehyde-O-methyltransferase activity, it may be useful in engineering strategies for the synthesis of natural vanillin from alternate sources.

  18. The Cj0588 protein is a Campylobacter jejuni RNA methyltransferase.

    Science.gov (United States)

    Sałamaszyńska-Guz, Agnieszka; Taciak, Bartłomiej; Kwiatek, Agnieszka; Klimuszko, Danuta

    2014-06-06

    TlyA proteins belong to 2'-O-methyltransferases. Methylation is a common posttranscriptional RNA modification. The Campylobacter jejuni Cj0588 protein belongs to the TlyA(I) protein family and is a rRNA methyltransferase. Methylation of ribosomal RNA catalyzed by Cj0588 appears to have an impact on the biology of the cell. Presence of the cj0588 gene in bacteria appears to be important for ribosome stability and virulence properties. Absence of the Cj0588 protein causes accumulation of the 50S ribosomal subunits, reduction in the amount of functional 70S ribosomes and confers increase resistance to capreomycin.

  19. Rapid estimation of glucosinolate thermal degradation rate constants in leaves of Chinese kale and broccoli (Brassica oleracea) in two seasons.

    Science.gov (United States)

    Hennig, Kristin; Verkerk, Ruud; Bonnema, Guusje; Dekker, Matthijs

    2012-08-15

    Kinetic modeling was used as a tool to quantitatively estimate glucosinolate thermal degradation rate constants. Literature shows that thermal degradation rates differ in different vegetables. Well-characterized plant material, leaves of broccoli and Chinese kale plants grown in two seasons, was used in the study. It was shown that a first-order reaction is appropriate to model glucosinolate degradation independent from the season. No difference in degradation rate constants of structurally identical glucosinolates was found between broccoli and Chinese kale leaves when grown in the same season. However, glucosinolate degradation rate constants were highly affected by the season (20-80% increase in spring compared to autumn). These results suggest that differences in glucosinolate degradation rate constants can be due to variation in environmental as well as genetic factors. Furthermore, a methodology to estimate rate constants rapidly is provided to enable the analysis of high sample numbers for future studies.

  20. Continued selenium biofortification of carrots and broccoli grown in soils once amended with Se-enriched S. pinnata

    Directory of Open Access Journals (Sweden)

    Gary S. Bañuelos

    2016-08-01

    Full Text Available Selenium (Se biofortification has been practiced in Se-deficient regions throughout the world primarily by adding inorganic sources of Se to the soil. Considering the use of adding organic sources of Se could be useful as an alternative Se amendment for the production of Se-biofortified food crops. In this multi-year micro-plot study, we investigate growing carrots and broccoli in soils that had been previously amended with Se-enriched Stanleya pinnata Pursh (Britton three and four years prior to planting one and two, respectively. Results showed that total and extractable Se concentrations in soils (0-30 cm were 1.65 mg kg-1 and 88 µg L-1, and 0.92 mg kg-1 and 48.6 µg L-1 at the beginning of the growing season for planting one and two, respectively. After each respective growing season, total Se concentrations in the broccoli florets and carrots ranged from 6.99 to 7.83 mg kg-1 and 3.15 to 6.25 mg kg-1 in planting one and two, respectively. In broccoli and carrot plant tissues, SeMet (selenomethionine was the predominant selenoamino acid identified in Se aqueous extracts. In postharvest soils from planting one, phospholipid analyses (PLFA showed that amending the soil with S. pinnata exerted no effect on the microbial biomass, AMF (arbuscular mycorrhizal fungi, actinomycetes and Gram-positive and bacterial PLFA at both 0-5 and 0-30 cm, respectively, three years later. Successfully producing Se-enriched broccoli and carrots three and four years later after amending soil with Se-enriched S. pinnata clearly demonstrates its potential source as an organic Se enriched fertilizer for Se-deficient regions.

  1. Continued Selenium Biofortification of Carrots and Broccoli Grown in Soils Once Amended with Se-enriched S. pinnata

    Science.gov (United States)

    Bañuelos, Gary S.; Arroyo, Irvin S.; Dangi, Sadikshya R.; Zambrano, Maria C.

    2016-01-01

    Selenium (Se) biofortification has been practiced in Se-deficient regions throughout the world primarily by adding inorganic sources of Se to the soil. Considering the use of adding organic sources of Se could be useful as an alternative Se amendment for the production of Se-biofortified food crops. In this multi-year micro-plot study, we investigate growing carrots and broccoli in soils that had been previously amended with Se-enriched Stanleya pinnata Pursh (Britton) three and 4 years prior to planting one and two, respectively. Results showed that total and extractable Se concentrations in soils (0–30 cm) were 1.65 mg kg-1 and 88 μg L-1, and 0.92 mg kg-1 and 48.6 μg L-1 at the beginning of the growing season for planting one and two, respectively. After each respective growing season, total Se concentrations in the broccoli florets and carrots ranged from 6.99 to 7.83 mg kg-1 and 3.15 to 6.25 mg kg-1 in planting one and two, respectively. In broccoli and carrot plant tissues, SeMet (selenomethionine) was the predominant selenoamino acid identified in Se aqueous extracts. In postharvest soils from planting one, phospholipid fatty acid (PLFA) analyses showed that amending the soil with S. pinnata exerted no effect on the microbial biomass, AMF (arbuscular mycorrhizal fungi), actinomycetes and Gram-positive and bacterial PLFA at both 0–5 and 0–30 cm, respectively, 3 years later. Successfully producing Se-enriched broccoli and carrots 3 and 4 years later after amending soil with Se-enriched S. pinnata clearly demonstrates its potential source as an organic Se enriched fertilizer for Se-deficient regions. PMID:27602038

  2. Continued Selenium Biofortification of Carrots and Broccoli Grown in Soils Once Amended with Se-enriched S. pinnata.

    Science.gov (United States)

    Bañuelos, Gary S; Arroyo, Irvin S; Dangi, Sadikshya R; Zambrano, Maria C

    2016-01-01

    Selenium (Se) biofortification has been practiced in Se-deficient regions throughout the world primarily by adding inorganic sources of Se to the soil. Considering the use of adding organic sources of Se could be useful as an alternative Se amendment for the production of Se-biofortified food crops. In this multi-year micro-plot study, we investigate growing carrots and broccoli in soils that had been previously amended with Se-enriched Stanleya pinnata Pursh (Britton) three and 4 years prior to planting one and two, respectively. Results showed that total and extractable Se concentrations in soils (0-30 cm) were 1.65 mg kg(-1) and 88 μg L(-1), and 0.92 mg kg(-1) and 48.6 μg L(-1) at the beginning of the growing season for planting one and two, respectively. After each respective growing season, total Se concentrations in the broccoli florets and carrots ranged from 6.99 to 7.83 mg kg(-1) and 3.15 to 6.25 mg kg(-1) in planting one and two, respectively. In broccoli and carrot plant tissues, SeMet (selenomethionine) was the predominant selenoamino acid identified in Se aqueous extracts. In postharvest soils from planting one, phospholipid fatty acid (PLFA) analyses showed that amending the soil with S. pinnata exerted no effect on the microbial biomass, AMF (arbuscular mycorrhizal fungi), actinomycetes and Gram-positive and bacterial PLFA at both 0-5 and 0-30 cm, respectively, 3 years later. Successfully producing Se-enriched broccoli and carrots 3 and 4 years later after amending soil with Se-enriched S. pinnata clearly demonstrates its potential source as an organic Se enriched fertilizer for Se-deficient regions.

  3. Small RNA Sequencing Reveals Differential miRNA Expression in the Early Development of Broccoli (Brassica oleracea var. italica) Pollen

    Science.gov (United States)

    Li, Hui; Wang, Yu; Wu, Mei; Li, Lihong; Jin, Chuan; Zhang, Qingli; Chen, Chengbin; Song, Wenqin; Wang, Chunguo

    2017-01-01

    Pollen development is an important and complex biological process in the sexual reproduction of flowering plants. Although the cytological characteristics of pollen development are well defined, the regulation of its early stages remains largely unknown. In the present study, miRNAs were explored in the early development of broccoli (Brassica oleracea var. italica) pollen. A total of 333 known miRNAs that originated from 235 miRNA families were detected. Fifty-five novel miRNA candidates were identified. Sixty of the 333 known miRNAs and 49 of the 55 predicted novel miRNAs exhibited significantly differential expression profiling in the three distinct developmental stages of broccoli pollen. Among these differentially expressed miRNAs, miRNAs that would be involved in the developmental phase transition from uninucleate microspores to binucleate pollen grains or from binucleate to trinucleate pollen grains were identified. miRNAs that showed significantly enriched expression in a specific early stage of broccoli pollen development were also observed. In addition, 552 targets for 127 known miRNAs and 69 targets for 40 predicted novel miRNAs were bioinformatically identified. Functional annotation and GO (Gene Ontology) analysis indicated that the putative miRNA targets showed significant enrichment in GO terms that were related to plant organ formation and morphogenesis. Some of enriched GO terms were detected for the targets directly involved in plant male reproduction development. These findings provided new insights into the functions of miRNA-mediated regulatory networks in broccoli pollen development.

  4. Effects of broccoli extract on biodistribution and labeling blood components with {sup 99m}Tc-GH

    Energy Technology Data Exchange (ETDEWEB)

    Cekic, Betul; Muftuler, Fazilet Zumrut Biber; Kilcar, Ayfer Yurt; Ichedef, Cigdem; Unak, Perihan [Ege University, Izmir (Turkey). Inst. of Nuclear Sciences. Dept. of Nuclear Applications

    2011-09-15

    Purpose: people consume vegetables without the knowledge of the side effects of the biological and chemical contents and interactions between radiopharmaceuticals and herbal extract. To this end, current study is focused on the effects of broccoli extract on biodistribution of radiolabeled glucoheptonate ({sup 99m}Tc-GH) and radiolabeling of blood components. Methods: GH was labeled with {sup 99m}Tc. Quality control studies were done utilizing TLC method. Biodistribution studies were performed on male rats which were treated via gavage with either broccoli extract or SF as control group for 15 days. Blood samples were withdrawn from rats' heart. Radiolabeling of blood constituents performed incubating with GH, SnCl{sub 2} and {sup 99m} Tc. Results: radiochemical yield of {sup 99m}Tc-GH is 98.46{+-}1.48 % (n=8). Biodistribution studies have shown that according to the control, the treated group with broccoli has approximately 10 times less uptake in kidney. The percentage of the radioactivity ratios of the blood components is found to be same in both groups. Conclusions: although there is no considerable effect on the radiolabeling of blood components, there is an outstanding change on the biodistribution studies especially on kidneys. The knowledge of this change on kidney uptake may contribute to reduce the risk of misdiagnosis and/or repetition of the examinations in Nuclear Medicine. (author)

  5. IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE

    Science.gov (United States)

    Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes methylation of inorganic arsenic to mono, di, and trimethylated arsenicals. Orthologous AS3MT genes in genomes ranging from simple echinoderm to human predict a protein with five conserved cysteine (C) residues. In ...

  6. Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

    DEFF Research Database (Denmark)

    Freiesleben, Ulrik Von

    1996-01-01

    Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome...

  7. Diversity in mechanism and function of tRNA methyltransferases

    Science.gov (United States)

    Swinehart, William E; Jackman, Jane E

    2015-01-01

    tRNA molecules undergo extensive post-transcriptional processing to generate the mature functional tRNA species that are essential for translation in all organisms. These processing steps include the introduction of numerous specific chemical modifications to nucleotide bases and sugars; among these modifications, methylation reactions are by far the most abundant. The tRNA methyltransferases comprise a diverse enzyme superfamily, including members of multiple structural classes that appear to have arisen independently during evolution. Even among closely related family members, examples of unusual substrate specificity and chemistry have been observed. Here we review recent advances in tRNA methyltransferase mechanism and function with a particular emphasis on discoveries of alternative substrate specificities and chemistry associated with some methyltransferases. Although the molecular function for a specific tRNA methylation may not always be clear, mutations in tRNA methyltransferases have been increasingly associated with human disease. The impact of tRNA methylation on human biology is also discussed. PMID:25626150

  8. Yorkie Promotes Transcription by Recruiting a Histone Methyltransferase Complex

    Directory of Open Access Journals (Sweden)

    Hyangyee Oh

    2014-07-01

    Full Text Available Hippo signaling limits organ growth by inhibiting the transcriptional coactivator Yorkie. Despite the key role of Yorkie in both normal and oncogenic growth, the mechanism by which it activates transcription has not been defined. We report that Yorkie binding to chromatin correlates with histone H3K4 methylation and is sufficient to locally increase it. We show that Yorkie can recruit a histone methyltransferase complex through binding between WW domains of Yorkie and PPxY sequence motifs of NcoA6, a subunit of the Trithorax-related (Trr methyltransferase complex. Cell culture and in vivo assays establish that this recruitment of NcoA6 contributes to Yorkie’s ability to activate transcription. Mammalian NcoA6, a subunit of Trr-homologous methyltransferase complexes, can similarly interact with Yorkie’s mammalian homolog YAP. Our results implicate direct recruitment of a histone methyltransferase complex as central to transcriptional activation by Yorkie, linking the control of cell proliferation by Hippo signaling to chromatin modification.

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

    Science.gov (United States)

    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.

  10. LC-MS/MS quantification of sulforaphane and indole-3-carbinol metabolites in human plasma and urine after dietary intake of selenium-fortified broccoli.

    Science.gov (United States)

    Hauder, Johanna; Winkler, Stefanie; Bub, Achim; Rüfer, Corinna E; Pignitter, Marc; Somoza, Veronika

    2011-08-10

    This study aimed at developing a sensitive LC-MS/MS method for the quantification of sulforaphane (SFN) and indole-3-carbinol metabolites in plasma and urine after dietary intake of regular and selenium-fertilized broccoli using stable isotope dilution analysis. In a three-armed, placebo-controlled, randomized human intervention study with 76 healthy volunteers, 200 g of regular (485 μg of total glucosinolates and <0.01 μg of selenium per gram fresh weight) or selenium-fertilized broccoli (589 μg of total glucosinolates and 0.25 μg of selenium per gram fresh weight) was administered daily for 4 weeks. Glucoraphanin and glucobrassicin metabolites quantified in plasma and urine were SFN-glutathione, SFN-cysteine, SFN-cysteinylglycine, SFN-acetylcysteine, and indole-3-carboxaldehyde, indole-3-carboxylic acid, and ascorbigen, respectively. Dietary intake of selenium-fertilized broccoli increased serum selenium concentration analyzed by means of atomic absorption spectroscopy by up to 25% (p < 0.001), but affected neither glucosinolate concentrations in broccoli nor their metabolite concentrations in plasma and urine compared to regular broccoli.

  11. Natural dye sensitized TiO2 nanorods assembly of broccoli shape based solar cells.

    Science.gov (United States)

    Yuvapragasam, Akila; Muthukumarasamy, N; Agilan, S; Velauthapillai, Dhayalan; Senthil, T S; Sundaram, Senthilarasu

    2015-07-01

    TiO2 nanorods based thin films with rutile phase have been synthesized using template free low temperature hydrothermal method. The scanning electron microscope images showed that the prepared TiO2 samples were made of TiO2 nanorods and the nanorods had arranged by itself to form a broccoli like shape. The X-ray diffraction studies revealed that the prepared TiO2 samples exhibit rutile phase. The grown TiO2 nanorods had been sensitized using the flowers of Sesbania (S) grandiflora, leaves of Camellia (C) sinensis and roots of Rubia (R) tinctorum. Dye sensitized solar cells had been fabricated using the natural dye sensitized TiO2 nanorods based thin film photoelectrode and the open circuit voltage and short circuit current density were found to lie in the range of 0.45-0.6 V and 5.6-6.4 mA/cm(2) respectively. The photovoltaic performance of all the fabricated natural dye sensitized TiO2 solar cells indicate that natural dyes have the potential to be used as effective sensitizer in dye sensitized solar cells.

  12. Protein Methyltransferases: A Distinct, Diverse, and Dynamic Family of Enzymes.

    Science.gov (United States)

    Boriack-Sjodin, P Ann; Swinger, Kerren K

    2016-03-22

    Methyltransferase proteins make up a superfamily of enzymes that add one or more methyl groups to substrates that include protein, DNA, RNA, and small molecules. The subset of proteins that act upon arginine and lysine side chains are characterized as epigenetic targets because of their activity on histone molecules and their ability to affect transcriptional regulation. However, it is now clear that these enzymes target other protein substrates, as well, greatly expanding their potential impact on normal and disease biology. Protein methyltransferases are well-characterized structurally. In addition to revealing the overall architecture of the subfamilies of enzymes, structures of complexes with substrates and ligands have permitted detailed analysis of biochemical mechanism, substrate recognition, and design of potent and selective inhibitors. This review focuses on how knowledge gained from structural studies has impacted the understanding of this large class of epigenetic enzymes.

  13. Plant isoflavone and isoflavanone O-methyltransferase genes

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  15. Potency of microfiltration membrane process in purifying broccoli (Brassica oleracea L.) fermented by lactic acid bacteria (LAB) as functional food

    Science.gov (United States)

    Susilowati, Agustine; Aspiyanto, Maryati, Yati; Melanie, Hakiki; Lotulung, Puspa D.

    2017-01-01

    Purifying broccoli (Brassica oleracea L.) fermented by Lactic Acid Bacteria (LAB) using mixture of L. bulgaricus, S. thermopillus, L. acidophillusand Bifidobacteriumbifidum and fructooligosaccharides (FOS) as carbon source have been performed to recover biomass concentrate for probiotic and antioxidant. Purification of fermented broccoli was conducted through microfiltration (MF) membrane of 0.15 µm at stirrer rotation speed 400 rpm, room temperature and pressure 40 psia for 30 minutes. Fermented broccoli produced via fermentation process with fermentation time 0 (initial) and 48 hours, and LAB concentration 10% and 20% (v/v) represented as biomass of A, B, C and D. The experimental result showed that based on selectivity of total organic acids, separating optimization was achieved at biomass D (fermentation time 48 hours and mixed LAB culture concentration 20%). Concentrate composition produced in this condition were total acids 6.04%, total solids 24.31%, total polyphenol 0.0252%, reducing sugar 68.25 mg/mL, total sugars 30.89 mg/mL, and dissolved protein 28.54 mg/mL with pH 3.94. In this condition, recovery of biomass concentrate of D for total acids 5.64 folds, total solids 1.82 folds, total polyphenol 3.03 folds, reducing sugar 1.16 folds, total sugars 1.19 folds, and dissolved protein 0.67 folds compared with feed (initial process). Identification of monomer of biomass concentrate D as polyphenol derivatives at T2,01 and T3.01 gave monomer with molecular weight (MW) 192.78 Dalton (Da.), and monomer with MW 191.08, 191.49 and 192.07 Da., while lactic acid derivatives showed MW 251.13, 251.6 and 252.14, and monomer with MW 250.63, 252.14 and 254.22 Da.

  16. Effects of combined treatments of irradiation and antimicrobial coatings on reduction of food pathogens in broccoli florets

    Energy Technology Data Exchange (ETDEWEB)

    Takala, P.N.; Salmieri, S.; Vu, K.D. [INRS-Institut Armand-Frappier, Research Laboratories in Sciences Applied to Food, Canadian Irradiation Centre, 531, Blvd des Prairies, Laval, QC, H7V 1B7 (Canada); Lacroix, M., E-mail: Monique.Lacroix@iaf.inrs.ca [INRS-Institut Armand-Frappier, Research Laboratories in Sciences Applied to Food, Canadian Irradiation Centre, 531, Blvd des Prairies, Laval, QC, H7V 1B7 (Canada)

    2011-12-15

    The effect of combined treatment of antimicrobial coatings and {gamma}-radiation on reduction of food pathogens such as Listeria monocytogenes, Escherichia coli, and Salmonella Typhimurium was evaluated in broccoli florets. Broccoli florets were inoculated with pathogenic bacteria at 10{sup 6} CFU/g. Inoculated florets were then coated with methylcellulose-based coating containing various mixtures of antimicrobial agents: organic acids (OAs) plus lactic acid bacteria metabolites (LABs), OA plus citrus extract (CE), OA plus CE plus spice mixture (SM), and OA plus rosemary extract (RE). Coated florets were irradiated with various doses (0-3.3 kGy), and microbial analyses were used to calculate the D{sub 10} value and radiosensitive relative. The coating containing OA plus CE was the most effective formulation for increasing the sensitization of Escherichia coli by 2.4 times as compared to the control without the antimicrobial coating. For Salmonella Typhimurium, coating containing OA plus LAB was the most effective formulation, increasing radiosensitivity by 2.4 times as well. All antimicrobial coatings had almost the same effect of increasing the sensitivity of Listeria monocytogenes (from 1.31 to 1.45 times) to {gamma}-irradiation. - Highlights: > Demonstrate scientifically the synergistic effect of the combined treatment of gamma-irradiation and natural antimicrobial coating in reduction of food pathogens in broccoli. > The coating containing organic acids plus citrus extract was the most efficient formulation for increasing the sensitization of E. coli by 2.40 times as compared to the control. > The coating containing organic acids plus lactic acid bacteria metabolites was the most effective formulation causing the sensitization of S. Typhimurium to {gamma}-irradiation by 2.4 times. > Potential in application of developed formulations to protect food products against food pathogens.

  17. Effect of broccoli sprouts on nasal response to live attenuated influenza virus in smokers: a randomized, double-blind study.

    Directory of Open Access Journals (Sweden)

    Terry L Noah

    Full Text Available Smokers have increased susceptibility and altered innate host defense responses to influenza virus infection. Broccoli sprouts are a source of the Nrf2 activating agentsulforaphane, and short term ingestion of broccoli sprout homogenates (BSH has been shown to reduce nasal inflammatory responses to oxidant pollutants.Assess the effects of BSH on nasal cytokines, virus replication, and Nrf2-dependent enzyme expression in smokers and nonsmokers.We conducted a randomized, double-blind, placebo-controlled trial comparing the effects of BSH on serially sampled nasal lavage fluid (NLF cytokines, viral sequence quantity, and Nrf2-dependent enzyme expression in NLF cells and biopsied epithelium. Healthy young adult smokers and nonsmokers ingested BSH or placebo (alfalfa sprout homogenate for 4 days, designated Days -1, 0, 1, 2. On Day 0 they received standard vaccine dose of live attenuated influenza virus (LAIV intranasally. Nasal lavage fluids and nasal biopsies were collected serially to assess response to LAIV.In area under curve analyses, post-LAIV IL-6 responses (P = 0.03 and influenza sequences (P = 0.01 were significantly reduced in NLF from BSH-treated smokers, whilequinoneoxidoreductasein NLF cells was significantly increased. In nonsmokers, a similar trend for reduction in virus quantity with BSH did not reach statistical significance.In smokers, short term ingestion of broccoli sprout homogenates appears to significantly reduce some virus-induced markers of inflammation, as well as reducing virus quantity. Nutritional antioxidant interventions have promise as a safe, low-cost strategy for reducing influenza risk among smokers and other at risk populations.ClinicalTrials.gov NCT01269723.

  18. The Impact of Broccoli II & Tomato II on European patents in conventional breeding, GMO’s and Synthetic Biology:

    DEFF Research Database (Denmark)

    Minssen, Timo; Nordberg, Ana

    2015-01-01

    (GMOs) and Synthetic Biology remains a major obstacle to the industry, research and innovation in an increasingly significant area of science. This paper has also been published as a working paper on SSRN, see: Minssen, Timo and Nordberg, A., The Impact of Broccoli II & Tomatoes II on European Patents...... in Conventional Breeding, GMO's and Synthetic Biology: The Grand Finale of a Juicy Patents Tale? (May 19, 2015). Univ. of Copenhagen Dept. of Economics Discussion. Available at SSRN: http://ssrn.com/abstract=2607865 or http://dx.doi.org/10.2139/ssrn.2607865...

  19. Gold nanoparticles synthesized by Brassica oleracea (Broccoli) acting as antimicrobial agents against human pathogenic bacteria and fungi

    Science.gov (United States)

    Piruthiviraj, Prakash; Margret, Anita; Krishnamurthy, Poornima Priyadharsani

    2016-04-01

    Production of antimicrobial agents through the synthesis of gold nanoparticles using green technology has been extensively made consistent by various researchers; yet, this study uses the flower bud's aqueous extracts of Brassica oleracea (Broccoli) as a reducing agent for chloroauric acid (1 mM). After 30 min of incubation, synthesis of gold nanoparticles (AuNps) was observed by a change in extract color from pale yellow to purple color. Synthesis of AuNps was confirmed in UV-visible spectroscopy at the range of approximately 560 nm. The SEM analysis showed the average nanoparticles size of 12-22 nm. The antimicrobial activity of AuNps was analyzed by subjecting it to human pathogenic bacteria (Gram-positive Staphylococcus aureus and Gram-negative Klebsiella pneumonia) and fungi (Aspergillus flavus, Aspergillus niger and Candida albicans) using disc diffusion method. The broccoli-synthesized AuNps showed the efficient antibacterial and antifungal activity of above-mentioned microbes. It was confirmed that AuNps have the best antimicrobial agent compared to the standard antibiotics (Gentamicin and Fluconazole). When the concentrations of AuNps were increased (10, 25, and 50 µg/ml), the sensitivity zone also increased for all the tested microbes. The synthesized AuNps are capable of rendering high antimicrobial efficacy and, hence, have a great potential in the preparation of drugs used against major bacterial and fungal diseases in humans.

  20. Enhanced resistance to Botrytis cinerea and Rhizoctonia solani in transgenic broccoli with a Trichoderma viride endochitinase gene

    Institute of Scientific and Technical Information of China (English)

    YU Ya; REN Shu-xin; GUO Yang-dong; ZHANG Lei; LIAN Wei-ran; XU Feng-feng; LI Shuang-tao; XIANG Juan; ZHANG Guo-zhen; HU Zan-min; ZHAO Bing

    2015-01-01

    A endochitinase gene (Tch ) from the fungus Trichoderma viride was introduced into broccoli (Brassica oleracea var. italica) by Agrobacterium-mediated transformation. Sixty-eight putative transformants were obtained and the presence of the Tch gene was conifrmed by both PCR and Southern blot analysis. RT-PCR analysis showed an accumulation of the transcript encoding the endochitinase protein in the transgenic plants. Using real-time quantitative PCR, the expression proifling of endochitinase gene was analyzed. Primary transformants and selfed progeny were examined for expression of the endo-chitinase using a lfuorometric assay and for their resistance to the pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The endochitinase activities in T0 in vitro plants, T0 mature plants and T1 mature plants were correlated with leaf lesions, and the transgenic line T618 had high endochitinse activities of 102.68, 114.53 and 120.27 nmol L–1 MU min–1 mg–1 protein in the three kinds of plants, respectively. The endochitinase activity showed a positive correlation with the resistance to the pathogens. Most transgenic T0 broccoli had increased resistance to the pathogens of B. cinerea and R. solani in leaf assays and this resistance was conifrmed to be inheritable. These ifndings suggested that expression of the Tch gene from T. viride could enhance resistance to pathogenic fungi in Brassica species.

  1. Effects of combined treatments of irradiation and antimicrobial coatings on reduction of food pathogens in broccoli florets

    Science.gov (United States)

    Takala, P. N.; Salmieri, S.; Vu, K. D.; Lacroix, M.

    2011-12-01

    The effect of combined treatment of antimicrobial coatings and γ-radiation on reduction of food pathogens such as Listeria monocytogenes, Escherichia coli, and Salmonella Typhimurium was evaluated in broccoli florets. Broccoli florets were inoculated with pathogenic bacteria at 10 6 CFU/g. Inoculated florets were then coated with methylcellulose-based coating containing various mixtures of antimicrobial agents: organic acids (OAs) plus lactic acid bacteria metabolites (LABs), OA plus citrus extract (CE), OA plus CE plus spice mixture (SM), and OA plus rosemary extract (RE). Coated florets were irradiated with various doses (0-3.3 kGy), and microbial analyses were used to calculate the D10 value and radiosensitive relative. The coating containing OA plus CE was the most effective formulation for increasing the sensitization of Escherichia coli by 2.4 times as compared to the control without the antimicrobial coating. For Salmonella Typhimurium, coating containing OA plus LAB was the most effective formulation, increasing radiosensitivity by 2.4 times as well. All antimicrobial coatings had almost the same effect of increasing the sensitivity of Listeria monocytogenes (from 1.31 to 1.45 times) to γ-irradiation.

  2. Differential Responses of Two Broccoli (Brassica oleracea L. var Italica Cultivars to Salinity and Nutritional Quality Improvement

    Directory of Open Access Journals (Sweden)

    Chokri Zaghdoud

    2012-01-01

    Full Text Available The comparative responses of two broccoli cultivars (Brassica oleracea var. Italica, cv. Parthenon and cv. Naxos to a 15 d exposure to different NaCl levels were investigated. Salinity led to increased concentrations of Na+ and Cl− ions in both cultivars, a disruption of the endogenous minerals levels in the shoots and roots—that varied with the cultivar and salt concentration—and decreases in the osmotic potential (Ψπ, root hydraulic conductance (L0, and stomatal conductance (Gs. The reduced biomass of Naxos at moderate NaCl indicates greater sensitivity to salinity, compared with Parthenon. Parthenon accumulated more soluble sugars, for osmotic adjustment, whereas Naxos accumulated proline, which gave the two cultivars differing nutritional characteristics. The total glucosinolates (GSLs content was not affected by salinity in Parthenon while it decreased significantly in Naxos as a consequence of the decrease in the indole GSL. However, Naxos accumulated more aliphatic GSLs under salt stress than Parthenon, which confers on this cultivar a greater nutritional value when cultivated under salinity.These results suggest that, at distinct salinity levels, each broccoli cultivar adopts a specific strategy, indicating the crucial role of the genetic background on the organoleptic and nutritional properties that each cultivar acquires.

  3. Evaluation of Image Analysis to Determine the N-Fertilizer Demand of Broccoli Plants (Brassica oleracea convar. botrytis var. italica

    Directory of Open Access Journals (Sweden)

    Simone Graeff

    2008-01-01

    Full Text Available Numerous models have been developed for calculating optimum decision rules for nitrogen fertilization based on remote sensing techniques. New technologies related to digital image analysis may provide an alternative method to estimate nutrient status faster and more efficiently than current techniques. A series of field studies was conducted to determine the applicability of digital image analysis for nitrogen demand estimates in broccoli plants. Digital images were taken under constant light conditions in various wavelength ranges (380–1300 nm using a digital imager. Images were processed for the parameters ∗ and ∗ in the ∗∗∗ color system. The image analysis showed a close correlation between the nitrogen status of broccoli plants and the parameter ∗ of the ∗∗∗ color system especially in the wavelength ranges 510780 and 516780 nm. The relationship between nutrient concentration in leaf dry matter and the parameters ∗ was used to determine the N fertilizer demand within the cultivation period. Estimated N amounts were applied as top dressing four weeks after setting and were 100 kg ha−1 lower than the standard fertilizer rate. Calculated N balances indicated a total uptake of applied N amounts in the image-based N treatments without considerable yield loss. Thus, digital image analysis proved to be an effective means of determining nitrogen status and adjusting fertilizer applications to preserve or enhance crop quality and yield considering sustainability.

  4. Inhibitory effects of broccoli extract on Escherichia coli O157:H7 quorum sensing and in vivo virulence.

    Science.gov (United States)

    Lee, Kang-Mu; Lim, Jeesun; Nam, Sunyoung; Yoon, Mi Young; Kwon, Yong-Kuk; Jung, Byeong Yeal; Park, Yongjin; Park, Sungsu; Yoon, Sang Sun

    2011-08-01

    Broccoli extract (BE) has numerous beneficial effects on human health including anticancer activity. Quorum sensing (QS), mediated by self-produced autoinducer (AI) molecules, is a key process for the production of virulence determinants in pathogenic bacteria. BE suppressed AI-2 synthesis and AI-2-mediated bacterial motility in a dose-dependent manner in Escherichia coli O157:H7. In addition, expression of the ler gene that regulates AI-3 QS system was also diminished in response to treatment with BE. Furthermore, in an in vivo efficacy test using Caenorhabditis elegans as a host organism, C. elegans fed on E. coli O157:H7 in the presence of BE survived longer than those fed solely on the pathogenic bacteria. Quantitative real-time PCR analysis indicated that quercetin was the most active among the tested broccoli-derived compounds in downregulating virulence gene expression, while treatment with myricetin significantly suppressed the expression of the eae gene involved in type III secretion system. These data suggest that BE and its flavonoid constituents can inhibit expression of QS-associated genes, thereby downregulating the virulence attributes of E. coli O157:H7 both in vitro and in vivo. This study clearly elucidates BE's QS-inhibitory activity and suggests that BE has the potential to be developed as an anti-infective agent.

  5. Discriminating between cultivars and treatments of broccoli using mass spectral fingerprinting and analysis of variance-principal component analysis.

    Science.gov (United States)

    Luthria, Devanand L; Lin, Long-Ze; Robbins, Rebecca J; Finley, John W; Banuelos, Gary S; Harnly, James M

    2008-11-12

    Metabolite fingerprints, obtained with direct injection mass spectrometry (MS) with both positive and negative ionization, were used with analysis of variance-principal components analysis (ANOVA-PCA) to discriminate between cultivars and growing treatments of broccoli. The sample set consisted of two cultivars of broccoli, Majestic and Legacy, the first grown with four different levels of Se and the second grown organically and conventionally with two rates of irrigation. Chemical composition differences in the two cultivars and seven treatments produced patterns that were visually and statistically distinguishable using ANOVA-PCA. PCA loadings allowed identification of the molecular and fragment ions that provided the most significant chemical differences. A standardized profiling method for phenolic compounds showed that important discriminating ions were not phenolic compounds. The elution times of the discriminating ions and previous results suggest that they were common sugars and organic acids. ANOVA calculations of the positive and negative ionization MS fingerprints showed that 33% of the variance came from the cultivar, 59% from the growing treatment, and 8% from analytical uncertainty. Although the positive and negative ionization fingerprints differed significantly, there was no difference in the distribution of variance. High variance of individual masses with cultivars or growing treatment was correlated with high PCA loadings. The ANOVA data suggest that only variables with high variance for analytical uncertainty should be deleted. All other variables represent discriminating masses that allow separation of the samples with respect to cultivar and treatment.

  6. VEGETATIVE GROWTH AND PRODUCTIVITY OF A NUMBER OF INTRODUCED BROCCOLI CULTIVARS (Brassica oleracea L. var. italica Plenck. IN BATUR VILLAGE, KINTAMANI DISTRICT, BANGLI REGENCY, BALI

    Directory of Open Access Journals (Sweden)

    Ni Kadek Raleni

    2015-09-01

    Full Text Available Broccoli (Brassica oleracea L. var. italica Plenck. is a vegetable crop belongs to Brassicaceae family. Broccoli has high nutrition, high in fiber and contains isotiacyanate that has anticancer activity. Broccoli market in Indonesia, particularly in modern supermarkets, increases 15-20% per year, however, productivity was still low, therefore effort to increase broccoli production in Indonesia need to be investigated. Field trial was conducted at Batur Village, Kintamani District, Bangli Regency, Bali, to find out cultivars that were adapted in tropical region. Introduced cultivars being trialed were ‘Alborada’, ‘Belstar’, ‘Fiesta’, ‘Sarasota’, ‘Bay Meadows’, ‘Castle Dome’, ‘Liutenant’, ‘Iron Man’, ‘Marathon’, ‘Green Gold’, ‘Imperial’, ‘Green Magic’ and ‘Lucky’ as control. Variable observed were vegetative growth, curding period, and productivity of each cultivar. This study employed Randomized Completely Block Design with 3 replicates (plots and 8 plants each plot. Data were analyzed using ANOVA (Analysis of variance on Costat program, followed by Duncan’s Multiple Range Test (DMRT on 1% level. Results show that each cultivar varied in adaptability in tropical region. ‘Castle Dome’ has the highest productivity, while ‘Fiesta’ was the lowest.

  7. The effect of the rate and method of nitrogen application on nitrogen uptake and utilization by broccoli (Brassica oleracea var. italica)

    NARCIS (Netherlands)

    Everaarts, A.P.; Willigen, de P.

    1999-01-01

    The effect of the rate and method of nitrogen application on nitrogen uptake and utilization by broccoli (Brassica oleracea var. italica) was studied in four field experiments. The methods of application were broadcast application vs band placement and split application. Maximum uptake of nitrogen b

  8. YccW is the m5C methyltransferase specific for 23S rRNA nucleotide 1962

    DEFF Research Database (Denmark)

    Purta, Elzbieta; O'Connor, Michelle; Bujnicki, Janusz M

    2008-01-01

    . coli marginally reduces its growth rate. YccW had previously eluded identification because it displays only limited sequence similarity to the m(5)C methyltransferases RsmB and RsmF and is in fact more similar to known m(5)U (5-methyluridine) RNA methyltransferases. In keeping with the previously...... proposed nomenclature system for bacterial rRNA methyltransferases, yccW is now designated as the rRNA large subunit methyltransferase gene rlmI....

  9. Association of myasthenia gravis with polymorphisms in the gene of histamine N-methyltransferase

    DEFF Research Database (Denmark)

    Kellermayer, Blanka; Polgar, Noemi; Pal, Jozsef

    2013-01-01

    Histamine N-methyltransferase (HNMT) is the main metabolizing enzyme of histamine. Histamine modulates immune responses and plays a role in the pathogenesis of autoimmune disorders.......Histamine N-methyltransferase (HNMT) is the main metabolizing enzyme of histamine. Histamine modulates immune responses and plays a role in the pathogenesis of autoimmune disorders....

  10. Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin

    Science.gov (United States)

    Chahine, Sarah; Okafor, Darius; Ong, Ana C.; Maybank, Rosslyn; Kwak, Yoon I.; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

    2015-01-01

    16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. PMID:26537447

  11. Negative in vitro selection identifies the rRNA recognition motif for ErmE methyltransferase

    DEFF Research Database (Denmark)

    Nielsen, A K; Douthwaite, S; Vester, B

    1999-01-01

    Erm methyltransferases modify bacterial 23S ribosomal RNA at adenosine 2058 (A2058, Escherichia coli numbering) conferring resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics. The motif that is recognized by Erm methyltransferases is contained within helix 73 of 23S r...

  12. Protein arginine N-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells.

    Science.gov (United States)

    Gou, Qing; He, ShuJiao; Zhou, ZeJian

    2017-02-01

    Hepatocellular carcinoma is the most common subtype of liver cancer. Protein arginine N-methyltransferase 1 was shown to be upregulated in various cancers. However, the role of protein arginine N-methyltransferase 1 in hepatocellular carcinoma progression remains incompletely understood. We investigated the clinical and functional significance of protein arginine N-methyltransferase 1 in a series of clinical hepatocellular carcinoma samples and a panel of hepatocellular carcinoma cell lines. We performed suppression analysis of protein arginine N-methyltransferase 1 using small interfering RNA to determine the biological roles of protein arginine N-methyltransferase 1 in hepatocellular carcinoma. In addition, the expression of epithelial-mesenchymal transition indicators was verified by western blotting in hepatocellular carcinoma cell lines after small interfering RNA treatment. Protein arginine N-methyltransferase 1 expression was found to be significantly upregulated in hepatocellular carcinoma cell lines and clinical tissues. Moreover, downregulation of protein arginine N-methyltransferase 1 in hepatocellular carcinoma cells by small interfering RNA could inhibit cell proliferation, migration, and invasion in vitro. These results indicate that protein arginine N-methyltransferase 1 may contribute to hepatocellular carcinoma progression and serves as a promising target for the treatment of hepatocellular carcinoma patients.

  13. Coordinate regulation of DNA methyltransferase expression during oogenesis

    Directory of Open Access Journals (Sweden)

    Bestor Timothy H

    2007-04-01

    Full Text Available Abstract Background Normal mammalian development requires the action of DNA methyltransferases (DNMTs for the establishment and maintenance of DNA methylation within repeat elements and imprinted genes. Here we report the expression dynamics of Dnmt3a and Dnmt3b, as well as a regulator of DNA methylation, Dnmt3L, in isolated female germ cells. Results Our results indicate that these enzymes are coordinately regulated and that their expression peaks during the stage of postnatal oocyte development when maternal methylation imprints are established. We find that Dnmt3a, Dnmt3b, Dnmt3L and Dnmt1o transcript accumulation is related to oocyte diameter. Furthermore, DNMT3L deficient 15 dpp oocytes have aberrantly methylated Snrpn, Peg3 and Igf2r DMRs, but normal IAP and LINE-1 methylation levels, thereby highlighting a male germ cell specific role for DNMT3L in the establishment of DNA methylation at repeat elements. Finally, real-time RT-PCR analysis indicates that the depletion of either DNMT3L or DNMT1o in growing oocytes results in the increased expression of the de novo methyltransferase Dnmt3b, suggesting a potential compensation mechanism by this enzyme for the loss of one of the other DNA methyltransferases. Conclusion Together these results provide a better understanding of the developmental regulation of Dnmt3a, Dnmt3b and Dnmt3L at the time of de novo methylation during oogenesis and demonstrate that the involvement of DNMT3L in retrotransposon silencing is restricted to the male germ line. This in turn suggests the existence of other factors in the oocyte that direct DNA methylation to transposons.

  14. A SABATH Methyltransferase from the moss Physcomitrella patens catalyzes

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Nan [ORNL; Ferrer, Jean-Luc [Universite Joseph Fourier, France; Moon, Hong S [Department of Plant Sciences, University of Tennessee; Kapteyn, Jeremy [Institute of Biological Chemistry, Washington State University; Zhuang, Xiaofeng [Department of Plant Sciences, University of Tennessee; Hasebe, Mitsuyasu [Laboratory of Evolutionary Biology, National Institute for Biology, 38 Nishigounaka; Stewart, Neal C. [Department of Plant Sciences, University of Tennessee; Gang, David R. [Institute of Biological Chemistry, Washington State University; Chen, Feng [University of Tennessee, Knoxville (UTK)

    2012-01-01

    Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested for methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5 lM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.

  15. Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana.

    Science.gov (United States)

    Feng, Chen-Zhuo; Zhu, Xiao-Jing; Dai, Zhong-Min; Liu, Feng-Qi; Xiang, Jian-Hai; Yang, Wei-Jun

    2007-06-01

    DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32% approximately 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, AfDnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana.

  16. Chromosome Doubling of Microspore-Derived Plants from Cabbage (Brassica oleracea var. capitata L.) and Broccoli (Brassica oleracea var. italica L.).

    Science.gov (United States)

    Yuan, Suxia; Su, Yanbin; Liu, Yumei; Li, Zhansheng; Fang, Zhiyuan; Yang, Limei; Zhuang, Mu; Zhang, Yangyong; Lv, Honghao; Sun, Peitian

    2015-01-01

    Chromosome doubling of microspore-derived plants is an important factor in the practical application of microspore culture technology because breeding programs require a large number of genetically stable, homozygous doubled haploid plants with a high level of fertility. In the present paper, 29 populations of microspore-derived plantlets from cabbage (Brassica oleracea var. capitata) and broccoli (Brassica oleracea var. italica) were used to study the ploidy level and spontaneous chromosome doubling of these populations, the artificial chromosome doubling induced by colchicine, and the influence of tissue culture duration on the chromosomal ploidy of the microspore-derived regenerants. Spontaneous chromosome doubling occurred randomly and was genotype dependent. In the plant populations derived from microspores, there were haploids, diploids, and even a low frequency of polyploids and mixed-ploidy plantlets. The total spontaneous doubling in the 14 cabbage populations ranged from 0 to 76.9%, compared with 52.2 to 100% in the 15 broccoli populations. To improve the rate of chromosome doubling, an efficient and reliable artificial chromosome doubling protocol (i.e., the immersion of haploid plantlet roots in a colchicine solution) was developed for cabbage and broccoli microspore-derived haploids. The optimal chromosome doubling of the haploids was obtained with a solution of 0.2% colchicine for 9-12 h or 0.4% colchicine for 3-9 h for cabbage and 0.05% colchicine for 6-12 h for broccoli. This protocol produced chromosome doubling in over 50% of the haploid genotypes for most of the populations derived from cabbage and broccoli. Notably, after 1 or more years in tissue culture, the chromosomes of the haploids were doubled, and most of the haploids turned into doubled haploid or mixed-ploidy plants. This is the first report indicating that tissue culture duration can change the chromosomal ploidy of microspore-derived regenerants.

  17. Plants as Biofactories: Postharvest Stress-Induced Accumulation of Phenolic Compounds and Glucosinolates in Broccoli Subjected to Wounding Stress and Exogenous Phytohormones.

    Science.gov (United States)

    Villarreal-García, Daniel; Nair, Vimal; Cisneros-Zevallos, Luis; Jacobo-Velázquez, Daniel A

    2016-01-01

    Broccoli contains high levels of bioactive molecules and is considered a functional food. In this study, postharvest treatments to enhance the concentration of glucosinolates and phenolic compounds were evaluated. Broccoli whole heads were wounded to obtain florets and wounded florets (florets cut into four even pieces) and stored for 24 h at 20 °C with or without exogenous ethylene (ET, 1000 ppm) or methyl jasmonate (MeJA, 250 ppm). Whole heads were used as a control for wounding treatments. Regarding glucosinolate accumulation, ET selectively induced the 4-hydroxylation of glucobrassicin in whole heads, resulting in ∼223% higher 4-hydroxyglucobrassicin than time 0 h samples. Additionally, glucoraphanin was increased by ∼53% in whole heads treated with ET, while neoglucobrassicin was greatly accumulated in wounded florets treated with ET or MeJA, showing increases of ∼193 and ∼286%, respectively. On the other hand, although only whole heads stored without phytohormones showed higher concentrations of phenolic compounds, which was reflected in ∼33, ∼30, and ∼46% higher levels of 1,2,2-trisinapoylgentiobose, 1,2-diferulolylgentiobiose, and 1,2-disinapoyl-2-ferulolylgentiobiose, respectively; broccoli florets stored under air control conditions showed enhanced concentrations of 3-O-caffeoylquinic acid, 1,2-disinapoylgentiobiose, and 1,2-disinapoyl-2-ferulolylgentiobiose (∼22, ∼185, and ∼65% more, respectively). Furthermore, exogenous ET and MeJA impeded individual phenolics accumulation. Results allowed the elucidation of simple and effective postharvest treatment to enhance the content of individual glucosinolates and phenolic compounds in broccoli. The stressed-broccoli tissue could be subjected to downstream processing in order to extract and purify bioactive molecules with applications in the dietary supplements, agrochemical and cosmetics markets.

  18. Putrescine N-methyltransferase--the start for alkaloids.

    Science.gov (United States)

    Biastoff, Stefan; Brandt, Wolfgang; Dräger, Birgit

    2009-01-01

    Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development.

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

    Directory of Open Access Journals (Sweden)

    Silvano ePresciuttini

    2014-01-01

    Full Text Available Higher brain dopamine content depending on lower activity of Catechol-O-Methyltransferase (COMT in subjects with high hypnotisability scores (highs has been considered responsible for their attentional characteristics. However, the results of the previous genetic studies on association between hypnotisability and the Catechol-O-Methyltransferase (COMT single nucleotide polymorphism (SNP rs4680 (Val158Met were inconsistent. Here, we used a selective genotyping approach to re-evaluate the association between hypnotisability 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 hypnotisability and focused attention abilities.

  20. Structural biology of human H3K9 methyltransferases.

    Directory of Open Access Journals (Sweden)

    Hong Wu

    Full Text Available UNLABELLED: SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  1. An allosteric inhibitor of protein arginine methyltransferase 3.

    Science.gov (United States)

    Siarheyeva, Alena; Senisterra, Guillermo; Allali-Hassani, Abdellah; Dong, Aiping; Dobrovetsky, Elena; Wasney, Gregory A; Chau, Irene; Marcellus, Richard; Hajian, Taraneh; Liu, Feng; Korboukh, Ilia; Smil, David; Bolshan, Yuri; Min, Jinrong; Wu, Hong; Zeng, Hong; Loppnau, Peter; Poda, Gennadiy; Griffin, Carly; Aman, Ahmed; Brown, Peter J; Jin, Jian; Al-Awar, Rima; Arrowsmith, Cheryl H; Schapira, Matthieu; Vedadi, Masoud

    2012-08-01

    PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets.

  2. Influence of cooking conditions on organoleptic and health-related properties of artichokes, green beans, broccoli and carrots.

    Science.gov (United States)

    Guillén, Sofía; Mir-Bel, Jorge; Oria, Rosa; Salvador, María L

    2017-02-15

    Colour, pigments, total phenolic content and antioxidant activity were investigated in artichokes, green beans, broccoli and carrots cooked under different conditions. Domestic induction hobs with temperature control were used to evaluate the effect of boiling, sous-vide cooking and water immersion cooking at temperatures below 100°C on the properties of each vegetable. Sous-vide cooking preserved chlorophyll, carotenoids, phenolic content and antioxidant activity to a greater extent than boiling for all of the vegetables tested and retained colour better, as determined by a(∗). A reduction of only 10-15°C in the cooking temperature was enough to improve the properties of the samples cooked by water immersion, except for green beans. Artichokes and carrots suffered pronounced losses of antioxidant activity during boiling (17.0 and 9.2% retention, respectively), but the stability of this parameter significantly increased with sous-vide cooking (84.9 and 55.3% retention, respectively).

  3. EFFECT OF SOIL SULFUR FERTILIZER AND SOME FOLIAR FERTILIZERS ON GROWTH AND YIELD OF BROCCOLI IN SALINE SOIL

    Directory of Open Access Journals (Sweden)

    Ali Husain JASIM

    2015-12-01

    Full Text Available Factorial experiment was conducted in the open fields of Agricultural College, Al-Qasim Green University during the agricultural seasons of 2013/2014 and 2014/2015 to study the effect of adding two levels of agricultural sulfur (control and add 100 kg.ha-1 and four levels of nutrient spray (without spray, high-potash fertilizer, high-phosphorus fertilizer and humic acid on growth and yield of broccoli under drip irrigation and polyethylene soil mulching in saline soil (9.6 dS.m-1. Randomized complete block design with three replicates was used. The results showed that agricultural sulfur led to increase number of leaves, leaf area, leaves chlorophyll content, diameter and weight of flower head compared to control. Spraying foliar fertilizer and its interaction with sulfur fertilizer also led to increase all of parameters above (except leaves chlorophyll content significantly compared to control treatment.

  4. Variation in broccoli cultivar phytochemical content under organic and conventional management systems: implications in breeding for nutrition.

    Science.gov (United States)

    Renaud, Erica N C; Lammerts van Bueren, Edith T; Myers, James R; Paulo, Maria João; van Eeuwijk, Fred A; Zhu, Ning; Juvik, John A

    2014-01-01

    Organic agriculture requires cultivars that can adapt to organic crop management systems without the use of synthetic pesticides as well as genotypes with improved nutritional value. The aim of this study encompassing 16 experiments was to compare 23 broccoli cultivars for the content of phytochemicals associated with health promotion grown under organic and conventional management in spring and fall plantings in two broccoli growing regions in the US (Oregon and Maine). The phytochemicals quantified included: glucosinolates (glucoraphanin, glucobrassicin, neoglucobrassin), tocopherols (δ-, γ-, α-tocopherol) and carotenoids (lutein, zeaxanthin, β-carotene). For glucoraphanin (17.5%) and lutein (13%), genotype was the major source of total variation; for glucobrassicin, region (36%) and the interaction of location and season (27.5%); and for neoglucobrassicin, both genotype (36.8%) and its interactions (34.4%) with season were important. For δ- and γ-tocopherols, season played the largest role in the total variation followed by location and genotype; for total carotenoids, genotype (8.41-13.03%) was the largest source of variation and its interactions with location and season. Overall, phytochemicals were not significantly influenced by management system. We observed that the cultivars with the highest concentrations of glucoraphanin had the lowest for glucobrassicin and neoglucobrassicin. The genotypes with high concentrations of glucobrassicin and neoglucobrassicin were the same cultivars and were early maturing F1 hybrids. Cultivars highest in tocopherols and carotenoids were open pollinated or early maturing F1 hybrids. We identified distinct locations and seasons where phytochemical performance was higher for each compound. Correlations among horticulture traits and phytochemicals demonstrated that glucoraphanin was negatively correlated with the carotenoids and the carotenoids were correlated with one another. Little or no association between

  5. RNA methyltransferase NSUN2 promotes stress-induced HUVEC senescence.

    Science.gov (United States)

    Cai, Xiaoyu; Hu, Yuanyuan; Tang, Hao; Hu, Han; Pang, Lijun; Xing, Junyue; Liu, Zhenyun; Luo, Yuhong; Jiang, Bin; Liu, Te; Gorospe, Myriam; Chen, Chuan; Wang, Wengong

    2016-04-12

    The tRNA methyltransferase NSUN2 delays replicative senescence by regulating the translation of CDK1 and CDKN1B mRNAs. However, whether NSUN2 influences premature cellular senescence remains untested. Here we show that NSUN2 methylates SHC mRNA in vitro and in cells, thereby enhancing the translation of the three SHC proteins, p66SHC, p52SHC, and p46SHC. Our results further show that the elevation of SHC expression by NSUN2-mediated mRNA methylation increased the levels of ROS, activated p38MAPK, thereby accelerating oxidative stress- and high-glucose-induced senescence of human vascular endothelial cells (HUVEC). Our findings highlight the critical impact of NSUN2-mediated mRNA methylation in promoting premature senescence.

  6. Cell and molecular biology of DNA methyltransferase 1.

    Science.gov (United States)

    Mohan, K Naga; Chaillet, J Richard

    2013-01-01

    The DNA cytosine methyltransferase 1 (DNMT1) is a ubiquitous nuclear enzyme that catalyzes the well-established reaction of placing methyl groups on the unmethylated cytosines in methyl-CpG:CpG base pairs in the hemimethylated DNA formed by methylated parent and unmethylated daughter strands. This activity regenerates fully methylated methyl-CpG:methyl-CpG pairs. Despite the straightforward nature of its catalytic activity, detailed biochemical, genetic, and developmental studies revealed intricate details of the central regulatory role of DNMT1 in governing the epigenetic makeup of the nuclear genome. DNMT1 mediates demethylation and also participates in seemingly wide cellular functions unrelated to maintenance DNA methylation. This review brings together mechanistic details of maintenance methylation by DNMT1, its regulation at transcriptional and posttranscriptional levels, and the seemingly unexpected functions of DNMT1 in the context of DNA methylation which is central to epigenetic changes that occur during development and the process of cell differentiation.

  7. Proteome identification of proteins interacting with histone methyltransferase SET8

    Institute of Scientific and Technical Information of China (English)

    Yi Qin; Huafang Ouyang; Jing Liu; Youhua Xie

    2013-01-01

    SET8 (also known as PR-Set7/9,SETD8,KMT5A),a member of the SET domain containing methyltransferase family,which specifically catalyzes mono-methylation of K20 on histone H4 (H4K20me1),has been implicated in multiple biological processes,such as gene transcriptional regulation,cell cycle control,genomic integrity maintenance and development.In this study,we used GST-SET8 fusion protein as bait to search for SET8 interaction partners to elucidate physiological functions of SET8.In combination with mass spectrometry,we identified 40 proteins that potentially interact with SET8.DDX21,a nucleolar protein,was further confirmed to associate with SET8.Furthermore,we discovered a novel function of SET8 in the regulation of rRNA transcription.

  8. Clinical utility of thiopurine S-methyltransferase genotyping.

    Science.gov (United States)

    Corominas, Hèctor; Baiget, Montserrat

    2004-01-01

    Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that plays a major role in the metabolism of thiopurine drugs such as mercaptopurine and azathioprine. The interindividual differences in response to thiopurine administration is in part due to the presence of genetic polymorphisms in the gene that regulates TPMT activity. TPMT genotype correlates well with the in vivo enzyme activity within erythrocytes. Patients with genetically determined decreased TPMT activity develop severe myelosuppression when treated with standard doses of thiopurine drugs because an excess of thioguanine nucleotides accumulates in hematopoietic tissues. TPMT genotyping provides clinicians with a reliable method for identifying TPMT-deficient patients who can benefit from low doses of thiopurine drugs in order to reduce the risk of developing adverse effects. Moreover, the administration of higher doses of the drug could improve therapeutic response in patients in whom the TPMT genotyping demonstrates the absence of mutated alleles.

  9. Aqueous extracts of selenium-fertilized broccoli increase selenoprotein activity and inhibit DNA single-strand breaks, but decrease the activity of quinone reductase in Hepa 1c1c7 cells.

    Science.gov (United States)

    Keck, Anna-Sigrid; Finley, John W

    2006-05-01

    Depending on growth conditions, broccoli may be enriched in the isothiocyanate sulforaphane and/or the mineral selenium (Se); both compounds may play an important role in the reduction of intracellular oxidative stress and chronic disease prevention. Sulforaphane up-regulates transcription of Phase II detoxification proteins (e.g. quinone reductase [QR]), whereas Se is needed for the production of thioredoxin reductase (TR) and glutathione peroxidase-1 (GPx1), both of which exhibit antioxidant activity. The objective of the present study was to determine whether the fertilization of broccoli with Se increases the antioxidant ability of broccoli. Hydrogen peroxide-induced DNA single-strand breaks (measured by single cell electrophoresis, Comet assay) and activity of antioxidant enzymes (GPx, TR and QR) were measured in mouse hepatoma cells (Hepa 1c1c7 cells) treated with purified sulforaphane, sodium selenite or extracts of selenized broccoli. When supplied separately as chemically pure substances, sodium selenite was more effective than sulforaphane for reduction of single-strand breaks. Se-fertilized broccoli extracts were the most effective for reduction of DNA single-strand breaks, and extracts that contained 0.71 microM Se and 0.08 microM sulforaphane inhibited 94% of DNA single-strand breaks. A significant positive association (r = 0.81, p = 0.009) between GPx1 activity and inhibition of DNA single-strand breaks as well as a 24h lag time between addition of Se, sulforaphane or broccoli extract and inhibition of single-strand breaks suggests that some of the antioxidant protection is mediated through selenoproteins. Conversely, fertilization of broccoli with Se decreased the ability of broccoli extract to induce QR activity. These results demonstrate that Se and sulforaphane, alone or as a component of broccoli, may help decrease oxidative stress. They further suggest that Se is the most important for decreasing oxidative stress, but maximizing the Se content

  10. Betaine-homocysteine methyltransferase (BHMT) : genomic sequencing and relevance to hyperhomocysteinemia and vascular disease in humans

    NARCIS (Netherlands)

    Heil, S.G.; Lievers, K.J.A.; Boers, G.H.; Verhoef, P.; Heijer, den M.; Trijbels, F.J.M.; Blom, H.J.

    2000-01-01

    Elevated homocysteine levels have been associated with arteriosclerosis and thrombosis. Hyperhomocysteinemia is caused by altered functioning of enzymes of its metabolism due to either inherited or acquired factors. Betaine-homocysteine methyltransferase (BHMT) serves, next to methionine synthase, a

  11. Successful treatment of a guanidinoacetate methyltransferase deficient patient : Findings with relevance to treatment strategy and pathophysiology

    NARCIS (Netherlands)

    Verbruggen, Krijn T.; Sijens, Paul E.; Schulze, Andreas; Lunsing, Roelineke J.; Jakobs, Cornelis; Salomons, Gajja S.; van Spronsen, Francian J.

    2007-01-01

    Biochemical and developmental results of treatment of a guanidinoacetate methyltransferase (GAMT) deficient patient with a mild clinical presentation and remarkable developmental improvement after treatment are presented. Treatment with creatine (Cr) supplementation resulted in partial normalization

  12. The effect of the rate and method of nitrogen application on nitrogen uptake and utilization by broccoli (Brassica oleracea var. italica)

    OpenAIRE

    Everaarts, A.P.; Willigen, de, P.

    1999-01-01

    The effect of the rate and method of nitrogen application on nitrogen uptake and utilization by broccoli (Brassica oleracea var. italica) was studied in four field experiments. The methods of application were broadcast application vs band placement and split application. Maximum uptake of nitrogen by the crop was around 300 kg ha-1. In one experiment, band placement positively influenced nitrogen uptake. Split application did not influence nitrogen uptake. Nitrogen application resulted in a h...

  13. Pre-soaking of seeds enhances pressure inactivation of E. coli O157:H7 and Salmonella spp. on crimson clover, red clover, radish and broccoli seeds.

    Science.gov (United States)

    Neetoo, Hudaa; Chen, Haiqiang

    2010-02-28

    The application of high hydrostatic pressure (HHP) at a level of 600 MPa at 20 degrees C to decontaminate crimson clover, red clover, radish and broccoli seeds inoculated with E. coli O157:H7 and Salmonella were evaluated. Salmonella was generally more pressure-resistant than E. coli O157:H7 on clover and radish seeds except on broccoli seeds where the trend was reversed. In addition, the application of HHP differentially affected seeds' germinability and the order of pressure tolerance of the seeds was such that red clover>crimson clover approximately broccoli >radish seeds with final germination percentages ranging from 85-100% while their untreated counterparts had final germination percentages of 99-100%. Pre-soaking the different types of seeds in water for 30, 60 or 90 min at ambient temperature followed by HHP at 600 MPa for 2 or 5 min at 20 degrees C significantly (Pseeds to germinate also varied as a function of the pre-soaking duration and the seed type. Pre-soaking radish and broccoli seeds for 30 min prior to HHP (2 or 5 min) resulted in germination percentages of seeds displayed higher germination potential when pre-soaked for 60 min at 20 degrees C prior to HPP (5 min) with final germination percentages of 94%, although their yield was substantially lower than their untreated counterparts. Red clover seeds pre-soaked for 60 min at 4 degrees C followed by HPP at 600 MPa for 5 min at 20 degrees C produced germination percentages of 91 and 95% after 3 and 8 days of sprouting compared to 99 and 100% respectively for untreated seeds. In addition, this condition did not significantly (P>0.05) reduce the sprout yield. The treatment also resulted in a reduction of a 5 log initial load of E. coli O157:H7 and Salmonella to an undetectable level (neither pathogen was detected in 2-g seed samples after enrichment).

  14. Assessing Competence of Broccoli Consumption on Inflammatory and Antioxidant Pathways in Restraint-Induced Models: Estimation in Rat Hippocampus and Prefrontal Cortex

    Directory of Open Access Journals (Sweden)

    Leila Khalaj

    2013-01-01

    Full Text Available A growing body of evidence advocated the protective and therapeutic potential of natural compounds and phytochemicals used in diets against pathological conditions. Herein, the outcome of dietary whole broccoli consumption prior to restraint stress has been investigated in the hippocampus and prefrontal cortex of male rats, two important regions involved in the processing of responses to stressful events. Interestingly, a region-specific effect was detected regarding some of antioxidant defense system factors: nuclear factor erythroid-derived 2-related factor 2 (Nrf-2 antioxidant pathway, mitochondrial prosurvival proteins involved in mitochondrial biogenesis, and apoptotic cell death proteins. Dietary broccoli supplementation modulated the restraint-induced changes towards a consistent overall protection in the hippocampus. In the prefrontal cortex, however, despite activation of most of the protective factors, presumably as an attempt to save the system against the stress insult, some detrimental outcomes such as induced malate dehydrogenase (MDA level and cleaved form of caspase-3 were detectable. Such diversity may be attributed in one hand to the different basic levels and/or availability of defensive mechanisms within the two studied cerebral regions, and on the other hand to the probable dose-dependent and hormetic effects of whole broccoli. More experiments are essential to demonstrate these assumptions.

  15. 西兰花甜玉米复合蔬菜纸的研制%Study on Compound Vegetable Paper of Broccoli and Sweet Corn

    Institute of Scientific and Technical Information of China (English)

    汤慧民; 尚海英

    2015-01-01

    With broccoli and sweet corn as raw material, sodium alginate, carrageenan,starch as adhesive, based on single factor experiment, the production process conditions of vegetable paper of broccoli and sweet corn was optimized through orthogonal experiment.The result showed that the acquired vegetable paper was best in color, flavor, taste and texture adopting the condition of the quality ratio of broccoli slurry and sweet corn pulp was 2:1,sodium alginate 0.5%, carrageenan 0.6%, starch 8%, film thickness 1.5 mm, drying 4 h at 65℃.%以西兰花和甜玉米为原料,海藻酸钠、卡拉胶、淀粉为胶黏剂,在单因素的基础上,采用正交试验法确定西兰花甜玉米复合蔬菜纸的最佳生产工艺条件:西兰花浆和甜玉米浆的质量比例是2:1、0.5%的海藻酸钠、0.6%卡拉胶、8%淀粉,涂膜厚度1.5 mm,65℃干燥4 h.所制得的蔬菜纸色、香、味、形俱佳.

  16. Effects of the 3D-clinorotation on endogenous substances of broccoli sprout (Brassica oleracea var. italica) and its food safety

    Science.gov (United States)

    Hiraishi, K.; Tomita-Yokotani, K.; Wakabayashi, K.; Hashimoto, H.; Miyagawa, T.; Yamashita, M.

    Habitation in outer space is one of our challenges in this century We are studying on space agriculture to provide foods for space living people However careful assessment should be made on the effects of exotic environment on the endogenous production of biologically active substances and food safety of plants cultivated in space Broccoli sprout Brassica oleracea var italica is known to produce sulforaphane 4-methylsulfinybutyl isothiocyanate which is effective to function as an antioxidant and enhance immunity Because of such substance it is recognized to be good food materials Broccoli sprouts were then cultivated for 3 days under the 3D-clinorotation The amount of sulforaphane produced by this treatment showed no significant difference compared to the ground control Secondly we examined population of microorganisms adhered on the surface of sprout cultivated under the 3D-clinorotation Number of the microorganisms colony formed was statistically higher than the control Mold species was identified to penicillium sp based on the microscopic observation Poor construction of plant cell wall elements cellulose lignin etc is well known effects of microgravity Defense function of the broccoli plant cells might be weakened against microorganism We also speculate other possible causes for the high rate of contamination such as photosynthetic activity of the plant or microclimate air flow heat transport and humidity around the seedling affected by pseudo-microgravity or the 3D-clinorotation Those factors may relate to the difference in proliferation

  17. Enhancement of phenolics, flavonoids and glucosinolates of Broccoli (Brassica olaracea, var. Italica as antioxidants in response to organic and bio-organic fertilizers

    Directory of Open Access Journals (Sweden)

    Abd El-Moniem M. Naguib

    2012-06-01

    Full Text Available A field experiment was carried out to study the effect of organic and bioorganic fertilizers on growth parameters, yield and the quality of two broccoli cultivars (Calabrese and Southern star. Bio-organic fertilizers gave better results for all vegetative growth parameters for Southern star cultivar compared to Calabrese cultivar. Total phenolic content (TPC, total flavonoid content (TFC and total glucosinolates content (TGsC were almost higher in Calabrese cultivar as a result of organic fertilizer treatment. The antioxidant activities of both cultivars were evaluated and Calabrese cultivar showed the higher1, 1-diphenyl-2-picrylhydrazyl DPPH. scavenging activity expressed as IC50 (its concentrations for 50% inhibition (16.56 μg/ml compared to Southern star (19.42 μg/ml. In addition, Calabrese cultivar showed the higher chelating power (75.36 μg/ml than Southern star (72.43 μg/ml at (30 μg/ml when the organic fertilizer was applied. The results indicated that there is a good margin for enhancing antioxidant compounds of broccoli for economic production using organic fertilization. This study indicated the potential application of broccoli as a potent natural source of antioxidants as nutraceuticals.

  18. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    Science.gov (United States)

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation.

  19. Crystal structure of dengue virus methyltransferase without S-adenosyl-L-methionine.

    Science.gov (United States)

    Noble, Christian G; Li, Shi-Hua; Dong, Hongping; Chew, Sock Hui; Shi, Pei-Yong

    2014-11-01

    Flavivirus methyltransferase is a genetically-validated antiviral target. Crystal structures of almost all available flavivirus methyltransferases contain S-adenosyl-L-methionine (SAM), the methyl donor molecule that co-purifies with the enzymes. This raises a possibility that SAM is an integral structural component required for the folding of dengue virus (DENV) methyltransferase. Here we exclude this possibility by solving the crystal structure of DENV methyltransferase without SAM. The SAM ligand was removed from the enzyme through a urea-mediated denaturation-and-renaturation protocol. The crystal structure of the SAM-depleted enzyme exhibits a vacant SAM-binding pocket, with a conformation identical to that of the SAM-enzyme co-crystal structure. Functionally, equivalent enzymatic activities (N-7 methylation, 2'-O methylation, and GMP-enzyme complex formation) were detected for the SAM-depleted and SAM-containing recombinant proteins. These results clearly indicate that the SAM molecule is not an essential component for the correct folding of DENV methyltransferase. Furthermore, the results imply a potential antiviral approach to search for inhibitors that can bind to the SAM-binding pocket and compete against SAM binding. To demonstrate this potential, we have soaked crystals of DENV methyltransferase without a bound SAM with the natural product Sinefungin and show that preformed crystals are capable of binding ligands in this pocket.

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

    Science.gov (United States)

    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

  1. The Role of Protein Arginine Methyltransferases in Inflammatory Responses

    Directory of Open Access Journals (Sweden)

    Ji Hye Kim

    2016-01-01

    Full Text Available Protein arginine methyltransferases (PRMTs mediate the methylation of a number of protein substrates of arginine residues and serve critical functions in many cellular responses, including cancer development, progression, and aggressiveness, T-lymphocyte activation, and hepatic gluconeogenesis. There are nine members of the PRMT family, which are divided into 4 types (types I–IV. Although most PRMTs do not require posttranslational modification (PTM to be activated, fine-tuning modifications, such as interactions between cofactor proteins, subcellular compartmentalization, and regulation of RNA, via micro-RNAs, seem to be required. Inflammation is an essential defense reaction of the body to eliminate harmful stimuli, including damaged cells, irritants, or pathogens. However, chronic inflammation can eventually cause several types of diseases, including some cancers, atherosclerosis, rheumatoid arthritis, and periodontitis. Therefore, inflammation responses should be well modulated. In this review, we briefly discuss the role of PRMTs in the control of inflammation. More specifically, we review the roles of four PRMTs (CARM1, PRMT1, PRMT5, and PRMT6 in modulating inflammation responses, particularly in terms of modulating the transcriptional factors or cofactors related to inflammation. Based on the regulatory roles known so far, we propose that PRMTs should be considered one of the target molecule groups that modulate inflammatory responses.

  2. Theoretical insights into catalytic mechanism of protein arginine methyltransferase 1.

    Directory of Open Access Journals (Sweden)

    Ruihan Zhang

    Full Text Available Protein arginine methyltransferase 1 (PRMT1, the major arginine asymmetric dimethylation enzyme in mammals, is emerging as a potential drug target for cancer and cardiovascular disease. Understanding the catalytic mechanism of PRMT1 will facilitate inhibitor design. However, detailed mechanisms of the methyl transfer process and substrate deprotonation of PRMT1 remain unclear. In this study, we present a theoretical study on PRMT1 catalyzed arginine dimethylation by employing molecular dynamics (MD simulation and quantum mechanics/molecular mechanics (QM/MM calculation. Ternary complex models, composed of PRMT1, peptide substrate, and S-adenosyl-methionine (AdoMet as cofactor, were constructed and verified by 30-ns MD simulation. The snapshots selected from the MD trajectory were applied for the QM/MM calculation. The typical SN2-favored transition states of the first and second methyl transfers were identified from the potential energy profile. Deprotonation of substrate arginine occurs immediately after methyl transfer, and the carboxylate group of E144 acts as proton acceptor. Furthermore, natural bond orbital analysis and electrostatic potential calculation showed that E144 facilitates the charge redistribution during the reaction and reduces the energy barrier. In this study, we propose the detailed mechanism of PRMT1-catalyzed asymmetric dimethylation, which increases insight on the small-molecule effectors design, and enables further investigations into the physiological function of this family.

  3. Methyltransferase and demethylase profiling studies during brown adipocyte differentiation.

    Science.gov (United States)

    Son, Min Jeong; Kim, Won Kon; Oh, Kyoung-Jin; Park, Anna; Lee, Da Som; Han, Baek Soo; Lee, Sang Chul; Bae, Kwang-Hee

    2016-07-01

    Although brown adipose tissue is important with regard to energy balance, the molecular mechanism of brown adipocyte differentiation has not been extensively studied. Specifically, regulation factors at the level of protein modification are largely unknown. In this study, we examine the changes in the expression level of enzymes which are involved in protein lysine methylation during brown adipocyte differentiation. Several enzymes, in this case SUV420H2, PRDM9, MLL3 and JHDM1D, were found to be up-regulated. On the other hand, Set7/9 was significantly down-regulated. In the case of SUV420H2, the expression level increased sharply during brown adipocyte differentiation, whereas the expression of SUV420H2 was marginally enhanced during the white adipocyte differentiation. The knock-down of SUV420H2 caused the suppression of brown adipocyte differentiation, as compared to a scrambled control. These results suggest that SUV420H2, a methyltransferase, is involved in brown adipocyte differentiation, and that the methylation of protein lysine is important in brown adipocyte differentiation. [BMB Reports 2016; 49(7): 388-393].

  4. Euchromatin histone methyltransferase 1 regulates cortical neuronal network development

    Science.gov (United States)

    Bart Martens, Marijn; Frega, Monica; Classen, Jessica; Epping, Lisa; Bijvank, Elske; Benevento, Marco; van Bokhoven, Hans; Tiesinga, Paul; Schubert, Dirk; Nadif Kasri, Nael

    2016-01-01

    Heterozygous mutations or deletions in the human Euchromatin histone methyltransferase 1 (EHMT1) gene cause Kleefstra syndrome, a neurodevelopmental disorder that is characterized by autistic-like features and severe intellectual disability (ID). Neurodevelopmental disorders including ID and autism may be related to deficits in activity-dependent wiring of brain circuits during development. Although Kleefstra syndrome has been associated with dendritic and synaptic defects in mice and Drosophila, little is known about the role of EHMT1 in the development of cortical neuronal networks. Here we used micro-electrode arrays and whole-cell patch-clamp recordings to investigate the impact of EHMT1 deficiency at the network and single cell level. We show that EHMT1 deficiency impaired neural network activity during the transition from uncorrelated background action potential firing to synchronized network bursting. Spontaneous bursting and excitatory synaptic currents were transiently reduced, whereas miniature excitatory postsynaptic currents were not affected. Finally, we show that loss of function of EHMT1 ultimately resulted in less regular network bursting patterns later in development. These data suggest that the developmental impairments observed in EHMT1-deficient networks may result in a temporal misalignment between activity-dependent developmental processes thereby contributing to the pathophysiology of Kleefstra syndrome. PMID:27767173

  5. Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

    Science.gov (United States)

    Yap, Damian B.; Lewis, M.E. Suzanne; Chijiwa, Chieko; Ramos‐Arroyo, Maria A.; Tkachenko, Natália; Milano, Valentina; Fradin, Mélanie; McKinnon, Margaret L.; Townsend, Katelin N.; Xu, Jieqing; Van Allen, M.I.; Ross, Colin J.D.; Dobyns, William B.; Weaver, David D.; Gibson, William T.

    2016-01-01

    ABSTRACT Weaver syndrome (WS) is a rare congenital disorder characterized by generalized overgrowth, macrocephaly, specific facial features, accelerated bone age, intellectual disability, and susceptibility to cancers. De novo mutations in the enhancer of zeste homolog 2 (EZH2) have been shown to cause WS. EZH2 is a histone methyltransferase that acts as the catalytic agent of the polycomb‐repressive complex 2 (PRC2) to maintain gene repression via methylation of lysine 27 on histone H3 (H3K27). Functional studies investigating histone methyltransferase activity of mutant EZH2 from various cancers have been reported, whereas WS‐associated mutations remain poorly characterized. To investigate the role of EZH2 in WS, we performed functional studies using artificially assembled PRC2 complexes containing mutagenized human EZH2 that reflected the codon changes predicted from patients with WS. We found that WS‐associated amino acid alterations reduce the histone methyltransferase function of EZH2 in this in vitro assay. Our results support the hypothesis that WS is caused by constitutional mutations in EZH2 that alter the histone methyltransferase function of PRC2. However, histone methyltransferase activities of different EZH2 variants do not appear to correlate directly with the phenotypic variability between WS patients and individuals with a common c.553G>C (p.Asp185His) polymorphism in EZH2. PMID:26694085

  6. O-Methyltransferases involved in biphenyl and dibenzofuran biosynthesis.

    Science.gov (United States)

    Khalil, Mohammed N A; Brandt, Wolfgang; Beuerle, Till; Reckwell, Dennis; Groeneveld, Josephine; Hänsch, Robert; Gaid, Mariam M; Liu, Benye; Beerhues, Ludger

    2015-07-01

    Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O-methylation reactions. cDNAs encoding the O-methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab-causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5-dihydroxybiphenyl, supplied by the first pathway-specific enzyme, biphenyl synthase (BIS). 3,5-Dihydroxybiphenyl underwent a single methylation reaction in the presence of S-adenosyl-l-methionine (SAM). The second enzyme, SaOMT2, exhibited its highest affinity for noraucuparin, however the turnover rate was greater with 5-hydroxyferulic acid. Both substrates were only methylated at the meta-positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of the substrates with SAM also took place at a position other than the sulfur group. Expression of SaOMT1, SaOMT2 and SaBIS3 was transiently induced in rowan cell cultures by the addition of the fungal elicitor. While the immediate SaOMT1 products were not detectable in elicitor-treated cell cultures, noraucuparin and noreriobofuran accumulated transiently, followed by increasing levels of the SaOMT2 products aucuparin and eriobofuran. SaOMT1, SaOMT2 and SaBIS3 were N- and C-terminally fused with the super cyan fluorescent protein and a modified yellow fluorescent protein, respectively. All the fluorescent reporter fusions were localized to the cytoplasm of Nicotiana benthamiana leaf epidermis cells. A revised biosynthetic pathway of biphenyls and dibenzofurans in the Malinae is presented.

  7. Mechanistic studies on transcriptional coactivator protein arginine methyltransferase 1.

    Science.gov (United States)

    Rust, Heather L; Zurita-Lopez, Cecilia I; Clarke, Steven; Thompson, Paul R

    2011-04-26

    Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to the guanidinium group of arginine residues in a number of important cell signaling proteins. PRMT1 is the founding member of this family, and its activity appears to be dysregulated in heart disease and cancer. To begin to characterize the catalytic mechanism of this isozyme, we assessed the effects of mutating a number of highly conserved active site residues (i.e., Y39, R54, E100, E144, E153, M155, and H293), which are believed to play key roles in SAM recognition, substrate binding, and catalysis. The results of these studies, as well as pH-rate studies, and the determination of solvent isotope effects (SIEs) indicate that M155 plays a critical role in both SAM binding and the processivity of the reaction but is not responsible for the regiospecific formation of asymmetrically dimethylated arginine (ADMA). Additionally, mutagenesis studies on H293, combined with pH studies and the lack of a normal SIE, do not support a role for this residue as a general base. Furthermore, the lack of a normal SIE with either the wild type or catalytically impaired mutants suggests that general acid/base catalysis is not important for promoting methyl transfer. This result, combined with the fact that the E144A/E153A double mutant retains considerably more activity then the single mutants alone, suggests that the PRMT1-catalyzed reaction is primarily driven by bringing the substrate guanidinium into the proximity of the S-methyl group of SAM and that the prior deprotonation of the substrate guanidinium is not required for methyl transfer.

  8. 辽西地区露地西兰花(绿花椰菜)栽培技术要点%Cultivation Technology of Broccoli in Western Area of Liaoning

    Institute of Scientific and Technical Information of China (English)

    颜津宁; 胡新颖; 杨迎东; 裴新辉; 商旭文; 颜范悦

    2012-01-01

    Planting technology of broccoli in autumn, including variety selection, seedling breeding, planting and managements, prevention and cure for plant diseases and insect pests, harvest were introduced. These techniques would provide references for the production of broccoli in autumn.%该文从品种选择、秧苗培育、整地定植、病虫害防治、收获及贮藏等方面介绍了辽西地区西兰花秋茬栽培技术,以期指导露地秋茬西兰花生产。

  9. Virtual screening and biological characterization of novel histone arginine methyltransferase PRMT1 inhibitors.

    Science.gov (United States)

    Heinke, Ralf; Spannhoff, Astrid; Meier, Rene; Trojer, Patrick; Bauer, Ingo; Jung, Manfred; Sippl, Wolfgang

    2009-01-01

    Lysine and arginine methyltransferases participate in the posttranslational modification of histones and regulate key cellular functions. Protein arginine methyltransferase 1 (PRMT1) has been identified as an essential component of mixed lineage leukemia (MLL) oncogenic complexes, revealing its potential as a novel therapeutic target in human cancer. The first potent arginine methyltransferase inhibitors were recently discovered by random- and target-based screening approaches. Herein we report virtual and biological screening for novel inhibitors of PRMT1. Structure-based virtual screening (VS) of the Chembridge database composed of 328 000 molecules was performed with a combination of ligand- and target-based in silico approaches. Nine inhibitors were identified from the top-scored docking solutions; these were experimentally tested using human PRMT1 and an antibody-based assay with a time-resolved fluorescence readout. Among several aromatic amines, an aliphatic amine and an amide were also found to be active in the micromolar range.

  10. Discovery of a Potent Class I Protein Arginine Methyltransferase Fragment Inhibitor.

    Science.gov (United States)

    Ferreira de Freitas, Renato; Eram, Mohammad S; Szewczyk, Magdalena M; Steuber, Holger; Smil, David; Wu, Hong; Li, Fengling; Senisterra, Guillermo; Dong, Aiping; Brown, Peter J; Hitchcock, Marion; Moosmayer, Dieter; Stegmann, Christian M; Egner, Ursula; Arrowsmith, Cheryl; Barsyte-Lovejoy, Dalia; Vedadi, Masoud; Schapira, Matthieu

    2016-02-11

    Protein methyltransferases (PMTs) are a promising target class in oncology and other disease areas. They are composed of SET domain methyltransferases and structurally unrelated Rossman-fold enzymes that include protein arginine methyltransferases (PRMTs). In the absence of a well-defined medicinal chemistry tool-kit focused on PMTs, most current inhibitors were identified by screening large and diverse libraries of leadlike molecules. So far, no successful fragment-based approach was reported against this target class. Here, by deconstructing potent PRMT inhibitors, we find that chemical moieties occupying the substrate arginine-binding site can act as efficient fragment inhibitors. Screening a fragment library against PRMT6 produced numerous hits, including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased global histone 3 arginine 2 methylation in cells, and can serve as a warhead for the development of PRMT chemical probes.

  11. UV-B irradiation changes specifically the secondary metabolite profile in broccoli sprouts: induced signaling overlaps with defense response to biotic stressors.

    Science.gov (United States)

    Mewis, Inga; Schreiner, Monika; Nguyen, Chau Nhi; Krumbein, Angelika; Ulrichs, Christian; Lohse, Marc; Zrenner, Rita

    2012-09-01

    Only a few environmental factors have such a pronounced effect on plant growth and development as ultraviolet light (UV). Concerns have arisen due to increased UV-B radiation reaching the Earth's surface as a result of stratospheric ozone depletion. Ecologically relevant low to moderate UV-B doses (0.3-1 kJ m(-2) d(-1)) were applied to sprouts of the important vegetable crop Brassica oleracea var. italica (broccoli), and eco-physiological responses such as accumulation of non-volatile secondary metabolites were related to transcriptional responses with Agilent One-Color Gene Expression Microarray analysis using the 2×204 k format Brassica microarray. UV-B radiation effects have usually been linked to increases in phenolic compounds. As expected, the flavonoids kaempferol and quercetin accumulated in broccoli sprouts (the aerial part of the seedlings) 24 h after UV-B treatment. A new finding is the specific UV-B-mediated induction of glucosinolates (GS), especially of 4-methylsulfinylbutyl GS and 4-methoxy-indol-3-ylmethyl GS, while carotenoids and Chl levels remained unaffected. Accumulation of defensive GS metabolites was accompanied by increased expression of genes associated with salicylate and jasmonic acid signaling defense pathways and up-regulation of genes responsive to fungal and bacterial pathogens. Concomitantly, plant pre-exposure to moderate UV-B doses had negative effects on the performance of the caterpillar Pieris brassicae (L.) and on the population growth of the aphid Myzus persicae (Sulzer). Moreover, insect-specific induction of GS in broccoli sprouts was affected by UV-B pre-treatment.

  12. 青花菜流通过程中营养品质变化的研究%Study on changes of nutritional quality in circulation of broccoli

    Institute of Scientific and Technical Information of China (English)

    郭李维; 王清; 杨娜; 高丽朴

    2011-01-01

    研究了不同流通温度和包装方式对青花菜外观品质与营养成分的影响。实验结果表明:青花菜在流通过程中随着组织的衰老,其叶绿素、VC、可溶性糖及可溶性蛋白质含量均呈下降趋势。青花菜在5℃中流通比在20℃中能更好地保持其营养成分。利用0.03mm厚的聚乙烯薄膜包装既可避免青花菜失水萎蔫,又可抑制黄化率。20℃无包装处理的青花菜最佳流通时间为1d,20℃PE处理的为2d,5℃无包装处理的为6d,5℃PE处理的为12d。%The effect of different temperature and packaging on the quality and nutrients of broccoli was studied.The results showed that the contents of chlorophyll,ascorbic acid,soluble sugars were decreased with senescence of broccoli in the circulation process.In addition,The broccoli were circulated at 5℃ that could better maintain the quality and nutrients than circulation at 20℃.Packaged with 0.03mm PE could not only prevent the loss of water,but also inhibit the rate of yellowing.Therefore,the broccoli PE packaging could keep a better appearance for 2 days,1 day longer than those directly exposed to the air at 20℃,while cold chain could show great advantage during transportation,the brocoli packed by PE film could store 12d at 5℃,and the unpackaged might keep better quality for 6 days as well.

  13. UV-B Irradiation Changes Specifically the Secondary Metabolite Profile in Broccoli Sprouts: Induced Signaling Overlaps with Defense Response to Biotic Stressors

    OpenAIRE

    Mewis, Inga; Schreiner, Monika; Nguyen, Chau Nhi; Krumbein, Angelika; Ulrichs, Christian; Lohse, Marc; Zrenner, Rita

    2012-01-01

    Only a few environmental factors have such a pronounced effect on plant growth and development as ultraviolet light (UV). Concerns have arisen due to increased UV-B radiation reaching the Earth’s surface as a result of stratospheric ozone depletion. Ecologically relevant low to moderate UV-B doses (0.3–1 kJ m–2 d–1) were applied to sprouts of the important vegetable crop Brassica oleracea var. italica (broccoli), and eco-physiological responses such as accumulation of non-volatile secondary m...

  14. Research Progress of Broccoli in the Prevention and Treatment of Diseases%西兰花预防和治疗肿瘤研究的新进展

    Institute of Scientific and Technical Information of China (English)

    洪姣; 王佳蕊; 黄春琦; 徐殿琴; 张婷; 许健

    2014-01-01

    In recent years, the morbidity of cancer, cardiovascular disease, diabetes and other diseases were increasing constantly. Meanwhile, people gradually realized that diet played an important role in the prevention or treatment of chronic disease by providing useful elements. Many papers have confirmed that glucosinolates, anthocyanin, vitamins and other beneficial ingredients in broccoli had the function of preventing cancer, diabetes and eye disease, and protecting cardio-vascular and antioxidation. This suggested that increasing our daily diet intake of broccoli could promote the health, prevent diseases. In this paper, we reviewed the medicinal value of broccoli and the influence of cooking and temperature on its useful elements, and illustrated different roles of different effective components of broccoli in the treatment of diseases.%近年来,肿瘤、心血管疾病及糖尿病等疾病的发病率不断上升,人们意识到可以在日常饮食中通过提供有益成分而起到预防或治疗疾病的作用。大量文献证实西兰花中所含有的芥子油苷、花青素、维生素等有益成分具有抗癌、预防糖尿病及眼部疾病、保护心血管抗氧化等作用,提示在日常饮食中增加西兰花的摄入可以促进身体健康,起到防病、治病和改善体质的功效。本文主要综述了西兰花的药用价值以及烹饪、温度等对其有效成分的影响,并总结了西兰花中不同的有效成分治疗疾病的作用。

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7.

    Science.gov (United States)

    Abuladze, Tamar; Li, Manrong; Menetrez, Marc Y; Dean, Timothy; Senecal, Andre; Sulakvelidze, Alexander

    2008-10-01

    A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for Escherichia coli O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three E. coli O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable E. coli organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (10(10), 10(9), and 10(8) PFU/ml) resulted in statistically significant reductions (P = E. coli O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of E. coli O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 10(9) PFU/ml) also significantly reduced the number of viable E. coli O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 +/- 4 h posttreatment of tomato samples) to 100% (at 24 +/- 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.

  17. Facile synthesis of nano cauliflower and nano broccoli like hierarchical superhydrophobic composite coating using PVDF/carbon soot particles via gelation technique.

    Science.gov (United States)

    Sahoo, Bichitra Nanda; Balasubramanian, Kandasubramanian

    2014-12-15

    We have elucidated a cost effective fabrication technique to produce superhydrophobic polyvinylidene fluoride (PVDF/DMF/candle soot particle and PVDF/DMF/camphor soot particle composite) porous materials. The water repellent dry composite was formed by the interaction of non-solvent (methanol) into PVDF/carbon soot particles suspension in N,N-dimethylformamide (DMF). It is seen that longer quenching time effectively changes the surface morphology of dry composites. The nano broccoli like hierarchical microstructure with micro or nano scaled roughen surface was obtained for PVDF/DMF/camphor soot particle, which reveals water contact angle of 172° with roll off angle of 2°. However, composite coating of PVDF/DMF/candle soot particle shows nano cauliflower like hierarchical, which illustrates water contact angle of 169° with roll off angle of 3°. To elucidate the enhancement of water repellent property of PVDF composites, we further divulge the evolution mechanism of nano cauliflower and nano broccoli structure. In order to evaluate the water contact angle of PVDF composites, surface diffusion of water inside the pores is investigated. Furthermore, the addition of small amount of carbon soot particles in composite not only provides the crystallization of PVDF, but also leads to dramatical amendment of surface morphology which increases the surface texture and roughness for superhydrophobicity.

  18. Study of Cleome Gynandra Extract on Broccoli Preservation%白花菜提取物对花椰菜保鲜效果的研究

    Institute of Scientific and Technical Information of China (English)

    曾维丽; 张臻; 刘少阳; 王飞

    2015-01-01

    论文研究了白花菜提取物对花椰菜中微生物、褐变指数和VC含量的影响。结果表明,白花菜提取物能有效抑制微生物的生长,有效减缓花椰菜的褐变速度和VC的降解速度,较好地保持花椰菜的品质,其中1.0 g/L白花菜提取物的保鲜效果最好。%The effects of Cleome gynandra extract on total number of colony, as well as browning index and vitamin C content of Broccoli were studied. The results showed that Cleome gynandra extract could effectively eliminate the microorganism, delay the browning rate and slow down the degradation of vitamin C. 1.0g/L Cleome gynandra extract was shown to be the best to fresh-keeping and storage quality of broccoli.

  19. Antiamnesic Effect of Broccoli (Brassica oleracea var. italica) Leaves on Amyloid Beta (Aβ)1-42-Induced Learning and Memory Impairment.

    Science.gov (United States)

    Park, Seon Kyeong; Ha, Jeong Su; Kim, Jong Min; Kang, Jin Yong; Lee, Du Sang; Guo, Tian Jiao; Lee, Uk; Kim, Dae-Ok; Heo, Ho Jin

    2016-05-04

    To examine the antiamnesic effects of broccoli (Brassica oleracea var. italica) leaves, we performed in vitro and in vivo tests on amyloid beta (Aβ)-induced neurotoxicity. The chloroform fraction from broccoli leaves (CBL) showed a remarkable neuronal cell-protective effect and an inhibition against acetylcholinesterase (AChE). The ameliorating effect of CBL on Aβ1-42-induced learning and memory impairment was evaluated by Y-maze, passive avoidance, and Morris water maze tests. The results indicated improving cognitive function in the CBL group. After the behavioral tests, antioxidant effects were detected by superoxide dismutase (SOD), oxidized glutathione (GSH)/total GSH, and malondialdehyde (MDA) assays, and inhibition against AChE was also presented in the brain. Finally, oxo-dihydroxy-octadecenoic acid (oxo-DHODE) and trihydroxy-octadecenoic acid (THODE) as main compounds were identified by quadrupole time-of-flight ultraperformance liquid chromatography (Q-TOF UPLC-MS) analysis. Therefore, our studies suggest that CBL could be used as a natural resource for ameliorating Aβ1-42-induced learning and memory impairment.

  20. Study on Ultrasonic Extraction Technique of Broccoli Polysaccharide%超声波法提取西兰花多糖的工艺研究

    Institute of Scientific and Technical Information of China (English)

    郎艳婷; 刘波; 张振宇; 郝明月; 苏万平

    2012-01-01

    采用超声波辅助法提取西兰花多糖,通过单因素试验与正交试验对提取工艺进行优化,以期确定西兰花多糖的最佳提取工艺.结果表明,超声波辅助法提取西兰花多糖的最佳工艺参数为:超声时间50min,超声波功率80W,提取温度60℃,料液比1∶50(mL/g),多糖的得率为8.24%.该试验方法简单、易行,可为西兰花多糖的工业化生产提供参考.%To determine the best broccoli polysaccharide extraction process with the ultrasonic assisted-extraction technology. The technique was optimized by a single factor experiments and orthogonal test. The results showed that the optimum extraction parameters of polysaccharides was that ultrasonic time for50 min, 80 W ultrasonic power at 60℃, ratio of liquid feed 1:50 (ml/g), and polysaccharide yield was 8.24%. The test method is simple, easy, and can provide a reference for the industrial production of broccoli polysaccharide.

  1. Effects of Organic and Waste-Derived Fertilizers on Yield, Nitrogen and Glucosinolate Contents, and Sensory Quality of Broccoli (Brassica oleracea L. var. italica).

    Science.gov (United States)

    Øvsthus, Ingunn; Breland, Tor Arvid; Hagen, Sidsel Fiskaa; Brandt, Kirsten; Wold, Anne-Berit; Bengtsson, Gunnar B; Seljåsen, Randi

    2015-12-23

    Organic vegetable production attempts to pursue multiple goals concerning influence on environment, production resources, and human health. In areas with limited availability of animal manure, there is a need for considering various off-farm nutrient resources for such production. Different organic and waste-derived fertilizer materials were used for broccoli production at two latitudes (58° and 67°) in Norway during two years. The fertilizer materials were applied at two rates of total N (80 and 170 kg ha(-1)) and compared with mineral fertilizer (170 kg ha(-1)) and no fertilizer. Broccoli yield was strongly influenced by fertilizer materials (algae meal < unfertilized control < sheep manure < extruded shrimp shell < anaerobically digested food waste < mineral fertilizer). Yield, but not glucosinolate content, was linearly correlated with estimated potentially plant-available N. However, extruded shrimp shell and mineral NPK fertilizer gave higher glucosinolate contents than sheep manure and no fertilizer. Sensory attributes were less affected by fertilizer material and plant-available N.

  2. Structural mechanism of S-adenosyl methionine binding to catechol O-methyltransferase.

    Directory of Open Access Journals (Sweden)

    Douglas Tsao

    Full Text Available Methyltransferases possess a homologous domain that requires both a divalent metal cation and S-adenosyl-L-methionine (SAM to catalyze its reactions. The kinetics of several methyltransferases has been well characterized; however, the details regarding their structural mechanisms have remained unclear to date. Using catechol O-methyltransferase (COMT as a model, we perform discrete molecular dynamics and computational docking simulations to elucidate the initial stages of cofactor binding. We find that COMT binds SAM via an induced-fit mechanism, where SAM adopts a different docking pose in the absence of metal and substrate in comparison to the holoenzyme. Flexible modeling of the active site side-chains is essential for observing the lowest energy state in the apoenzyme; rigid docking tools are unable to recapitulate the pose unless the appropriate side-chain conformations are given a priori. From our docking results, we hypothesize that the metal reorients SAM in a conformation suitable for donating its methyl substituent to the recipient ligand. The proposed mechanism enables a general understanding of how divalent metal cations contribute to methyltransferase function.

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

    Science.gov (United States)

    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…

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Guanidinoacetate Methyltransferase (GAMT) Deficiency: Late Onset of Movement Disorder and Preserved Expressive Language

    Science.gov (United States)

    O'Rourke, Declan J.; Ryan, Stephanie; Salomons, Gajja; Jakobs, Cornelis; Monavari, Ahmad; King, Mary D.

    2009-01-01

    Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine biosynthesis, characterized by early-onset learning disability and epilepsy in most affected children. Severe expressive language delay is a constant feature even in the mildest clinical phenotypes. We report the clinical, biochemical, imaging, and treatment data of two…

  6. DNA methyltransferase and alcohol dehydrogenase: gene-nutrient interactions in relation to risk of colorectal polyps.

    NARCIS (Netherlands)

    Jung, A.Y.; Poole, E.M.; Bigler, J.; Whitton, J.; Potter, J.D.; Ulrich, C.M.

    2008-01-01

    Disturbances in DNA methylation are a characteristic of colorectal carcinogenesis. Folate-mediated one-carbon metabolism is essential for providing one-carbon groups for DNA methylation via DNA methyltransferases (DNMTs). Alcohol, a folate antagonist, could adversely affect one-carbon metabolism. In

  7. Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus.

    Science.gov (United States)

    Lukacin, Richard; Matern, Ulrich; Specker, Silvia; Vogt, Thomas

    2004-11-19

    Caffeoyl-coenzyme A O-methyltransferase cDNA was cloned from dark-grown Ammi majus L. (Apiaceae) cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli. The translated polypeptide of 27.1-kDa shared significant identity to other members of this highly conserved class of proteins and was 98.8% identical to the corresponding O-methyltransferase from parsley. For biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal-affinity chromatography, although the recombinant enzyme did not contain any affinity tag. Based on sequence analysis and substrate specificity, the enzyme classifies as a cation-dependent O-methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of Mg2+-ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg2+ was replaced by Mn2+ or Co2+ in the assays. This effect could point to yet unknown functions and substrate specificities in situ and suggests promiscuous roles for the lignin specific cluster of plant O-methyltransferases.

  8. Local chromatin microenvironment determines DNMT activity : from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

    NARCIS (Netherlands)

    van der Wijst, Monique G. P.; Venkiteswaran, Muralidhar; Chen, Hui; Xu, Guo-Liang; Plosch, Torsten; Rots, Marianne G.

    2015-01-01

    Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known fun

  9. rmtA, encoding a putative anginine methyltransferase, regulates secondary metabolism and development in Aspergillus flavus

    Science.gov (United States)

    Aspergillus flavus is found colonizing numerous oil seed crops such as corn, peanuts, sorghum, treenuts and cotton worldwide, contaminating them with aflatoxin and other harmful potent toxins. In the phylogenetically related model fungus Aspergillus nidulans, the methyltransferase, RmtA, has been de...

  10. The histone methyltransferase and putative oncoprotein MMSET is overexpressed in a large variety of human tumors

    DEFF Research Database (Denmark)

    Hudlebusch, Heidi Rye; Santoni-Rugiu, Eric; Simon, Ronald

    2011-01-01

    Multiple myeloma SET (Suppressor of variegation, Enhancer of zeste, and Trithorax) domain (MMSET) is a histone lysine methyltransferase deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation and poor prognosis. With the aim of understanding, if MMSET can be involved...

  11. Catechol-O-methyltransferase gene methylation and substance use in adolescents : the TRAILS study

    NARCIS (Netherlands)

    van der Knaap, L. J.; Schaefer, J. M.; Franken, I. H. A.; Verhulst, F. C.; van Oort, F. V. A.; Riese, H.

    2014-01-01

    Substance use often starts in adolescence and poses a major problem for society and individual health. The dopamine system plays a role in substance use, and catechol-O-methyltransferase (COMT) is an important enzyme that degrades dopamine. The Val(108/158)Met polymorphism modulates COMT activity an

  12. Loss of the histone methyltransferase EZH2 induces resistance to multiple drugs in acute myeloid leukemia

    DEFF Research Database (Denmark)

    Göllner, Stefanie; Oellerich, Thomas; Agrawal-Singh, Shuchi

    2017-01-01

    In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction of h...

  13. A fluorescence resonance energy transfer-based method for histone methyltransferases

    DEFF Research Database (Denmark)

    Devkota, Kanchan; Lohse, Brian; Nyby Jakobsen, Camilla;

    2015-01-01

    A simple dye–quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye...

  14. The histone methyltransferase SET8 is required for S-phase progression

    DEFF Research Database (Denmark)

    Jørgensen, Stine; Elvers, Ingegerd; Trelle, Morten Beck;

    2008-01-01

    Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show...

  15. Recognition elements in rRNA for the tylosin resistance methyltransferase RlmA(II)

    DEFF Research Database (Denmark)

    Lebars, Isabelle; Husson, Clotilde; Yoshizawa, Satoko

    2007-01-01

    antibiotics. We have previously solved the solution structure of hairpin 35 in the conformation that is recognized by the RlmA(II) methyltransferase from Streptococcus pneumoniae. It was shown that while essential recognition elements are located in hairpin 35, the interactions between RlmA(II) and hairpin 35...

  16. DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents.

    NARCIS (Netherlands)

    B.J. Glassner (Brian); G. Weeda (Geert); J.M. Allan (James); J.L.M. Broekhof (Jose'); N.H.E. Carls (Nick); I. Donker (Ingrid); B.P. Engelward (Bevin); R.J. Hampson (Richard); R. Hersmus (Remko); M.J. Hickman (Mark); R.B. Roth (Richard); H.B. Warren (Henry); M.M. Wu (Mavis); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

    1999-01-01

    textabstractWe have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA methyltransf

  17. DNA methyltransferase DNMT3A associates with viral proteins and impacts HSV-1 infection.

    Science.gov (United States)

    Rowles, Daniell L; Tsai, Yuan-Chin; Greco, Todd M; Lin, Aaron E; Li, Minghao; Yeh, Justin; Cristea, Ileana M

    2015-06-01

    Viral infections can alter the cellular epigenetic landscape, through modulation of either DNA methylation profiles or chromatin remodeling enzymes and histone modifications. These changes can act to promote viral replication or host defense. Herpes simplex virus type 1 (HSV-1) is a prominent human pathogen, which relies on interactions with host factors for efficient replication and spread. Nevertheless, the knowledge regarding its modulation of epigenetic factors remains limited. Here, we used fluorescently-labeled viruses in conjunction with immunoaffinity purification and MS to study virus-virus and virus-host protein interactions during HSV-1 infection in primary human fibroblasts. We identified interactions among viral capsid and tegument proteins, detecting phosphorylation of the capsid protein VP26 at sites within its UL37-binding domain, and an acetylation within the major capsid protein VP5. Interestingly, we found a nuclear association between viral capsid proteins and the de novo DNA methyltransferase DNA (cytosine-5)-methyltransferase 3A (DNMT3A), which we confirmed by reciprocal isolations and microscopy. We show that drug-induced inhibition of DNA methyltransferase activity, as well as siRNA- and shRNA-mediated DNMT3A knockdowns trigger reductions in virus titers. Altogether, our results highlight a functional association of viral proteins with the mammalian DNA methyltransferase machinery, pointing to DNMT3A as a host factor required for effective HSV-1 infection.

  18. Structures of the m(6)A Methyltransferase Complex: Two Subunits with Distinct but Coordinated Roles.

    Science.gov (United States)

    Zhou, Katherine I; Pan, Tao

    2016-07-21

    In this issue of Molecular Cell, Wang et al. (2016a) report crystal structures of the core of the METTL3/METTL14 m(6)A methyltransferase complex and propose how the two subunits interact and cooperate to bind and methylate RNA.

  19. CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

    Science.gov (United States)

    CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

  20. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Jensen, Michael R

    2004-01-01

    SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show tha...

  1. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing

    DEFF Research Database (Denmark)

    Relling, M V; Gardner, E E; Sandborn, W J;

    2011-01-01

    Thiopurine methyltransferase (TPMT) activity exhibits monogenic co-dominant inheritance, with ethnic differences in the frequency of occurrence of variant alleles. With conventional thiopurine doses, homozygous TPMT-deficient patients (~1 in 178 to 1 in 3,736 individuals with two nonfunctional TP...

  2. Guanidinoacetate methyltransferase (GAMT) deficiency : Outcomes in 48 individuals and recommendations for diagnosis, treatment and monitoring

    NARCIS (Netherlands)

    Stockler-Ipsiroglu, Sylvia; van Karnebeek, Clara; Longo, Nicola; Korenke, G. Christoph; Mercimek-Mahmutoglu, Saadet; Marquart, Iris; Barshop, Bruce; Grolik, Christiane; Schlune, Andrea; Angle, Brad; Araujo, Helena Caldeira; Coskun, Turgay; Diogo, Luisa; Geraghty, Michael; Haliloglu, Goknur; Konstantopoulou, Vassiliki; Leuzzi, Vincenzo; Levtova, Alina; MacKenzie, Jennifer; Maranda, Bruno; Mhanni, Aizeddin A.; Mitchell, Grant; Morris, Andrew; Newlove, Theresa; Renaud, Deborah; Scaglia, Fernando; Valayannopoulos, Vassili; van Spronsen, Francjan J.; Verbruggen, Krijn T.; Yuskiv, Nataliya; Nyhan, William; Schulze, Andreas

    2014-01-01

    We collected data on 48 patients from 38 families with guanidinoacetate methyltransferase (GAMT) deficiency. Global developmental delay/intellectual disability (DD/ID) with speech/language delay and behavioral problems as the most affected domains was present in 44 participants, with additional epil

  3. LED复合光处理对西兰花低温保鲜效果的影响%Effect of LED Composite Light on Preservation of Broccoli during Cold Storage

    Institute of Scientific and Technical Information of China (English)

    李宁; 阎瑞香; 张娜

    2015-01-01

    In order to study on effect of different treat of LED composite light on preservation of broccoli ,fresh broccoli was used as the main materials .By measuring the indicators at 4 ℃ low temperature conditions , such as sensory quality ,vitamin C content ,chlorophyll content and broccoli ethylene production rate ,effect of LED compos-ite light on preservation for broccoli was determined .The results showed that treatment with LED composite of red and blue was better for the quality of broccoli compared with non-light treatment.The former could extend storage time by 10-15 d.Meanwhile,it could got a higher score of sensory evaluation ,prevented the rapid decline in im-portant nutrient substance vitamin C content of broccoli ,delayed the time of the evolution of ethylene and the ap-pearance of respiration peak , decreased the respiration variable peak , and reduced membrane lipid peroxidation damage of broccoli during storage .%为研究不同LED复合光处理对西兰花保鲜效果的影响。以新鲜西兰花为材料,通过测定4℃低温条件下,西兰花的感官品质、维生素C含量、叶绿素含量、乙烯生成速率等各项指标,研究LED红蓝、LED红绿复合光处理对西兰花保鲜效果的影响。结果表明,与无光处理方式相比, LED红蓝复合光处理效果显著,不仅延长了西兰花保鲜期10~15 d左右,而且较好地保持了西兰花原有的外观品质,阻止了西兰花贮藏期间重要营养物质Vc含量的快速流失,延缓了贮藏期间乙烯释放量峰值和呼吸跃变出现的时间,显著降低了呼吸跃变的峰值,减少了膜脂过氧化对西兰花造成的损伤。

  4. Floral Benzenoid Carboxyl Methyltransferases: From in Vitro to in Planta Function

    Energy Technology Data Exchange (ETDEWEB)

    Effmert,U.; Saschenbrecker, S.; Ross, J.; Negre, F.; Fraser, C.; Noel, J.; Dudareva, N.; Piechulla, B.

    2005-01-01

    Benzenoid carboxyl methyltransferases synthesize methyl esters (e.g., methyl benzoate and methyl salicylate), which are constituents of aromas and scents of many plant species and play important roles in plant communication with the surrounding environment. Within the past five years, eleven such carboxyl methyltransferases were isolated and most of them were comprehensively investigated at the biochemical, molecular and structural level. Two types of enzymes can be distinguished according to their substrate preferences: the SAMT-type enzymes isolated from Clarkia breweri, Stephanotis floribunda, Antirrhinum majus, Hoya carnosa, and Petunia hybrida, which have a higher catalytic efficiency and preference for salicylic acid, while BAMT-type enzymes from A. majus, Arabidopsis thaliana, Arabidopsis lyrata, and Nicotiana suaveolens prefer benzoic acid. The elucidation of C. breweri SAMT's three-dimensional structure allowed a detailed modelling of the active sites of the carboxyl methyltransferases and revealed that the SAM binding pocket is highly conserved among these enzymes while the methyl acceptor binding site exhibits some variability, allowing a classification into SAMT-type and BAMT-type enzymes. The analysis of expression patterns coupled with biochemical characterization showed that these carboxyl methyltransferases are involved either in floral scent biosynthesis or in plant defense responses. While the latter can be induced by biotic or abiotic stress, the genes responsible for floral scent synthesis exhibit developmental and rhythmic expression pattern. The nature of the product and efficiency of its formation in plants depend on the availability of substrates, the catalytic efficiency of the enzyme toward benzoic acid and/or salicylic acid, and the transcriptional, translational, and post-translational regulation at the enzyme level. The biochemical properties of benzenoid carboxyl methyltransferases suggest that the genes involved in plant defenses

  5. Genomic survey, gene expression analysis and structural modeling suggest diverse roles of DNA methyltransferases in legumes.

    Science.gov (United States)

    Garg, Rohini; Kumari, Romika; Tiwari, Sneha; Goyal, Shweta

    2014-01-01

    DNA methylation plays a crucial role in development through inheritable gene silencing. Plants possess three types of DNA methyltransferases (MTases), namely Methyltransferase (MET), Chromomethylase (CMT) and Domains Rearranged Methyltransferase (DRM), which maintain methylation at CG, CHG and CHH sites. DNA MTases have not been studied in legumes so far. Here, we report the identification and analysis of putative DNA MTases in five legumes, including chickpea, soybean, pigeonpea, Medicago and Lotus. MTases in legumes could be classified in known MET, CMT, DRM and DNA nucleotide methyltransferases (DNMT2) subfamilies based on their domain organization. First three MTases represent DNA MTases, whereas DNMT2 represents a transfer RNA (tRNA) MTase. Structural comparison of all the MTases in plants with known MTases in mammalian and plant systems have been reported to assign structural features in context of biological functions of these proteins. The structure analysis clearly specified regions crucial for protein-protein interactions and regions important for nucleosome binding in various domains of CMT and MET proteins. In addition, structural model of DRM suggested that circular permutation of motifs does not have any effect on overall structure of DNA methyltransferase domain. These results provide valuable insights into role of various domains in molecular recognition and should facilitate mechanistic understanding of their function in mediating specific methylation patterns. Further, the comprehensive gene expression analyses of MTases in legumes provided evidence of their role in various developmental processes throughout the plant life cycle and response to various abiotic stresses. Overall, our study will be very helpful in establishing the specific functions of DNA MTases in legumes.

  6. Effects of Different Pollination Periods on Seed Yield of Broccoli Self-incompatible Lines%不同授粉时期对青花菜自交不亲和系种子产量的影响

    Institute of Scientific and Technical Information of China (English)

    李易蓉; 木万福; 袁建民; 但忠; 苏银玲; 杨龙

    2013-01-01

    为了提高青花菜自交不亲和系种子产量,研究了授粉时期对不同熟性青花菜自交不亲和系结荚率、平均单荚籽粒数、种子千粒质量和种子产量的影响。研究结果表明,不同授粉时期对青花菜自交不亲和系种子生产有显著差异,早熟青花菜自交不亲和系在末花期授粉的结荚率、单荚籽粒数、种子千粒质量、种子产量最高,而中熟、晚熟青花菜自交不亲和系在盛花期授粉结荚率、单荚籽粒数、种子千粒质量、种子产量最高。%In order to increase the seed yield of self-incompatible lines of broccoli, we studied the effects of different pollination periods on podding rate, grain number of a single pod, thousand seed weight and seed yield of the self -incompatible lines of broccoli in this paper. The results showed that, different pollination periods affected the seed production of the self-incompatible lines of broccoli significantly. The late flowering period was the best pollination period for early-maturing broccoli cultivar, while the full-bloom period was best for mid-maturing and late-maturing cultivars, by doing so, we could obtain the highest podding rate, grain number of a single pod, thousand seed weight and seed yield of the three different kinds of broccoli cultivars.

  7. Callus cell proliferation from broccoli leaf slice using IBA and BAP in vitro culture: Its biochemical and antioxidant properties.

    Science.gov (United States)

    Sharif Hossain, A B M; Haq, Imdadul; Ibrahim, Nasir A; Aleissa, Mohammed Saad

    2016-03-01

    Plant tissue or cell culture keeps a significant role in micro-propagation in the plant production industry. Combination of 6-Benzylaminopurine (BAP) and other plant growth regulators like 1-Naphthaleneacetic acid (NAA) or Indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) was used in the most of the research in tissue culture. The study was carried out to investigate the optimization of the concentration of IBA and BAP combination (0, 0.25, 0.50, 1.0, 1.50, 2.0, 2.5, 3.0 and 3.5 mg/l) for the root, callus and leaf proliferation from the leaf cutting slice. The highest number (6.75) of root proliferation was observed in the concentration of 2.0 mg/l IBA+0.25 mg/l BAP combination. The callus initiation was found in the concentration of IBA 1.0-3.5 mg/l+BAP 1.0-2.0 mg/l. However, the highest callus weight was observed at the concentration of IBA 1.5 mg/l+BAP 1.0 mg/l combination than other combination of concentrations. Positively leaf initiation and formation was better in the concentration of IBA 1-3.5 mg/l+BAP 1.0-2.0 mg/l combination. In addition, the 2,2-diphenyl-2-picrylhydarzyl (DPPH) free radical scavenging potential was higher (70.1%) in leaves extract than in callus extracts (46.3%) at the concentration of 10 mg/ml though both extracts had lower DPPH free radical scavenging activity compared to the positive control, vitamin C and BHT. Theresults conclude that the optimum concentration was IBA 1.5 mg/l+BAP 1.0 mg/l combination to produce callus cell proliferation and concentration of 2.0 mg/l IBA+0.25 mg/l BAP combination was the optimum for root proliferation of broccoli in vitro.

  8. The use of biodegradable mulch for tomato and broccoli production: Crop yield and quality, mulch deterioration, and growers' perceptions

    Science.gov (United States)

    Cowan, Jeremy Scott

    Biodegradable mulch may offer the benefits of polyethylene mulch for crop production with the added benefit of biodegradability. Four studies were carried out in Mount Vernon, WA to evaluate biodegradable mulch for tomato (Solanum lycopersicum L.) and broccoli (Brassica oleracea var. italica) production. The first study compared four biodegradable mulch treatments: BioAgri, BioTelo, WeedGuardPlus (cellulose product), and SB-PLA-10/11/12 (experimental, non-woven fabric), to polyethylene mulch and bare ground in high tunnels and in the open field for tomato yield and fruit quality over three growing seasons. Biodegradable plastic films produced yields and fruit quality comparable to polyethylene. Moreover, high tunnels increased total and marketable fruit weight five and eight times, respectively, compared to the open field. The second study quantified relationships among visual assessment parameters and mulch mechanical properties. Visual assessments and mechanical property tests of polyethylene, BioAgri, BioTelo, WeedGuardPlus, and SB-PLA-10/11/12, were made over three growing seasons. Regression analyses found the strongest relationship overall (r2 = 0.41) to be between the percent of initial breaking force in the machine direction and log 10 of percent visual deterioration. However, evaluating mulch products individually and increasing sample frequency are recommended for future research. The third study evaluated three biodegradable mulch products, BioAgri, Crown 1, and SB-PLA-11, after soil-incorporation. The average area of recovered mulch fragments decreased for all mulch products over time. The number of mulch fragments initially increased for all mulch products, with the greatest number of Crown 1 and BioAgri fragments recovered 132 and 299 days after incorporation, respectively. At 397 days after soil-incorporation, the total area of recovered fragments of Crown 1 and BioAgri was 0% and 34% of the theoretical maximum area, respectively. The fourth study

  9. Callus cell proliferation from broccoli leaf slice using IBA and BAP in vitro culture: Its biochemical and antioxidant properties

    Directory of Open Access Journals (Sweden)

    A.B.M. Sharif Hossain

    2016-03-01

    Full Text Available Plant tissue or cell culture keeps a significant role in micro-propagation in the plant production industry. Combination of 6-Benzylaminopurine (BAP and other plant growth regulators like 1-Naphthaleneacetic acid (NAA or Indole-3-acetic acid (IAA or indole-3-butyric acid (IBA was used in the most of the research in tissue culture. The study was carried out to investigate the optimization of the concentration of IBA and BAP combination (0, 0.25, 0.50, 1.0, 1.50, 2.0, 2.5, 3.0 and 3.5 mg/l for the root, callus and leaf proliferation from the leaf cutting slice. The highest number (6.75 of root proliferation was observed in the concentration of 2.0 mg/l IBA+0.25 mg/l BAP combination. The callus initiation was found in the concentration of IBA 1.0–3.5 mg/l+BAP 1.0–2.0 mg/l. However, the highest callus weight was observed at the concentration of IBA 1.5 mg/l+BAP 1.0 mg/l combination than other combination of concentrations. Positively leaf initiation and formation was better in the concentration of IBA 1–3.5 mg/l+BAP 1.0–2.0 mg/l combination. In addition, the 2,2-diphenyl-2-picrylhydarzyl (DPPH free radical scavenging potential was higher (70.1% in leaves extract than in callus extracts (46.3% at the concentration of 10 mg/ml though both extracts had lower DPPH free radical scavenging activity compared to the positive control, vitamin C and BHT. Theresults conclude that the optimum concentration was IBA 1.5 mg/l+BAP 1.0 mg/l combination to produce callus cell proliferation and concentration of 2.0 mg/l IBA+0.25 mg/l BAP combination was the optimum for root proliferation of broccoli in vitro.

  10. A conformational switch in the active site of BT_2972, a methyltransferase from an antibiotic resistant pathogen B. thetaiotaomicron.

    Directory of Open Access Journals (Sweden)

    Veerendra Kumar

    Full Text Available Methylation is one of the most common biochemical reactions involved in cellular and metabolic functions and is catalysed by the action of methyltransferases. Bacteroides thetaiotaomicron is an antibiotic-resistant bacterium that confers resistance through methylation, and as yet, there is no report on the structure of methyltransferases from this bacterium. Here, we report the crystal structure of an AdoMet-dependent methyltransferase, BT_2972 and its complex with AdoMet and AdoHcy for B. thetaiotaomicron VPI-5482 strain along with isothermal titration calorimetric assessment of the binding affinities. Comparison of the apo and complexed BT_2972 structures reveals a significant conformational change between open and closed forms of the active site that presumably regulates the association with cofactors and may aid interaction with substrate. Together, our analysis suggests that BT_2972 is a small molecule methyltransferase and might catalyze two O-methylation reaction steps involved in the ubiquinone biosynthesis pathway.

  11. The effect of soil mulching with organic mulches, on weed infestation in broccoli and tomato cultivated under polypropylene fibre, and without a cover

    Directory of Open Access Journals (Sweden)

    Kosterna Edyta

    2014-07-01

    Full Text Available An investigation was done on the effect of different types of organic mulches that were applied in form of straw to the soil mulching process, on the weed infestation, number, and fresh mass of weeds in broccoli cv. Milady F1 (Brassica oleracea L. var. italica Plenck and tomato cv. Polfast F1 (Lycopersicon esculentum Mill. grown under polypropylene fibre as a covering, or grown without a covering. The different types of organic straw mulches were: rye (Secale cereale L., corn (Zea mays L., rape (Brassica napus L. subsp. napus, and buckwheat (Fagopyrum esculentum Moench.. All the organic mulches were applied at a dose of 10 t/ha. The effect of the mulches was compared to a control plot which had no mulch. The type of organic mulch applied to the soil mulching process influenced species composition, number, and fresh mass of weeds. This effect could be the result of the properties of the mulch (colour, structure, etc. or the allelopathic effect on the germination and growth of individual weeds species. Irrespective of the investigated factors, 24 and 25 weeds species, respectively, were observed immediately after cover removal and before broccoli and tomato harvest. In the first date of estimation Chenopodium album L., Echinochloa crus-galli (L. P. Beauv., Fallopia convolvulus (L. Á. Löve, Stellaria media (L. Vill., and Viola tricolor L. dominated, however, before the vegetables were harvested Ch. album, V. tricolor, Veronica arvensis L., and E. crus-galli dominated. An application of polypropylene fibre contributed to an increase in the number and fresh mass of weeds in both vegetables in the first date of estimation (after cover removal. During this period, vegetables cannot compete with weeds. It is important to note, though, that before the vegetables were harvested, a decrease was found in the number and fresh mass of weeds in the covered plots. The most efficient weed limiter, both after cover removal and also before the broccoli and

  12. Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

    DEFF Research Database (Denmark)

    Vranken, Charlotte; Deen, Jochem; Dirix, Lieve

    2014-01-01

    We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore...... to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay...... the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes...

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

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, M. L. [Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. República, Apt. 127, 2781-901 Oeiras (Portugal); Bonifácio, M. J.; Soares-da-Silva, P. [Department of Research and Development, BIAL, 4785 S. Mamede do Coronado (Portugal); Carrondo, M. A.; Archer, M., E-mail: archer@itqb.unl.pt [Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. República, Apt. 127, 2781-901 Oeiras (Portugal)

    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°.

  14. Discovery of sphingosine 1-O-methyltransferase in rat kidney and liver homogenates

    Institute of Scientific and Technical Information of China (English)

    Santosh J SACKET; Dong-soon IM

    2008-01-01

    Aim:To characterize sphingosine methyltransferase in rat tissues.Methods:By using S-adenosyl-L-(methyl-3H) methionine,enzymatic activity was measured in the rat liver and kidney homogenates.Results:The optimum pH and reaction time for the enzyme assay were pH 7.8 and 1 h.ZnCl2 inhibited the activity,but not MgCl2,CaCl2,CoCl2,or NiCl2.In the kidney homogenate,enzymatic activity was detectable in the cytosol and all membrane fractions from the plasma membrane and other organelles; however,in the liver homogenate,enzymatic activity was detectable in all membrane fractions,but not in the cytosol.We also tested the enzymatic activity with structurally-modified sphingosine derivatives.Conclusion:We found sphingosine l-O-methyltransferase activity in the rat liver and kidney homogenates.

  15. Effects of Trichoderma harzianum Rifai over Plasmodiophora brassicae Woronin in broccoli, in Escagüey, municipality of Rangel, Mérida State

    Directory of Open Access Journals (Sweden)

    Mirna Labrador Morales

    2016-03-01

    Full Text Available The effectiveness of Trichoderma harzianum in suppressing clubroot of brassicas, which is caused by Plasmodiophora brassicae, was tested on broccoli (Brassica oleracea var. italica Plenck, in field conditions, in Escagüey, municipality of Rangel, Merida State, Venezuela. The experiment showed that the applications of a biopesticide based on this antagonist had a significant effect on the percentage of health plants and the crop yields, in dependence of the dose used. In addition, the relation benefits/cost was also favorable. These results showed that, for these particular conditions, P. brassicae is an adequate biological alternative to control the clubroot of brassicas, no aggressive to environment and human beings, useful for the transition phase toward a sustainable agriculture, without chemical pesticides.

  16. Vigor de sementes de brócolos submetidas a coberturas biodegradáveis e micronutrientes Vigor of broccoli seeds submitted to biodegradable coatings and micronutrients

    Directory of Open Access Journals (Sweden)

    Juliana A. Batista

    2005-07-01

    Full Text Available O desenvolvimento e a aplicação de filmes ou coberturas biodegradáveis na horticultura são técnicas praticadas há vários anos, que visam promover melhoria na qualidade do produto. no presente trabalho desenvolveu-se e caracterizou-se biofilmes de pectina, gelatina e ácidos graxos e verificou-se sua eficiência como cobertura para sementes de brócolos (Brassica oleracea L. var. italica. Os filmes foram caracterizados quanto à solubilidade em água. A contagem e a observação de plantas de brócolos, a partir da germinação de sementes cobertas e não cobertas foi realizada em média a cada três dias, aos 27 dias fêz-se a quantificação da matéria fresca e seca. Os filmes de pectina e ácido esteárico foram 100% solúveis em água, enquanto os elaborados com pectina e gelatina (1/1 foram apenas 18%. A aplicação das coberturas filmogênicas nas sementes de brócolos não afetou a emergência das plantas, demonstrando assim sua potencialidade para uso comercial. Os fertilizantes molibdato de sódio (0,2 mg L-1 e ácido bórico (1mg L-1, aplicados juntamente com os biofilmes, também não afetaram a emergência e o desenvolvimento das plantas.The development and application of biodegradable coatings and films have been used for many years in order to improve quality of the coated or packed product. The development and characterization of pectin and pectin/gelatin-based biofilms with fatty acids was evaluated and verified their efficiency as coatings to broccoli seeds (Brassica oleracea L. var. italica. The solubility in water of the films was determined. The counting and visual observations of the germinated broccoli seeds were done each three days and fresh and dried weight of the plants were determined at the end of the experiment. Pectin-based films with stearic acid were 100% soluble in water and composite films of pectin and gelatin were 18%. The application of the biodegradable coatings on broccoli seeds had no effect on the

  17. Green pests and diseases-controlling techniques and efficacy of broccoli%花椰菜病虫害绿色防控技术及其效果

    Institute of Scientific and Technical Information of China (English)

    张明真

    2012-01-01

    福建省云霄县佳州村“佳田农民专业合作社”蔬菜生产基地推广实施花椰菜病虫害绿色防控技术,取得显著成效。该文介绍了绿色防控技术的示范效果及主要措施。%Green pests and diseases-controlling techniques of broccoli were popularized and applied to vegetable production bases of Jia tian farmers' professional co-operatives in Jia zhou village, Yunxiao county, Fujian province. This demonstration obtained significant effect. This paper introduced the demonstration effect and main measures of green controlling techniques.

  18. De Novo DNA Methyltransferase DNMT3b Interacts with NEDD8-modified Proteins*

    OpenAIRE

    Shamay, Meir; Greenway, Melanie; Liao, Gangling; AMBINDER, RICHARD F; Hayward, S. Diane

    2010-01-01

    DNA methylation and histone modifications play an important role in transcription regulation. In cancer cells, many promoters become aberrantly methylated through the activity of the de novo DNA methyltransferases DNMT3a and DNMT3b and acquire repressive chromatin marks. NEDD8 is a ubiquitin-like protein modifier that is conjugated to target proteins, such as cullins, to regulate their activity, and cullin 4A (CUL4A) in its NEDD8-modified form is essential for repressive chromatin formation. ...

  19. Catecholamine-o-methyltransferase polymorphisms are associated with postoperative pain intensity.

    LENUS (Irish Health Repository)

    Lee, Peter J

    2011-02-01

    single nucleotide polymorphisms (SNPs) in the genes for catecholamine-O-methyltransferase (COMT), μ-opioid receptor and GTP cyclohydrolase (GCH1) have been linked to acute and chronic pain states. COMT polymorphisms are associated with experimental pain sensitivity and a chronic pain state. No such association has been identified perioperatively. We carried out a prospective observational clinical trial to examine associations between these parameters and the development of postoperative pain in patients undergoing third molar (M3) extraction.

  20. Thiopurine S-methyltransferase polymorphisms and thiopurine toxicity in treatment of inflammatory bowel disease

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To evaluate the relationship between thiopu- rine S-methyltransferase (TPMT) polymorphisms and thiopurine-induced adverse drug reactions (ADRs) in inflammatory bowel disease (IBD). METHODS: Eligible articles that compared the frequency of TPMT polymorphisms among thiopurine-tolerant and-intolerant adult IBD patients were included. Statistical analysis was performed with Review Manager 5.0. Sub-analysis/sensitivity analysis was also performed. RESULTS: Nine studies that investigated a total of 1309 part...

  1. Survival and tumorigenesis in O6-methylguanine DNA methyltransferase-deficient mice following cyclophosphamide exposure

    OpenAIRE

    Nagasubramanian, Ramamoorthy; Hansen, Ryan J.; Delaney, Shannon M.; Cherian, Mathew M.; Samson, Leona D.; Kogan, Scott C.; Dolan, M Eileen

    2008-01-01

    O6-methylguanine DNA methyltransferase (MGMT) deficiency is associated with an increased susceptibility to alkylating agent toxicity. To understand the contribution of MGMT in protecting against cyclophosphamide (CP)-induced toxicity, mutagenesis and tumorigenesis, we compared the biological effects of this agent in transgenic Mgmt knockout and wild-type mice. In addition, neurofibromin (Nf1)+/− background was used to increase the likelihood of CP-induced tumorigenesis. Cohorts of Mgmt-profic...

  2. Dietary Intake of Sulforaphane-Rich Broccoli Sprout Extracts during Juvenile and Adolescence Can Prevent Phencyclidine-Induced Cognitive Deficits at Adulthood.

    Directory of Open Access Journals (Sweden)

    Yumi Shirai

    Full Text Available Oxidative stress and inflammation play a role in cognitive impairment, which is a core symptom of schizophrenia. Furthermore, a hallmark of the pathophysiology of this disease is the dysfunction of cortical inhibitory γ-aminobutyric acid (GABA neurons expressing parvalbumin (PV, which is also involved in cognitive impairment. Sulforaphane (SFN, an isothiocyanate derived from broccoli, is a potent activator of the transcription factor Nrf2, which plays a central role in the inducible expressions of many cytoprotective genes in response to oxidative stress. Keap1 is a cytoplasmic protein that is essential for the regulation of Nrf2 activity. Here, we found that pretreatment with SFN attenuated cognitive deficits, the increase in 8-oxo-dG-positive cells, and the decrease in PV-positive cells in the medial prefrontal cortex and hippocampus after repeated administration of phencyclidine (PCP. Furthermore, PCP-induced cognitive deficits were improved by the subsequent subchronic administration of SFN. Interestingly, the dietary intake of glucoraphanin (a glucosinolate precursor of SFN during the juvenile and adolescence prevented the onset of PCP-induced cognitive deficits as well as the increase in 8-oxo-dG-positive cells and the decrease in PV-positive cells in the brain at adulthood. Moreover, the NRF2 gene and the KEAP1 gene had an epistatic effect on cognitive impairment (e.g., working memory and processing speed in patients with schizophrenia. These findings suggest that SFN may have prophylactic and therapeutic effects on cognitive impairment in schizophrenia. Therefore, the dietary intake of SFN-rich broccoli sprouts during the juvenile and adolescence may prevent the onset of psychosis at adulthood.

  3. Water balance and N-metabolism in broccoli (Brassica oleracea L. var. Italica) plants depending on nitrogen source under salt stress and elevated CO2.

    Science.gov (United States)

    Zaghdoud, Chokri; Carvajal, Micaela; Ferchichi, Ali; Del Carmen Martínez-Ballesta, María

    2016-11-15

    Elevated [CO2] and salinity in the soils are considered part of the effects of future environmental conditions in arid and semi-arid areas. While it is known that soil salinization decreases plant growth, an increased atmospheric [CO2] may ameliorate the negative effects of salt stress. However, there is a lack of information about the form in which inorganic nitrogen source may influence plant performance under both conditions. Single factor responses and the interactive effects of two [CO2] (380 and 800ppm), three different NO3(-)/NH4(+) ratios in the nutrient solution (100/0, 50/50 and 0/100, with a total N concentration of 3.5mM) and two NaCl concentrations (0 and 80mM) on growth, leaf gas exchange parameters in relation to root hydraulic conductance and N-assimilating enzymes of broccoli (Brassica oleracea L. var. Italica) plants were determined. The results showed that a reduced NO3(-) or co-provision of NO3(-) and NH4(+) could be an optimal source of inorganic N for broccoli plants. In addition, elevated [CO2] ameliorated the effect of salt exposure on the plant growth through an enhanced rate of photosynthesis, even at low N-concentration. However, NO3(-) or NO3(-)/NH4(+) co-provision display differential plant response to salt stress regarding water balance, which was associated to N metabolism. The results may contribute to our understanding of N-fertilization modes under increasing atmospheric [CO2] to cope with salt stress, where variations in N nutrition significantly influenced plant response.

  4. Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation.

    Science.gov (United States)

    Baubec, Tuncay; Colombo, Daniele F; Wirbelauer, Christiane; Schmidt, Juliane; Burger, Lukas; Krebs, Arnaud R; Akalin, Altuna; Schübeler, Dirk

    2015-04-09

    DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined genomic binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG-dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.

  5. miR-29 Represses the Activities of DNA Methyltransferases and DNA Demethylases

    Directory of Open Access Journals (Sweden)

    Izuho Hatada

    2013-07-01

    Full Text Available Members of the microRNA-29 (miR-29 family directly target the DNA methyltransferases, DNMT3A and DNMT3B. Disturbances in the expression levels of miR-29 have been linked to tumorigenesis and tumor aggressiveness. Members of the miR-29 family are currently thought to repress DNA methylation and suppress tumorigenesis by protecting against de novo methylation. Here, we report that members of the miR-29 family repress the activities of DNA methyltransferases and DNA demethylases, which have opposing roles in control of DNA methylation status. Members of the miR-29 family directly inhibited DNA methyltransferases and two major factors involved in DNA demethylation, namely tet methylcytosine dioxygenase 1 (TET1 and thymine DNA glycosylase (TDG. Overexpression of miR-29 upregulated the global DNA methylation level in some cancer cells and downregulated DNA methylation in other cancer cells, suggesting that miR-29 suppresses tumorigenesis by protecting against changes in the existing DNA methylation status rather than by preventing de novo methylation of DNA.

  6. Chromatin Targeting of de Novo DNA Methyltransferases by the PWWP Domain

    Institute of Scientific and Technical Information of China (English)

    Ying-ZiGe; Min-TiePu; HumairaGowher; Hai-PingWu; Jian-PingDing; AlbertJeltsch; Guo-LiangXu

    2005-01-01

    DNA methylation patterns of mammalian genomes are generated in gametogenesis and early embryonic development. Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the process. Both en-zymes contain a long N-terminal regulatory region linked to a conserved C-terminal domain responsible forthe catalytic activity. Although a PWWP domain in the N-terminal region has been shown to bind DNA in vitro, it is unclear how the DNA methyltransferases access their substrate in chromatin in vivo. We show here that the two proteins are associated with chromatin including mitotic chromosomes in mammalian cells, and the PWWP domain is essential for the chromatin targeting of the enzymes. The functional significance of PWWPmediated chromatin targeting is suggested by the fact that a missense mutation in this domain of human DNMT3B causes immunodeficiency, centromeric heterochromatin instability, facial anomalies (ICF) syndrome, which is characterized by loss of methylation insatellite DNA, pericentromeric instability, and immunodeficiency. We demonstrate that the mutant protein completely loses its chromatin targeting capacity. Our data establish the PWWP domain as a novel chromatin/chromosome-targeting module and suggest that the PWWP-mediated chromatin association is essential for the function of the de novo methyltransferases during development.

  7. Regulation of expression and activity of DNA (cytosine-5) methyltransferases in mammalian cells.

    Science.gov (United States)

    Kinney, Shannon R Morey; Pradhan, Sriharsa

    2011-01-01

    Three active DNA (cytosine-5) methyltransferases (DNMTs) have been identified in mammalian cells, Dnmt1, Dnmt3a, and Dnmt3b. DNMT1 is primarily a maintenance methyltransferase, as it prefers to methylate hemimethylated DNA during DNA replication and in vitro. DNMT3A and DNMT3B are de novo methyltransferases and show similar activity on unmethylated and hemimethylated DNA. DNMT3L, which lacks the catalytic domain, binds to DNMT3A and DNMT3B variants and facilitates their chromatin targeting, presumably for de novo methylation. There are several mechanisms by which mammalian cells regulate DNMT levels, including varied transcriptional activation of the respective genes and posttranslational modifications of the enzymes that can affect catalytic activity, targeting, and enzyme degradation. In addition, binding of miRNAs or RNA-binding proteins can also alter the expression of DNMTs. These regulatory processes can be disrupted in disease or by environmental factors, resulting in altered DNMT expression and aberrant DNA methylation patterns.

  8. An essential role for DNA methyltransferase DNMT3B in cancer cell survival.

    Science.gov (United States)

    Beaulieu, Normand; Morin, Steves; Chute, Ian C; Robert, Marie-France; Nguyen, Hannah; MacLeod, A Robert

    2002-08-02

    Abnormal methylation and associated silencing of tumor suppressor genes is a common feature of many types of cancers. The observation of persistent methylation in human cancer cells lacking the maintenance methyltransferase DNMT1 suggests the involvement of other DNA methyltransferases in gene silencing in cancer. To test this hypothesis, we have evaluated methylation and gene expression in cancer cells specifically depleted of DNMT3A or DNMT3B, de novo methyltransferases that are expressed in adult tissues. Here we have shown that depletion of DNMT3B, but not DNMT3A, induced apoptosis of human cancer cells but not normal cells. DNMT3B depletion reactivated methylation-silenced gene expression but did not induce global or juxtacentromeric satellite demethylation as did specific depletion of DNMT1. Furthermore, the effect of DNMT3B depletion was rescued by exogenous expression of either of the splice variants DNMT3B2 or DNMT3B3 but not DNMT1. These results indicate that DNMT3B has significant site selectivity that is distinct from DNMT1, regulates aberrant gene silencing, and is essential for cancer cell survival.

  9. Cooperativity between DNA Methyltransferases in the Maintenance Methylation of Repetitive Elements

    Science.gov (United States)

    Liang, Gangning; Chan, Matilda F.; Tomigahara, Yoshitaka; Tsai, Yvonne C.; Gonzales, Felicidad A.; Li, En; Laird, Peter W.; Jones, Peter A.

    2002-01-01

    We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells. PMID:11756544

  10. Transcriptome profiling of Set5 and Set1 methyltransferases: Tools for visualization of gene expression

    Directory of Open Access Journals (Sweden)

    Glòria Mas Martín

    2014-12-01

    Full Text Available Cells regulate transcription by coordinating the activities of multiple histone modifying complexes. We recently identified the yeast histone H4 methyltransferase Set5 and discovered functional overlap with the histone H3 methyltransferase Set1 in gene expression. Specifically, using next-generation RNA sequencing (RNA-Seq, we found that Set5 and Set1 function synergistically to regulate specific transcriptional programs at subtelomeres and transposable elements. Here we provide a comprehensive description of the methodology and analysis tools corresponding to the data deposited in NCBI's Gene Expression Omnibus (GEO under the accession number GSE52086. This data complements the experimental methods described in Mas Martín G et al. (2014 and provides the means to explore the cooperative functions of histone H3 and H4 methyltransferases in the regulation of transcription. Furthermore, a fully annotated R code is included to enable researchers to use the following computational tools: comparison of significant differential expression (SDE profiles; gene ontology enrichment of SDE; and enrichment of SDE relative to chromosomal features, such as centromeres, telomeres, and transposable elements. Overall, we present a bioinformatics platform that can be generally implemented for similar analyses with different datasets and in different organisms.

  11. Highly Iterated Palindromic Sequences (HIPs and Their Relationship to DNA Methyltransferases

    Directory of Open Access Journals (Sweden)

    Jeff Elhai

    2015-03-01

    Full Text Available The sequence GCGATCGC (Highly Iterated Palindrome, HIP1 is commonly found in high frequency in cyanobacterial genomes. An important clue to its function may be the presence of two orphan DNA methyltransferases that recognize internal sequences GATC and CGATCG. An examination of genomes from 97 cyanobacteria, both free-living and obligate symbionts, showed that there are exceptional cases in which HIP1 is at a low frequency or nearly absent. In some of these cases, it appears to have been replaced by a different GC-rich palindromic sequence, alternate HIPs. When HIP1 is at a high frequency, GATC- and CGATCG-specific methyltransferases are generally present in the genome. When an alternate HIP is at high frequency, a methyltransferase specific for that sequence is present. The pattern of 1-nt deviations from HIP1 sequences is biased towards the first and last nucleotides, i.e., those distinguish CGATCG from HIP1. Taken together, the results point to a role of DNA methylation in the creation or functioning of HIP sites. A model is presented that postulates the existence of a GmeC-dependent mismatch repair system whose activity creates and maintains HIP sequences.

  12. The Eukaryotic DNMT2 Genes Encode a New Class of Cytosine-5 DNA Methyltransferases

    Institute of Scientific and Technical Information of China (English)

    Lin-YaTang; M.NarsaReddy; VanyaRasheva; Tai-LinLee; Meng-JauLin; Ming-ShiuHung; C.-K.JamesShen

    2005-01-01

    DNMT2 is a subgroup of the eukaryotic cytosine-5 DNA methyltransferase gene family. Unlike the other family members, proteins encoded by DNMT2 genes were not known before to possess DNA methyltransferase activities. Most recently, we have showm that thegenome of Drosophila S2 cells stably expressing an exogenous Drosophila dDNMT2 cDNA became anoma-lously methylated at the 5'-positions of cytosines(Reddy, M. N., Tang, L. Y., Lee, T. L., and Shen, C.-K. J.(2003) Oncogene, in press). We present evidence here that the genomes of transgenic flies overexpressing the dDnmt2 protein also became hypermethylated at specific regions. Furthermore, transient transfection studies in combination with sodium bisulfite sequencing demonstrated that dDnmt2 as well as its mousc ortholog, mDnmt2, are capable of methylating a cotrans-fected plasmid DNA. These data provide solid evidence that the fly and mouse DNMT2 gene products are genuine cytosine-5 DNA methyltransferases.

  13. Kinetic analysis of Yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Robert J Wood

    Full Text Available BACKGROUND: DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics. METHODOLOGY/PRINCIPAL FINDINGS: Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein and quencher (dabcyl and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71+/-0.07 indicating that it is a sensitive assay for the identification of inhibitors. CONCLUSIONS/SIGNIFICANCE: The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

  14. Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase

    Institute of Scientific and Technical Information of China (English)

    Bin-Zhong Li; Guo-Liang Xu; Zheng Huang; Qing-Yan Cui; Xue-Hui Song; Lin Du; Albert Jeltsch; Ping Chen; Guohong Li; En Li

    2011-01-01

    Cytosine methylation of genomic DNA controls gene expression and maintains genome stability. How a specific DNA sequence is targeted for methylation by a methyltransferase is largely unknown. Here, we show that histone H3 tails lacking lysine 4 (K4) methylation function as an allosteric activator for methyltransferase Dnmt3a by binding to its plant homeodomain (PHD). In vitro, histone H3 peptides stimulated the methylation activity of Dnmt3a up to 8-fold, in a manner reversely correlated with the level of K4 methylation. The biological significance of allosteric regulation was manifested by molecular modeling and identification of key residues in both the PHD and the catalytic domain of Dnmt3a whose mutations impaired the stimulation of methylation activity by H3 peptides but not the binding of H3 peptides. Significantly, these mutant Dnmt3a proteins were almost inactive in DNA methylation when expressed in mouse embryonic stem cells while their recruitment to genomic targets was unaltered. We therefore propose a two-step mechanism for de novo DNA methylation - first recruitment of the methyltransferase probably assisted by a chromatin- or DNA-binding factor, and then allosteric activation depending on the interaction between Dnmt3a and the histone tails - the latter might serve as a checkpoint for the methylation activity.

  15. A novel methyltransferase from the intracellular pathogen Plasmodiophora brassicae methylates salicylic acid.

    Science.gov (United States)

    Ludwig-Müller, Jutta; Jülke, Sabine; Geiß, Kathleen; Richter, Franziska; Mithöfer, Axel; Šola, Ivana; Rusak, Gordana; Keenan, Sandi; Bulman, Simon

    2015-05-01

    The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana, which is characterized by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. Plasmodiophora brassicae seems to suppress plant defence reactions, but information on how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease. The accumulation of SA and the emission of methylated SA (methyl salicylate, MeSA) were observed in P. brassicae-infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the mutant Atbsmt1, deficient in the methylation of SA, indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. We found that P. brassicae possesses a methyltransferase (PbBSMT) with homology to plant methyltransferases. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses, we showed that PbBSMT can methylate SA, benzoic and anthranilic acids.

  16. Methylated nucleosides in tRNA and tRNA methyltransferases

    Directory of Open Access Journals (Sweden)

    Hiroyuki eHori

    2014-05-01

    Full Text Available To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s. Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon–anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed.

  17. S-Adenosyl-L-methionine: macrocin O-methyltransferase activities in a series of Streptomyces fradiae mutants that produce different levels of the macrolide antibiotic tylosin.

    OpenAIRE

    Seno, E T; Baltz, R H

    1982-01-01

    A series of mutants of Streptomyces fradiae selected for increased production of the macrolide antibiotic tylosin was analyzed for levels of expression of macrocin O-methyltransferase, the enzyme which catalyzes the final step in the biosynthesis of tylosin. Increased tylosin production was accompanied by increased macrocin O-methyltransferase in some of the mutants. Increased expression of macrocin O-methyltransferase was due to more rapid early biosynthesis of the enzyme, to reduced decay o...

  18. Yunnan-Guizhou Plateau in Summer and Autumn Broccoli Standardized Cultivation Techniques%云贵高原反季青花菜标准化栽培技术

    Institute of Scientific and Technical Information of China (English)

    刘兴平

    2012-01-01

    云贵高原夏秋季节气候温凉,适于种植反季青花菜,本文结合多年生产实践,从品种选择、培育壮苗、规格定植、肥水管理、病虫防治、适时采收等措施总结介绍了一套云贵高原冷凉山区反季青花菜标准化栽培技术。%Yunnan-Guizhou Plateau in summer and autumn climate was cool and suitable for growing-season broccoli, the paper summary description is a combination of years of production practices, species selection, nurturing seedlings, specifications planting, fertilizer and water management, pest control, timely harvest measuresYunnan-Guizhou Plateau in cold mountain-season broccoli standardization of cultivation techniques.

  19. Active packaging for fresh-cut broccoli using 1-methylcyclopropene in biodegradable sachet/ Embalagem ativa para brócolis minimamente processado utilizando 1-metilciclopropeno em sachê biodegradável

    Directory of Open Access Journals (Sweden)

    Marta de Toledo Benassi

    2006-07-01

    Full Text Available Fresh-cut broccoli florets were packed in polypropylene pots containing a sachet with 1- methylcyclopropene (1-MCP and sealed with biodegradable starch-based film. Broccoli was stored for 8 days at 12oC and after this time the color and the texture of the product were similar of the fresh broccoli, with no off-flavor development or decay. Active packaging with 1-MCP in a sachet was efficient to extent shelf life of broccoli florets retarding yellowing and vitamin C losses. It is an alternative of 1-MCP treatment for fresh-cut products and besides, the sachet can absorbs condensed water reducing spoilage and off-odor development.Brócolis minimamente processados foram embalados em bandejas de polipropileno com sachê contendo 1-metilciclopropeno (1-MCP e selado com filme biodegradável de amido. Os brócolis foram armazenados por 8 dias a 12oC e após este tempo a cor e a textura mantiveram-se similares às do produto fresco, sem desenvolvimento de odor não característico ou podridão. A embalagem ativa contendo 1-MCP na forma de sachê foi eficiente no aumento da vida de prateleira de brócolis, retardando o amarelecimento e a perda de vitamina C. Esta é uma alternativa para o tratamento com 1-MCP para produtos minimamente processados e além disso, o sachê pode absorver a água condensada, reduzindo a deterioração e o desenvolvimento de odor não característico.

  20. Effect of Different Drying Methods on the Sulforaphane Content of Broccoli%不同干燥方法对西兰花中萝卜硫素含量影响

    Institute of Scientific and Technical Information of China (English)

    谢述琼; 何珺; 杨佳年; 陈震

    2013-01-01

    Drying Broccoli's flower,stalk and leaf with three usual drying methods which were drying in the shade,50 ℃-90 ℃drying and drying in the sun,indentifying sulforaphane content with HPLC. Sulforaphane content in broccoli's different parts was seed>flower>stalk>leaf,with different drying methods,drying was the best and drying in the shade was the worst,with the five drying temperature it was 90 ℃ that there were the highest sulforaphane content of broccoli. with the different parts the sulforaphane content of broccoli's seed was the highest,it had 10.48 mg/g;the best drying methods was 90℃drying.%  采用阴干、50℃~90℃烘干及晒干三种常用干燥方式干燥西兰花的花、茎、叶,采用HPLC测定萝卜硫素含量。西兰花不同部位中萝卜硫素含量是种子>花>茎>叶;而不同干燥方法中烘干最好,阴干最差,5种烘干温度中以90℃烘干时萝卜硫素含量最高。西兰花不同部位中种子萝卜硫素含量最高,有10.48 mg/g;而干燥方式采用90℃烘干最佳。

  1. Research of Controling Freezing Point of Broccoli by Using Freezing Point Regulators%冰点调节剂对西兰花冰点的控制研究

    Institute of Scientific and Technical Information of China (English)

    林本芳; 鲁晓翔; 李江阔

    2012-01-01

    本文研究不同冰点调节剂对西兰花冰点的影响.实验选取葡萄糖、维生素C、氯化钙和山梨醇四种冰点调节剂,利用单因素比较实验法,系统研究了冰点调节剂种类及其使用条件对西兰花冰点的影响.结果表明,氯化钙是较好的冰点调节剂;3%氯化钙浸泡30min对西兰花的冰点调节效果较好,使西兰花冰点从-0.8℃降到-1℃.%In this research, the effects of different freezing point regulators on broccoli freezing point were investigated. Four kinds of freezing point regulators, including glucose, vitamin C, calcium chloride and sorbitol, were used for research on the influence of freezing point of broccoli by the single factor comparison experiment method. The results indicated that calcium chloride and glucose were the better freezing point regulators. When broccoli was soaked for 30 minutes with calcium chloride content being of 3%, its' freezing point was decreased from -0.8℃to-1 ℃.

  2. The catechol-O-methyltransferase inhibitory potential of Z-vallesiachotamine by in silicoand in vitro approaches

    Directory of Open Access Journals (Sweden)

    Carolina dos Santos Passos

    2015-08-01

    Full Text Available AbstractZ-Vallesiachotamine is a monoterpene indole alkaloid that has a β-N-acrylate group in its structure. This class of compounds has already been described in different Psychotriaspecies. Our research group observed that E/Z-vallesiachotamine exhibits a multifunctional feature, being able to inhibit targets related to neurodegeneration, such as monoamine oxidase A, sirtuins 1 and 2, and butyrylcholinesterase enzymes. Aiming at better characterizing the multifunctional profile of this compound, its effect on cathecol-O-methyltransferase activity was investigated. The cathecol-O-methyltransferase activity was evaluated in vitro by a fluorescence-based method, using S-(5′-adenosyl-l-methionine as methyl donor and aesculetin as substrate. The assay optimization was performed varying the concentrations of methyl donor (S-(5′-adenosyl-l-methionine and enzyme. It was observed that the highest concentrations of both factors (2.25 U of the enzyme and 100 µM of S-(5′-adenosyl-l-methionine afforded the more reproducible results. The in vitro assay demonstrated that Z-vallesiachotamine was able to inhibit the cathecol-O-methyltransferase activity with an IC50 close to 200 µM. Molecular docking studies indicated that Z-vallesiachotamine can bind the catechol pocket of catechol-O-methyltransferase enzyme. The present work demonstrated for the first time the inhibitory properties of Z-vallesiachotamine on cathecol-O-methyltransferase enzyme, affording additional evidence regarding its multifunctional effects in targets related to neurodegenerative diseases.

  3. Selenium-based S-adenosylmethionine analog reveals the mammalian seven-beta-strand methyltransferase METTL10 to be an EF1A1 lysine methyltransferase.

    Directory of Open Access Journals (Sweden)

    Tadahiro Shimazu

    Full Text Available Lysine methylation has been extensively studied in histones, where it has been shown to provide specific epigenetic marks for the regulation of gene expression; however, the molecular mechanism and physiological function of lysine methylation in proteins other than histones remains to be fully addressed. To better understand the substrate diversity of lysine methylation, S-adenosylmethionine (SAM derivatives with alkyne-moieties have been synthesized. A selenium-based SAM analog, propargylic Se-adenosyl-l-selenomethionine (ProSeAM, has a wide spectrum of reactivity against various lysine methyltransferases (KMTs with sufficient stability to support enzymatic reactions in vitro. By using ProSeAM as a chemical probe for lysine methylation, we identified substrates for two seven-beta-strand KMTs, METTL21A and METTL10, on a proteomic scale in mammalian cells. METTL21A has been characterized as a heat shock protein (HSP-70 methyltransferase. Mammalian METTL10 remains functionally uncharacterized, although its ortholog in yeast, See1, has been shown to methylate the translation elongation factor eEF1A. By using ProSeAM-mediated alkylation followed by purification and quantitative MS analysis, we confirmed that METTL21A labels HSP70 family proteins. Furthermore, we demonstrated that METTL10 also methylates the eukaryotic elongation factor EF1A1 in mammalian cells. Subsequent biochemical characterization revealed that METTL10 specifically trimethylates EF1A1 at lysine 318 and that siRNA-mediated knockdown of METTL10 decreases EF1A1 methylation levels in vivo. Thus, our study emphasizes the utility of the synthetic cofactor ProSeAM as a chemical probe for the identification of non-histone substrates of KMTs.

  4. Application of Tissue Culture, Molecular Marker and QTL Analysis in Broccoli Breeding%组织培养、分子标记和QT L技术在青花菜育种中的应用

    Institute of Scientific and Technical Information of China (English)

    张志仙; 朱长志; 檀国印; 王峰; 何道根

    2015-01-01

    育种是优异种质资源基因的重组。除传统新种质产生的方法外,通过组织培养、分子标记等技术来扩大物种的遗传基础已成为新种质资源形成的重要手段。青花菜,芸薹属甘蓝中以绿色花球为食用器官的一个变种,近年来在中国的种植面积越来越大,利用现代生物技术手段进行青花菜育种研究工作也越来越受到关注和重视。本文综述了游离小孢子培养、原生质体融合和转基因技术在青花菜种质资源创制中的应用,分析了各种方法存在的问题,并对青花菜遗传图谱构建、花球相关农艺性状及抗病性QTL/基因定位研究进行了探讨,并对其未来研究方向做了展望。%Breeding is kind of the genomic combination of excellent germplasms. Apart from conventional breeding methods generating new germplasms, approaches based on tissue culture and the molecular marker assisted breeding have become very popular biotechnologies to create the new breeding materials. Broccoli (Brassica oleracea L. var. italica Plenck), is an important vegetable worldwilde. In recent decades, broccoli production and consumption has risen significantly and the usage of biotechnologies on broccoli breeding has made great progress in China. This paper reviewed the uses of microspore culture, protoplast culture and fusion, transgenic technologies in improvement breeding of broccoli, and the limitation in those researches was mentioned as well. Moreover, construction of linkage maps, QTLs/genes for agronomically important broccoli traits including head morphologies and diseases resistance were analyzed in this review.

  5. Crystal structure of the Escherichia coli 23S rRNA:m5C methyltransferase RlmI (YccW) reveals evolutionary links between RNA modification enzymes

    DEFF Research Database (Denmark)

    Sunita, S; Tkaczuk, Karolina L; Purta, Elzbieta;

    2008-01-01

    Methylation is the most common RNA modification in the three domains of life. Transfer of the methyl group from S-adenosyl-l-methionine (AdoMet) to specific atoms of RNA nucleotides is catalyzed by methyltransferase (MTase) enzymes. The rRNA MTase RlmI (rRNA large subunit methyltransferase gene I...

  6. Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2012-05-15

    DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

  7. Furanocoumarin biosynthesis in Ammi majus L. Cloning of bergaptol O-methyltransferase.

    Science.gov (United States)

    Hehmann, Marc; Lukacin, Richard; Ekiert, Halina; Matern, Ulrich

    2004-03-01

    Plants belonging to the Apiaceae or Rutaceae accumulate methoxylated psoralens, such as bergapten or xanthotoxin, as the final products of their furanocoumarin biosynthesis, and the rate of accumulation depends on environmental and other cues. Distinct O-methyltransferase activities had been reported to methylate bergaptol to bergapten and xanthotoxol to xanthotoxin, from induced cell cultures of Ruta graveolens, Petroselinum crispum and Ammi majus. Bergaptol 5-O-methyltransferase (BMT) cDNA was cloned from dark-grown Ammi majus L. cells treated with a crude fungal elicitor. The translated polypeptide of 38.7 kDa, composed of 354 amino acids, revealed considerable sequence similarity to heterologous caffeic acid 3-O-methyltransferases (COMTs). For homologous comparison, COMT was cloned from A. majus plants and shown to share 64% identity and about 79% similarity with the BMT sequence at the polypeptide level. Functional expression of both enzymes in Escherichia coli revealed that the BMT activity in the bacterial extracts was labile and rapidly lost on purification, whereas the COMT activity remained stable. Furthermore, the recombinant AmBMT, which was most active in potassium phosphate buffer of pH 8 at 42 degrees C, showed narrow substrate specificity for bergaptol (Km SAM 6.5 micro m; Km Bergaptol 2.8 micro m) when assayed with a variety of substrates, including xanthotoxol, while the AmCOMT accepted 5-hydroxyferulic acid, esculetin and other substrates. Dark-grown A. majus cells expressed significant BMT activity which nevertheless increased sevenfold within 8 h upon the addition of elicitor and reached a transient maximum at 8-11 h, whereas the COMT activity was rather low and did not respond to the elicitation. Complementary Northern blotting revealed that the BMT transcript abundance increased to a maximum at 7 h, while only a weak constitutive signal was observed for the COMT transcript. The AmBMT sequence thus represents a novel database accession

  8. 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase.

    Directory of Open Access Journals (Sweden)

    Hongping Dong

    Full Text Available RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2'-O methyltransferase activities that are required for the formation of 5' type I cap (m(7GpppAm of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4 specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2'-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2'-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2'-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2'-O-methyladenosine. The 2'-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2'-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2'-O methylation of internal adenosine of

  9. NRMT is an alpha-N-methyltransferase that methylates RCC1 and retinoblastoma protein.

    Science.gov (United States)

    Tooley, Christine E Schaner; Petkowski, Janusz J; Muratore-Schroeder, Tara L; Balsbaugh, Jeremy L; Shabanowitz, Jeffrey; Sabat, Michal; Minor, Wladek; Hunt, Donald F; Macara, Ian G

    2010-08-26

    The post-translational methylation of alpha-amino groups was first discovered over 30 years ago on the bacterial ribosomal proteins L16 and L33 (refs 1, 2), but almost nothing is known about the function or enzymology of this modification. Several other bacterial and eukaryotic proteins have since been shown to be alpha-N-methylated. However, the Ran guanine nucleotide-exchange factor, RCC1, is the only protein for which any biological function of alpha-N-methylation has been identified. Methylation-defective mutants of RCC1 have reduced affinity for DNA and cause mitotic defects, but further characterization of this modification has been hindered by ignorance of the responsible methyltransferase. All fungal and animal N-terminally methylated proteins contain a unique N-terminal motif, Met-(Ala/Pro/Ser)-Pro-Lys, indicating that they may be targets of the same, unknown enzyme. The initiating Met is cleaved, and the exposed alpha-amino group is mono-, di- or trimethylated. Here we report the discovery of the first alpha-N-methyltransferase, which we named N-terminal RCC1 methyltransferase (NRMT). Substrate docking and mutational analysis of RCC1 defined the NRMT recognition sequence and enabled the identification of numerous new methylation targets, including SET (also known as TAF-I or PHAPII) and the retinoblastoma protein, RB. Knockdown of NRMT recapitulates the multi-spindle phenotype seen with methylation-defective RCC1 mutants, demonstrating the importance of alpha-N-methylation for normal bipolar spindle formation and chromosome segregation.

  10. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yuan [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); Wu, Keqiang [Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan (China); Dhaubhadel, Sangeeta [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); An, Lizhe, E-mail: lizhean@lzu.edu.cn [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Tian, Lining, E-mail: tianl@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada)

    2010-05-28

    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  13. Crystal structure of phosphoethanolamine methyltransferase from Plasmodium falciparum in complex with amodiaquine

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Soon Goo; Alpert, Tara D.; Jez, Joseph M. (WU)

    2012-07-17

    Phosphoethanolamine N-methyltransferase (PMT) is essential for phospholipid biogenesis in the malarial parasite Plasmodium falciparum. PfPMT catalyzes the triple methylation of phosphoethanolamine to produce phosphocholine, which is then used for phosphatidylcholine synthesis. Here we describe the 2.0 {angstrom} resolution X-ray crystal structure of PfPMT in complex with amodiaquine. To better characterize inhibition of PfPMT by amodiaquine, we determined the IC{sub 50} values of a series of aminoquinolines using a direct radiochemical assay. Both structural and functional analyses provide a possible approach for the development of new small molecule inhibitors of PfPMT.

  14. The 'de novo' DNA methyltransferase Dnmt3b compensates the Dnmt1-deficient intestinal epithelium.

    Science.gov (United States)

    Elliott, Ellen N; Sheaffer, Karyn L; Kaestner, Klaus H

    2016-01-25

    Dnmt1 is critical for immediate postnatal intestinal development, but is not required for the survival of the adult intestinal epithelium, the only rapidly dividing somatic tissue for which this has been shown. Acute Dnmt1 deletion elicits dramatic hypomethylation and genomic instability. Recovery of DNA methylation state and intestinal health is dependent on the de novo methyltransferase Dnmt3b. Ablation of both Dnmt1 and Dnmt3b in the intestinal epithelium is lethal, while deletion of either Dnmt1 or Dnmt3b has no effect on survival. These results demonstrate that Dnmt1 and Dnmt3b cooperate to maintain DNA methylation and genomic integrity in the intestinal epithelium.

  15. Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound.

    Science.gov (United States)

    Mitchell, Lorna H; Drew, Allison E; Ribich, Scott A; Rioux, Nathalie; Swinger, Kerren K; Jacques, Suzanne L; Lingaraj, Trupti; Boriack-Sjodin, P Ann; Waters, Nigel J; Wigle, Tim J; Moradei, Oscar; Jin, Lei; Riera, Tom; Porter-Scott, Margaret; Moyer, Mikel P; Smith, Jesse J; Chesworth, Richard; Copeland, Robert A

    2015-06-11

    A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies.

  16. Queen pheromones modulate DNA methyltransferase activity in bee and ant workers.

    Science.gov (United States)

    Holman, Luke; Trontti, Kalevi; Helanterä, Heikki

    2016-01-01

    DNA methylation is emerging as an important regulator of polyphenism in the social insects. Research has concentrated on differences in methylation between queens and workers, though we hypothesized that methylation is involved in mediating other flexible phenotypes, including pheromone-dependent changes in worker behaviour and physiology. Here, we find that exposure to queen pheromone affects the expression of two DNA methyltransferase genes in Apis mellifera honeybees and in two species of Lasius ants, but not in Bombus terrestris bumblebees. These results suggest that queen pheromones influence the worker methylome, pointing to a novel proximate mechanism for these key social signals.

  17. Histone methyltransferase G9a contributes to H3K27 methylation in vivo

    Institute of Scientific and Technical Information of China (English)

    Hui Wu; Bing Zhu; Xiuzhen Chen; Jun Xiong; Yingfeng Li; Hong Li; Xiaojun Ding; Sheng Liu; She Chen; Shaorong Gao

    2011-01-01

    @@ Dear Editor, Histone modifications play a vital role in the conformation and function of their associated chromatin templates[1].Histone H3K27 methylation mediated by the PRC2 complex is critical for transcriptional regulation,Polycomb silencing,Drosophila segmentation,mammalian X inactivation and cancer[1].Interestingly,H3K27me1(H3 mono-methylated at residue K27)levels in vivo remain unaffected after PRC2 disruption[2,3],which is an indication for the existence of other contributing histone methyltransferase(s)to H3K27me1.

  18. DNA Methyltransferase Gene dDnmt2 and Longevity of Drosophila

    Institute of Scientific and Technical Information of China (English)

    Meng-JauLin; Lin-YaTang; M.NarsaReddy; C.K.JamesShen

    2005-01-01

    The DNA methylation program of the fruit fly Drosophila melanogaster is carried out by the single DNA methyltransferase gene dDnmt2, the function of which is unknown before. We present evidence that intactness of the gene is required for maintenance of the normal life span of the fruit flies. In contrast, overexpression of dDnmt2 could extend Drosophila life span. The study links the Drosophila DNA methylation program with the small heatshock proteins and longevity/aging and has interesting implication on the eukaryotic DNA methyl-ation programs in general.

  19. Protein lysine methyltransferase G9a acts on non-histone targets

    Science.gov (United States)

    Rathert, Philipp; Dhayalan, Arunkumar; Murakami, Marie; Zhang, Xing; Tamas, Raluca; Jurkowska, Renata; Komatsu, Yasuhiko; Shinkai, Yoichi; Cheng, Xiaodong; Jeltsch, Albert

    2009-01-01

    By methylation of peptide arrays, we determined the specificity profile of the protein methyltransferase G9a. We show that it mostly recognizes an Arg-Lys sequence and that its activity is inhibited by methylation of the arginine residue. Using the specificity profile, we identified new non-histone protein targets of G9a, including CDYL1, WIZ, ACINUS and G9a (automethylation), as well as peptides derived from CSB. We demonstrate potential downstream signaling pathways for methylation of non-histone proteins. PMID:18438403

  20. Discovery and development of DNA methyltransferase inhibitors using in silico approaches.

    Science.gov (United States)

    Medina-Franco, José L; Méndez-Lucio, Oscar; Dueñas-González, Alfonso; Yoo, Jakyung

    2015-05-01

    Multiple strategies have evolved during the past few years to advance epigenetic compounds targeting DNA methyltransferases (DNMTs). Significant progress has been made in HTS, lead optimization and determination of 3D structures of DNMTs. In light of the emerging concept of epi-informatics, computational approaches are employed to accelerate the development of DNMT inhibitors helping to screen chemical databases, mine the DNMT-relevant chemical space, uncover SAR and design focused libraries. Computational methods also synergize with natural-product-based drug discovery and drug repurposing. Herein, we survey the latest developments of in silico approaches to advance epigenetic drug and probe discovery targeting DNMTs.

  1. Regulation of DNA replication and chromosomal polyploidy by the MLL-WDR5-RBBP5 methyltransferases

    Science.gov (United States)

    Lu, Fei; Wu, Xiaojun; Yin, Feng; Chia-Fang Lee, Christina; Yu, Min; Mihaylov, Ivailo S.; Yu, Jiekai; Sun, Hong

    2016-01-01

    ABSTRACT DNA replication licensing occurs on chromatin, but how the chromatin template is regulated for replication remains mostly unclear. Here, we have analyzed the requirement of histone methyltransferases for a specific type of replication: the DNA re-replication induced by the downregulation of either Geminin, an inhibitor of replication licensing protein CDT1, or the CRL4CDT2 ubiquitin E3 ligase. We found that siRNA-mediated reduction of essential components of the MLL-WDR5-RBBP5 methyltransferase complexes including WDR5 or RBBP5, which transfer methyl groups to histone H3 at K4 (H3K4), suppressed DNA re-replication and chromosomal polyploidy. Reduction of WDR5/RBBP5 also prevented the activation of H2AX checkpoint caused by re-replication, but not by ultraviolet or X-ray irradiation; and the components of MLL complexes co-localized with the origin recognition complex (ORC) and MCM2-7 replicative helicase complexes at replication origins to control the levels of methylated H3K4. Downregulation of WDR5 or RBBP5 reduced the methylated H3K4 and suppressed the recruitment of MCM2-7 complexes onto replication origins. Our studies indicate that the MLL complexes and H3K4 methylation are required for DNA replication but not for DNA damage repair. PMID:27744293

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

    Energy Technology Data Exchange (ETDEWEB)

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P. (F. Hoffmann-La Roche Ltd., Basel (Switzerland))

    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.

  3. Antiproliferative effects of DNA methyltransferase 3B depletion are not associated with DNA demethylation.

    Directory of Open Access Journals (Sweden)

    Sabine Hagemann

    Full Text Available Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs.

  4. An association between overexpression of DNA methyltransferase 3B4 and clear cell renal cell carcinoma.

    Science.gov (United States)

    Liu, You; Sun, Liantao; Fong, Peter; Yang, Jie; Zhang, Zhuxia; Yin, Shuihui; Jiang, Shuyuan; Liu, Xiaolei; Ju, Hongge; Huang, Lihua; Bai, Jing; Gong, Kerui; Yan, Shaochun; Zhang, Chunyang; Shao, Guo

    2017-02-01

    It is well known that abnormal DNA methylations occur frequently in kidney cancer. However, it remains unclear exactly which types of DNA methyltransferases (DNMT) contribute to the pathologies of kidney cancers. In order to determine the functions of DNA methyltransferase in kidney tumorigenesis on the molecular level, we examined the mRNA expression levels of DNMT1, DNMT3A, DNMT3B, and DNMT3B variants in renal cell carcinoma tissue. Both mRNA and protein levels of DNMT3B4, a splice variant of DNMT3B, were increased in renal cell carcinoma tissue compared with adjacent control tissues. Additionally, Alu elements and long interspersed nuclear elements (LINE-1) were hypomethylated in renal cell carcinoma tissue. Meanwhile, methylation of the promoter for RASSF1A, a tumor suppressor gene, was moderately increased in renal cell carcinoma tissue, while RASSF1A expression was decreased. Thus, our data suggest that the overexpression of DNMT3B4 may play an important role in human kidney tumorigenesis through chromosomal instability and methylation of RASSF1A.

  5. Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm, Bombyx mori.

    Science.gov (United States)

    Mitsudome, Takumi; Mon, Hiroaki; Xu, Jian; Li, Zhiqing; Lee, Jae Man; Patil, Anandrao Ashok; Masuda, Atsushi; Iiyama, Kazuhiro; Morokuma, Daisuke; Kusakabe, Takahiro

    2015-03-01

    DNA methylation is an important epigenetic mechanism involved in gene expression of vertebrates and invertebrates. In general, DNA methylation profile is established by de novo DNA methyltransferases (DNMT-3A, -3B) and maintainance DNA methyltransferase (DNMT-1). DNMT-1 has a strong substrate preference for hemimethylated DNA over the unmethylated one. Because the silkworm genome lacks an apparent homologue of de novo DNMT, it is still unclear that how silkworm chromosome establishes and maintains its DNA methylation profile. As the first step to unravel this enigma, we purified recombinant BmDNMT-1 using baculovirus expression system and characterized its DNA-binding and DNA methylation activity. We found that the BmDNMT-1 preferentially methylates hemimethylated DNA despite binding to both unmethylated and hemimethylated DNA. Interestingly, BmDNMT-1 formed a complex with DNA in the presence or absence of methyl group donor, S-Adenosylmethionine (AdoMet) and the AdoMet-dependent complex formation was facilitated by Zn(2+) and Mn(2+). Our results provide clear evidence that BmDNMT-1 retained the function as maintenance DNMT but its sensitivity to metal ions is different from mammalian DNMT-1.

  6. The Ca2+-induced methyltransferase xPRMT1b controls neural fate in amphibian embryo.

    Science.gov (United States)

    Batut, Julie; Vandel, Laurence; Leclerc, Catherine; Daguzan, Christiane; Moreau, Marc; Néant, Isabelle

    2005-10-18

    We have previously shown that an increase in intracellular Ca2+ is both necessary and sufficient to commit ectoderm to a neural fate in Xenopus embryos. However, the relationship between this Ca2+ increase and the expression of early neural genes has yet to be defined. Using a subtractive cDNA library between untreated and caffeine-treated animal caps, i.e., control ectoderm and ectoderm induced toward a neural fate by a release of Ca2+, we have isolated the arginine N-methyltransferase, xPRMT1b, a Ca2+-induced target gene, which plays a pivotal role in this process. First, we show in embryo and in animal cap that xPRMT1b expression is Ca2+-regulated. Second, overexpression of xPRMT1b induces the expression of early neural genes such as Zic3. Finally, in the whole embryo, antisense approach with morpholino oligonucleotide against xPRMT1b impairs neural development and in animal caps blocks the expression of neural markers induced by a release of internal Ca2+. Our results implicate an instructive role of an enzyme, an arginine methyltransferase protein, in the embryonic choice of determination between epidermal and neural fate. The results presented provide insights by which a Ca2+ increase induces neural fate.

  7. Overexpression of INCREASED CAMBIAL ACTIVITY, a putative methyltransferase, increases cambial activity and plant growth

    Institute of Scientific and Technical Information of China (English)

    Hyunsook Kim; Mikiko Kojima; Daeseok Choi; Soyoung Park; Minami Matsui; Hitoshi Sakakibara; Ildoo Hwang

    2016-01-01

    Cambial activity is a prerequisite for secondary growth in plants; however, regulatory factors control ing the activity of the secondary meristem in radial growth remain elusive. Here, we identified INCREASED CAMBIAL ACTIVITY (ICA), a gene encoding a putative pectin methyltransferase, which could function as a modulator for the meristematic activity of fascicular and interfascicular cambium in Arabidopsis. An overexpressing transgenic line, 35S::ICA, showed accelerated stem elongation and radial thickening, resulting in increased accumulation of biomass, and increased levels of cytokinins (CKs) and gibberel ins (GAs). Expression of genes encoding pectin methylesterases involved in pectin modification together with pectin methyltransferases was highly induced in 35S::ICA, which might contribute to an increase of methanol emission as a byproduct in 35S::ICA. Methanol treatment induced the expression of GA-or CK-responsive genes and stimulated plant growth. Overal , we propose that ectopic expression of ICA increases cambial activity by regulating CK and GA homeostasis, and methanol emission, eventual y leading to stem elongation and radial growth in the inflorescence stem.

  8. The Histone Methyltransferase Activity of MLL1 Is Dispensable for Hematopoiesis and Leukemogenesis

    Directory of Open Access Journals (Sweden)

    Bibhu P. Mishra

    2014-05-01

    Full Text Available Despite correlations between histone methyltransferase (HMT activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4 methyltransferase critical for maintaining hematopoietic stem cells (HSCs. Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16 acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function.

  9. A Remodeled Protein Arginine Methyltransferase 1 (PRMT1) Generates Symmetric Dimethylarginine*

    Science.gov (United States)

    Gui, Shanying; Gathiaka, Symon; Li, Jun; Qu, Jun; Acevedo, Orlando; Hevel, Joan M.

    2014-01-01

    Protein arginine methylation is emerging as a significant post-translational modification involved in various cell processes and human diseases. As the major arginine methylation enzyme, protein arginine methyltransferase 1 (PRMT1) strictly generates monomethylarginine and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA). The two types of dimethylarginines can lead to distinct biological outputs, as highlighted in the PRMT-dependent epigenetic control of transcription. However, it remains unclear how PRMT1 product specificity is regulated. We discovered that a single amino acid mutation (Met-48 to Phe) in the PRMT1 active site enables PRMT1 to generate both ADMA and SDMA. Due to the limited amount of SDMA formed, we carried out quantum mechanical calculations to determine the free energies of activation of ADMA and SDMA synthesis. Our results indicate that the higher energy barrier of SDMA formation (ΔΔG‡ = 3.2 kcal/mol as compared with ADMA) may explain the small amount of SDMA generated by M48F-PRMT1. Our study reveals unique energetic challenges for SDMA-forming methyltransferases and highlights the exquisite control of product formation by active site residues in the PRMTs. PMID:24478314

  10. X-ray crystal structure of N-6 adenine deoxyribose nucleic acid methyltransferase from Streptococcus pneumoniae

    Science.gov (United States)

    Tran, Phidung Hong

    X-ray diffraction by using resonant anomalous scattering has become a popular tool for solving crystal structures in the last ten years with the expanded availability of tunable synchrotron radiation for protein crystallography. Mercury atoms were used for phasing. The crystal structure of N-6 deoxyribose nucleic acid methyltransferase from Streptoccocus pneumoniae (DpnM) was solved by using the Multiple Anomalous Diffraction technique. The crystal structure reveals the formation of mercaptide between the mercury ion and the thiol group on the cysteine amino acid in a hydrophobic environment. The crystal structure contains the bound ligand, S- adenosyl-l-methionine on the surface of the concave opening. The direction of the β-strands on the beta sheets are identical to other solved methyltransferases. The highly conserved motifs, DPPY and the FxGxG, are found to be important in ligand binding and possibly in methyl group transfer. The structure has a concave cleft with an opening on the order of 30 Å that can accommodate a DNA duplex. By molecular modelling coupled to sequence alignment, two other highly conserved residues Arg21 and Gly19 are found to be important in catalysis.

  11. Comparative analysis of DNA methyltransferase gene family in fungi: a focus on Basidiomycota

    Directory of Open Access Journals (Sweden)

    Ruirui Huang

    2016-10-01

    Full Text Available DNA methylation plays a crucial role in the regulation of gene expression in eukaryotes. Mushrooms belonging to the phylum Basidiomycota are highly valued for both nutritional and pharmaceutical uses. A growing number of studies have demonstrated the significance of DNA methylation in the development of plants and animals. However, our understanding of DNA methylation in mushrooms is limited. In this study, we identified and conducted comprehensive analyses on DNA methyltransferases (DNMtases in representative species from Basidiomycota and Ascomycota, and obtained new insights into their classification and characterization in fungi. Our results revealed that DNMtases in basidiomycetes can be divided into two classes, the Dnmt1 class and the newly defined Rad8 class. We also demonstrated that the fusion event between the characteristic domains of the DNMtases family and Snf2 family in the Rad8 class is fungi-specific, possibly indicating a functional novelty of Rad8 DNMtases in fungi. Additionally, expression profiles of DNMtases in the edible mushroom Pleurotus ostreatus revealed diverse expression patterns in various organs and developmental stages. For example, DNMtase genes displayed higher expression levels in dikaryons than in monokaryons. Consistent with the expression profiles, we found that dikaryons are more susceptible to the DNA methyltransferase inhibitor 5-azacytidine. Taken together, our findings pinpoint an important role of DNA methylation during the growth of mushrooms and provide a foundation for understanding of DNMtases in basidiomycetes.

  12. Comparative Analysis of DNA Methyltransferase Gene Family in Fungi: A Focus on Basidiomycota

    Science.gov (United States)

    Huang, Ruirui; Ding, Qiangqiang; Xiang, Yanan; Gu, Tingting; Li, Yi

    2016-01-01

    DNA methylation plays a crucial role in the regulation of gene expression in eukaryotes. Mushrooms belonging to the phylum Basidiomycota are highly valued for both nutritional and pharmaceutical uses. A growing number of studies have demonstrated the significance of DNA methylation in the development of plants and animals. However, our understanding of DNA methylation in mushrooms is limited. In this study, we identified and conducted comprehensive analyses on DNA methyltransferases (DNMtases) in representative species from Basidiomycota and Ascomycota, and obtained new insights into their classification and characterization in fungi. Our results revealed that DNMtases in basidiomycetes can be divided into two classes, the Dnmt1 class and the newly defined Rad8 class. We also demonstrated that the fusion event between the characteristic domains of the DNMtases family and Snf2 family in the Rad8 class is fungi-specific, possibly indicating a functional novelty of Rad8 DNMtases in fungi. Additionally, expression profiles of DNMtases in the edible mushroom Pleurotus ostreatus revealed diverse expression patterns in various organs and developmental stages. For example, DNMtase genes displayed higher expression levels in dikaryons than in monokaryons. Consistent with the expression profiles, we found that dikaryons are more susceptible to the DNA methyltransferase inhibitor 5-azacytidine. Taken together, our findings pinpoint an important role of DNA methylation during the growth of mushrooms and provide a foundation for understanding of DNMtases in basidiomycetes. PMID:27818666

  13. Cloning and expression analysis of an o-methyltransferase (OMT) gene from Chinese shrimp, Fenneropenaeus chinensis.

    Science.gov (United States)

    Li, Dian-Xiang; Du, Xin-Jun; Zhao, Xiao-Fan; Wang, Jin-Xing

    2006-09-01

    O-methyltransferase (OMT) is ubiquitously present in diverse organisms and plays an important regulatory role in plant and animal growth, development, reproduction and defence and has also been implicated in human emotion and disease. A putative o-methyltransferase (OMT) gene has been cloned from the haemocytes of bacteria-infected Chinese shrimp (Fenneropenaeus chinensis) by suppression subtractive hybridisation (SSH) coupled with the SMART cDNA method. The isolated 944 bp full-length cDNA contains a single 666bp open reading frame (ORF) encoding a putative OMT protein of 221 amino acids. The predicted protein has a molecular weight of 24,572.06 Da and a pI of 5.27 as well as ten phosphorylation sites. Northern blot and in situ hybridisation analyses demonstrated that the OMT transcripts were constitutively expressed in tissue of shrimp challenged by bacterial infection and in unchallenged shrimp tissue. Constitutive OMT transcript was found in areas such as haemocytes, heart, hepatopancreas, stomach, gill, intestine and ovary. However, the OMT transcripts were upregulated in hepatopancreas and stomach in challenged shrimp.

  14. Induction of phase 2 antioxidant enzymes by broccoli sulforaphane: Perspectives in maintaining the antioxidant activity of vitamins A, C, and E

    Directory of Open Access Journals (Sweden)

    Sekhar s Boddupalli

    2012-01-01

    Full Text Available Consumption of fruits and vegetables is recognized as an important part of a healthy diet. Increased consumption of cruciferous vegetables in particular has been associated with a decreased risk of several degenerative and chronic diseases, including cardiovascular disease and certain cancers. Members of the cruciferous vegetable family, which includes broccoli, Brussels sprouts, cauliflower, and cabbage, accumulate significant concentrations of glucosinolates, which are metabolized in vivo to biologically active isothiocyanates (ITCs. The ITC sulforaphane, which is derived from glucoraphanin, has garnered particular interest due to its extraordinary ability to induce expression of several enzymes via the Keap1/Nrf2/ARE pathway. Nrf2/ARE gene products are typically characterized as Phase II detoxification enzymes and/or antioxidant (AO enzymes. Over the last decade, human clinical studies have begun to provide in vivo evidence of both Phase II and AO enzyme induction by SF. Many AO enzymes are redox cycling enzymes that maintain redox homeostasis and activity of free radical scavengers such as such as vitamins A, C and E. In this review, we present the existing evidence for induction of PII and AO enzymes by SF, the interactions of SF-induced AO enzymes and proposed maintenance of the essential vitamins A, C and E, and, finally, the current view of genotypic effects on ITC metabolism and AO enzyme induction and function.

  15. Characterization of fermented broccoli (Brassica oleracea L. and spinach (Amaranthus sp. produced using microfiltration membrane as folic acid source for smart food formula

    Directory of Open Access Journals (Sweden)

    Susilowati Agustine

    2017-01-01

    Full Text Available Purifying and drying both fermented biomasses of broccoli (Brassica oleracea L. and spinach (Amaranthus sp. by Kombucha culture has been conducted to recover concentrate and powder of folic acid. The aims of this study is to determine the differences of particles characteristics and compositions of concentrate and powder from both mentioned folic acid source through Micro Filtration (MF membrane and without MF membrane. The best folic acid produced by MF membrane process (room temperature, stirrer rotation speed 400 rpm, pressure 40 psia and 30 minutes and drying (30 °C, 22 cm Hg and 24 hours were resulted in biomass of the concentrate and powder with compositions of total solids 6.29 % and 96.91 %, total polyphenol 0.25 % and 0.06 %, folic acid 58.8 μg/mL and 54.33 μg/mL, reducing sugar 105.34 mg/mL and 441.39 mg/mL, and total acids 0.57 % and 2.33 %, respectively. In optimum condition, fermented spinach concentrate contributed to particles distribution with diameter size (Ø between 0,4 and 100 μm (75.45 %, and with Ø between 100 and 1000 μm (26.3 %, otherwise, the process without MF membrane was resulted the particles distribution respectively 74.1 % and 25.9% by each interval of Ø.

  16. Glucoraphanin, the bioprecursor of the widely extolled chemopreventive agent sulforaphane found in broccoli, induces Phase-I xenobiotic metabolizing enzymes and increases free radical generation in rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Perocco, Paolo [Department of Experimental Pathology, Cancerology Section, viale Filopanti 22, I-40126, University of Bologna, Bologna (Italy); Bronzetti, Giorgio [Institute of Biology and Agricultural Biotechnology - CNR Research Area, via Moruzzi, I-56124 Pisa (Italy); Canistro, Donatella; Sapone, Andrea; Affatato, Alessandra; Pozzetti, Laura; Broccoli, Massimiliano [Department of Pharmacology, Molecular Toxicology Unit, via Irnerio 48, I-40126, University of Bologna, Bologna (Italy); Valgimigli, Luca [Department of Organic Chemistry ' A. Mangini' , Viale Risorgimento 4, I-40127, Alma-Mater Studiorum, University of Bologna, Bologna (Italy); Pedulli, Gian Franco [Department of Organic Chemistry ' A. Mangini' , Viale Risorgimento 4, I-40127, Alma-Mater Studiorum, University of Bologna, Bologna (Italy); Iori, Renato [C.R.A - Research Institute for Industrial Crops, via di Corticella 133, I-40129 Bologna (Italy); Barillari, Jessica [Institute of Biology and Agricultural Biotechnology - CNR Research Area, via Moruzzi, I-56124 Pisa (Italy)]|[C.R.A - Research Institute for Industrial Crops, via di Corticella 133, I-40129 Bologna (Italy); Sblendorio, Valeriana [Department of Pharmacology, Molecular Toxicology Unit, via Irnerio 48, I-40126, University of Bologna, Bologna (Italy); Legator, Marvin S. [Department of Preventive Medicine and Community Health, Division of Environmental Toxicology, The University of Texas Medical Branch at Galveston, 700 Harborside Drive, Galveston, TX 77555-1110 (United States); Paolini, Moreno [Department of Pharmacology, Molecular Toxicology Unit, via Irnerio 48, I-40126, University of Bologna, Bologna (Italy); Abdel-Rahman, Sherif Z. [Department of Preventive Medicine and Community Health, Division of Environmental Toxicology, The University of Texas Medical Branch at Galveston, 700 Harborside Drive, Galveston, TX 77555-1110 (United States)]. E-mail: sabdelra@utmb.edu

    2006-03-20

    Epidemiological and animal studies linking high fruit and vegetable consumption to lower cancer risk have strengthened the belief that long-term administration of isolated naturally occurring dietary constituents could reduce the risk of cancer. In recent years, metabolites derived from phytoalexins, such as glucoraphanin found in broccoli and other cruciferous vegetables (Brassicaceae), have gained much attention as potential cancer chemopreventive agents. The protective effect of these micronutrients is assumed to be due to the inhibition of Phase-I carcinogen-bioactivating enzymes and/or induction of Phase-II detoxifying enzymes, an assumption that still remains uncertain. The protective effect of glucoraphanin is thought to be due to sulforaphane, an isothiocyanate metabolite produced from glucoraphanin by myrosinase. Here we show, in rat liver, that while glucoraphanin slightly induces Phase-II enzymes, it powerfully boosts Phase-I enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), nitrosamines and olefins. Induction of the cytochrome P450 (CYP) isoforms CYP1A1/2, CYP3A1/2 and CYP2E1 was confirmed by Western immunoblotting. CYP induction was paralleled by an increase in the corresponding mRNA levels. Concomitant with this Phase-I induction, we also found that glucoraphanin generated large amount of various reactive radical species, as determined by electron paramagnetic resonance (EPR) spectrometry coupled to a radical-probe technique. This suggests that long-term uncontrolled administration of glucoraphanin could actually pose a potential health hazard.

  17. Growth inhibition and antioxidative status induced by selenium-enriched broccoli extract and selenocompounds in DNA mismatch repair-deficient human colon cancer cells.

    Science.gov (United States)

    Tsai, Cheng-Fang; Ou, Bor-Rung; Liang, Yu-Chuan; Yeh, Jan-Ying

    2013-08-15

    The effects of enzymatic-digested Se-enriched broccoli extracts (SeB) and selenocompounds on growth and antioxidative status in human colon cancer cells was investigated in this study. HCT116 and HCT116+Chr.3 cells were treated with selenocompounds (sodium selenite, sodium selenate, Se-Met, MeSeCys) or SeB [high-Se (H-SeB) or low-Se (L-SeB)]. The cytotoxicity induced by selenocompounds in HCT116 cells was not associated with cellular H2O2 level, while the differential cytotoxicity observed by sodium selenite between HCT116 and HCT116+Chr.3 cell lines was related to cellular H2O2 production with the change in antioxidative enzyme activity, and the restoration of chromosome 3. H-SeB was found to reduce the cellular H2O2 content in HCT116+Chr.3 cells. The results in this study indicate that regardless of Se content, the cytotoxicity in HCT116 cells of both SeB forms appeared to be H2O2-independent, whereas the cytotoxicity in HCT116+Chr.3 of either SeB form appeared to be H2O2-dependent with an increase in antioxidative ability for H-SeB.

  18. Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

    Energy Technology Data Exchange (ETDEWEB)

    Mizuno, Kouichi, E-mail: koumno@akita-pu.ac.jp [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Matsuzaki, Masahiro [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Kanazawa, Shiho [Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610 (Japan); Tokiwano, Tetsuo; Yoshizawa, Yuko [Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita 010-0195 (Japan); Kato, Misako [Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610 (Japan)

    2014-10-03

    Graphical abstract: Trigonelline synthase catalyzes the conversion of nicotinic acid to trigonelline. We isolated and characterized trigonelline synthase gene(s) from Coffea arabica. - Highlights: • Trigonelline is a major compound in coffee been same as caffeine is. • We isolated and characterized trigonelline synthase gene. • Coffee trigonelline synthases are highly homologous with coffee caffeine synthases. • This study contributes the fully understanding of pyridine alkaloid metabolism. - Abstract: Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-{sup 14}C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or

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

    NARCIS (Netherlands)

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

    2012-01-01

    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)

  20. An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

    Science.gov (United States)

    Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their rela...

  1. Polymorphisms in O-methyltransferase genes are associated with stover cell wall digestibility in European maize (Zea mays L.)

    DEFF Research Database (Denmark)

    Brenner, Everton A; Zein, Imad; Chen, Yongsheng

    2010-01-01

    Background OMT (O-methyltransferase) genes are involved in lignin biosynthesis, which relates to stover cell wall digestibility. Reduced lignin content is an important determinant of both forage quality and ethanol conversion efficiency of maize stover. Results Variation in genomic sequences codi...

  2. The effect of Ecstasy on memory is moderated by a functional polymorphism in the cathechol-O-methyltransferase (COMT) gene

    NARCIS (Netherlands)

    T. Schilt; M.W.J. Koeter; M.M.L. de Win; J.R. Zinkstok; T.A. van Amelsvoort; B. Schmand; W. van den Brink

    2009-01-01

    There is ample evidence for decreased verbal memory in heavy Ecstasy users. However, findings on the presence of a dose-response relation are inconsistent, possibly due to individual differences in genetic vulnerability. Catechol-O-methyltransferase (COMT) is involved in the catabolism of Ecstasy. T

  3. Thirteen new patients with guanidinoacetate methyltransferase deficiency and functional characterization of nineteen novel missense variants in the GAMT gene

    DEFF Research Database (Denmark)

    Mercimek-Mahmutoglu, Saadet; Ndika, Joseph; Kanhai, Warsha

    2014-01-01

    Guanidinoacetate methyltransferase deficiency (GAMT-D) is an autosomal recessively inherited disorder of creatine biosynthesis. Creatine deficiency on cranial proton magnetic resonance spectroscopy, and elevated guanidinoacetate levels in body fluids are the biomarkers of GAMT-D. In 74 patients 5...

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

    Science.gov (United States)

    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. Characterization of cytosine methylated regions and 5-cytosine DNA methyltransferase (Ehmeth) in the protozoan parasite Entamoeba histolytica.

    Science.gov (United States)

    Fisher, Ohad; Siman-Tov, Rama; Ankri, Serge

    2004-01-01

    The DNA methylation status of the protozoan parasite Entamoeba histolytica was heretofore unknown. In the present study, we developed a new technique, based on the affinity of methylated DNA to 5-methylcytosine antibodies, to identify methylated DNA in this parasite. Ribosomal DNA and ribosomal DNA circles were isolated by this method and we confirmed the validity of our approach by sodium bisulfite sequencing. We also report the identification and the characterization of a gene, Ehmeth, encoding a DNA methyltransferase strongly homologous to the human DNA methyltransferase 2 (Dnmt2). Immunofluorescence microscopy using an antibody raised against a recombinant Ehmeth showed that Ehmeth is concentrated in the nuclei of trophozoites. The recombinant Ehmeth has a weak but significant methyltransferase activity when E.histolytica genomic DNA is used as substrate. 5-Azacytidine (5-AzaC), an inhibitor of DNA methyltransferase, was used to study in vivo the role of DNA methylation in E.histolytica. Genomic DNA of trophozoites grown with 5-AzaC (23 microM) was undermethylated and the ability of 5-AzaC-treated trophozoites to kill mammalian cells or to cause liver abscess in hamsters was strongly impaired.

  6. Vagus nerve contributes to the development of steatohepatitis and obesity in phosphatidylethanolamine N-methyltransferase deficient mice

    NARCIS (Netherlands)

    Gao, Xia; van der Veen, Jelske N.; Zhu, Linfu; Chaba, Todd; Ordonez, Marta; Lingrell, Susanne; Koonen, Debby P. Y.; Dyck, Jason R. B.; Gomez-Munoz, Antonio; Vance, Dennis E.; Jacobs, Rene L.

    2015-01-01

    BACKGROUND & AIMS: Phosphatidylethanolamine N-methyltransferase (PEMT), a liver enriched enzyme, is responsible for approximately one third of hepatic phosphatidylcholine biosynthesis. When fed a high-fat diet (HFD), Pemt(-/-) mice are protected from HF-induced obesity; however, they develop steatoh

  7. Crystal Structure and Catalytic Mechanism of CouO, a Versatile C-Methyltransferase from Streptomyces rishiriensis

    Science.gov (United States)

    Pavkov-Keller, Tea; Steiner, Kerstin; Faber, Mario; Tengg, Martin; Schwab, Helmut; Gruber-Khadjawi, Mandana

    2017-01-01

    Friedel–Crafts alkylation of aromatic systems is a classic reaction in organic chemistry, for which regiospecific mono-alkylation, however, is generally difficult to achieve. In nature, methyltransferases catalyze the addition of methyl groups to a wide range of biomolecules thereby modulating the physico-chemical properties of these compounds. Specifically, S-adenosyl-L-methionine dependent C-methyltransferases possess a high potential to serve as biocatalysts in environmentally benign organic syntheses. Here, we report on the high resolution crystal structure of CouO, a C-methyltransferase from Streptomyces rishiriensis involved in the biosynthesis of the antibiotic coumermycin A1. Through molecular docking calculations, site-directed mutagenesis and the comparison with homologous enzymes we identified His120 and Arg121 as key functional residues for the enzymatic activity of this group of C-methyltransferases. The elucidation of the atomic structure and the insight into the catalytic mechanism provide the basis for the (semi)-rational engineering of the enzyme in order to increase the substrate scope as well as to facilitate the acceptance of SAM-analogues as alternative cofactors. PMID:28152088

  8. MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases

    Science.gov (United States)

    Yang, Ya-Ling; Wang, Feng-Sheng; Li, Sung-Chou; Tiao, Mao-Meng; Huang, Ying-Hsien

    2017-01-01

    MicroRNA-29 (miR-29) is found to modulate hepatic stellate cells’ (HSCs) activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates were subjected to bile duct-ligation (BDL) to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A) expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development. PMID:28106784

  9. MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases

    Directory of Open Access Journals (Sweden)

    Ya-Ling Yang

    2017-01-01

    Full Text Available MicroRNA-29 (miR-29 is found to modulate hepatic stellate cells’ (HSCs activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice and wild-type littermates were subjected to bile duct-ligation (BDL to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development.

  10. Fitness cost and interference of Arm/Rmt aminoglycoside resistance with the RsmF housekeeping methyltransferases.

    Science.gov (United States)

    Gutierrez, Belen; Escudero, Jose A; San Millan, Alvaro; Hidalgo, Laura; Carrilero, Laura; Ovejero, Cristina M; Santos-Lopez, Alfonso; Thomas-Lopez, Daniel; Gonzalez-Zorn, Bruno

    2012-05-01

    Arm/Rmt methyltransferases have emerged recently in pathogenic bacteria as enzymes that confer high-level resistance to 4,6-disubstituted aminoglycosides through methylation of the G1405 residue in the 16S rRNA (like ArmA and RmtA to -E). In prokaryotes, nucleotide methylations are the most common type of rRNA modification, and they are introduced posttranscriptionally by a variety of site-specific housekeeping enzymes to optimize ribosomal function. Here we show that while the aminoglycoside resistance methyltransferase RmtC methylates G1405, it impedes methylation of the housekeeping methyltransferase RsmF at position C1407, a nucleotide that, like G1405, forms part of the aminoglycoside binding pocket of the 16S rRNA. To understand the origin and consequences of this phenomenon, we constructed a series of in-frame knockout and knock-in mutants of Escherichia coli, corresponding to the genotypes rsmF(+), ΔrsmF, rsmF(+) rmtC(+), and ΔrsmF rmtC(+). When analyzed for the antimicrobial resistance pattern, the ΔrsmF bacteria had a decreased susceptibility to aminoglycosides, including 4,6- and 4,5-deoxystreptamine aminoglycosides, showing that the housekeeping methylation at C1407 is involved in intrinsic aminoglycoside susceptibility in E. coli. Competition experiments between the isogenic E. coli strains showed that, contrary to expectation, acquisition of rmtC does not entail a fitness cost for the bacterium. Finally, matrix-assisted laser desorption ionization (MALDI) mass spectrometry allowed us to determine that RmtC methylates the G1405 residue not only in presence but also in the absence of aminoglycoside antibiotics. Thus, the coupling between housekeeping and acquired methyltransferases subverts the methylation architecture of the 16S rRNA but elicits Arm/Rmt methyltransferases to be selected and retained, posing an important threat to the usefulness of aminoglycosides worldwide.

  11. Identification of an S-adenosylmethionine (SAM) dependent arsenic methyltransferase in Danio rerio

    Energy Technology Data Exchange (ETDEWEB)

    Hamdi, Mohamad [Department of Biological Sciences, Oakland University, Rochester, MI 48309 (United States); Yoshinaga, Masafumi; Packianathan, Charles; Qin, Jie [Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, FL33199 (United States); Hallauer, Janell; McDermott, Joseph R. [Department of Biological Sciences, Oakland University, Rochester, MI 48309 (United States); Yang, Hung-Chi [Department of Medical Biotechnology and Laboratory Sciences, Chang-Gung University, Tao-Yuan, Kwei-San 333, Taiwan (China); Tsai, Kan-Jen [School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Liu, Zijuan, E-mail: liu2345@oakland.edu [Department of Biological Sciences, Oakland University, Rochester, MI 48309 (United States)

    2012-07-15

    Arsenic methylation is an important cellular metabolic process that modulates arsenic toxicity and carcinogenicity. Biomethylation of arsenic produces a series of mono-, di- and tri-methylated arsenic metabolites that can be detected in tissues and excretions. Here we report that zebrafish exposed to arsenite (As{sup III}) produces organic arsenicals, including MMA{sup III}, MMA{sup V} and DMA{sup V} with characteristic tissue ratios, demonstrating that an arsenic methylation pathway exists in zebrafish. In mammals, cellular inorganic arsenic is methylated by a SAM-dependent arsenic methyltransferase, AS3MT. A zebrafish arsenic methyltransferase homolog, As3mt, was identified by sequence alignment. Western blotting analysis showed that As3mt was universally expressed in zebrafish tissues. Prominent expression in liver and intestine correlated with methylated arsenic metabolites detected in those tissues. As3mt was expressed in and purified from Escherichia coli for in vitro functional studies. Our results demonstrated that As3mt methylated As{sup III} to DMA{sup V} as an end product and produced MMA{sup III} and MMA{sup V} as intermediates. The activity of As3mt was inhibited by elevated concentrations of the substrate As{sup III} as well as the metalloid selenite, which is a well-known antagonistic micronutrient of arsenic toxicity. The activity As3mt was abolished by substitution of either Cys160 or Cys210, which corresponds to conserved cysteine residues in AS3MT homologs, suggesting that they are involved in catalysis. Expression in zebrafish of an enzyme that has a similar function to human and rodent orthologs in catalyzing intracellular arsenic biomethylation validates the applicability of zebrafish as a valuable vertebrate model for understanding arsenic-associated diseases in humans. -- Highlights: ► Zebrafish methylated As{sup III} to MMA{sup III}, MMA{sup V} and DMA{sup V}. ► A zebrafish arsenic methyltransferase (As3mt) was purified in E. coli.

  12. 不同质量浓度臭氧化水对鲜切西兰花贮藏品质的影响%Effect of Ozonated Water with Various Concentrations on Storage Quality of Fresh-cut Broccoli

    Institute of Scientific and Technical Information of China (English)

    王宏延; 曾凯芳; 贾凝; 陈存坤; 王文生

    2012-01-01

    采用不同质量浓度臭氧化水处理鲜切西兰花。结果表明:质量浓度2.0mg/L的臭氧化水处理组保鲜效果最优,在贮藏12d时与对照组相比,质量损失率降低24.37%,VC含量提高40.7%,多酚氧化酶酶活降低23.5%,过氧化物酶酶活降低25.6%;在4℃的条件下,质量浓度2.0mg/L的臭氧化水处理组鲜切西兰花贮藏期可达到15d。%Ozonated water can be used for disinfection,sterilization,deodorization and preservation with multiple advantages such as rapid sterilization rate,simple operation and without residues.It can be used directly for the sterilization of fresh-cut vegetables so that it is a potential technology of cold sterilization and green sterilization.The results showed that the best preservation effect on fresh-cut broccoli was observed at the condition of 2 mg/L ozonated water.Compared with the control,ozonated water-treated fresh-cut broccoli after storage for 12 days revealed a reduction by 24.37% for weight loss rate,an improvement by 40.7% for vitamin C,a decline by 23.5% for polyphenol oxidase activity and a decrease by 25.6% for peroxidase activity.The storage life of fresh-cut broccoli subjected to treatment with 2 mg/L ozonated water can be extended to 15 days at the storage temperature of 4 ℃.

  13. 细胞分裂素类生长调节剂对青花菜采后衰老的影响%Effects of Cytokinin Growth Regulators on Senescence of Harvested Broccoli (Brassica oleracea L. var. italica) Florets

    Institute of Scientific and Technical Information of China (English)

    王云莉; 王成荣; 王然; 杨绍兰; 刘宪路

    2009-01-01

    Application of cytokinin growth regulators, KT (Kinetin), 6-BA (Benzyladenine), and CPPU [N-(2-chloro-4-pyridyl)-N'-pheuylurea] on delaying senescence of harvested broccoli florets was studied. The results showed that use of KT, 6-BA or CPPU on harvested broccoli delayed floret yellowing and chlorophyll degradation, decreased respiration and ethylene production rate, postponed climacteric time, and retarded membrane permeability loss. The content of endogenous ABA was lower and the levels of IAA and GA were higher in the broccoli treated with 6-BA and CPPU than those in the control during 12-84 h. The treated effects enhanced with increasing concentration in the study. The CPPU treatment (40 mg·L~(-1)) was most effective in delaying the broccoli yellowing, followed by 6-BA and KT.%以青花菜'绿皇后'品种为试材,研究了25℃条件下细胞分裂素类生长调节剂KT、6-BA和CPPU对延缓青花菜采后衰老的效果.结果表明:3种细胞分裂素类生长调节剂处理均在不同程度上延缓了青花菜花蕾黄化和叶绿素分解,推迟了呼吸和乙烯跃变峰出现的时间,降低了峰值,并维持了细胞膜稳定性.内源激素测定结果表明,6-BA和CPPU处理使青花菜花蕾在试验的前期阶段(12~84 h)一直保持相对较高的内源IAA和GA水平,相对较低的内源ABA水平.3种细胞分裂素类生长调节剂以CPPU延缓青花菜衰老效果最为明显,其次是6-BA,KT效果最弱.延缓青花菜采后衰老效果随着处理的浓度增加而增加,以40 mg·L~(-1)CPPU延缓青花菜衰老效果最佳.

  14. Control Efficacy of Insecticides Mixture on Broccolis%几种药剂混配防治西兰花根部病害综合效果试验

    Institute of Scientific and Technical Information of China (English)

    方辉; 王会福; 余山红; 毛肇蓬

    2013-01-01

      为了有效防治西兰花根肿病、根腐病、根结线虫病等根部病害,提升西兰花的产量和品质,采用灌根法进行几种药剂混配防治西兰花根部病害田间药效试验,结果表明:68%精甲霜•锰锌WG 500倍+3.2%阿维菌素EC 1000倍+微补精力600倍+微补根力钙600倍对西兰花根部病害具有较好的综合防治效果,其中连续2次灌根后50 d的校正防效达82.1%,分别比68%精甲霜•锰锌WG 500倍+3.2%阿维菌素EC 1000倍、5%石灰氮GR 1200 kg/hm2高出5.6%、21.3%,持效期较长,安全性好,因此可以在生产上加以推广应用。%  The purpose of the trial was to control clubroot disease, ceitocybe bescens, root knot nematode disease of broccoli, in order to promote the broccoli production and quality. Mixture of several insecticides was used on broccoli by root­irrigation method in this trail. The result showed that the mixture of 68%Mefenoxam­Mancozeb WG 1:500 plus 3.2%Avermectins EC 1:1 000 plus 425 g/L MNPK­38 g/L Calcium 1:600 and 160 g/L Calcium 1:600 had better control effect on Broccoli root diseases, and the 50 days’ correction control effect after two consecutive root­pouring reached 82.1%, which was higher than that of the mixture of 68%Mefenoxam­Mancozeb WG 1:500 plus 3.2%Avermectins EC 1:1 000 and that of 5%Calcium Cyanamide GR 1 200kg/hm2 by 5.6% and 21.3%, respectively. With longer effective lasting period and better security, this mixture can be demonstrated to apply to the practical production.

  15. Catechol-O-Methyltransferase gene val158met polymorphism and depressive symptoms during early childhood

    Science.gov (United States)

    Sheikh, Haroon I.; Kryski, Katie R.; Smith, Heather J.; Dougherty, Lea R.; Klein, Daniel N.; Bufferd, Sara J.; Singh, Shiva M.; Hayden, Elizabeth P.

    2017-01-01

    Catechol-O-Methyltransferase (COMT) is a critical regulator of catecholamine levels in the brain. A functional polymorphism of the COMT gene, val158met, has been linked to internalizing symptoms (i.e., depression and anxiety) in adolescents and adults. We extended this research by investigating whether the val158met polymorphism was associated with childhood symptoms of depression and anxiety in two independent samples of young children (Ns = 476 and 409). In both samples, preschool-aged children were genotyped for the COMT val158met polymorphism. Symptoms of psychopathology were assessed via parent interviews and primary caregiver reports. In both samples, children homozygous for the val allele had higher levels of depressive symptoms compared to children with at least one copy of the met allele. Our findings extend previous research in older participants by showing links between the COMT val158met polymorphism and internalizing symptoms in early childhood. PMID:23475824

  16. Role of the EZH2 histone methyltransferase as a therapeutic target in cancer.

    Science.gov (United States)

    Italiano, Antoine

    2016-09-01

    Besides being a genetic disease, cancer is also an epigenetic disease. The histone methyltransferase EZH2 is the catalytic subunit of PRC2, a highly conserved protein complex that regulates gene expression by methylating lysine 27 on histone H3. Given its role in tumorigenesis and its prognostic value in several tumor types, this protein appears a relevant therapeutic target. This review focuses on the preclinical and preliminary clinical results of studies investigating EZH2 inhibitors in human malignancies. These emerging data suggest that EZH2 inhibitors represent a very promising class of drugs, which will probably have a major impact on improving outcome and reducing toxicity for patients with indolent and aggressive B-cell lymphomas and other specific solid tumors.

  17. Rationalization of Activity Cliffs of a Sulfonamide Inhibitor of DNA Methyltransferases with Induced-Fit Docking

    Directory of Open Access Journals (Sweden)

    José L. Medina-Franco

    2014-02-01

    Full Text Available Inhibitors of human DNA methyltransferases (DNMT are of increasing interest to develop novel epi-drugs for the treatment of cancer and other diseases. As the number of compounds with reported DNMT inhibition is increasing, molecular docking is shedding light to elucidate their mechanism of action and further interpret structure–activity relationships. Herein, we present a structure-based rationalization of the activity of SW155246, a distinct sulfonamide compound recently reported as an inhibitor of human DNMT1 obtained from high-throughput screening. We used flexible and induce-fit docking to develop a binding model of SW155246 with a crystallographic structure of human DNMT1. Results were in excellent agreement with experimental information providing a three-dimensional structural interpretation of ‘activity cliffs’, e.g., analogues of SW155246 with a high structural similarity to the sulfonamide compound, but with no activity in the enzymatic assay.

  18. Strategy to target the substrate binding site of SET domain protein methyltransferases.

    Science.gov (United States)

    Nguyen, Kong T; Li, Fengling; Poda, Gennadiy; Smil, David; Vedadi, Masoud; Schapira, Matthieu

    2013-03-25

    Protein methyltransferases (PMTs) are a novel gene family of therapeutic relevance involved in chromatin-mediated signaling and other biological mechanisms. Most PMTs are organized around the structurally conserved SET domain that catalyzes the methylation of a substrate lysine. A few potent chemical inhibitors compete with the protein substrate, and all are anchored in the channel recruiting the methyl-accepting lysine. We propose a novel strategy to design focused chemical libraries targeting the substrate binding site, where a limited number of warheads each occupying the lysine-channel of multiple enzymes would be decorated by different substituents. A variety of sequence and structure-based approaches used to analyze the diversity of the lysine channel of SET domain PMTs support the relevance of this strategy. We show that chemical fragments derived from published inhibitors are valid warheads that can be used in the design of novel focused libraries targeting other PMTs.

  19. Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases

    DEFF Research Database (Denmark)

    Ntokou, Eleni; Hansen, Lykke Haastrup; Kongsted, Jacob;

    2015-01-01

    homology and may be evolutionarily linked to a common ancestor. To explore their individual specificity and similarity we performed two sets of experiments. We created a homology model of Cfr and explored the C2/C8 specificity using docking and binding energy calculations on the Cfr homology model and an X......Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering). RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence......-ray structure of RlmN. We used a trinucleotide as target sequence and assessed its positioning at the active site for methylation. The calculations are in accordance with different poses of the trinucleotide in the two enzymes indicating major evolutionary changes to shift the C2/C8 specificities. To explore...

  20. Miniaturization of High-Throughput Epigenetic Methyltransferase Assays with Acoustic Liquid Handling.

    Science.gov (United States)

    Edwards, Bonnie; Lesnick, John; Wang, Jing; Tang, Nga; Peters, Carl

    2016-02-01

    Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter volumes to high-density assay formats using only acoustic energy-no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio.

  1. DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation

    DEFF Research Database (Denmark)

    Shaknovich, Rita; Cerchietti, Leandro; Tsikitas, Lucas;

    2011-01-01

    The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation and t......, the GC B cells of Dnmt1 hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair.......The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation...... and the role of DNA methyltransferases in the formation of GCs. DNA methylation profiling revealed a marked shift in DNA methylation patterning in GC B cells versus resting/naive B cells. This shift included significant differential methylation of 235 genes, with concordant inverse changes in gene expression...

  2. Human nicotinamide N-methyltransferase gene: Molecular cloning, structural characterization and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Aksoy, S.; Weinshilboum, R.M. [Mayo Medical School/Mayo Clinic/Mayo Foundation, Rochester, MN (United States); Brandriff, B.F. [Lawrence Livermore National Lab., CA (United States); Ward, A.; Little, P.F.R. [Imperial College of Science, Technology and Medicine, London (United Kingdom)

    1995-10-10

    Genomic DNA clones for nicotinamide N-methyltransferase (NNMT), an enzyme that catalyzes drug and xenobiotic metabolism, were isolated from a human chromosome 11-specific DNA library. Study of one of those clones, when combined with PCR-based experiments performed with human genomic DNA, made it possible to determine the structure of the human NNMT gene. The gene was approximately 16.5 kb in length and consisted of 3 exons and 2 introns. Transcription initiation for the NNMT gene occurred 105-109 nucleotides 5{prime}-upstream from the cDNA translation initiation codon on the basis of the results of both primer extension and 5{prime}-rapid amplification of cDNA ends. NNMT mapped to chromosome band 11q23.1 by fluorescence in situ hybridization.

  3. Brain Histamine N-Methyltransferase As a Possible Target of Treatment for Methamphetamine Overdose

    Science.gov (United States)

    Kitanaka, Junichi; Kitanaka, Nobue; Hall, F. Scott; Uhl, George R.; Takemura, Motohiko

    2016-01-01

    Stereotypical behaviors induced by methamphetamine (METH) overdose are one of the overt symptoms of METH abuse, which can be easily assessed in animal models. Currently, there is no successful treatment for METH overdose. There is increasing evidence that elevated levels of brain histamine can attenuate METH-induced behavioral abnormalities, which might therefore constitute a novel therapeutic treatment for METH abuse and METH overdose. In mammals, histamine N-methyltransferase (HMT) is the sole enzyme responsible for degrading histamine in the brain. Metoprine, one of the most potent HMT inhibitors, can cross the blood–brain barrier and increase brain histamine levels by inhibiting HMT. Consequently, this compound can be a candidate for a prototype of drugs for the treatment of METH overdose. PMID:26966348

  4. DNA methyltransferases are required to induce heterochromatic re-replication in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Hume Stroud

    2012-07-01

    Full Text Available The relationship between epigenetic marks on chromatin and the regulation of DNA replication is poorly understood. Mutations of the H3K27 methyltransferase genes, Arabidopsis trithorax-related protein5 (ATXR5 and ATXR6, result in re-replication (repeated origin firing within the same cell cycle. Here we show that mutations that reduce DNA methylation act to suppress the re-replication phenotype of atxr5 atxr6 mutants. This suggests that DNA methylation, a mark enriched at the same heterochromatic regions that re-replicate in atxr5/6 mutants, is required for aberrant re-replication. In contrast, RNA sequencing analyses suggest that ATXR5/6 and DNA methylation cooperatively transcriptionally silence transposable elements (TEs. Hence our results suggest a complex relationship between ATXR5/6 and DNA methylation in the regulation of DNA replication and transcription of TEs.

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

    Science.gov (United States)

    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.

  6. Histone methyltransferases and demethylases:regulators in balancing osteogenic and adipogenic differentiation of mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Peng Deng; Qian-Ming Chen; Christine Hong; Cun-Yu Wang

    2015-01-01

    Mesenchymal stem cells (MSCs) are characterized by their self-renewing capacity and differentiation potential into multiple tissues. Thus, management of the differentiation capacities of MSCs is important for MSC-based regenerative medicine, such as craniofacial bone regeneration, and in new treatments for metabolic bone diseases, such as osteoporosis. In recent years, histone modification has been a growing topic in the field of MSC lineage specification, in which the Su(var)3–9, enhancer-of-zeste, trithorax (SET) domain-containing family and the Jumonji C (JmjC) domain-containing family represent the major histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs), respectively. In this review, we summarize the current understanding of the epigenetic mechanisms by which SET domain-containing KMTs and JmjC domain-containing KDMs balance the osteogenic and adipogenic differentiation of MSCs.

  7. Ash2 acts as an ecdysone receptor coactivator by stabilizing the histone methyltransferase Trr.

    Science.gov (United States)

    Carbonell, Albert; Mazo, Alexander; Serras, Florenci; Corominas, Montserrat

    2013-02-01

    The molting hormone ecdysone triggers chromatin changes via histone modifications that are important for gene regulation. On hormone activation, the ecdysone receptor (EcR) binds to the SET domain-containing histone H3 methyltransferase trithorax-related protein (Trr). Methylation of histone H3 at lysine 4 (H3K4me), which is associated with transcriptional activation, requires several cofactors, including Ash2. We find that ash2 mutants have severe defects in pupariation and metamorphosis due to a lack of activation of ecdysone-responsive genes. This transcriptional defect is caused by the absence of the H3K4me3 marks set by Trr in these genes. We present evidence that Ash2 interacts with Trr and is required for its stabilization. Thus we propose that Ash2 functions together with Trr as an ecdysone receptor coactivator.

  8. Human calmodulin methyltransferase: expression, activity on calmodulin, and Hsp90 dependence.

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    Sophia Magen

    Full Text Available Deletion of the first exon of calmodulin-lysine N-methyltransferase (CaM KMT, previously C2orf34 has been reported in two multigene deletion syndromes, but additional studies on the gene have not been reported. Here we show that in the cells from 2p21 deletion patients the loss of CaM KMT expression results in accumulation of hypomethylated calmodulin compared to normal controls, suggesting that CaM KMT is essential for calmodulin methylation and there are no compensatory mechanisms for CaM methylation in humans. We have further studied the expression of this gene at the transcript and protein levels. We have identified 2 additional transcripts in cells of the 2p21 deletion syndrome patients that start from alternative exons positioned outside the deletion region. One of them starts in the 2(nd known exon, the other in a novel exon. The transcript starting from the novel exon was also identified in a variety of tissues from normal individuals. These new transcripts are not expected to produce proteins. Immunofluorescent localization of tagged CaM KMT in HeLa cells indicates that it is present in both the cytoplasm and nucleus of cells whereas the short isoform is localized to the Golgi apparatus. Using Western blot analysis we show that the CaM KMT protein is broadly expressed in mouse tissues. Finally we demonstrate that the CaM KMT interacts with the middle portion of the Hsp90 molecular chaperon and is probably a client protein since it is degraded upon treatment of cells with the Hsp90 inhibitor geldanamycin. These findings suggest that the CaM KMT is the major, possibly the single, methyltransferase of calmodulin in human cells with a wide tissue distribution and is a novel Hsp90 client protein. Thus our data provides basic information for a gene potentially contributing to the patient phenotype of two contiguous gene deletion syndromes.

  9. Inhibition of H3K9 methyltransferase G9a ameliorates methylglyoxal-induced peritoneal fibrosis

    Science.gov (United States)

    Maeda, Kazuya; Doi, Shigehiro; Nakashima, Ayumu; Nagai, Takuo; Irifuku, Taisuke; Ueno, Toshinori; Masaki, Takao

    2017-01-01

    Activity of H3K9 histone methyltransferase G9a is reportedly induced by transforming growth factor-β1 (TGF-β1) and plays an important role in the progression of cancer and fibrosis. In this study, we investigated whether inhibition of G9a-mediated H3K9 methylation attenuates peritoneal fibrosis in mice and human peritoneal mesothelial cells (HPMCs). Nonadherent cells of peritoneal dialysis (PD) patients were isolated from PD effluent to examine expression of G9a. Peritoneal fibrosis was induced by peritoneal injection of methylglyoxal (MGO) in male C57/B6 mice for 3 weeks. BIX01294, a G9a inhibitor, was administered by subcutaneous injection. Effects of BIX01294 on MGO-induced pathological and functional changes in mice were evaluated by immunohistochemistry and a peritoneal equilibration test. HPMCs were isolated from human omentum, and the inhibitory effect of BIX01294 on TGF-β1-induced fibrotic changes was investigated in the HPMCs by western blotting. G9a was upregulated in nonadherent cells of human PD effluent, the peritoneum of MGO-injected mice, and TGF-β1-stimulated HPMCs. BIX01294 significantly reduced the submesothelial zone thickness and cell density in MGO-injected mice. Immunohistochemical staining revealed that BIX01294 treatment decreased not only mono-methylation of H3K9 (H3K9me1), but also the number of mesenchymal cells, accumulation of collagen, and infiltration of monocytes. In addition to the pathological changes, BIX01294 reduced the level of TGF-β1 in peritoneal fluid and improved peritoneal functions. Furthermore, BIX01294 inhibited TGF-β1-induced fibrotic changes along with suppression of H3K9me1 in HPMCs. Therefore, inhibition of H3K9 methyltransferase G9a suppresses peritoneal fibrosis through a reduction of H3K9me1. PMID:28278257

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

    Science.gov (United States)

    Dang, Thu-Thuy T; Facchini, Peter J

    2012-06-01

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

  11. A disulfide-bond cascade mechanism for arsenic(III) S-adenosylmethionine methyltransferase

    Science.gov (United States)

    Marapakala, Kavitha; Packianathan, Charles; Ajees, A. Abdul; Dheeman, Dharmendra S.; Sankaran, Banumathi; Kandavelu, Palani; Rosen, Barry P.

    2015-01-01

    Methylation of the toxic metalloid arsenic is widespread in nature. Members of every kingdom have arsenic(III) S-adenosylmethionine (SAM) methyltransferase enzymes, which are termed ArsM in microbes and AS3MT in animals, including humans. Trivalent arsenic(III) is methylated up to three times to form methylarsenite [MAs(III)], dimethylarsenite [DMAs(III)] and the volatile trimethylarsine [TMAs(III)]. In microbes, arsenic methylation is a detoxification process. In humans, MAs(III) and DMAs(III) are more toxic and carcinogenic than either inorganic arsenate or arsenite. Here, new crystal structures are reported of ArsM from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508 (CmArsM) with the bound aromatic arsenicals phenylarsenite [PhAs(III)] at 1.80 Å resolution and reduced roxarsone [Rox(III)] at 2.25 Å resolution. These organoarsenicals are bound to two of four conserved cysteine residues: Cys174 and Cys224. The electron density extends the structure to include a newly identified conserved cysteine residue, Cys44, which is disulfide-bonded to the fourth conserved cysteine residue, Cys72. A second disulfide bond between Cys72 and Cys174 had been observed previously in a structure with bound SAM. The loop containing Cys44 and Cys72 shifts by nearly 6.5 Å in the arsenic(III)-bound structures compared with the SAM-bound structure, which suggests that this movement leads to formation of the Cys72–Cys174 disulfide bond. A model is proposed for the catalytic mechanism of arsenic(III) SAM methyltransferases in which a disulfide-bond cascade maintains the products in the trivalent state. PMID:25760600

  12. Functional characterisation of three o-methyltransferases involved in the biosynthesis of phenolglycolipids in Mycobacterium tuberculosis.

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    Roxane Simeone

    Full Text Available Phenolic glycolipids are produced by a very limited number of slow-growing mycobacterial species, most of which are pathogen for humans. In Mycobacterium tuberculosis, the etiologic agent of tuberculosis, these molecules play a role in the pathogenicity by modulating the host immune response during infection. The major variant of phenolic glycolipids produced by M. tuberculosis, named PGL-tb, consists of a large lipid core terminated by a glycosylated aromatic nucleus. The carbohydrate part is composed of three sugar residues, two rhamnosyl units and a terminal fucosyl residue, which is per-O-methylated, and seems to be important for pathogenicity. While most of the genes responsible for the synthesis of the lipid core domain and the saccharide appendage of PGL-tb have been characterized, the enzymes involved in the O-methylation of the fucosyl residue of PGL-tb remain unknown. In this study we report the identification and characterization of the methyltransferases required for the O-methylation of the terminal fucosyl residue of PGL-tb. These enzymes are encoded by genes Rv2954c, Rv2955c and Rv2956. Mutants of M. tuberculosis harboring deletion within these genes were constructed. Purification and analysis of the phenolglycolipids produced by these strains, using a combination of mass spectrometry and NMR spectroscopy, revealed that Rv2954c, Rv2955c and Rv2956 encode the methyltransferases that respectively catalysed the O-methylation of the hydroxyl groups located at positions 3, 4 and 2 of the terminal fucosyl residue of PGL-tb. Our data also suggest that methylation at these positions is a sequential process, starting with position 2, followed by positions 4 and 3.

  13. Metabolomic profiles of arsenic (+3 oxidation state) methyltransferase knockout mice: effect of sex and arsenic exposure.

    Science.gov (United States)

    Huang, Madelyn C; Douillet, Christelle; Su, Mingming; Zhou, Kejun; Wu, Tao; Chen, Wenlian; Galanko, Joseph A; Drobná, Zuzana; Saunders, R Jesse; Martin, Elizabeth; Fry, Rebecca C; Jia, Wei; Stýblo, Miroslav

    2017-01-01

    Arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in the pathway for methylation of inorganic arsenic (iAs). Altered As3mt expression and AS3MT polymorphism have been linked to changes in iAs metabolism and in susceptibility to iAs toxicity in laboratory models and in humans. As3mt-knockout mice have been used to study the association between iAs metabolism and adverse effects of iAs exposure. However, little is known about systemic changes in metabolism of these mice and how these changes lead to their increased susceptibility to iAs toxicity. Here, we compared plasma and urinary metabolomes of male and female wild-type (WT) and As3mt-KO (KO) C57BL/6 mice and examined metabolomic shifts associated with iAs exposure in drinking water. Surprisingly, exposure to 1 ppm As elicited only small changes in the metabolite profiles of either WT or KO mice. In contrast, comparisons of KO mice with WT mice revealed significant differences in plasma and urinary metabolites associated with lipid (phosphatidylcholines, cytidine, acyl-carnitine), amino acid (hippuric acid, acetylglycine, urea), and carbohydrate (L-sorbose, galactonic acid, gluconic acid) metabolism. Notably, most of these differences were sex specific. Sex-specific differences were also found between WT and KO mice in plasma triglyceride and lipoprotein cholesterol levels. Some of the differentially changed metabolites (phosphatidylcholines, carnosine, and sarcosine) are substrates or products of reactions catalyzed by other methyltransferases. These results suggest that As3mt KO alters major metabolic pathways in a sex-specific manner, independent of iAs treatment, and that As3mt may be involved in other cellular processes beyond iAs methylation.

  14. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    Energy Technology Data Exchange (ETDEWEB)

    Kanno, Yuichiro, E-mail: ykanno@phar.toho-u.ac.jp; Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-03-27

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.

  15. The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond.

    Science.gov (United States)

    Stopa, Nicole; Krebs, Jocelyn E; Shechter, David

    2015-06-01

    Post-translational arginine methylation is responsible for regulation of many biological processes. The protein arginine methyltransferase 5 (PRMT5, also known as Hsl7, Jbp1, Skb1, Capsuleen, or Dart5) is the major enzyme responsible for mono- and symmetric dimethylation of arginine. An expanding literature demonstrates its critical biological function in a wide range of cellular processes. Histone and other protein methylation by PRMT5 regulate genome organization, transcription, stem cells, primordial germ cells, differentiation, the cell cycle, and spliceosome assembly. Metazoan PRMT5 is found in complex with the WD-repeat protein MEP50 (also known as Wdr77, androgen receptor coactivator p44, or Valois). PRMT5 also directly associates with a range of other protein factors, including pICln, Menin, CoPR5 and RioK1 that may alter its subcellular localization and protein substrate selection. Protein substrate and PRMT5-MEP50 post-translation modifications induce crosstalk to regulate PRMT5 activity. Crystal structures of C. elegans PRMT5 and human and frog PRMT5-MEP50 complexes provide substantial insight into the mechanisms of substrate recognition and procession to dimethylation. Enzymological studies of PRMT5 have uncovered compelling insights essential for future development of specific PRMT5 inhibitors. In addition, newly accumulating evidence implicates PRMT5 and MEP50 expression levels and their methyltransferase activity in cancer tumorigenesis, and, significantly, as markers of poor clinical outcome, marking them as potential oncogenes. Here, we review the substantial new literature on PRMT5 and its partners to highlight the significance of understanding this essential enzyme in health and disease.

  16. Molecular and biochemical characterization of the jasmonic acid methyltransferase gene from black cottonwood (Populus trichocarpa)

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Nan [ORNL; Yao, Jianzhuang [University of Tennessee, Knoxville (UTK); Chaiprasongsuk, Minta [University of Tennessee, Knoxville (UTK); Li, Guanglin [University of Tennessee, Knoxville (UTK); Guan, Ju [University of Tennessee, Knoxville (UTK); Tschaplinski, Timothy J [ORNL; Guo, Hong [University of Tennessee, Knoxville (UTK); Chen, Feng [University of Tennessee, Knoxville (UTK)

    2013-01-01

    Methyl jasmonate is a metabolite known to be produced by many plants and has roles in diverse biological processes. It is biosynthesized by the action of S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase (JMT), which belongs to the SABATH family of methyltransferases. Herein is reported the isolation and biochemical characterization of a JMT gene from black cottonwood (Populus trichocarpa). The genome of P. trichocarpa contains 28 SABATH genes (PtSABATH1 to PtSABATH28). Recombinant PtSABATH3 expressed in Escherichia coli showed the highest level of activity with jasmonic acid (JA) among carboxylic acids tested. It was therefore renamed PtJMT1. PtJMT1 also displayed activity with benzoic acid (BA), with which the activity was about 22% of that with JA. PtSABATH2 and PtSABATH4 were most similar to PtJMT1 among all PtSABATHs. However, neither of them had activity with JA. The apparent Km values of PtJMT1 using JA and BA as substrate were 175 lM and 341 lM, respectively. Mutation of Ser-153 and Asn-361, two residues in the active site of PtJMT1, to Tyr and Ser respectively, led to higher specific activity with BA than with JA. Homology-based structural modeling indicated that substrate alignment, in which Asn-361 is involved, plays a role in determining the substrate specificity of PtJMT1. In the leaves of young seedlings of black cottonwood, the expression of PtJMT1 was induced by plant defense signal molecules methyl jasmonate and salicylic acid and a fungal elicitor alamethicin, suggesting that PtJMT1 may have a role in plant defense against biotic stresses. Phylogenetic analysis suggests that PtJMT1 shares a common ancestor with the Arabidopsis JMT, and functional divergence of these two apparent JMT orthologs has occurred since the split of poplar and Arabidopsis lineages.

  17. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode.

    Science.gov (United States)

    Lin, Jingyu; Mazarei, Mitra; Zhao, Nan; Zhu, Junwei J; Zhuang, Xiaofeng; Liu, Wusheng; Pantalone, Vincent R; Arelli, Prakash R; Stewart, Charles N; Chen, Feng

    2013-12-01

    Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence-related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full-length cDNAs of GmSAMT1 from a SCN-resistant soybean line and from a SCN-susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli-expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μM. To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN-susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.

  18. Atomic structure of a folate/FAD-dependent tRNA T54 methyltransferase.

    Science.gov (United States)

    Nishimasu, Hiroshi; Ishitani, Ryuichiro; Yamashita, Koki; Iwashita, Chikako; Hirata, Akira; Hori, Hiroyuki; Nureki, Osamu

    2009-05-19

    tRNAs from all 3 phylogenetic domains have a 5-methyluridine at position 54 (T54) in the T-loop. The methyl group is transferred from S-adenosylmethionine by TrmA methyltransferase in most Gram-negative bacteria and some archaea and eukaryotes, whereas it is transferred from 5,10-methylenetetrahydrofolate (MTHF) by TrmFO, a folate/FAD-dependent methyltransferase, in most Gram-positive bacteria and some Gram-negative bacteria. However, the catalytic mechanism remains unclear, because the crystal structure of TrmFO has not been solved. Here, we report the crystal structures of Thermus thermophilus TrmFO in its free form, tetrahydrofolate (THF)-bound form, and glutathione-bound form at 2.1-, 1.6-, and 1.05-A resolutions, respectively. TrmFO consists of an FAD-binding domain and an insertion domain, which both share structural similarity with those of GidA, an enzyme involved in the 5-carboxymethylaminomethylation of U34 of some tRNAs. However, the overall structures of TrmFO and GidA are basically different because of their distinct domain orientations, which are consistent with their respective functional specificities. In the THF complex, the pteridin ring of THF is sandwiched between the flavin ring of FAD and the imidazole ring of a His residue. This structure provides a snapshot of the folate/FAD-dependent methyl transfer, suggesting that the transferring methylene group of MTHF is located close to the redox-active N5 atom of FAD. Furthermore, we established an in vitro system to measure the methylation activity. Our TrmFO-tRNA docking model, in combination with mutational analyses, suggests a catalytic mechanism, in which the methylene of MTHF is directly transferred onto U54, and then the exocyclic methylene of U54 is reduced by FADH(2).

  19. Composto orgânico na produção e qualidade de sementes de brócolis Organic compost in broccoli seed yield and quality

    Directory of Open Access Journals (Sweden)

    Felipe Oliveira Magro

    2010-06-01

    from high physiological potential seeds. Although there are studies about nutrition and recommendation of fertilization to broccoli (Brassica oleracea L. var. italica Plenk, rarely it finds out works that approach the nutrients effects in seed yield and quality. The objective of this work was to evaluate the organic compost influence in broccoli seed quality and yield. The experiment was lead at São Manuel Experimental Farm and the evaluations at Horticulture Sector in Agronomic Science School (FCA/UNESP in Botucatu. The treatments were four organic compost levels (30, 60, 90 and 120 t ha-1, and control without organic compost. The experimental design was randomized blocks with four replications. The characteristics evaluated were seed yield and number of seed per plant further the characteristics related with seed quality: one thousand seed mass, germination test, first germination counting, index of germination speed and electrical conductivity. The regression showed a linear response in function of organic compost levels, where larger levels resulted higher yield despite the seed quality is not affected.

  20. Bacillus thuringiensis protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants.

    Science.gov (United States)

    Cao, Jun; Bates, Sarah L; Zhao, Jian-Zhou; Shelton, Anthony M; Earle, Elizabeth D

    2006-06-01

    In an effort to develop a chemically inducible system for insect management, we studied production of Cry1Ab Bacillus thuringiensis (Bt) protein and control of the diamondback moth (DBM), Plutella xylostella L., in inducer-treated and untreated tissues of a broccoli line transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-S-methyl (= 1,2,3 benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester) (ASM) triggered expression of the cry1Ab gene and produced a high level of Cry1Ab protein within 2-3 days. Cry1Ab protein persisted in leaves for at least 8 weeks, providing prolonged protection from P. xylostella attack. Signals generated in inducer-treated leaves were transferred to untreated newly emerged leaves or heads, as seen by production of Cry1Ab protein and/or protection from insect damage in these plant parts. Signal transduction proceeded in an attenuated manner up to the sixth newly emerged leaf. No Cry1Ab protein was detectable by ELISA in uninduced young leaves, but small amounts of the protein were present in uninduced leaves older than 3 weeks and caused some insect mortality. Such basal expression of Bt genes without induction may favor the evolution of resistant insect populations and therefore limits the application of the PR-1a/cry1Ab system for insect management. However, the rapid production and steady maintenance of a high level of transgenic protein upon induction, the signal transduction observed, and the fact that the chemical inducer can be used in field conditions make the PR-1a promoter attractive for chemical regulation of other agriculturally or pharmaceutically important genes for which low expression in the absence of induction is not a concern.

  1. Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study.

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    Loretta Müller

    Full Text Available Enhancing antiviral host defense responses through nutritional supplementation would be an attractive strategy in the fight against influenza. Using inoculation with live attenuated influenza virus (LAIV as an infection model, we have recently shown that ingestion of sulforaphane-containing broccoli sprout homogenates (BSH reduces markers of viral load in the nose. To investigate the systemic effects of short-term BSH supplementation in the context of LAIV-inoculation, we examined peripheral blood immune cell populations in non-smoking subjects from this study, with a particular focus on NK cells. We carried out a randomized, double-blinded, placebo-controlled study measuring the effects of BSH (N = 13 or placebo (alfalfa sprout homogenate, ASH; N = 16 on peripheral blood mononuclear cell responses to a standard nasal vaccine dose of LAIV in healthy volunteers. Blood was drawn prior to (day-1 and post (day2, day21 LAIV inoculation and analyzed for neutrophils, monocytes, macrophages, T cells, NKT cells, and NK cells. In addition, NK cells were enriched, stimulated, and assessed for surface markers, intracellular markers, and cytotoxic potential by flow cytometry. Overall, LAIV significantly reduced NKT (day2 and day21 and T cell (day2 populations. LAIV decreased NK cell CD56 and CD158b expression, while significantly increasing CD16 expression and cytotoxic potential (on day2. BSH supplementation further increased LAIV-induced granzyme B production (day2 in NK cells compared to ASH and in the BSH group granzyme B levels appeared to be negatively associated with influenza RNA levels in nasal lavage fluid cells. We conclude that nasal influenza infection may induce complex changes in peripheral blood NK cell activation, and that BSH increases virus-induced peripheral blood NK cell granzyme B production, an effect that may be important for enhanced antiviral defense responses.ClinicalTrials.gov NCT01269723.

  2. A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

    Science.gov (United States)

    Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

    2014-05-01

    The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

  3. 不同干燥方式对西兰花蔬菜粉品质的影响%Effect of Different Drying Methods on Vegetable Powder Quality of the Broccoli (Brassica Oleracea)

    Institute of Scientific and Technical Information of China (English)

    杨华; 杨性民; 孙金才

    2013-01-01

    In this paper,the broccoli vegetable powder was as the research object.The changes of the main components and product quality of the broccoli vegetable powder by spray drying or vacuum freeze-drying (FD) treatment were studied in this paper.The results showed that:the main components after drying got enrichment,moisture content was greatly reduced.The moisture content of the broccoli powder by spray drying treatment was lower than vacuum FD's;But protein and vitamin C,the spray drying treatment was below the FD's.The colour,rehydration rate,dispersion,solubility,etc were analysised.The results showed that:in the colour,the spray drying powder was emerald green,but the FD's powder was thick green; In rehydration rate,FD's powder being the more effective; In dispersion,FD's powder was little better than the spray drying's; But in the solubility,powder,aroma,delicate degree,etc,the spray drying powder was better than the FD's broccoli vegetable powder.After the studying,found that the quality of the broccoli powder produced by vacuum freeze-drying treatmengt was better than by spray drying's.%以西兰花蔬菜粉为研究对象,研究西兰花蔬菜粉真空冷冻干燥、喷雾干燥前后产品的主要成分和品质变化.结果表明:干燥后主要成分得到浓缩,水分含量大大降低,其中喷雾干燥的西兰花粉的水分含量低于真空冷冻干燥的;在蛋白质和维生素C含量方面,喷雾干燥的低于冻干的西兰花粉.不同干燥方式对色泽、复水比、分散性、溶解度等品质的影响:在色差上,喷雾干燥的粉末为鲜绿色,而冻干的为浓绿色;在复水比上,冻干的蔬菜粉明显较喷雾干燥的优越;在分散性方面,冻干的粉末略好于喷雾干燥的;而在溶解度、粉性、香气、细腻度等方面,喷雾干燥的优于冻干的西兰花蔬菜粉.真空冷冻干燥方式制得的西兰花粉品质比喷雾干燥的好.

  4. Resposta de brócolis, couve-flor e repolho à adubação com boro em solo arenoso Response of boron fertilization on broccoli, cauliflower and cabbage planted in sandy soil

    Directory of Open Access Journals (Sweden)

    Luiz Carlos Pizetta

    2005-03-01

    Full Text Available Foram avaliados em condições de campo, em solo arenoso, com baixo teor de boro, os efeitos da adubação com cinco doses de boro (0; 2; 4; 6 e 8 kg ha-1 de B na forma de bórax na produção de brócolis, couve-flor e repolho. O experimento obedeceu a um esquema fatorial com delineamento experimental de blocos ao acaso com três repetições. As adubações orgânica e química, inclusive o bórax, foram feitas no sulco antes do transplantio das mudas e a colheita foi feita entre 63 e 93 dias após o transplantio. A produtividade de brócolis variou de 16,9 a 20,5 t ha-1; a de couve-flor de 21,6 a 29,6 t ha-1 e a de repolho de 40,5 a 46,4 t ha-1. O aumento observado na produtividade de brócolis e de repolho foi linear e o efeito das doses de boro na produtividade de couve-flor foi quadrático, sendo necessários 5,1 kg ha-1 de B para atingir a produtividade máxima de 30 t ha-1. Brócolis e repolho mostraram-se menos sensíveis do que a couve-flor tanto à deficiência quanto ao excesso de boro. No caso da couve-flor, com a aplicação de 2 kg ha-1 ou de 6 kg ha-1 de B houve significativa perda de qualidade do produto.The effects of boron fertilization on yield of broccoli, cauliflower and cabbage were evaluated through a field experiment carried out on a sandy soil low in available boron. Five boron levels (0; 2; 4; 6; and 8 kg ha-1 B as borax were applied in broccoli, cauliflower and cabbage using a factorial scheme and a randomized block design with three replicates. Organic manure and chemical fertilizers, including borax, were applied in the planting furrow before seedlings transplant and plants were harvested 63 to 93 days after planting date. The yield intervals obtained with broccoli, cauliflower and cabbage varied according to the following intervals: 16.9 to 20.5 t ha-1, 21.6 to 29.6 t ha-1 and 40.5 to 46.3 t ha-1, respectively. The increase in production observed in broccoli and cabbage yield was linear with boron levels and the

  5. The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics

    DEFF Research Database (Denmark)

    Long, K. S.; Poehlsgaard, Jacob; Kehrenberg, C.;

    2006-01-01

    A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin...... and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPSA for resistance to the following...... drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPSA drugs, which bind...

  6. Crystallization and preliminary crystallographic analysis of tRNA (m7G46) methyltransferase from Escherichia coli

    Science.gov (United States)

    Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun; Niu, Liwen

    2008-01-01

    Transfer RNA (tRNA) (m7G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N 7-­methylguanosine (m7G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His6 tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P21. PMID:18678947

  7. Crystallization and preliminary crystallographic analysis of tRNA (m(7)G46) methyltransferase from Escherichia coli.

    Science.gov (United States)

    Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun; Niu, Liwen

    2008-08-01

    Transfer RNA (tRNA) (m(7)G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-L-methionine (SAM) as the methyl-group donor to catalyze the formation of N(7)-methylguanosine (m(7)G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His(6) tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2(1).

  8. Reduced Expression of Histone Methyltransferases KMT2C and KMT2D Correlates with Improved Outcome in Pancreatic Ductal Adenocarcinoma.

    Science.gov (United States)

    Dawkins, Joshua B N; Wang, Jun; Maniati, Eleni; Heward, James A; Koniali, Lola; Kocher, Hemant M; Martin, Sarah A; Chelala, Claude; Balkwill, Frances R; Fitzgibbon, Jude; Grose, Richard P

    2016-08-15

    Genes encoding the histone H3 lysine 4 methyltransferases KMT2C and KMT2D are subject to deletion and mutation in pancreatic ductal adenocarcinoma (PDAC), where these lesions identify a group of patients with a more favorable prognosis. In this study, we demonstrate that low KMT2C and KMT2D expression in biopsies also defines better outcome groups, with median survivals of 15.9 versus 9.2 months (P = 0.029) and 19.9 versus 11.8 months (P = 0.001), respectively. Experiments with eight human pancreatic cell lines showed attenuated cell proliferation when these methyltransferases were depleted, suggesting that this improved outcome may reflect a cell-cycle block with diminished progression from G0-G1 RNA-seq analysis of PDAC cell lines following KMT2C or KMT2D knockdown identified 31 and 124 differentially expressed genes, respectively, with 19 genes in common. Gene-set enrichment analysis revealed significant downregulation of genes related to cell-cycle and growth. These data were corroborated independently by examining KMT2C/D signatures extracted from the International Cancer Genome Consortium and The Cancer Genome Atlas datasets. Furthermore, these experiments highlighted a potential role for NCAPD3, a condensin II complex subunit, as an outcome predictor in PDAC using existing gene expression series. Kmt2d depletion in KC/KPC cell lines also led to an increased response to the nucleoside analogue 5-fluorouracil, suggesting that lower levels of this methyltransferase may mediate the sensitivity of PDAC to particular treatments. Therefore, it may also be therapeutically beneficial to target these methyltransferases in PDAC, especially in those patients demonstrating higher KTM2C/D expression. Cancer Res; 76(16); 4861-71. ©2016 AACR.

  9. Reduced Expression of Histone Methyltransferases KMT2C and KMT2D Correlates with Improved Outcome in Pancreatic Ductal Adenocarcinoma

    Science.gov (United States)

    Dawkins, Joshua B.N.; Wang, Jun; Maniati, Eleni; Heward, James A.; Koniali, Lola; Kocher, Hemant M.; Martin, Sarah A.; Chelala, Claude; Balkwill, Frances R.; Fitzgibbon, Jude; Grose, Richard P.

    2017-01-01

    Genes encoding the histone H3 lysine 4 methyltransferases KMT2C and KMT2D are subject to deletion and mutation in pancreatic ductal adenocarcinoma (PDAC), where these lesions identify a group of patients with a more favorable prognosis. In this study, we demonstrate that low KMT2C and KMT2D expression in biopsies also defines better outcome groups, with median survivals of 15.9 versus 9.2 months (P = 0.029) and 19.9 versus 11.8 months (P = 0.001), respectively. Experiments with eight human pancreatic cell lines showed attenuated cell proliferation when these methyltransferases were depleted, suggesting that this improved outcome may reflect a cell-cycle block with diminished progression from G0–G1. RNA-seq analysis of PDAC cell lines following KMT2C or KMT2D knockdown identified 31 and 124 differentially expressed genes, respectively, with 19 genes in common. Gene-set enrichment analysis revealed significant downregulation of genes related to cell-cycle and growth. These data were corroborated independently by examining KMT2C/D signatures extracted from the International Cancer Genome Consortium and The Cancer Genome Atlas datasets. Furthermore, these experiments highlighted a potential role for NCAPD3, a condensin II complex subunit, as an outcome predictor in PDAC using existing gene expression series. Kmt2d depletion in KC/KPC cell lines also led to an increased response to the nucleoside analogue 5-fluorouracil, suggesting that lower levels of this methyltransferase may mediate the sensitivity of PDAC to particular treatments. Therefore, it may also be therapeutically beneficial to target these methyltransferases in PDAC, especially in those patients demonstrating higher KTM2C/D expression. PMID:27280393

  10. Facile synthesis of N-6 adenosine modified analogue toward S-adenosyl methionine derived probe for protein arginine methyltransferases

    Institute of Scientific and Technical Information of China (English)

    Wei Hong; James Dowden

    2011-01-01

    Chemically modified cellular co-factors that provide function, such as immobilization or incorporation of fluorescent dyes, are valuable probes of biological activity. A convenient route to obtain S-adenosyl methionine (AdoMet) analogues modified at N-6 adenosine to feature a linker terminating in azide functionality is described herein. Subsequent decoration of such AdoMet analogues with guanidinium terminated linkers leads to novel potential bisubstrate inhibitors for protein arginine methyltransferases, PRMTs.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis

    Directory of Open Access Journals (Sweden)

    Obarska Agnieszka

    2006-01-01

    Full Text Available Abstract Background Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM. However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin. Results Extensive database searches were carried out to identify orthologs and close paralogs of HEN1. Based on the multiple sequence alignment a phylogenetic tree of the HEN1 family was constructed. The fold-recognition approach was used to identify related methyltransferases with experimentally solved structures and to guide the homology modeling of the HEN1 catalytic domain. Additionally, we identified a La-like predicted RNA binding domain located C-terminally to the DSRM domain and a domain with a peptide prolyl cis/trans isomerase (PPIase fold, but without the conserved PPIase active site, located N-terminally to the catalytic domain. Conclusion The bioinformatics analysis revealed that the catalytic domain of HEN1 is not closely related to any known RNA:2'-OH methyltransferases (e.g. to the RrmJ/fibrillarin superfamily, but rather to small-molecule methyltransferases. The structural model was used as a platform to identify the putative active site and substrate-binding residues of HEN and to propose its mechanism of action.

  13. GAMT2 Encodes a Methyltransferase of Gibberellic Acid That is Involved in Seed Maturation and Germination in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Shufan Xing; Genji Qin; Yan Shi; Zhiqiang Ma; Zhangliang Chen; Hongya Gu; Li-Jia Qu

    2007-01-01

    Salicylic acid methyltransferase (SAMT), benzoic acid methyltransferase (BAMT) and theobromine methyltransferase (TH) (henceforth, SABATH) family proteins belong to a unique class of methyltransferase that can methylate small molecular compounds including indole-3-acidic acid (IAA), salicylic acid (SA) and jasmonic acid (JA), in plants. Here we report that the GAMT2 protein, which has 34.2% similarity with IAMT1 in the amino acid sequence, can methylate gibberellic acid (GA). Bioinformatics analysis suggests that GAMT2 may be able to methylate one molecule larger than SA. GAMT2 is predominantly expressed in the developing seed embryo and endosperm in Arabidopsis.During seed germination, the expression of GAMT2 decreases until the cotyledons expand out of the seed coat.Overexpression of GAMT2 in Arabidopsis resulted in multiple phenotypes, including dwarfism, retarded growth,late flowering, and reduced fertility, which are similar to the phenotypes of GA-deficient mutants. Seed germination assay showed that GAMT2 overexpression in plants was hypersensitive to GA biosynthesis inhibitor (ancymidol)and abscisic acid (ABA) treatments, whereas the GAMT2 null mutant (SALK_075450) was slightly insensitive to such treatments, suggesting that GAMT2 may methylate GA or ABA. Enzyme activity analysis indicated that GAMT2 was able to methylate GA3 into Methyl-GA3 in vitro, but could not methylate ABA. Microarray analysis on GAMT2overexpression plants suggested that Methyl-GA may be an inactive form of GA in Arabidopsis. These data suggest that GAMT2 is involved in seed maturation and germination by modulating GA activity.

  14. O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients

    OpenAIRE

    Spiegl-Kreinecker, Sabine; Pirker, Christine; Filipits, Martin; Lötsch, Daniela; Buchroithner, Johanna; Pichler, Josef; Silye, Rene; Weis, Serge; Micksche, Michael; Fischer, Johannes; Berger, Walter

    2009-01-01

    O6-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in t...

  15. Deoxyribonucleic acid (DNA) methyltransferase contributes to p16 promoter CpG island methylation in lung adenocarcinoma with smoking.

    Science.gov (United States)

    Sun, Rongju; Liu, Jiahong; Wang, Bo; Ma, Lingyun; Quan, Xiaojiao; Chu, Zhixiang; Li, Tanshi

    2015-01-01

    In this study, the relationship between CpG island methylation and smoking and DNA methyltransferase in the occurrence and development of lung adenocarcinoma was explored by detecting p16 promoter methylation status. Protein and mRNA levels of p16 were detected by immunohistochemistry and in situ hybridization assays. p16 gene promoter and exon 1 CpG island locus Hap II sites methylation status was analyzed with the methylation-specific PCR. Only 4 of 40 p16-positive cases were detected to methylate on CpG islands with 10% methylating rate whereas 18 of p16-negative cases were methylated up to 36.73% of methylating rate. The methylating rates of both p16-positive and p16-negative groups were significantly different. 17 of 50 cases with smoking from total 89 lung adenocarcinoma cases were detected to methylate on CpG islands while only 5 of the remaining 39 non-smokers to methylate. The difference of the methylating rates in both smokers and non-smokers was significant to suggest the closely association of CpG island methylation of p16 with smoking. Furthermore, p16 promoter CpG islands were detected to methylate in 15 of 35 cases with higher DNA methyltransferase activity whereas only 7 detected to methylate in the remaining 54 cases with lower DNA methyltransferase activity. p16 promoter CpG island methylation likely made p16 expressing silence thus contributed to the tumorigenesis of lung adenocarcinoma. Smoking is likely to promote p16 CpG island methylation or by its effect of the activity and metabolism of DNA methyltransferase 1 (DNMT) on CpG island methylation status.

  16. The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia.

    Directory of Open Access Journals (Sweden)

    William N Pappano

    Full Text Available Histone methyltransferases are epigenetic regulators that modify key lysine and arginine residues on histones and are believed to play an important role in cancer development and maintenance. These epigenetic modifications are potentially reversible and as a result this class of enzymes has drawn great interest as potential therapeutic targets of small molecule inhibitors. Previous studies have suggested that the histone lysine methyltransferase G9a (EHMT2 is required to perpetuate malignant phenotypes through multiple mechanisms in a variety of cancer types. To further elucidate the enzymatic role of G9a in cancer, we describe herein the biological activities of a novel peptide-competitive histone methyltransferase inhibitor, A-366, that selectively inhibits G9a and the closely related GLP (EHMT1, but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2. Additionally, the selectivity profile of A-366 has aided in the discovery of a potentially important role for G9a/GLP in maintenance of leukemia. Treatment of various leukemia cell lines in vitro resulted in marked differentiation and morphological changes of these tumor cell lines. Furthermore, treatment of a flank xenograft leukemia model with A-366 resulted in growth inhibition in vivo consistent with the profile of H3K9me2 reduction observed. In summary, A-366 is a novel and highly selective inhibitor of G9a/GLP that has enabled the discovery of a role for G9a/GLP enzymatic activity in the growth and differentiation status of leukemia cells.

  17. Effects of Nitrogen and Carbon Sources on Transcription of Soluble Methyltransferases in Methanosarcina mazei Strain Gö1†

    OpenAIRE

    Veit, Katharina; Ehlers, Claudia; Schmitz, Ruth A.

    2005-01-01

    The methanogenic archaeon Methanosarcina mazei strain Gö1 uses versatile carbon sources and is able to fix molecular nitrogen with methanol as carbon and energy sources. Here, we demonstrate that when growing on trimethylamine (TMA), nitrogen fixation does not occur, indicating that ammonium released during TMA degradation is sufficient to serve as a nitrogen source and represses nif gene induction. We further report on the transcriptional regulation of soluble methyltransferases, which catal...

  18. Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence.

    Directory of Open Access Journals (Sweden)

    Allison E James

    Full Text Available DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam. To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence.

  19. Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence.

    Science.gov (United States)

    James, Allison E; Rogovskyy, Artem S; Crowley, Michael A; Bankhead, Troy

    2016-01-01

    DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam). To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence.

  20. Crystallization and preliminary crystallographic analysis of tRNA (m{sup 7}G46) methyltransferase from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

    2008-08-01

    tRNA (m{sup 7}G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution. Transfer RNA (tRNA) (m{sup 7}G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N{sup 7}-methylguanosine (m{sup 7}G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His{sub 6} tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2{sub 1}.

  1. Cloning and expression of two sterol C-24 methyltransferase genes from upland cotton (Gossypium hirsuturm L.)

    Institute of Scientific and Technical Information of China (English)

    Ming Luo; Kunling Tan; Zhongyi Xiao; Mingyu Hu; Peng Liao; Kuijun Chen

    2008-01-01

    Brassinosteroids (BRs) are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. Two kinds of intermediates, sitosterol and campesterol, play a crucial role in cell elongation, cellulose biosynthesis, and accumulation. To illuminate the effects of sitosterol and campesterol on the development of cotton (Gossypium hirsuturm L.) fibers through screening cotton fiber EST database and contigging the candidate ESTs, two key genes GhSMT2-1 and GhSMT2-2 controlling the sitosterol biosynthesis were cloned from developing fibers of upland cotton cv. Xuzhou 142. The full length of GhSMT2-1 was 1, 151bp, including an 8bp 5'-untranslated region (UTR), a 1, 086bp open reading frame (ORF), and a 57bp 3'-UTR. GhSMT2-1 gene encoded a polypeptide of 361 amino acid residues with a predicted molecular mass of 40kDa. The full length of GhSMT2-2 was 1, 166bp, including an 18bp 5'-UTR, a 1, 086bp ORF, and a 62bp 3'-UTR. GhSMT2-2 gene encoded a polypeptide of 361 amino acid residues with a predicted molecular mass of 40kDa. The two deduced amino acid sequences had high homology with the SMT2 from Arabidopsis thaliana and Nicotiana tabacum. Furthermore, the typical conserved structures characterized by the sterol C-24 methyltransferase, such as region I (LDVGCGVGGPIVIRAI), region Ⅱ (IEATCHAP), and region Ⅲ (YEWGWGQSFHF), were present in both deduced proteins. Southern blotting analysis indicated that GhSMT2-1 or GhSMT2-2 was a single copy in upland cotton genome. Quantitative real-time RT-PCR analysis revealed that the highest expression levels of both genes were detected in 10 DPA (day post anthesis) fibers, while the lowest levels were observed in cotyledon and leaves. The expression level of GhSMT2-1 was 10 times higher than that of GhSMT2-2 in all the organs and tissues detected. These results indicate that the homologue of sterol C-24 methyltransferase gene was cloned from upland cotton and both GhSMT2 genes play a crucial

  2. Recobrimento de sementes de brócolos e salsa com coberturas e filmes biodegradáveis Covering broccoli and parsley seeds with biodegradable films and coatings

    Directory of Open Access Journals (Sweden)

    Patrícia Sayuri Tanada-Palmu

    2005-01-01

    Full Text Available O objetivo deste trabalho foi a comparação do desempenho de sementes de brócolos e de salsa cobertas ou aderidas a filmes biodegradáveis de quitosana e gelatina. Inicialmente, determinou-se o número ótimo de camadas de cobertura e a espessura do filme, para não comprometer a germinação das sementes. O desempenho foi avaliado por meio da capacidade de germinação e do vigor, e pelas massas de material fresco e seco de plantas com cerca de 30 dias. Observou-se germinação inferior ao controle nas sementes inseridas às fitas. O recobrimento de sementes obteve bons resultados em termos de vigor e desenvolvimento das plantas. Pelos resultados indicados, verificou-se que o recobrimento de sementes, com coberturas biodegradáveis, pode ser promissor, devido à melhoria na germinação das sementes recobertas e também no desenvolvimento das plantas quando comparadas às sementes sem tratamento.The objective of this work was to compare the performance of broccoli and parsley seeds coated or adhered to biodegradable films of gelatin and chitosan. Initially, the optimum number of coating layers and the thickness of the film were determined in order not to affect the germination of seeds. The performance was evaluated by germination capacity and vigor, and by fresh and dry weight of plants with 30 days. The seeds inserted into the films of gelatin and chitosan showed lower germination results than the control seeds. The coating of the seeds with gelatin and chitosan coatings of had good results in terms of vigor and development of plants. The results indicated that coating the seeds with biodegradable coatings can be promising due to the improvement of the germination of the coated seeds and the development of the plants when compared to the seeds with not treated.

  3. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  4. A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity.

    Science.gov (United States)

    Zhang, Hui; Yang, Yin; Dong, Huilei; Cai, Chenxin

    2016-12-15

    DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a superstructure-based electrochemical assay for signal-amplified detection of DNA MTase activity using M.SssI as an example. First, low-density coverage of DNA duplexes on the surface of the gold electrode was achieved by immobilized mercaptohexanol, followed by immobilization of DNA duplexes. The duplex can be cleaved by BstUI endonuclease in the absence of DNA superstructures. However, the cleavage is blocked after the DNA is methylated by M.SssI. The DNA superstructures are formed with the addition of helper DNA. By using an electroactive complex, RuHex, which can bind to DNA double strands, the activity of M.SssI can be quantitatively detected by differential pulse voltammetry. Due to the high site-specific cleavage by BstUI and signal amplification by the DNA superstructure, the biosensor can achieve ultrasensitive detection of DNA MTase activity down to 0.025U/mL. The method can be used for evaluation and screening of the inhibitors of MTase, and thus has potential in the discovery of methylation-related anticancer drugs.

  5. Association of DNA methyltransferase polymorphisms with susceptibility to primary gouty arthritis

    Science.gov (United States)

    Zhong, Xiaowu; Peng, Yuanhong; Yao, Chengjiao; Qing, Yufeng; Yang, Qibin; Guo, Xiaolan; Xie, Wenguang; Zhao, Mingcai; Cai, Xiaoming; Zhou, Jing-Guo

    2016-01-01

    Gouty arthritis is the most common type of inflammatory and immune disease, and the prevalence and incidence of gout increases annually. Genetic variations in the DNA methyltransferases (DNMTs) gene have not, to the best of our knowledge, been reported to influence gene expression and to participate in the pathogenesis of gout. The aim of the present study was to investigate whether the DNMT1, DNMT3A and DNMT3B polymorphisms contribute to gout susceptibility. These polymorphisms were screened for in 336 gout patients and 306 healthy control subjects (from a South China population) for association with gout. The distribution frequencies of DNMT1 rs2228611 AA genotype (P=0.007) and A allele (P=0.002; odds ratio=1.508, 95% confidence interval=1.158–1.964) were found to be significantly increased in the gout patients when compared with those in the healthy control subjects. The rs1550117 in DNMT3A and rs2424913 in DNMT3B exhibited no significant associations with gout susceptibility between the patients and control subjects. These results demonstrated that the DNMT1 rs2228611 polymorphism may be involved in the pathogenesis of gout, while DNMT3A rs1550117 and DNMT3B rs2424913 did not show any obvious significance in the current study; thus, may not be used as risk factors to predict the susceptibility to gout. However, further studies are required to investigate the functions and regulatory mechanism of the polymorphisms of DNMTs in gout. PMID:27699015

  6. Association of DNA methyltransferase polymorphisms with susceptibility to primary gouty arthritis.

    Science.gov (United States)

    Zhong, Xiaowu; Peng, Yuanhong; Yao, Chengjiao; Qing, Yufeng; Yang, Qibin; Guo, Xiaolan; Xie, Wenguang; Zhao, Mingcai; Cai, Xiaoming; Zhou, Jing-Guo

    2016-10-01

    Gouty arthritis is the most common type of inflammatory and immune disease, and the prevalence and incidence of gout increases annually. Genetic variations in the DNA methyltransferases (DNMTs) gene have not, to the best of our knowledge, been reported to influence gene expression and to participate in the pathogenesis of gout. The aim of the present study was to investigate whether the DNMT1, DNMT3A and DNMT3B polymorphisms contribute to gout susceptibility. These polymorphisms were screened for in 336 gout patients and 306 healthy control subjects (from a South China population) for association with gout. The distribution frequencies of DNMT1 rs2228611 AA genotype (P=0.007) and A allele (P=0.002; odds ratio=1.508, 95% confidence interval=1.158-1.964) were found to be significantly increased in the gout patients when compared with those in the healthy control subjects. The rs1550117 in DNMT3A and rs2424913 in DNMT3B exhibited no significant associations with gout susceptibility between the patients and control subjects. These results demonstrated that the DNMT1 rs2228611 polymorphism may be involved in the pathogenesis of gout, while DNMT3A rs1550117 and DNMT3B rs2424913 did not show any obvious significance in the current study; thus, may not be used as risk factors to predict the susceptibility to gout. However, further studies are required to investigate the functions and regulatory mechanism of the polymorphisms of DNMTs in gout.

  7. Evolution of the key alkaloid enzyme putrescine N-methyltransferase from spermidine synthase.

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    Anne eJunker

    2013-07-01

    Full Text Available Putrescine N-methyltransferases (PMTs are the first specific enzymes of the biosynthesis of nicotine and tropane alkaloids. PMTs transfer a methyl group onto the diamine putrescine from S-adenosyl-L-methionine (SAM as coenzyme. PMT proteins have presumably evolved from spermidine synthases (SPDSs, which are ubiquitous enzymes of polyamine metabolism. SPDS use decarboxylated SAM as coenzyme to transfer an aminopropyl group onto putrescine. In an attempt to identify possible and necessary steps in the evolution of PMT from SPDS, homology based modeling of Datura stramonium SPDS1 and PMT was employed to gain deeper insight in the preferred binding positions and conformations of the substrate and the alternative coenzymes. Based on predictions of amino acids responsible for the change of enzyme specificities, sites of mutagenesis were derived. PMT activity was generated in Datura stramonium SPDS1 after few amino acid exchanges. Concordantly, Arabidopsis thaliana SPDS1 was mutated and yielded enzymes with both, PMT and SPDS activities. Kinetic parameters were measured for enzymatic characterization. The switch from aminopropyl to methyl transfer depends on conformational changes of the methionine part of the coenzyme in the binding cavity of the enzyme. The rapid generation of PMT activity in SPDS proteins and the wide-spread occurrence of putative products of N-methylputrescine suggest that PMT activity is present frequently in the plant kingdom.

  8. Development and media regulate alternative splicing of a methyltransferase pre-mRNA in Monascus pilosus.

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    Zhang, Ming-Yong; Miyake, Tsuyoshi

    2009-05-27

    Two alternatively spliced mRNAs (d- and l-MpLaeA) of a methyltransferase gene (MpLaeA) were identified from Monascus pilosus IFO4520 and its mutant MK-1. Alternative splicing of the MpLaeA pre-mRNA occurred in the 5'-untranslated region (5'-UTR). The alternative splicing patterns of MpLaeA were regulated by the fungal growth stage and the principal nutrients: that is, the short l-MpLaeA mRNA was a constitutive transcript at all growth stages and different carbon or nitrogen sources, but the glutamate and NaNO(3) as main nitrogen source could up-regulate the long d-MpLaeA mRNA form. The long spliced 5'-UTR of d-MpLaeA blocked GFP expression in Escherichia coli , suggesting that d-MpLaeA mRNA was an ineffective spliced mRNA. Down-regulation of MpLaeA by transgenic antisense d-MpLaeA cDNA resulted in decreasing synthesis of monacolin K in M. pilosus. This suggested that the alternative splicing of MpLaeA mRNA might regulate the synthesis of monacolin K.

  9. RNA-mediated epigenetic heredity requires the cytosine methyltransferase Dnmt2.

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    Jafar Kiani

    2013-05-01

    Full Text Available RNA-mediated transmission of phenotypes is an important way to explain non-Mendelian heredity. We have previously shown that small non-coding RNAs can induce hereditary epigenetic variations in mice and act as the transgenerational signalling molecules. Two prominent examples for these paramutations include the epigenetic modulation of the Kit gene, resulting in altered fur coloration, and the modulation of the Sox9 gene, resulting in an overgrowth phenotype. We now report that expression of the Dnmt2 RNA methyltransferase is required for the establishment and hereditary maintenance of both paramutations. Our data show that the Kit paramutant phenotype was not transmitted to the progeny of Dnmt2(-/- mice and that the Sox9 paramutation was also not established in Dnmt2(-/- embryos. Similarly, RNA from Dnmt2-negative Kit heterozygotes did not induce the paramutant phenotype when microinjected into Dnmt2-deficient fertilized eggs and microinjection of the miR-124 microRNA failed to induce the characteristic giant phenotype. In agreement with an RNA-mediated mechanism of inheritance, no change was observed in the DNA methylation profiles of the Kit locus between the wild-type and paramutant mice. RNA bisulfite sequencing confirmed Dnmt2-dependent tRNA methylation in mouse sperm and also indicated Dnmt2-dependent cytosine methylation in Kit RNA in paramutant embryos. Together, these findings uncover a novel function of Dnmt2 in RNA-mediated epigenetic heredity.

  10. The Pseudomonas aeruginosa chemotaxis methyltransferase CheR1 impacts on bacterial surface sampling.

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    Juliane Schmidt

    Full Text Available The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa.

  11. Serotonin-Induced Hypersensitivity via Inhibition of Catechol O-Methyltransferase Activity

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    Tsao Douglas

    2012-04-01

    Full Text Available Abstract The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol O-methyltransferase (COMT, an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself (Ki = 44 μM. Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for β2- and β3-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions.

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

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    Kathryn T Hall

    Full Text Available 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.

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

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    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 Is Associated with Somatosensory Amplification and Nocebo Responses

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    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

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

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    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.

  16. Polymorphism of the catechol-O-methyltransferase gene in Han Chinese patients with psoriasis vulgaris

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    Lin Gao

    2009-01-01

    Full Text Available Psoriasis vulgaris is defined by a series of linked cellular changes in the skin: hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia, and infiltration of T lymphocytes, neutrophils and other types of leukocytes in the affected skin. Catechol-O-methyltransferase ( COMT 158 polymorphism can reduce the activity of the COMT enzyme that may trigger defective differentiation of keratinocytes in psoriasis. Immunocytes can degrade and inactivate catecholamines via monamine oxidase (MAO and COMT in the cells. We hypothesized that the COMT-158 G > A polymorphism was associated with the risk of psoriasis vulgaris in Han Chinese people. In a hospital-based case-control study, 524 patients with psoriasis vulgaris and 549 psoriasis-free controls were studied. COMT-158 G > A polymorphism was genotyped using the PCR sequence-specific primer (PCR-SSP technique. We found no statistically significant association between the COMT-158 allele A and the risk of psoriasis vulgaris (p = 0.739 adjusted OR = 1.03; 95% CI = 0.81-1.31. This suggests that the COMT-158 G > A polymorphism may not contribute to the etiology of psoriasis vulgaris in the Han Chinese population.

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

    Science.gov (United States)

    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.

  18. Genetic influences on insight problem solving: The role of catechol-o-methyltransferase (COMT gene polymorphisms

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    Weili eJiang

    2015-10-01

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

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

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    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.

  20. Development of fed-batch profiles for efficient biosynthesis of catechol-O-methyltransferase

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    G.M. Espírito Santo

    2014-09-01

    Full Text Available Catechol-O-methyltransferase (COMT, EC 2.1.1.6 plays a crucial role in dopamine metabolism which has intimately linked this enzyme to some neurodegenerative diseases, such as Parkinson's disease. In recent years, in the attempt of developing new therapeutic strategies for Parkinson's disease, there has been a growing interest in the search for effective COMT inhibitors. In order to do so, large amounts of COMT in an active form are needed, and the best way to achieve this is by up-scaling its production through biotechnological processes. In this work, a fed-batch process for the biosynthesis of the soluble isoform of COMT in Escherichia coli is proposed. This final process was selected through the evaluation of the effect of different dissolved oxygen concentrations, carbon and nitrogen source concentrations and feeding profiles on enzymatic production and cell viability, while controlling various parameters (pH, temperature, starting time of the feeding and induction phases and carbon source concentration during the process. After several batch and fed-batch experiments, a final specific COMT activity of 442.34 nmol/h/mg with approximately 80% of viable cells at the end of the fermentation were achieved. Overall, the results described herein provide a great improvement on hSCOMT production in recombinant bacteria and provide a new and viable option for the use of a fed-batch fermentation with a constant feeding profile to the large scale production of this enzyme.

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

    Science.gov (United States)

    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

  2. Catechol-O-methyltransferase Val158Met polymorphism is associated with somatosensory amplification and nocebo responses.

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    Laura Wendt

    Full Text Available 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.

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

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    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.

  4. Sequential Inactivation of Gliotoxin by the S-Methyltransferase TmtA.

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    Duell, Elke R; Glaser, Manuel; Le Chapelain, Camille; Antes, Iris; Groll, Michael; Huber, Eva M

    2016-04-15

    The epipolythiodioxopiperazine (ETP) gliotoxin mediates toxicity via its reactive thiol groups and thereby contributes to virulence of the human pathogenic fungus Aspergillus fumigatus. Self-intoxication of the mold is prevented either by reversible oxidation of reduced gliotoxin or by irreversible conversion to bis(methylthio)gliotoxin. The latter is produced by the S-methyltransferase TmtA and attenuates ETP biosynthesis. Here, we report the crystal structure of TmtA in complex with S-(5'-adenosyl)-l-homocysteine. TmtA features one substrate and one cofactor binding pocket per protein, and thus, bis-thiomethylation of gliotoxin occurs sequentially. Molecular docking of substrates and products into the active site of TmtA reveals that gliotoxin forms specific interactions with the protein surroundings, and free energy calculations indicate that methylation of the C10a-SH group precedes alkylation of the C3-SH site. Altogether, TmtA is well suited to selectively convert gliotoxin and to control its biosynthesis, suggesting that homologous enzymes serve to regulate the production of their toxic natural sulfur compounds in a similar manner.

  5. Arsenic (+3 oxidation state methyltransferase is a specific but replaceable factor against arsenic toxicity

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    Maki Tokumoto

    2014-01-01

    Full Text Available Inorganic metalloids, such as arsenic (As, antimony (Sb, selenium (Se, and tellurium (Te, are methylated in biota. In particular, As, Se, and Te are methylated and excreted in urine. The biomethylation is thought to be a means to detoxify the metalloids. The methylation of As is catalyzed by arsenic (+3 oxidation state methyltransferase (AS3MT. However, it is still unclear whether AS3MT catalyzes the methylation of the other metalloids. It is also unclear whether other factors catalyze the As methylation instead of AS3MT. Recombinant human AS3MT (rhAS3MT was prepared and used in the in vitro methylation of As, Se, and Te. As, but not Se and Te, was specifically methylated in the presence of rhAS3MT. Then, siRNA targeting AS3MT was introduced into human hepatocarcinoma (HepG2 cells. Although AS3MT protein expression was completely silenced by the gene knockdown, no increase in As toxicity was found in the HepG2 cells transfected with AS3MT-targeting siRNA. We conclude that AS3MT catalyzes the methylation of As and not other biomethylatable metalloids, such as Se and Te. We speculate that other methylation enzyme(s also catalyze the methylation of As in HepG2 cells.

  6. 5-Methyltetrahydrofolate-homocysteine methyltransferase gene polymorphism (MTR and risk of head and neck cancer

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    A.L.S. Galbiatti

    2010-05-01

    Full Text Available The functional effect of the A>G transition at position 2756 on the MTR gene (5-methyltetrahydrofolate-homocysteine methyltransferase, involved in folate metabolism, may be a risk factor for head and neck squamous cell carcinoma (HNSCC. The frequency of MTR A2756G (rs1805087 polymorphism was compared between HNSCC patients and individuals without history of neoplasias. The association of this polymorphism with clinical histopathological parameters was evaluated. A total of 705 individuals were included in the study. The polymerase chain reaction-restriction fragment length polymorphism technique was used to genotype the polymorphism. For statistical analysis, the chi-square test (univariate analysis was used for comparisons between groups and multiple logistic regression (multivariate analysis was used for interactions between the polymorphism and risk factors and clinical histopathological parameters. Using univariate analysis, the results did not show significant differences in allelic or genotypic distributions. Multivariable analysis showed that tobacco and alcohol consumption (P < 0.05, AG genotype (P = 0.019 and G allele (P = 0.028 may be predictors of the disease and a higher frequency of the G polymorphic allele was detected in men with HNSCC compared to male controls (P = 0.008. The analysis of polymorphism regarding clinical histopathological parameters did not show any association with the primary site, aggressiveness, lymph node involvement or extension of the tumor. In conclusion, our data provide evidence that supports an association between the polymorphism and the risk of HNSCC.

  7. Structural Basis for Inhibition of Histamine N-Methyltransferase by Diverse Drugs

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    Horton,J.; Sawada, K.; Nishibori, M.; Cheng, X.

    2005-01-01

    In mammals, histamine action is terminated through metabolic inactivation by histamine N-methyltransferase (HNMT) and diamine oxidase. In addition to three well-studied pharmacological functions, smooth muscle contraction, increased vascular permeability, and stimulation of gastric acid secretion, histamine plays important roles in neurotransmission, immunomodulation, and regulation of cell proliferation. The histamine receptor H1 antagonist diphenhydramine, the antimalarial drug amodiaquine, the antifolate drug metoprine, and the anticholinesterase drug tacrine (an early drug for Alzheimer's disease) are surprisingly all potent HNMT inhibitors, having inhibition constants in the range of 10-100 nM. We have determined the structural mode of interaction of these four inhibitors with HNMT. Despite their structural diversity, they all occupy the histamine-binding site, thus blocking access to the enzyme's active site. Near the N terminus of HNMT, several aromatic residues (Phe9, Tyr15, and Phe19) adopt different rotamer conformations or become disordered in the enzyme-inhibitor complexes, accommodating the diverse, rigid hydrophobic groups of the inhibitors. The maximized shape complementarity between the protein aromatic side-chains and aromatic ring(s) of the inhibitors are responsible for the tight binding of these varied inhibitors.

  8. DNA methyltransferase 3b is dispensable for mouse neural crest development.

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    Bridget T Jacques-Fricke

    Full Text Available The neural crest is a population of multipotent cells that migrates extensively throughout vertebrate embryos to form diverse structures. Mice mutant for the de novo DNA methyltransferase DNMT3b exhibit defects in two neural crest derivatives, the craniofacial skeleton and cardiac ventricular septum, suggesting that DNMT3b activity is necessary for neural crest development. Nevertheless, the requirement for DNMT3b specifically in neural crest cells, as opposed to interacting cell types, has not been determined. Using a conditional DNMT3b allele crossed to the neural crest cre drivers Wnt1-cre and Sox10-cre, neural crest DNMT3b mutants were generated. In both neural crest-specific and fully DNMT3b-mutant embryos, cranial neural crest cells exhibited only subtle migration defects, with increased numbers of dispersed cells trailing organized streams in the head. In spite of this, the resulting cranial ganglia, craniofacial skeleton, and heart developed normally when neural crest cells lacked DNMT3b. This indicates that DNTM3b is not necessary in cranial neural crest cells for their development. We conclude that defects in neural crest derivatives in DNMT3b mutant mice reflect a requirement for DNMT3b in lineages such as the branchial arch mesendoderm or the cardiac mesoderm that interact with neural crest cells during formation of these structures.

  9. Prenatal Exposure to Lipopolysaccharide Alters Renal DNA Methyltransferase Expression in Rat Offspring

    Science.gov (United States)

    Chen, Rui; Deng, Youcai; Liao, Xi; Wei, Yanling; Li, Xiaohui; Su, Min; Yu, Jianhua; Yi, Ping

    2017-01-01

    Prenatal exposure to inflammation results in hypertension during adulthood but the mechanisms are not well understood. Maternal exposure to lipopolysaccharide (LPS) alters interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels in the fetal environment. As reported in many recent studies, IL-6 regulates DNA methyltransferases (DNMTs) through the transcription factor friend leukemia virus integration 1 (Fli-1). The present study explores the role of intrarenal DNMTs during development of hypertension induced by prenatal exposure to LPS. Pregnant rats were randomly divided into four treatment groups: control, LPS, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor), and the combination of LPS and PDTC. Expression of IL-6, Fli-1, TNF-α, DNMT1 and DNMT3B was significantly increased in the offspring of LPS-treated rats. Global DNA methylation level of renal cortex also increased dramatically in rat offspring of the LPS group. Prenatal PDTC administration reversed the increases in gene expression and global DNA methylation level. These findings suggest that prenatal exposure to LPS may result in changes of intrarenal DNMTs through the IL-6/Fli-1 pathway and TNF-α, which probably involves hypertension in offspring due to maternal exposure to inflammation. PMID:28103274

  10. Role of microRNAs and DNA methyltransferases in transmitting induced genomic instability between cell generations

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    Katriina eHuumonen

    2014-09-01

    Full Text Available There is limited understanding of how radiation or chemicals induce genomic instability, and how the instability is epigenetically transmitted to the progeny of exposed cells or organisms. Here we measured the expression of microRNAs (miRNAs and DNA methyltransferases (DNMTs in murine embryonal fibroblasts exposed to ionizing radiation or 2,3,7,8 -tetrachlorodibenzo-p-dioxin (TCDD, which were previously shown to induce genomic instability in this cell line. Cadmium was used as a reference agent that does not induce genomic instability in our experimental model. Measurements at 8 and 15 days after exposure did not identify any such persistent changes that could be considered as signals transmitting genomic instability to the progeny of exposed cells. However, measurements at 2 days after exposure revealed findings that may reflect initial stages of genomic instability. Changes that were common to TCDD and two doses of radiation (but not to cadmium included 5 candidate signature miRNAs and general up-regulation of miRNA expression. Expression of DNMT3a, DNMT3b and DNMT2 were suppressed by cadmium but not by TCDD or radiation, consistently with the hypothesis that sufficient expression of DNMTs is necessary in the initial phase of induced genomic instability.

  11. The Study of DNA Methyltransferase-3B Promoter Variant Genotype among Iranian Sporadic Breast Cancer Patients

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    Ebrahim Eftekhar

    2014-05-01

    Full Text Available Background: DNA methyltransferase-3B (DNMT3B is an important enzyme responsible for maintaining the DNA methylation pattern in eukaryotic cells. In this study we have investigated the correlation between the 46359C→T polymorphism in the DNMT3B gene and the risk of breast cancer incidence among sporadic breast cancer patients in Fars Province, Southern Iran. Methods: In this case-control study, 100 breast cancer patients and 138 healthy control subjects were genotyped for the DNMT3B gene by the polymerase chain reaction-restriction fragment length polymorphism method. Results: The genotype frequency in the case (CC 27%, CT 47%, TT 26% group significantly (P=0.008 differed from the control (CC 19.56%, CT 67.3%, TT 13% group. We observed a decreased association between the CT genotype and lymph node involvement in breast cancer patients. Our results have shown that in comparison to the homozygous CC genotype carriers the DNMT3B-CT genotype has a significantly lower risk for breast cancer (OR=0.515, 95% CI=0.267-0.994, P=0.048. Conclusion: Our case-control study showed that the CT genotype was significantly associated with decreased breast cancer risk. Consistent with these results, a significant decrease of CT genotype among lymph node positive breast cancer patients was observed. However, a larger study population with more clinical data is needed to confirm these results.

  12. De novo DNA methyltransferase DNMT3b interacts with NEDD8-modified proteins.

    Science.gov (United States)

    Shamay, Meir; Greenway, Melanie; Liao, Gangling; Ambinder, Richard F; Hayward, S Diane

    2010-11-19

    DNA methylation and histone modifications play an important role in transcription regulation. In cancer cells, many promoters become aberrantly methylated through the activity of the de novo DNA methyltransferases DNMT3a and DNMT3b and acquire repressive chromatin marks. NEDD8 is a ubiquitin-like protein modifier that is conjugated to target proteins, such as cullins, to regulate their activity, and cullin 4A (CUL4A) in its NEDD8-modified form is essential for repressive chromatin formation. We found that DNMT3b associates with NEDD8-modified proteins. Whereas DNMT3b interacts directly in vitro with NEDD8, conjugation of NEDD8 to target proteins enhances this interaction in vivo. DNMT3b immunoprecipitated two major bands of endogenously NEDDylated proteins at the size of NEDDylated cullins, and indeed DNMT3b interacted with CUL1, CUL2, CUL3, CUL4A, and CUL5. Moreover, DNMT3b preferentially immunoprecipitated the NEDDylated form of endogenous CUL4A. NEDD8 enhanced DNMT3b-dependent DNA methylation. Chromatin immunoprecipitation assays suggest that DNMT3b recruits CUL4A and NEDD8 to chromatin, whereas deletion of Dnmt3b reduces the association of CUL4A and NEDD8 at a repressed promoter in a cancer cell line.

  13. Protective effect of O6-methylguanine-DNA-methyltransferase on mammalian cells

    Institute of Scientific and Technical Information of China (English)

    LI Dong-bo; WANG Ji-shi; FANG Qin; SUN Hai-yang; XU Wei; LI Wei-da

    2007-01-01

    Background O6-methylguanine-DNA-methyltransferase (MGMT) is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells.Methods Mammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells (PBMCs) via liposome, then assayed for gene expression at RNA and protein levels. MTT assay was used to check the drug resistance of cells transfected with MGMT gene.Results MGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively.Results of Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC50 values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC.Conclusion The alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.

  14. New insights on the mechanism of quinoline-based DNA Methyltransferase inhibitors.

    Science.gov (United States)

    Gros, Christina; Fleury, Laurence; Nahoum, Virginie; Faux, Céline; Valente, Sergio; Labella, Donatella; Cantagrel, Frédéric; Rilova, Elodie; Bouhlel, Mohamed Amine; David-Cordonnier, Marie-Hélène; Dufau, Isabelle; Ausseil, Frédéric; Mai, Antonello; Mourey, Lionel; Lacroix, Laurent; Arimondo, Paola B

    2015-03-06

    Among the epigenetic marks, DNA methylation is one of the most studied. It is highly deregulated in numerous diseases, including cancer. Indeed, it has been shown that hypermethylation of tumor suppressor genes promoters is a common feature of cancer cells. Because DNA methylation is reversible, the DNA methyltransferases (DNMTs), responsible for this epigenetic mark, are considered promising therapeutic targets. Several molecules have been identified as DNMT inhibitors and, among the non-nucleoside inhibitors, 4-aminoquinoline-based inhibitors, such as SGI-1027 and its analogs, showed potent inhibitory activity. Here we characterized the in vitro mechanism of action of SGI-1027 and two analogs. Enzymatic competition studies with the DNA substrate and the methyl donor cofactor, S-adenosyl-l-methionine (AdoMet), displayed AdoMet non-competitive and DNA competitive behavior. In addition, deviations from the Michaelis-Menten model in DNA competition experiments suggested an interaction with DNA. Thus their ability to interact with DNA was established; although SGI-1027 was a weak DNA ligand, analog 5, the most potent inhibitor, strongly interacted with DNA. Finally, as 5 interacted with DNMT only when the DNA duplex was present, we hypothesize that this class of chemical compounds inhibit DNMTs by interacting with the DNA substrate.

  15. Structural analysis of a putative SAM-dependent methyltransferase, YtqB, from Bacillus subtilis.

    Science.gov (United States)

    Park, Sun Cheol; Song, Wan Seok; Yoon, Sung-il

    2014-04-18

    S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) methylate diverse biological molecules using a SAM cofactor. The ytqB gene of Bacillus subtilis encodes a putative MTase and its biological function has never been characterized. To reveal the structural features and the cofactor binding mode of YtqB, we have determined the crystal structures of YtqB alone and in complex with its cofactor, SAM, at 1.9 Å and 2.2 Å resolutions, respectively. YtqB folds into a β-sheet sandwiched by two α-helical layers, and assembles into a dimeric form. Each YtqB monomer contains one SAM binding site, which shapes SAM into a slightly curved conformation and exposes the reactive methyl group of SAM potentially to a substrate. Our comparative structural analysis of YtqB and its homologues indicates that YtqB is a SAM-dependent class I MTase, and provides insights into the substrate binding site of YtqB.

  16. Abnormal expression of DNA methyltransferases and genomic imprinting in cloned goat fibroblasts.

    Science.gov (United States)

    Wan, Yongjie; Deng, Mingtian; Zhang, Guomin; Ren, Caifang; Zhang, Hao; Zhang, Yanli; Wang, Lizhong; Wang, Feng

    2016-01-01

    Somatic cell nuclear transfer (SCNT) is a useful way to produce cloned animals. However, SCNT animals exhibit DNA methylation and genomic imprinting abnormalities. These abnormalities may be due to the faulty epigenetic reprogramming of donor cells. To investigate the consequence of SCNT on the genomic imprinting and global methylation in the donor cells, growth patterns and apoptosis of cloned goat fibroblast cells (CGFCs) at passage 7 were determined. Growth patterns in CGFCs were similar to the controls; however, the growth rate in log phase was lower and apoptosis in CGFCs were significantly higher (P < 0.01). In addition, quantitative expression analysis of three DNA methyltransferases (Dnmt) and two imprinted genes (H19, IGF2R) was conducted in CGFCs: Dnmt1 and Dnmt3b expression was significantly reduced (P < 0.01), and H19 expression was decreased sixfold (P < 0.01); however, the expression of Dnmt3a was unaltered and IGF2R expression was significantly increased (P < 0.05). Finally, we used bisulfite sequencing PCR to compare the DNA methylation patterns in differentially methylated regions (DMRs) of H19 and IGF2R. The DMRs of H19 (P < 0.01) and IGF2R (P < 0.01) were both highly methylated in CGFCs. These results indicate that the global genome might be hypomethylated. Moreover, there is an aberrant expression of imprinted genes and DMR methylation in CGFCs.

  17. Exploration of Cyanine Compounds as Selective Inhibitors of Protein Arginine Methyltransferases: Synthesis and Biological Evaluation

    Science.gov (United States)

    2016-01-01

    Protein arginine methyltransferase 1 (PRMT1) is involved in many biological activities, such as gene transcription, signal transduction, and RNA processing. Overexpression of PRMT1 is related to cardiovascular diseases, kidney diseases, and cancers; therefore, selective PRMT1 inhibitors serve as chemical probes to investigate the biological function of PRMT1 and drug candidates for disease treatment. Our previous work found trimethine cyanine compounds that effectively inhibit PRMT1 activity. In our present study, we systematically investigated the structure–activity relationship of cyanine structures. A pentamethine compound, E-84 (compound 50), showed inhibition on PRMT1 at the micromolar level and 6- to 25-fold selectivity over CARM1, PRMT5, and PRMT8. The cellular activity suggests that compound 50 permeated the cellular membrane, inhibited cellular PRMT1 activity, and blocked leukemia cell proliferation. Additionally, our molecular docking study suggested compound 50 might act by occupying the cofactor binding site, which provided a roadmap to guide further optimization of this lead compound. PMID:25559100

  18. Homocysteine homeostasis and betaine-homocysteine S-methyltransferase expression in the brain of hibernating bats.

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    Yijian Zhang

    Full Text Available Elevated homocysteine is an important risk factor that increases cerebrovascular and neurodegenerative disease morbidity. In mammals, B vitamin supplementation can reduce homocysteine levels. Whether, and how, hibernating mammals, that essentially stop ingesting B vitamins, maintain homocysteine metabolism and avoid cerebrovascular impacts and neurodegeneration remain unclear. Here, we compare homocysteine levels in the brains of torpid bats, active bats and rats to identify the molecules involved in homocysteine homeostasis. We found that homocysteine does not elevate in torpid brains, despite declining vitamin B levels. At low levels of vitamin B6 and B12, we found no change in total expression level of the two main enzymes involved in homocysteine metabolism (methionine synthase and cystathionine β-synthase, but a 1.85-fold increase in the expression of the coenzyme-independent betaine-homocysteine S-methyltransferase (BHMT. BHMT expression was observed in the amygdala of basal ganglia and the cerebral cortex where BHMT levels were clearly elevated during torpor. This is the first report of BHMT protein expression in the brain and suggests that BHMT modulates homocysteine in the brains of hibernating bats. BHMT may have a neuroprotective role in the brains of hibernating mammals and further research on this system could expand our biomedical understanding of certain cerebrovascular and neurodegenerative disease processes.

  19. Characterization of NF-kB-mediated inhibition of catechol-O-methyltransferase

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    Conrad Matthew

    2009-03-01

    Full Text Available Abstract Background Catechol-O-methyltransferase (COMT, an enzyme that metabolizes catecholamines, has recently been implicated in the modulation of pain. Specifically, low COMT activity is associated with heightened pain perception and development of musculoskeletal pain in humans as well as increased experimental pain sensitivity in rodents. Results We report that the proinflammatory cytokine tumor necrosis factor α (TNFα downregulates COMT mRNA and protein in astrocytes. Examination of the distal COMT promoter (P2-COMT reveals a putative binding site for nuclear factor κB (NF-κB, the pivotal regulator of inflammation and the target of TNFα. Cell culture assays and functional deletion analyses of the cloned P2-COMT promoter demonstrate that TNFα inhibits P2-COMT activity in astrocytes by inducing NF-κB complex recruitment to the specific κB binding site. Conclusion Collectively, our findings provide the first evidence for NF-κB-mediated inhibition of COMT expression in the central nervous system, suggesting that COMT contributes to the pathogenesis of inflammatory pain states.

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

    Science.gov (United States)

    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.

  1. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China); Zhao, Bo; Kieff, Elliott [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States); Peng, Chih-Wen, E-mail: pengcw@mail.tcu.edu.tw [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)

    2013-01-18

    Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

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

    Science.gov (United States)

    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.

  3. Single-Turnover Kinetics of Methyl Transfer to tRNA by Methyltransferases

    Science.gov (United States)

    Hou, Ya-Ming

    2016-01-01

    Summary Methyl transfer from S-adenosyl methionine (abbreviated as AdoMet) to biologically active molecules such as mRNAs and tRNAs is one of the most fundamental and widespread reactions in nature, occurring in all three domains of life. The measurement of kinetic constants of AdoMet-dependent methyl transfer is therefore important for understanding the reaction mechanism in the context of biology. When kinetic constants of methyl transfer are measured in steady state over multiple rounds of turnover, the meaning of these constants is difficult to define and is often limited by non-chemical steps of the reaction, such as product release after each turnover. Here the measurement of kinetic constants of methyl transfer by tRNA methyltransferases in rapid equilibrium binding condition for one methyl transfer is described. The advantage of such a measurement is that the meaning of kinetic constants can be directly assigned to the steps associated with the chemistry of methyl transfer, including the substrate binding affinity to the methyl transferase, the pre-chemistry re-arrangement of the active site, and the chemical step of methyl transfer. An additional advantage is that kinetic constants measured for one methyl transfer can be correlated with structural information of the methyl transferase to gain direct insight into its reaction mechanism. PMID:26965259

  4. Characterization of an O-methyltransferase from Streptomyces avermitilis MA-4680.

    Science.gov (United States)

    Yoon, Youngdae; Park, Younghee; Lee, Youngshim; Yi, Yong Sub; Jo, Geunhyeong; Park, Jun Cheol; Ahn, Joong-Hoon; Lim, Yoongho

    2010-09-01

    A search of the Streptomyces avermitilis genome reveals that its closest homologs are several O-methyltransferases. Among them, one gene (viz., saomt5) was cloned into the pET-15b expression vector by polymerase chain reaction using sequence-specific oligonucleotide primers. Biochemical characterization with the recombinant protein showed that SaOMT5 was S-adenosyl-L-methionine-dependent Omethyltransferase. Several compounds were tested as substrates of SaOMT5. As a result, SaOMT5 catalyzed Omethylation of flavonoids such as 6,7-dihydroxyflavone, 2',3'-dihydroxyflavone, 3',4'-dihydroxyflavone, quercetin, and 7,8-dihydroxyflavone, and phenolic compounds such as caffeic acid and caffeoyl Co-A. These reaction products were analyzed by TLC, HPLC, LC/MS, and NMR spectroscopy. In addition, SaOMT5 could convert phenolic compounds containing ortho-dihydroxy groups into Omethylated compounds, and 6,7-dihydroxyflavone was known to be the best substrate. SaOMT5 converted 6,7- dihydroxyflavone into 6-hydroxy-7-methoxyflavone and 7-hydroxy-6-methoxyflavone, and caffeic acid into ferulic acid and isoferulic acid, respectively. Moreover, SaOMT5 turned out to be a Mg2+-dependent OMT, and the effect of Mg2+ ion on its activity was five times greater than those of Ca2+, Fe2+, and Cu2+ ions, EDTA, and metal-free medium.

  5. Analysis of oxidative stress status, catalase and catechol-O-methyltransferase polymorphisms in Egyptian vitiligo patients.

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    Dina A Mehaney

    Full Text Available 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.

  6. Jasmonic acid carboxyl methyltransferase regulates development and herbivory-induced defense response in rice

    Institute of Scientific and Technical Information of China (English)

    Jinfeng Qi; Yonggen Lou; Jiancai Li; Xiu Han; Ran Li; Jianqiang Wu; Haixin Yu; Lingfei Hu; Yutao Xiao; Jing Lu

    2016-01-01

    Jasmonic acid (JA) and related metabolites play a key role in plant defense and growth. JA carboxyl methyltransferase (JMT) may be involved in plant defense and development by methylating JA to methyl jasmonate (MeJA) and thus influencing the concentrations of JA and related metabolites. However, no JMT gene has been well characterized in monocotyledon defense and development at the molecular level. After we cloned a rice JMT gene, OsJMT1, whose encoding protein was localized in the cytosol, we found that the recombinant OsJMT1 protein catalyzed JA to MeJA. OsJMT1 is up-regulated in response to infestation with the brown planthopper (BPH; Nilaparvata lugens). Plants in which OsJMT1 had been overexpressed (oe-JMT plants) showed reduced height and yield. These oe-JMT plants also exhibited increased MeJA levels but reduced levels of herbivore-induced JA and jasmonoyl-isoleucine (JA-Ile). The oe-JMT plants were more attractive to BPH female adults but showed increased resistance to BPH nymphs, probably owing to the different responses of BPH female adults and nymphs to the changes in levels of H2O2 and MeJA in oe-JMT plants. These results indicate that OsJMT1, by altering levels of JA and related metabolites, plays a role in regulating plant development and herbivore-induced defense responses in rice.

  7. Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis.

    Science.gov (United States)

    Ren, Jinqi; Wang, Yaqing; Liang, Yuheng; Zhang, Yongqing; Bao, Shilai; Xu, Zhiheng

    2010-04-23

    Modulation of ribosomal assembly is a fine tuning mechanism for cell number and organ size control. Many ribosomal proteins undergo post-translational modification, but their exact roles remain elusive. Here, we report that ribosomal protein s10 (RPS10) is a novel substrate of an oncoprotein, protein-arginine methyltransferase 5 (PRMT5). We show that PRMT5 interacts with RPS10 and catalyzes its methylation at the Arg(158) and Arg(160) residues. The methylation of RPS10 at Arg(158) and Arg(160) plays a role in the proper assembly of ribosomes, protein synthesis, and optimal cell proliferation. The RPS10-R158K/R160K mutant is not efficiently assembled into ribosomes and is unstable and prone to degradation by the proteasomal pathway. In nucleoli, RPS10 interacts with nucleophosmin/B23 and is predominantly concentrated in the granular component region, which is required for ribosome assembly. The RPS10 methylation mutant interacts weakly with nucleophosmin/B23 and fails to concentrate in the granular component region. Our results suggest that PRMT5 is likely to regulate cell proliferation through the methylation of ribosome proteins, and thus reveal a novel mechanism for PRMT5 in tumorigenesis.

  8. Substrate specificity of mammalian N-terminal α-amino methyltransferase

    Science.gov (United States)

    Petkowski, Janusz J.; Schaner Tooley, Christine E.; Anderson, Lissa C.; Shumilin, Igor A.; Balsbaugh, Jeremy L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Minor, Wladek; Macara, Ian G.

    2012-01-01

    N-terminal methylation of free α-amino-groups is a post-translational modification of proteins that has been known for 30 years but has been very little studied. In this modification, the initiating M residue is cleaved and the exposed α-amino group is mono- di- or trimethylated by NRMT, a recently identified N-terminal methyltransferase. Currently, all known eukaryotic α-aminomethylated proteins have a unique N-terminal motif, M-X-P-K, where X is A, P, or S. NRMT can also methylate artificial substrates in vitro in which X is G, F, Y, C, M, K, R, N, Q or H. Methylation efficiencies of N-terminal amino acids are variable with respect to the identity of X. Here we use in vitro peptide methylation assays and substrate immunoprecipitations to show that the canonical M-X-P-K methylation motif is not the only one recognized by NRMT. We predict that N-terminal methylation is a widespread post-translational modification, and that there is interplay between N-terminal acetylation and N-terminal methylation. We also use isothermal calorimetry experiments to demonstrate that NRMT can efficiently recognize and bind to its fully methylated products. PMID:22769851

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

    Science.gov (United States)

    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.

  10. Site specificity of the Arabidopsis METI DNA methyltransferase demonstrated through hypermethylation of the superman locus.

    Science.gov (United States)

    Kishimoto, N; Sakai, H; Jackson, J; Jacobsen, S E; Meyerowitz, E M; Dennis, E S; Finnegan, E J

    2001-05-01

    Plants with low levels of DNA methylation show a range of developmental abnormalities including homeotic transformation of floral organs. Two independent DNA METHYLTRANSFERASEI (METI) antisense transformants with low levels of DNA methylation had flowers with increased numbers of stamens which resembled flowers seen on the loss-of-function superman (sup) mutant plants and on transgenic plants that ectopically express APETALA3 (AP3). These METI antisense plants have both increased and decreased methylation in and around the sup gene, compared with untransformed controls. DNA from the antisense plants was demethylated at least 4 kb upstream of the sup gene, while there was dense methylation around the start of transcription and within the coding region of this gene; these regions were unmethylated in control DNA. Methylation within the sup gene was correlated with an absence of SUP transcripts. The pattern and density of methylation was heterogeneous among different DNA molecules from the same plant, with some molecules being completely unmethylated. Methylcytosine occurred in asymmetric sites and in symmetric CpA/TpG but rarely in CpG dinucleotides in the antisense plants. In contrast, segregants lacking the METI antisense construct and epimutants with a hypermethylated allele of sup (clark kent 3), both of which have active METI genes, showed a higher frequency of methylation of CpG dinucleotides and of asymmetric cytosines. We conclude that METI is the predominant CpG methyltransferase and directly or indirectly affects asymmetric methylation.

  11. ANTITUMOR EFFECT OF SARCNU IN A 06-METHYLGUANINE-DNA METHYLTRANSFERASE POSITIVE HUMAN GLIOMA XENOGRAFT MODEL

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To assess whether novel analogue of nitrosoureas, 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU), has antitumor effect to 06-methylguanine-DNA methyltransferase (MGMT) positive tumors in vivo. Methods: MGMT positive human glioma cell line SF-767 xenografts in nude mice were treated with SarCNU. The antitumor efficacy of SarCNU was compared with the results of 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment with or without 06-benzylguanine (06-BG) preadministration. Results: Since the SF-767 is MGMT strongly positive, BCNU treatment alone did not result in a satisfactory anticancer effect. As expected, 06-BG by depleting MGMT activity, significantly enhanced BCNU antitumor efficacy (p<0.001). More interestingly, SarCNU treatment alone had a better antitumor effect than 06-BG plus BCNU treatment (F=51.7, p=0.00036). Conclusion: Since SarCNU enters cells via extraneuronal monoamine transporter (EMT), the enhanced antitumor activity of SarCNU in this MGMT positive human tumor xenograft model may be due to the presence of EMT in SF-767.SarCNU may be used as an alternative treatment for MGMT positive tumors, specifically for tumors expressing EMT.

  12. Identification and characterization of DNAzymes targeting DNA methyltransferase I for suppressing bladder cancer proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xiangbo; Zhang, Lu; Ding, Nianhua; Yang, Xinghui; Zhang, Jin; He, Jiang; Li, Zhi; Sun, Lun-Quan, E-mail: lunquansun@csu.edu.cn

    2015-05-29

    Epigenetic inactivation of genes plays a critical role in many important human diseases, especially in cancer. A core mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA, which is catalyzed by DNA methyltransferases (DNMTs). The inhibition of DNMTs may lead to demethylation and expression of the silenced tumor suppressor genes. Although DNMT inhibitors are currently being developed as potential anticancer agents, only limited success is achieved due to substantial toxicity. Here, we utilized a multiplex selection system to generate efficient RNA-cleaving DNAzymes targeting DNMT1. The lead molecule from the selection was shown to possess efficient kinetic profiles and high efficiency in inhibiting the enzyme activity. Transfection of the DNAzyme caused significant down-regulation of DNMT1 expression and reactivation of p16 gene, resulting in reduced cell proliferation of bladder cancers. This study provides an alternative for targeting DNMTs for potential cancer therapy. - Highlights: • Identified DNMT1-targeted DNAzymes by multiplex selection system. • Biochemically characterized a lead DNAzyme with high kinetic efficiency. • Validated DNMT1-targeted DNAzyme in its enzymatic and cellular activities.

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

    Science.gov (United States)

    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

    2015-11-01

    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.

  14. Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs

    Science.gov (United States)

    Gayatri, Sitaram; Cowles, Martis W.; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T.

    2016-01-01

    Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245

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

    Science.gov (United States)

    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

  16. Disulfiram sensitizes pituitary adenoma cells to temozolomide by regulating O6-methylguanine-DNA methyltransferase expression.

    Science.gov (United States)

    Zhao, Yachao; Xiao, Zheng; Chen, Wenna; Yang, Jinsheng; Li, Tao; Fan, Bo

    2015-08-01

    O6-methylguanine-DNA methyltransferase (MGMT) activity is responsible for temozolomide (TMZ) resistance in patients harboring aggressive pituitary adenomas. Recently, disulfiram (DSF) has been shown to induce the loss of MGMT protein and increase TMZ efficacy in glioblastoma cells, while CD133+ nestin+ cells isolated from the cell population have been implicated as pituitary adenoma stem-like cells. However, whether DSF is able to potentiate the cytotoxic effects of TMZ on human pituitary adenoma cells has not been investigated to date. In the present study, CD133+ nestin+ phenotype cells were isolated from primary cultured human pituitary adenoma cells using microbeads. It was found that DSF reduced MGMT protein expression and sensitized human pituitary adenoma cells and stem-like cells to TMZ in vitro, while the proteasome inhibitor PS-341 abrogated the inhibitory effect of DSF on MGMT in vitro. The sensitizing effect of DSF was also verified in primary cultured human pituitary adenoma cells in vivo. The results of the present study suggested that DSF can increase the efficacy of the anti-tumor effect of TMZ on human pituitary adenoma cells and CD133+ nestin+ stem like cells via the ubiquitin-proteasomal MGMT protein elimination route. DSF combined with TMZ may be an effective therapeutic strategy against aggressive pituitary adenomas.

  17. Crystal structure of the homocysteine methyltransferase MmuM from Escherichia coli.

    Science.gov (United States)

    Li, Kunhua; Li, Gengnan; Bradbury, Louis M T; Hanson, Andrew D; Bruner, Steven D

    2016-02-01

    Homocysteine S-methyltransferases (HMTs, EC 2.1.1.0) catalyse the conversion of homocysteine to methionine using S-methylmethionine or S-adenosylmethionine as the methyl donor. HMTs play an important role in methionine biosynthesis and are widely distributed among micro-organisms, plants and animals. Additionally, HMTs play a role in metabolite repair of S-adenosylmethionine by removing an inactive diastereomer from the pool. The mmuM gene product from Escherichia coli is an archetypal HMT family protein and contains a predicted zinc-binding motif in the enzyme active site. In the present study, we demonstrate X-ray structures for MmuM in oxidized, apo and metallated forms, representing the first such structures for any member of the HMT family. The structures reveal a metal/substrate-binding pocket distinct from those in related enzymes. The presented structure analysis and modelling of co-substrate interactions provide valuable insight into the function of MmuM in both methionine biosynthesis and cofactor repair.

  18. Immunohistochemical evaluation of O6 -methylguanine DNA methyltransferase (MGMT) expression in 117 cases of glioblastoma.

    Science.gov (United States)

    Miyazaki, Masaya; Nishihara, Hiroshi; Terasaka, Shunsuke; Kobayashi, Hiroyuki; Yamaguchi, Shigeru; Ito, Tamio; Kamoshima, Yuuta; Fujimoto, Shin; Kaneko, Sadao; Katoh, Masahito; Ishii, Nobuaki; Mohri, Hiromi; Tanino, Mishie; Kimura, Taichi; Tanaka, Shinya

    2014-06-01

    Temozolomide (TMZ) is an oral alkylating agent which is widely used in the treatment of glioblastoma (GBM) and is composed of astrocytic and/or oligodendroglial tumors, and the evaluation of O(6) -methylguanine DNA methyltransferase (MGMT) expression is important to predict the response to TMZ therapy. In this study, we conducted immunohistochemical analysis of 117 cases of Japanese GBM including 19 cases of GBM with oligodendroglioma component (GBMO), using a scoring system for quantitative evaluation of staining intensity and proportion of MGMT, and performed survival analysis of these patients. Immunohistochemically, 55 cases (47%) were positive for MGMT with various intensities and proportions (total score (TS) ≥ 2), while 62 cases (53%) were negative (TS = 0). The distribution of MGMT expression pattern was not affected by any clinicopathological parameters such as the histological subtype (GBM vs. GBMO), age and gender. The survival analysis of these patients revealed that the minimal expression of MGMT (TS ≥ 2) was a significant unfavorable prognostic factor (P MGMT expression in GBM was the most potent independent predictor for progression free survival (P MGMT expression in GBM. In addition, our results emphases the clinicopathological values of the immunohistochemical approach for MGMT expression in glioma patients as a routine laboratory examination.

  19. O(6)-Methylguanine-DNA methyltransferase (MGMT): A drugable target in lung cancer?

    Science.gov (United States)

    Hiddinga, Birgitta I; Pauwels, Patrick; Janssens, Annelies; van Meerbeeck, Jan P

    2016-07-18

    This manuscript addresses the role of O(6)-methylguanine-DNA methyltransferase (MGMT) as a biomarker in the oncogenesis of cancer and the opportunity of turning this gene into a drugable target in neuroendocrine tumours of the lung. Studies in brain tumours conclude that MGMT promoter methylation is considered a strong predictive factor for a favourable outcome for treatment with temozolomide, e.g. alkylating agent. We conducted a systematic review of MGMT in non-small cell lung cancer (NSCLC), small-cell lung cancer (SCLC) and other pulmonary neuroendocrine tumours (NETs) to evaluate whether MGMT is a prognostic and/or predictive factor to select patients with lung cancer who can benefit from treatment with temozolomide. In NSCLC MGMT promoter methylation is not a prognostic and predictive factor, hence temozolomide has no place. In SCLC and NET patients with a MGMT promoter methylation benefit of temozolomide has to be confirmed.Temozolomide can be considered a 'personalized' treatment if the predictive role of MGMT is further confirmed.

  20. Expression of O(6)-methylguanine DNA methyltransferase (MGMT) and its clinical significance in gastroenteropancreatic neuroendocrine neoplasm.

    Science.gov (United States)

    Yang, Qiu-Chen; Wang, Yu-Hong; Lin, Yuan; Xue, Ling; Chen, Yuan-Jia; Chen, Min-Hu; Chen, Jie

    2014-01-01

    O(6)-methylguanine-DNA methyltransferase (MGMT) is a widespread DNA repair enzyme defending against mutation caused by guanine O(6)-alkylating agents. Until now, we know only little about the expression of MGMT in gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN). To study the expression of MGMT and its clinical significance in GEP-NEN, 174 specimens of GEP-NEN were examined, of which 152 specimens came from The First Affiliated Hospital, Sun Yat-sen University during October 1995 to November 2013, 22 specimens came from Peking Union Medical College Hospital during September 2004 to April 2010. MGMT protein was detected with EnVision immunohistochemical staining method. Clinicopathological factors were also collected and analyzed. We observed that the overall expression rate of MGMT was 83.9%. Over expression of MGMT protein was not associated with sex, age, functional status, primary tumor location, grading, classification, TNM stage and metastasis (P > 0.05). Kaplan-Meier analysis revealed that there was no significant difference in survival between MGMT-positive and MGMT-negative tumors of GEP-NEN patients (χ(2) = 0.887, P = 0.346). In multivariate analyses carried out by Cox proportional hazards regression model, MGMT expression was also not an independent predictors of survival. These results demonstrated that MGMT protein was highly expressed in GEP-NEN. MGMT deficiency rate was similar in pancreatic NEN and in gastrointestinal NEN. MGMT expression was not correlated with prognosis of GEP-NEN.

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

    Science.gov (United States)

    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.

  2. Role of type II protein arginine methyltransferase 5 in the regulation of Circadian Per1 gene.

    Directory of Open Access Journals (Sweden)

    Jungtae Na

    Full Text Available Circadian clocks are the endogenous oscillators that regulate rhythmic physiological and behavioral changes to correspond to daily light-dark cycles. Molecular dissections have revealed that transcriptional feedback loops of the circadian clock genes drive the molecular oscillation, in which PER/CRY complexes inhibit the transcriptional activity of the CLOCK/BMAL1 heterodimer to constitute a negative feedback loop. In this study, we identified the type II protein arginine methyltransferase 5 (PRMT5 as an interacting molecule of CRY1. Although the Prmt5 gene was constitutively expressed, increased interaction of PRMT5 with CRY1 was observed when the Per1 gene was repressed both in synchronized mouse liver and NIH3T3 cells. Moreover, rhythmic recruitment of PRMT5 and CRY1 to the Per1 gene promoter was found to be associated with an increased level of histone H4R3 dimethylation and Per1 gene repression. Consistently, decreased histone H4R3 dimethylation and altered rhythmic Per1 gene expression were observed in Prmt5-depleted cells. Taken together, these findings provide an insight into the link between histone arginine methylation by PRMT5 and transcriptional regulation of the circadian Per1 gene.

  3. A methylation-stimulated DNA machine: an autonomous isothermal route to methyltransferase activity and inhibition analysis.

    Science.gov (United States)

    Zhu, Changfeng; Wen, Yanqin; Peng, Hongzhen; Long, Yitao; He, Yao; Huang, Qing; Li, Di; Fan, Chunhai

    2011-04-01

    The operation of DNA nanomachines is generally triggered by either conformational changes of DNA nanostructure or external environmental stimuli. In the present study, we demonstrate an alternative driving force, DNA methylation, to stimulate DNA machine operation. DNA methylation changes neither DNA sequence and conformation nor external environment, however, blocks its cleavage by corresponding methylation-sensitive restriction endonuclease. We thus designed a strand displacement amplification DNA machine, which could be stimulated upon DNA methylation and then autonomously generates accumulated amounts of peroxidase-mimicking DNAzyme signaling machine products in an isothermal manner. The machine product DNAzyme could catalyze the H(2)O(2)-mediated oxidation of 2,2'-azino-bis(3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS(2-)) to a colored product ABTS(·-). This methylation-stimulated DNA machine was further used as a colorimetric assay for analysis of methyltransferases activities and screening of methylation inhibitors. As compared with classical methylation assay, this facile isothermal DNA machine avoids the introduction of methylation-specific polymerase chain reaction and radioactive labels, which might be employed as an effective tool for DNA methylation analysis.

  4. 微真空条件下复合保鲜剂对西兰花采后衰老的影响%Effects of Compound Preservatives on Senescence of Postharvest Broccoli under Micro-vacuum Conditions

    Institute of Scientific and Technical Information of China (English)

    王丽娇; 孙兴丽; 岳本芳; 李文香; 王士奎; 张圣杰

    2013-01-01

    为探讨微真空贮藏条件下不同浓度6-苄基腺嘌呤(6-BA)和0.2%苯甲酸钠(SBN)复合保鲜剂对西兰花采后衰老的影响,以西兰花为试材,贮藏于温度为3±-0.5℃、真空压力为400~500 torr的微真空贮藏条件下,通过对西兰花贮藏期间叶绿素、Vc及丙二醛含量(MDA)与PPO、POD活性的测定,探讨较佳的复合保鲜剂浓度.结果表明:3种浓度的复合保鲜剂均可不同程度地抑制西兰花叶绿素、Vc的降解,减少膜脂过氧化产物MDA的积累,降低PPO、POD活性,其中40 mg/L 6-BA+0.2% SBN处理对调控西兰花采后衰老的效果最佳.%In order to explore the effects of compound preservatives of 6-BA and 0.2% SBN on senescence of postharvest broccoli in micro-vacuum conditions,fleshly broccoli was stored at temperature of 3±0.5 ℃ with pressure of 400~500 torr.The contents of chlorophyll,Vc and MDA and the activities of PPO and POD were determined to explore better composite preservative concentration.The results showed that three concentrations can inhibit the degradation of chlorophyll and Vc,reduce the accumulation of membrane lipid peroxidation product MDA,lower the activities of PPO and POD in varying degrees.40 mg/L 6-BA and 0.2% SBN showed the best effect on inhibition of broccoli postharvest senescence.

  5. Isolation,identification and fungicide screening of the pathogen of broccoli flower-ball rot disease in laboratory%松花菜花球腐烂病病原分离鉴定及室内药剂筛选

    Institute of Scientific and Technical Information of China (English)

    王国荣; 刘晓曦; 吴金丹; 王文凤; 苏珍珠; 楼兵干

    2015-01-01

    松花菜花球腐烂病是近年来发生在浙江省松花菜种植区的一种严重病害,主要危害松花菜花球、花梗,引起花球变褐腐烂,花梗发黑枯萎,造成严重经济损失。通过对松花菜花球腐烂病典型症状样本的采集,病原菌的分离、纯化,致病性测定,形态观察,16S rDNA 序列分析,脂肪酸分析和 Biolog 鉴定,明确病原菌为野油菜黄单胞菌野油菜致病变种 Xanthomonas campestris pv.campestris (Pammel)Dye。选择8种杀菌剂采用 K-B 灭菌滤纸片法对病菌进行室内抑菌测定,结果表明,72%农用硫酸链霉素和 PHMB 对该病菌有较强的抑制作用,其次中生菌素和三氯异氰尿酸也有一定的抑制作用。%Broccoli flower-ball rot disease has become a serious disease in recent years in broccoli-producing areas of Zhejiang Province.It mainly infects the flower-ball and stalks of broccoli,causing the flower-ball brown and rot,and the stalks black and withered,and finally leads to a serious economic loss.In this study,we collected samples with typical symptoms of this disease,isolated and purified the pathogen,and then conducted a series of experiments,including the pathogenicity determination,observation on morphological characteristics,analysis of 1 6S rDNA gene sequences and fatty acid methyl esters (FAME),and Biolog identification.All of these results showed that the pathogen was Xanthomonas campestris pv.campestris (Pammel)Dye.Moreover,we selected 8 kinds of fungicides to do the bacterial inhibition assay in the laboratory with K-B sterile filter paper.The results showed that 72% agricultural streptomycin sulfate and PHMB had strong inhibition action to the pathogen,and zhongshengmycin and trichloroiso cyanuric acid also had inhibition action to the pathogen to a certain extent.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  8. Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins.

    Science.gov (United States)

    Anang, Saumya; Subramani, Chandru; Nair, Vidya P; Kaul, Sheetal; Kaushik, Nidhi; Sharma, Chandresh; Tiwari, Ashutosh; Ranjith-Kumar, C T; Surjit, Milan

    2016-04-26

    Hepatitis E virus (HEV) is a major cause of hepatitis in normal and organ transplant individuals. HEV open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (X-domain), V, DUF3729 and Y. The macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand RNA viruses, playing important roles in multiple cellular processes. Towards understanding the function of the HEV macro domain, we characterized its interaction partners among other HEV encoded proteins. Here, we report that the HEV X-domain directly interacts with the viral methyltransferase and the ORF3 proteins. ORF3 association with the X-domain was mediated through two independent motifs, located within its N-terminal 35aa (amino acids) and C-terminal 63-123aa. Methyltransferase interaction domain was mapped to N-terminal 30-90aa. The X-domain interacted with both ORF3 and methyltransferase through its C-terminal region, involving 66(th),67(th) isoleucine and 101(st),102(nd) leucine, conserved across HEV genotypes. Furthermore, ORF3 and methyltransferase competed with each other for associating with the X-domain. These findings provide molecular understanding of the interaction between the HEV macro domain, methyltransferase and ORF3, suggesting an important role of the macro domain in the life cycle of HEV.

  9. Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.

    Directory of Open Access Journals (Sweden)

    Matthew B Kilgore

    Full Text Available 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.

  10. 干燥方式对西兰花超微粉理化特性及抗氧化活性的影响%Effect of Drying Methods on Physicochemical Properties and Antioxidant Activity of Superfine Broccoli Powder

    Institute of Scientific and Technical Information of China (English)

    杨磊磊; 王然; 王风舞; 王成荣

    2012-01-01

    Broccoli was dried by three different methods, hot air drying, vacuum drying, vacuum freeze drying before superfine comminution to investigate the effects of these three drying methods on physicochemical properties and antioxidant activity of superfine broccoli powder. The results showed that hot air drying provided superfine broccoli powder with the smallest angle of repose and the highest mobility. Vacuum dried powder had the highest specific weight. Vacuum freeze dried powder the highest water-absorbing capacity, oil-holding capacity and dissolvability but the lowest mobility. The nutritional quality of vacuum freeze dried power was much better than that of hot air dried powder and vacuum dried powder. The IC50 values of vacuum freeze dried, vacuum dried and hot air dried powder samples were 0.130, 0.210 g/100 mL and 0.245 g/100 mL for DPPH radicals, and 0.650, 0.710 g/100 mL and 0.720 g/100 mL for superoxide anion radicals, respectively. These data demonstrate that vacuum freeze dried broccoli powder has the strongest antioxidant activity.%通过热风干燥、真空干燥、真空冷冻干燥对西兰花进行干燥后超微粉碎,研究不同方式干燥后西兰花超微粉的理化特性及其抗氧化活性。结果表明:热风干燥的西兰花超微粉休止角最小,流动性最大;真空干燥的西兰花超微粉容重最高;真空冷冻干燥的西兰花超微粉溶解性、持水力和持油力最大,容重最小,但其流动性最差。真空冷冻干燥的西兰花超微粉的营养品质明显优于热风干燥及真空干燥。真空冷冻干燥、真空干燥和热风干燥生产的西兰花超微粉的甲醇提取液对DPPH自由基的IC50值分别为0.130、0.210、0.245g/100mL;对O2^-·的IC50值分别为0.650、0.710、0.720g/100mL,真空冷冻干燥西兰花超微粉抗氧化活性最高。

  11. 闽东南沿海中部地区青花菜田间害虫发生动态及其可持续控制技术研究%Dynamics of Insect Pests in Broccoli (Brassica oleracea var.italica) Field in Middle Southeast Coastal Region of Fujian Province and Its Sustainable Control Technique

    Institute of Scientific and Technical Information of China (English)

    王再兴; 官德义; 刘志钦; 陈桂信; 何水林

    2011-01-01

    青花菜是近年闽东南沿海地区发展迅速的一种出口蔬菜,但其病虫害发生过重,农残超标已严重制约了该地区青花菜的可持续发展,因此,建立并推广应用可持续病虫控制技术已势在必行.本研究以福建省惠安县青花菜生产田块为研究对象,在跟踪监测种植季节(9月份到翌年3月份)主要虫害发生动态并确立重点防治对象及其关键时期的基础上,研究了灯诱、性诱和高效低毒农药对其主要虫害的防治效果,从而建立起一套青花菜田间虫害的可持续控制技术.这一研究成果对闽东南沿海青花菜及类似十字花科蔬菜生产的可持续发展具有一定的指导意义.%Broccoli is one of the most important vegetables for export that have expanded rapidly in the past decades in the southeast costal region of Fujian Province. Excessive pesticide residues due to the inappropriate usage of pesticides has become a big problem for the sustainable development of broccoli in this area, and the development of sustainable pests and diseases control techniques is currently an urgent task. In this project, study on the dynamics of insect pests in the field of broccoli and its control were carried out in Huian County, which located in the middle part of southeast costal region of Fujian Province. The dynamics of pests in broccoli fields from September to March of next year was monitored, the main insect pests and the key period for their control were determined. The effect of light trap and sexual attraction as well as the effect of environment friendly pesticides on the main pests of broccoli were determined, and a sustainable control technique was consequently established. The results of this study are important for the pollution free production of broccoli as well as other brassicaceous vegetables in southeast coastal region of Fujian.

  12. Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

    Science.gov (United States)

    Houtz, Robert L.

    1999-01-01

    The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

  13. Comparison of Sulforaphen Content in Seeds and Callus Induced From Different Organs of Broccoli Species%西兰花种子及其愈伤组织中萝卜硫素含量比较

    Institute of Scientific and Technical Information of China (English)

    张品南; 马绍英; 杨海荣; 杨柯; 刘会杰; 李胜; 周文政; 付瑞军

    2013-01-01

    The content and distribution of sulforaphen were investigated by HPLC in seeds and callus induced from different organs of Broccoli species.The results showed that there were significant differences in sulforaphen content among the 9 broccoli species.‘ Yu huang' seeds had the highest content of sulforaphen,up to 942.758 μg/g,while ‘ Zhong qing NO · 9' was the lowest one (172.808 μg/g).The order of sulforaphen contents among different callus was seed lobe > hypocotyls > radicle.The average content of seeds was 1.45,2.37 and 2.92 times as high as each organ callus.%采用高效液相色谱法对西兰花种子及其不同器官诱导的愈伤组织中萝卜硫素的含量进行比较.结果表明供试的9个品种的种子中萝卜硫素含量差异显著,“玉皇”含量最高,达942.758 μg/g;“中青9号”含量最低,为172.808 μg/g.不同器官诱导的愈伤组织中萝卜硫素平均含量子叶>胚轴>胚根,种子中的平均含量分别为各器官获得愈伤组织平均含量的1.45、2.37和2.92倍.

  14. Approaches to enzyme and substrate design of the murine Dnmt3a DNA methyltransferase.

    Science.gov (United States)

    Jurkowska, Renata Z; Siddique, Abu Nasar; Jurkowski, Tomasz P; Jeltsch, Albert

    2011-07-01

    Dnmt3a-C, the catalytic domain of the Dnmt3a DNA-(cytosine-C5)-methyltransferase, is active in an isolated form but, like the full-length Dnmt3a, shows only weak DNA methylation activity. To improve this activity by directed evolution, we set up a selection system in which Dnmt3a-C methylated its own expression plasmid in E. coli, and protected it from cleavage by methylation-sensitive restriction enzymes. However, despite screening about 400 clones that were selected in three rounds from a random mutagenesis library of 60 000 clones, we were not able to isolate a variant with improved activity, most likely because of a background of uncleaved plasmids and plasmids that had lost the restriction sites. To improve the catalytic activity of Dnmt3a-C by optimization of the sequence of the DNA substrate, we analyzed its flanking-sequence preference in detail by bisulfite DNA-methylation analysis and sequencing of individual clones. Based on the enrichment and depletion of certain bases in the positions flanking >1300 methylated CpG sites, we were able to define a sequence-preference profile for Dnmt3a-C from the -6 to the +6 position of the flanking sequence. This revealed preferences for T over a purine at position -2, A over G at -1, a pyrimidine at +1, and A and T over G at +3. We designed one "good" substrate optimized for methylation and one "bad" substrate designed not to be efficiently methylated, and showed that the optimized substrate is methylated >20 times more rapidly at its central CpG site. The optimized Dnmt3a-C substrate can be applied in enzymatic high-throughput assays with Dnmt3a-C (e.g., for inhibitor screening), because the increased activity provides an improved dynamic range and better signal/noise ratio.

  15. AS1411 alters the localization of a complex containing protein arginine methyltransferase 5 and nucleolin.

    Science.gov (United States)

    Teng, Yun; Girvan, Allicia C; Casson, Lavona K; Pierce, William M; Qian, Mingwei; Thomas, Shelia D; Bates, Paula J

    2007-11-01

    AS1411 is a quadruplex-forming oligonucleotide aptamer that targets nucleolin. It is currently in clinical trials as a treatment for various cancers. We have proposed that AS1411 inhibits cancer cell proliferation by affecting the activities of certain nucleolin-containing complexes. Here, we report that protein arginine methyltransferase 5 (PRMT5), an enzyme that catalyzes the formation of symmetrical dimethylarginine (sDMA), is a nucleolin-associated protein whose localization and activity are altered by AS1411. Levels of PRMT5 were found to be decreased in the nucleus of AS1411-treated DU145 human prostate cancer cells, but increased in the cytoplasm. These changes were dependent on nucleolin and were not observed in cells pretreated with nucleolin-specific small interfering RNA. Treatment with AS1411 altered levels of PRMT5 activity (assessed by sDMA levels) in accord with changes in its localization. In addition, our data indicate that nucleolin itself is a substrate for PRMT5 and that distribution of sDMA-modified nucleolin is altered by AS1411. Because histone arginine methylation by PRMT5 causes transcriptional repression, we also examined expression of selected PRMT5 target genes in AS1411-treated cells. For some genes, including cyclin E2 and tumor suppressor ST7, a significant up-regulation was noted, which corresponded with decreased PRMT5 association with the gene promoter. We conclude that nucleolin is a novel binding partner and substrate for PRMT5, and that AS1411 causes relocalization of the nucleolin-PRMT5 complex from the nucleus to the cytoplasm. Consequently, the nuclear activity of PRMT5 is decreased, leading to derepression of some PRMT5 target genes, which may contribute to the biological effects of AS1411.

  16. Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells

    Directory of Open Access Journals (Sweden)

    Yoda Satoshi

    2008-11-01

    Full Text Available Abstract Background Alterations in the processing of the genetic information in carcinogenesis result from stable genetic mutations or epigenetic modifications. It is becoming clear that nucleosomal histones are central to proper gene expression and that aberrant DNA methylation of genes and histone methylation plays important roles in tumor progression. To date, several histone lysine methyltransferases (HKMTs have been identified and histone lysine methylation is now considered to be a critical regulator of transcription. However, still relatively little is known about the role of HKMTs in tumorigenesis. Results We observed differential HKMT expression in a lung cancer model in which normal human bronchial epithelial (NHBE cells expressing telomerase, SV40 large T antigen, and Ras were immortal, formed colonies in soft agar, and expressed specific HKMTs for H3 lysine 9 and 27 residues but not for H3 lysine 4 residue. Modifications in the H3 tails affect the binding of proteins to the histone tails and regulate protein function and the position of lysine methylation marks a gene to be either activated or repressed. In the present study, suppression by siRNA of HKMTs (EZH2, G9A, SETDB1 and SUV39H1 that are over-expressed in immortalized and transformed cells lead to reduced cell proliferation and much less anchorage-independent colony growth. We also found that the suppression of H3-K9, G9A and SUV39H1 induced apoptosis and the suppression of H3-K27, EZH2 caused G1 arrest. Conclusion Our results indicate the potential of these HKMTs in addition to the other targets for epigenetics such as DNMTs and HDACs to be interesting therapeutic targets.

  17. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Science.gov (United States)

    Smadbeck, James; Peterson, Meghan B; Zee, Barry M; Garapaty, Shivani; Mago, Aashna; Lee, Christina; Giannis, Athanassios; Trojer, Patrick; Garcia, Benjamin A; Floudas, Christodoulos A

    2014-01-01

    Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2) maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2. These inhibitors should

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  1. Low enzymatic activity haplotypes of the human catechol-O-methyltransferase gene: enrichment for marker SNPs.

    Directory of Open Access Journals (Sweden)

    Andrea G Nackley

    Full Text Available Catechol-O-methyltransferase (COMT is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous (val(158met position, designated as low (LPS, average (APS, and high pain sensitive (HPS, are associated with experimental pain sensitivity and risk of developing chronic musculoskeletal pain conditions. APS and HPS haplotypes produce significant functional effects, coding for 3- and 20-fold reductions in COMT enzymatic activity, respectively. In the present study, we investigated whether additional minor single nucleotide polymorphisms (SNPs, accruing in 1 to 5% of the population, situated in the COMT transcript region contribute to haplotype-dependent enzymatic activity. Computer analysis of COMT ESTs showed that one synonymous minor SNP (rs769224 is linked to the APS haplotype and three minor SNPs (two synonymous: rs6267, rs740602 and one nonsynonymous: rs8192488 are linked to the HPS haplotype. Results from in silico and in vitro experiments revealed that inclusion of allelic variants of these minor SNPs in APS or HPS haplotypes did not modify COMT function at the level of mRNA folding, RNA transcription, protein translation, or enzymatic activity. These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation. Thus, both minor synonymous and nonsynonymous SNPs in the coding region are markers of functional APS and HPS haplotypes rather than independent contributors to COMT activity.

  2. How metal substitution affects the enzymatic activity of catechol-o-methyltransferase.

    Directory of Open Access Journals (Sweden)

    Manuel Sparta

    Full Text Available Catechol-O-methyltransferase (COMT degrades catecholamines, such as dopamine and epinephrine, by methylating them in the presence of a divalent metal cation (usually Mg(II, and S-adenosyl-L-methionine. The enzymatic activity of COMT is known to be vitally dependent on the nature of the bound metal: replacement of Mg(II with Ca(II leads to a complete deactivation of COMT; Fe(II is slightly less than potent Mg(II, and Fe(III is again an inhibitor. Considering the fairly modest role that the metal plays in the catalyzed reaction, this dependence is puzzling, and to date remains an enigma. Using a quantum mechanical / molecular mechanical dynamics method for extensive sampling of protein structure, and first principle quantum mechanical calculations for the subsequent mechanistic study, we explicate the effect of metal substitution on the rate determining step in the catalytic cycle of COMT, the methyl transfer. In full accord with experimental data, Mg(II bound to COMT is the most potent of the studied cations and it is closely followed by Fe(II, whereas Fe(III is unable to promote catalysis. In the case of Ca(II, a repacking of the protein binding site is observed, leading to a significant increase in the activation barrier and higher energy of reaction. Importantly, the origin of the effect of metal substitution is different for different metals: for Fe(III it is the electronic effect, whereas in the case of Ca(II it is instead the effect of suboptimal protein structure.

  3. Epigenetic remodeling of meiotic crossover frequency in Arabidopsis thaliana DNA methyltransferase mutants.

    Directory of Open Access Journals (Sweden)

    Nataliya E Yelina

    Full Text Available Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO. Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1 mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes.

  4. The Expression of Antibiotic Resistance Methyltransferase Correlates with mRNA Stability Independently of Ribosome Stalling.

    Science.gov (United States)

    Dzyubak, Ekaterina; Yap, M N

    2016-12-01

    Members of the Erm methyltransferase family modify 23S rRNA of the bacterial ribosome and render cross-resistance to macrolides and multiple distantly related antibiotics. Previous studies have shown that the expression of erm is activated when a macrolide-bound ribosome stalls the translation of the leader peptide preceding the cotranscribed erm Ribosome stalling is thought to destabilize the inhibitory stem-loop mRNA structure and exposes the erm Shine-Dalgarno (SD) sequence for translational initiation. Paradoxically, mutations that abolish ribosome stalling are routinely found in hyper-resistant clinical isolates; however, the significance of the stalling-dead leader sequence is largely unknown. Here, we show that nonsense mutations in the Staphylococcus aureus ErmB leader peptide (ErmBL) lead to high basal and induced expression of downstream ErmB in the absence or presence of macrolide concomitantly with elevated ribosome methylation and resistance. The overexpression of ErmB is associated with the reduced turnover of the ermBL-ermB transcript, and the macrolide appears to mitigate mRNA cleavage at a site immediately downstream of the ermBL SD sequence. The stabilizing effect of antibiotics on mRNA is not limited to ermBL-ermB; cationic antibiotics representing a ribosome-stalling inducer and a noninducer increase the half-life of specific transcripts. These data unveil a new layer of ermB regulation and imply that ErmBL translation or ribosome stalling serves as a "tuner" to suppress aberrant production of ErmB because methylated ribosome may impose a fitness cost on the bacterium as a result of misregulated translation.

  5. DNA Methyltransferase 3B Gene Promoter and Interleukin-1 Receptor Antagonist Polymorphisms in Childhood Immune Thrombocytopenia

    Directory of Open Access Journals (Sweden)

    Margarita Pesmatzoglou

    2012-01-01

    Full Text Available Primary immune thrombocytopenia (ITP is one of the most common blood diseases as well as the commonest acquired bleeding disorder in childhood. Although the etiology of ITP is unclear, in the pathogenesis of the disease, both environmental and genetic factors including polymorphisms of TNF-a, IL-10, and IL-4 genes have been suggested to be involved. In this study, we investigated the rs2424913 single-nucleotide polymorphism (SNP (C46359T in DNA methyltransferase 3B (DNMT3B gene promoter and the VNTR polymorphism of IL-1 receptor antagonist (IL-1 Ra intron-2 in 32 children (17 boys with the diagnosis of ITP and 64 healthy individuals. No significant differences were found in the genotype distribution of DNMT3B polymorphism between the children with ITP and the control group, whereas the frequency of allele T appeared significantly increased in children with ITP (P = 0.03, OR = 2, 95% CI: 1.06–3.94. In case of IL-1 Ra polymorphism, children with ITP had a significantly higher frequency of genotype I/II, compared to control group (P = 0.043, OR = 2.60, 95% CI: 1.02–6.50. Moreover, genotype I/I as well as allele I was overrepresented in the control group, suggesting that allele I may have a decreased risk for development of ITP. Our findings suggest that rs2424913 DNMT3B SNP as well as IL-1 Ra VNTR polymorphism may contribute to the susceptibility to ITP.

  6. Expression pattern and clinical significance of DNA methyltransferase 3B variants in gastric carcinoma.

    Science.gov (United States)

    Su, Xianwei; Lv, Chengyu; Qiao, Fengchang; Qiu, Xuemei; Huang, Wenbin; Wu, Qingxiang; Zhao, Zhujiang; Fan, Hong

    2010-03-01

    The aim of this study was to detect the expression pattern of DNA methyltransferase 3B (DNMT3B) variants in primary gastric cancer (GC) and to explore the clinical significance of DNMT3B variants in gastric carcinogenesis. Specific polymerase chain reaction (PCR) primer sets were designed to distinguish individual DNMT3B variants according to their splicing patterns. Expression levels of DNMT3B variants were assessed by quantitative real-time RT-PCR in gastric cancer tissue, normal gastric mucosae and GC cell lines. The relationship between the expression patterns of the DNMT3B variants and corresponding clinical information was analyzed by observing the expression levels of different variants in the tumors. These results demonstrate that DNMT3B overexpression is related to late phase invasion (P=0.029) and intestinal type (P=0.012) in GC. DNMT3B3 expression was higher in normal tissue, compared to tumor tissue (P=0.033). In contrast, only 18, 32 and 35% of the patient tumors overexpressed DNMT3B1, DNMT3B4 and DNMT3B5, respectively. While taking into account environmental factors (H. pylori, Epstein-Barr virus infection), H. pylori infection elevated DNMT3B1 and DNMT3B3 variants in tumors, while increasing DNMT3B4 in both tumor and non-cancerous tissues. Our findings indicated that the expression of DNMT3B3 is the major splice variant in normal gastric mucosae and may be affected by H. pylori infection. Elevated DNMT3B variants may influence the progression of gastric cancer and may possibly be a powerful indicator for the disease.

  7. Role of the DNA methyltransferase variant DNMT3b3 in DNA methylation.

    Science.gov (United States)

    Weisenberger, Daniel J; Velicescu, Mihaela; Cheng, Jonathan C; Gonzales, Felicidad A; Liang, Gangning; Jones, Peter A

    2004-01-01

    Several alternatively spliced variants of DNA methyltransferase (DNMT) 3b have been described. Here, we identified new murine Dnmt3b mRNA isoforms and found that mouse embryonic stem (ES) cells expressed only Dnmt3b transcripts that contained exons 10 and 11, whereas the Dnmt3b transcripts in somatic cells lacked these exons, suggesting that this region is important for embryonic development. DNMT3b2 and 3b3 were the major isoforms expressed in human cell lines and the mRNA levels of these isoforms closely correlated with their protein levels. Although DNMT3b3 may be catalytically inactive, it still may be biologically important because D4Z4 and satellites 2 and 3 repeat sequences, all known DNMT3b target sequences, were methylated in cells that predominantly expressed DNMT3b3. Treatment of cells with the mechanism-based inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) caused a complete depletion of DNMT1, 3a, 3b1, and 3b2 proteins. Human DNMT3b3 and the murine Dnmt3b3-like isoform, Dnmt3b6, were also depleted although less efficiently, suggesting that DNMT3b3 also may be capable of DNA binding. Moreover, de novo methylation of D4Z4 in T24 cancer cells after 5-Aza-CdR treatment only occurred when DNMT3b3 was expressed, reinforcing its role as a contributing factor of DNA methylation. The expression of either DNMT3b2 or 3b3, however, was not sufficient to explain the abnormal methylation of DNMT3b target sequences in human cancers, which may therefore be dependent on factors that affect DNMT3b targeting. Methylation analyses of immunodeficiency, chromosomal instabilities, and facial abnormalities cells revealed that an Alu repeat sequence was highly methylated, suggesting that Alu sequences are not DNMT3b targets.

  8. Identification and characterization of alternatively spliced variants of DNA methyltransferase 3a in mammalian cells.

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    Weisenberger, Daniel J; Velicescu, Mihaela; Preciado-Lopez, Miguel A; Gonzales, Felicidad A; Tsai, Yvonne C; Liang, Gangning; Jones, Peter A

    2002-09-18

    CpG methylation is mediated by the functions of at least three active DNA methyltransferases (DNMTs). While DNMT1 is thought to perform maintenance methylation, the more recently discovered DNMT3a and DNMT3b enzymes are thought to facilitate de novo methylation. Murine Dnmt3a and 3b are developmentally regulated and a new Dnmt3a isoform, Dnmt3a2, has been recently shown to be expressed preferentially in mouse embryonic stem (ES) cells. Here we have characterized four alternatively spliced variants of human and mouse DNMT3a. These transcripts included a novel exon 1 (1beta) that was spliced into the same exon 2 acceptor splice site used by the original exon 1 (1alpha). Cloning and sequencing of the 5' region of the human DNMT3a gene revealed that exon 1beta was situated upstream of exon 1alpha and that the entire region was contained within a CpG island. We also identified other alternatively spliced species containing intron 4 inclusions that were associated with either exon 1alpha or 1beta. These were expressed at low levels in mouse and human cells. All transcripts were highly conserved between human and mouse. The levels of Dnmt3a mRNA containing exon 1beta were 3-25-fold greater in mouse ES cells than in various somatic cells as determined by semiquantitative reverse transcription-polymerase chain reaction analysis, while the levels of exon 1alpha-containing transcripts were slightly higher in human and mouse somatic cells. The preferential expression of the beta transcript in ES cells suggests that this transcript, in addition to Dnmt3a2, may also be important for de novo methylation during development.

  9. Arsenic methylation by an arsenite S-adenosylmethionine methyltransferase from Spirulina platensis.

    Science.gov (United States)

    Guo, Yuqing; Xue, Ximei; Yan, Yu; Zhu, Yongguan; Yang, Guidi; Ye, Jun

    2016-11-01

    Arsenic-contaminated water is a serious hazard for human health. Plankton plays a critical role in the fate and toxicity of arsenic in water by accumulation and biotransformation. Spirulina platensis (S. platensis), a typical plankton, is often used as a supplement or feed for pharmacy and aquiculture, and may introduce arsenic into the food chain, resulting in a risk to human health. However, there are few studies about how S. platensis biotransforms arsenic. In this study, we investigated arsenic biotransformation by S. platensis. When exposed to arsenite (As(III)), S. platensis accumulated arsenic up to 4.1mg/kg dry weight. After exposure to As(III), arsenate (As(V)) was the predominant species making up 64% to 86% of the total arsenic. Monomethylarsenate (MMA(V)) and dimethylarsenate (DMA(V)) were also detected. An arsenite S-adenosylmethionine methyltransferase from S. platensis (SpArsM) was identified and characterized. SpArsM showed low identity with other reported ArsM enzymes. The Escherichia coli AW3110 bearing SparsM gene resulted in As(III) methylation and conferring resistance to As(III). The in vitro assay showed that SpArsM exhibited As(III) methylation activity. DMA(V) and a small amount of MMA(V) were detected in the reaction system within 0.5hr. A truncated SpArsM derivative lacking the last 34 residues still had the ability to methylate As(III). The three single mutants of SpArsM (C59S, C186S, and C238S) abolished the capability of As(III) methylation, suggesting the three cysteine residues are involved in catalysis. We propose that SpArsM is responsible for As methylation and detoxification of As(III) and may contribute to As biogeochemistry.

  10. DNA methyltransferase activity is required for memory-related neural plasticity in the lateral amygdala.

    Science.gov (United States)

    Maddox, Stephanie A; Watts, Casey S; Schafe, Glenn E

    2014-01-01

    We have previously shown that auditory Pavlovian fear conditioning is associated with an increase in DNA methyltransferase (DNMT) expression in the lateral amygdala (LA) and that intra-LA infusion or bath application of an inhibitor of DNMT activity impairs the consolidation of an auditory fear memory and long-term potentiation (LTP) at thalamic and cortical inputs to the LA, in vitro. In the present study, we use awake behaving neurophysiological techniques to examine the role of DNMT activity in memory-related neurophysiological changes accompanying fear memory consolidation and reconsolidation in the LA, in vivo. We show that auditory fear conditioning results in a training-related enhancement in the amplitude of short-latency auditory-evoked field potentials (AEFPs) in the LA. Intra-LA infusion of a DNMT inhibitor impairs both fear memory consolidation and, in parallel, the consolidation of training-related neural plasticity in the LA; that is, short-term memory (STM) and short-term training-related increases in AEFP amplitude in the LA are intact, while long-term memory (LTM) and long-term retention of training-related increases in AEFP amplitudes are impaired. In separate experiments, we show that intra-LA infusion of a DNMT inhibitor following retrieval of an auditory fear memory has no effect on post-retrieval STM or short-term retention of training-related changes in AEFP amplitude in the LA, but significantly impairs both post-retrieval LTM and long-term retention of AEFP amplitude changes in the LA. These findings are the first to demonstrate the necessity of DNMT activity in the consolidation and reconsolidation of memory-associated neural plasticity, in vivo.

  11. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

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    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2

  12. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

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    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA-protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 [Formula: see text]M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly

  13. DNA methyltransferase 1 mutations and mitochondrial pathology: is mtDNA methylated?

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    Alessandra eMaresca

    2015-03-01

    Full Text Available Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5-methyltransferase 1. DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is

  14. O-6-methylguanine-deoxyribonucleic acid methyltransferase methylation enhances response to temozolomide treatment in esophageal cancer

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    Rifat Hasina

    2013-01-01

    Full Text Available Background: World-wide, esophageal cancer is a growing epidemic and patients frequently present with advanced disease that is surgically inoperable. Hence, chemotherapy is the predominate treatment. Cytotoxic platinum compounds are mostly used, but their efficacy is only moderate. Newer alkylating agents have shown promise in other tumor types, but little is known about their utility in esophageal cancer. Methods: We utilized archived human esophageal cancer samples and esophageal cancer cell lines to evaluate O-6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT hypermethylation status and determined sensitivity to the alkylating drug temozolomide (TMZ. Immunoblot analysis was performed to determine MGMT protein expression in cell lines. To assess and confirm the effect of TMZ treatment in a methylated esophageal cancer cell line in vivo, a mouse flank xenograft tumor model was utilized. Results: Nearly 71% (12/17 of adenocarcinoma and 38% (3/8 of squamous cell carcinoma (SCC patient samples were MGMT hypermethylated. Out of four adenocarcinoma and nine SCC cell lines tested, one of each histology was hypermethylated. Immunoblot analyses confirmed that hypermethylated cell lines did not express the MGMT protein. In vitro cell viability assays showed the methylated Kyse-140 and FLO cells to be sensitive to TMZ at an IC 50 of 52-420 μM, whereas unmethylated cells Kyse-410 and SKGT-4 did not respond. In an in vivo xenograft tumor model with Kyse-140 cells, which are MGMT hypermethylated, TMZ treatment abrogated tumor growth by more than 60%. Conclusion: MGMT methylation may be an important biomarker in subsets of esophageal cancers and targeting by TMZ may be utilized to successfully treat these patients.

  15. The Role of Catechol-O-Methyltransferase (COMT Gene in the Etiopathogenesis of Schizophrenia

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    Ceren Acar

    2014-09-01

    Full Text Available Genetic factors in the risk of developing schizophrenia is of great importance. With the help of the advances in the field of genetics in recent years by using linkage analysis several genes have been identified that may be a risk factor in schizophrenia. Several association studies have been performed in many different populations on the candidate susceptibility genes that were defined in previous studies. However, these studies give controversial results in different countries with different populations, and there are problems in obtaining replicable results. In this review we aimed to focus on the genetic basis of schizophrenia and the relationship between schizophrenia and catechol-O-methyltransferase (COMT gene. COMT encodes an enzyme molecule which has an important function in dopamine pathways. It has great importance in catecholamine metabolism and pharmacology and genetic mechanism of catechol metabolism variations and their clinical consequences. COMT transfers the methyl group from S-adenosyl-methionine to the hydroxyl group of catechol nucleus (such as dopamine, norepinephrine or catechol estrogen. Genetic variations found in COMT gene are associated with a broad spectrum of clinical phenotype including psychiatric disorders or estrogen related cancers. Several groups have performed studies on the relationship between schizophrenia and COMT. The most commonly studied polymorphism in COMT gene is rs4680 and it causes a valine methionine conversion at codon 158. The association studies on this polymorphism in different populations gave both positive and negative results. Schizoprenia is a complex disease caused by the interaction of environmental and genetic factors, while interpreting the genetic data, this fact and the possibility of the presence of different gene products should be taken into account. [Psikiyatride Guncel Yaklasimlar - Current Approaches in Psychiatry 2014; 6(3.000: 217-226

  16. Deletion of gene encoding methyltransferase (gidB) confers high-level antimicrobial resistance in Salmonella.

    Science.gov (United States)

    Mikheil, Dareen M; Shippy, Daniel C; Eakley, Nicholas M; Okwumabua, Ogi E; Fadl, Amin A

    2012-04-01

    The glucose-inhibited division gene (gid)B, which resides in the gid operon, was thought to have a role in the modulation of genes similar to that of gidA. Recent studies have indicated that GidB is a methyltransferase enzyme that is involved in the methylation of the 16S ribosomal RNA (rRNA) in Escherichia coli. In this study, we investigated the role of GidB in susceptibility to antibiotics and the overall biology of Salmonella. A gidB isogenic mutant of Salmonella was constructed and subsequently characterized under different conditions. Our data indicated that growth and invasion characteristics of the gidB mutant were similar to those of the wild type (WT). The gidB mutant was outgrown by the WT in a competitive growth assay, indicating a compromised overall bacterial fitness. Under the stress of nalidixic acid, the gidB mutant's motility was significantly reduced. Similarly, the mutant showed a filamentous morphology and smaller colony size compared with the rod-shaped and large colonies of the WT in the presence of nalidixic acid. Most importantly, deletion of gidB conferred high-level resistance to the aminoglycoside antibiotics streptomycin and neomycin. A primer extension assay determined the methylation site for the WT to be at G527 of the 16S rRNA. A lack of methylation in the mutant indicated that GidB is required for this methylation. Taken together, these data indicate that the GidB enzyme has a significant role in the alteration of antibiotic susceptibility and the modulation of growth and morphology under stress conditions in Salmonella.

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

    Science.gov (United States)

    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.

  18. Common Polymorphism’s Analysis of Thiopurine S-Methyltransferase (TPMT in Iranian Population

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    Mehdi Azad

    2009-01-01

    Full Text Available Objective: Thiopurine S-methyltransferase (TPMT catalyses the S-methylation of thiopurinedrugs. Low activity phenotypes are correlated with several mutations in the TPMTgene and adverse drug reactions. The molecular basis for dissimilar enzymatic activityof TPMT has been established in Caucasians, African-Americans and Southwest Asians,but it remains to be elucidated in Iranian population. Until present, no study on Iranianpopulation has been performed on the known alleles of TPMT. The aim of this study wasto investigate the frequencies of four of the most common variants of this gene.Materials and Methods: This study was conducted during 2007 at the Department of Hematology,Tarbiat Modares University, Tehran, Iran. Using PCR-RFLP and allele specificPCR techniques, allelic variants of the TPMT gene TPMT*2(G238C, TPMT*3B (G460A,TPMT*3C (A719G and TPMT*3A (G460A and A719G were genotyped in a normal populationof 127 Iranians.Results: In this study TPMT*2 showed a prevalence of 7.08%. TPMT*3C and *3A werefound in 2.47% and 2.18% of the samples, respectively. TPMT*3B variant was not detectedin Iranian subjects. 112 out of 127 participants showed homozygote wild type allele.Conclusion: This study is the first to analyze TPMT allele frequencies in a sample ofIranian population and indicates that TPMT*2 is the most common allele (7.08% in thispopulation. These results can help to organize national pretreatment strategies in patientswith acute lympho blastic leukemia (ALL or other diseases requiring thiopurine medicationin their standard therapy.

  19. Atomic Insight into the Altered O6-Methylguanine-DNA Methyltransferase Protein Architecture in Gastric Cancer.

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    Naveed Anjum Chikan

    Full Text Available O6-methylguanine-DNA methyltransferase (MGMT is one of the major DNA repair protein that counteracts the alkalyting agent-induced DNA damage by replacing O6-methylguanine (mutagenic lesion back to guanine, eventually suppressing the mismatch errors and double strand crosslinks. Exonic alterations in the form of nucleotide polymorphism may result in altered protein structure that in turn can lead to the loss of function. In the present study, we focused on the population feared for high exposure to alkylating agents owing to their typical and specialized dietary habits. To this end, gastric cancer patients pooled out from the population were selected for the mutational screening of a specific error prone region of MGMT gene. We found that nearly 40% of the studied neoplastic samples harbored missense mutation at codon151 resulting into Serine to Isoleucine variation. This variation resulted in bringing about the structural disorder, subsequently ensuing into a major stoichiometric variance in recognition domain, substrate binding and selectivity loop of the active site of the MGMT protein, as observed under virtual microscope of molecular dynamics simulation (MDS. The atomic insight into MGMT protein by computational approach showed a significant change in the intra molecular hydrogen bond pattern, thus leading to the observed structural anomalies. To further examine the mutational implications on regulatory plugs of MGMT that holds the protein in a DNA-Binding position, a MDS based analysis was carried out on, all known physically interacting amino acids essentially clustered into groups based on their position and function. The results generated by physical-functional clustering of protein indicated that the identified mutation in the vicinity of the active site of MGMT protein causes the local and global destabilization of a protein by either eliminating the stabilizing salt bridges in cluster C3, C4, and C5 or by locally destabilizing the

  20. Cloning, functional characterization and catalytic mechanism of a bergaptol O-methyltransferase from Peucedanum praeruptorum Dunn

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    Yucheng eZhao

    2016-05-01

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

  1. An Unusual Co(I)-H Interaction: Structural and Mechanistic Ramifications for Methyltransferases

    Science.gov (United States)

    Kumar, Manoj

    2014-03-01

    The cob(II)alamin cob(I)alamin (Co2+/Co1+) reduction is a common chemical event in a broad family of cytoplasmic methyltransferases and ATP:corrinoid adenosyltransferases, respectively. Despite its broad and general chemical appeal, the Co2+/Co1+ reduction continues to remain one of the least understood aspects of corrinoid chemistry. This is due in part to the inaccessible redox chemistry of Co2+/Co1+ couple under cellular conditions i.e., the reduction potential of cob(II)alamin (-500 mV vs SHE) is more negative than that of the common physiological reductants (-280 mV to -440 mV vs SHE) present in the cellular environments. To gain better understanding about the Co2+/Co1+ reduction, we have utilized the density functional theory and quantum mechanics/molecular mechanics (QM/MM) computational methods. The calculations indicate that cob(I)alamin, a ubiquitous B12 intermediate, is not square planar as has been commonly accepted, but a square pyramidal species due to the unusual hydrogen bonding interaction between the Co1+ ion and its axial ligands (Angew. Chem. Int. Ed. 2011, 50, 8702-8705; Inorg. Chem.2012, 51, 5533-5538). The Co1+-H interaction exerts an anodic shift of 100 mV vs SHE upon the reduction potential of the Co2+/Co1+ couple, which explains why this redox process is observed inside transferases. Building upon these new insights, an alternate mechanism for the enzyme-bound Co2+/Co1+ redox process is suggested that is mediated by the square pyramidal cob(I)alamin rather than its commonly accepted square planar analogue.

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

    Science.gov (United States)

    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.

  3. Visualizing molecular juggling within a B[subscript 12]-dependent methyltransferase complex

    Energy Technology Data Exchange (ETDEWEB)

    Kung, Yan; Ando, Nozomi; Doukov, Tzanko I.; Blasiak, Leah C.; Bender, Güne; #351; ; Seravalli, Javier; Ragsdale, Stephen W.; Drennan, Catherine L. (MIT); (Michigan); (UNL)

    2013-04-08

    Derivatives of vitamin B{sub 12} are used in methyl group transfer in biological processes as diverse as methionine synthesis in humans and CO{sub 2} fixation in acetogenic bacteria. This seemingly straightforward reaction requires large, multimodular enzyme complexes that adopt multiple conformations to alternately activate, protect and perform catalysis on the reactive B{sub 12} cofactor. Crystal structures determined thus far have provided structural information for only fragments of these complexes, inspiring speculation about the overall protein assembly and conformational movements inherent to activity. Here we present X-ray crystal structures of a complete 220 kDa complex that contains all enzymes responsible for B{sub 12}-dependent methyl transfer, namely the corrinoid iron-sulphur protein and its methyltransferase from the model acetogen Moorella thermoacetica. These structures provide the first three-dimensional depiction of all protein modules required for the activation, protection and catalytic steps of B{sub 12}-dependent methyl transfer. In addition, the structures capture B{sub 12} at multiple locations between its 'resting' and catalytic positions, allowing visualization of the dramatic protein rearrangements that enable methyl transfer and identification of the trajectory for B{sub 12} movement within the large enzyme scaffold. The structures are also presented alongside in crystallo spectroscopic data, which confirm enzymatic activity within crystals and demonstrate the largest known conformational movements of proteins in a crystalline state. Taken together, this work provides a model for the molecular juggling that accompanies turnover and helps explain why such an elaborate protein framework is required for such a simple, yet biologically essential reaction.

  4. Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

  5. DNA methyltransferase DNMT3b protein overexpression as a prognostic factor in patients with diffuse large B-cell lymphomas.

    Science.gov (United States)

    Amara, Khaled; Ziadi, Sonia; Hachana, Mohamed; Soltani, Nabil; Korbi, Sadok; Trimeche, Mounir

    2010-07-01

    Diffuse large B-cell lymphomas (DLBCL) are the most common type of aggressive lymphomas, with considerable heterogeneity in clinical presentation, molecular characteristics, and outcome. Previous studies have showed significant correlations between DNA methyltransferase (DNMT) overexpression and unfavorable prognosis in human cancers. Therefore, we investigated in this study the biological and prognostic significance of DNMT1, DNMT3a, and DNMT3b protein expression in DLBCL. DNA methyltransferase (DNMT) expression was analyzed by immunohistochemistry in 81 DLBCL cases and correlated with clinicopathological parameters. Kaplan-Meier curves were used to estimate survival rates, and the Cox proportional hazard regression model was used to evaluate the prognostic impact of DNMT expression. Our results showed that overexpression of DNMT1, DNMT3a, and DNMT3b were detected in 48%, 13%, and 45% of investigated cases, respectively. DNA methyltransferase 1 (DNMT1) and DNMT3b overexpression was significantly correlated with advanced clinical stages (P = 0.028 and P = 0.016, respectively). Moreover, concomitant expression of DNMT1 and DNMT3b was significantly correlated with resistance to treatment (P = 0.015). With regard to survival rates, although data was available only for 40 patients, DNMT3b overexpression was significantly correlated with shorter overall survival (P = 0.006) and progression-free survival (P = 0.016). Interestingly, multivariate analysis demonstrated that DNMT3b overexpression was an independent prognostic factor for predicting shortened overall survival (P = 0.004) and progression-free survival (P = 0.024). In conclusion, DNMT3b overexpression was identified as an independent prognostic factor for predicting shortened survival of patients with DLBCL and could be, therefore, useful in identifying patients who would benefit from aggressive therapy.

  6. Isolation of DNA-methyltransferase genes from strawberry (Fragaria x ananassa Duch.) and their expression in relation to micropropagation.

    Science.gov (United States)

    Chang, Linlin; Zhang, Zhihong; Han, Baiming; Li, He; Dai, Hongyan; He, Ping; Tian, Hongzhe

    2009-09-01

    DNA methylation can control gene expression and may also play a role in plant development. Methylation of cytosine residues in DNA is enzymatically catalyzed by DNA methyltransferases. In this study, full-length genomic genes and cDNAs of methyltransferase (MET1) and domain-rearranged methyltransferase (DRM) were isolated from strawberry (Fragaria x ananassa Duch.). Two genomic clones (FaMET1a and FaMET1b) encoding MET1 had open-reading frame of 4,695 and 4,671 nucleotides with two introns, respectively. Amino acid sequence comparison indicated high similarity (98.72% identity) of strawberry MET1 protein to other plant MET1 sequences. The full-length cDNA of strawberry DRM genes (FaDRMa, FaDRMb and FaDRMc) were 2,273, 2,282 and 2,288 bp, respectively. Ten introns with different sizes were dispersed in FaDRM genes. Similarly, FaDRMa, FaDRMb and FaDRMc had high-sequence similarity overall. Expressions of strawberry MET1 and DRM genes were compared among in vitro-micropropagated plants, generations of micropropagated plants and conventionally propagated plants. The transcriptional expressions of both FaMET1 and FaDRM genes were downregulated in micropropagated plants, and they were recovered in the first and second runner generations of micropropagated plants. However, there was a slighter difference in global DNA methylation rates between micropropagated plants and conventionally propagated plants. Therefore, there was no positive relation between global DNA methylation rates and the expression levels of MET1 and DRM genes.

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

    Science.gov (United States)

    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. O 6 -methylguanine DNA methyltransferase gene promoter methylation in high-grade gliomas: A review of current status

    Directory of Open Access Journals (Sweden)

    Vaishali Suri

    2011-01-01

    Full Text Available Assessment of promoter methylation of the O 6 -methylguanine DNA methyltransferase (MGMT gene has recently gained importance in molecular profiling of high-grade gliomas. It has emerged not only as an important prognostic marker but also as a predictive marker for response to temozolomide in patients with newly diagnosed glioblastoma. Further, recent studies indicate that MGMT promoter methylation has strong prognostic relevance even in anaplastic (grade III gliomas, irrespective of therapy (chemotherapy or radiotherapy. This article provides an overview of its use as a predictive and prognostic biomarker, as well as the methods employed for its assessment and use in therapeutic decision making.

  9. De novo DNA methyltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells

    OpenAIRE

    Tadokoro, Yuko; Ema, Hideo; Okano, Masaki; Li, En; Nakauchi, Hiromitsu

    2007-01-01

    DNA methylation is an epigenetic modification essential for development. The DNA methyltransferases Dnmt3a and Dnmt3b execute de novo DNA methylation in gastrulating embryos and differentiating germline cells. It has been assumed that these enzymes generally play a role in regulating cell differentiation. To test this hypothesis, we examined the role of Dnmt3a and Dnmt3b in adult stem cells. CD34−/low, c-Kit+, Sca-1+, lineage marker− (CD34− KSL) cells, a fraction of mouse bone marrow cells hi...

  10. Knockout of arsenic (+3 oxidation state) methyltransferase results in sex-dependent changes in phosphatidylcholine metabolism in mice.

    Science.gov (United States)

    Huang, Madelyn C; Douillet, Christelle C; Stýblo, Miroslav

    2016-12-01

    Arsenic (+3 oxidation state) methyltransferase is the key enzyme in the methylation pathway for inorganic arsenic. We have recently shown that As3mt knockout (KO) has a profound effect on metabolomic profiles in mice. Phosphatidylcholine species (PCs) were the largest group of metabolites altered in both plasma and urine. The present study used targeted analysis to investigate the KO-associated changes in PC profiles in the liver, the site of PC synthesis. Results show that As3mt KO has a systemic effect on PC metabolism and that this effect is sex dependent.

  11. P16-specific DNA methylation by engineered zinc finger methyltransferase inactivates gene transcription and promotes cancer metastasis

    OpenAIRE

    Cui, Chenghua; Gan, Ying; Gu, Liankun; Wilson, James; Liu, Zhaojun; Zhang, Baozhen; Deng, Dajun

    2015-01-01

    Background P16 DNA methylation is well known to be the most frequent event in cancer development. It has been reported that genetic inactivation of P16 drives cancer growth and metastasis, however, whether P16 DNA methylation is truly a driver in cancer metastasis remains unknown. Results A P16-specific DNA methyltransferase (P16-dnmt) expression vector is designed using a P16 promoter-specific engineered zinc finger protein fused with the catalytic domain of dnmt3a. P16-dnmt transfection sig...

  12. Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria

    DEFF Research Database (Denmark)

    Giessing, Anders; Jensen, Søren Skov; Rasmussen, Anette

    2009-01-01

    The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding......,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance...

  13. Noncompetitive inhibition of indolethylamine-N-methyltransferase by N,N-dimethyltryptamine and N,N-dimethylaminopropyltryptamine.

    Science.gov (United States)

    Chu, Uyen B; Vorperian, Sevahn K; Satyshur, Kenneth; Eickstaedt, Kelsey; Cozzi, Nicholas V; Mavlyutov, Timur; Hajipour, Abdol R; Ruoho, Arnold E

    2014-05-13

    Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis-Menten and Lineweaver-Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N',N'-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 μM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14 ) identified an N-terminal helix-loop-helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were -6.34 and -7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.

  14. Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

    DEFF Research Database (Denmark)

    Karminska, K. H.; Purta, E.; Hansen, L .H.

    2010-01-01

    The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase...... of a 4Fe-4S cluster, a SAM molecule coordinated to the iron-sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis...

  15. Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants.

    Science.gov (United States)

    Jeltsch, A; Sobotta, T; Pingoud, A

    1996-05-01

    The EcoRV DNA methyltransferase (M.EcoRV) is an alpha-adenine methyltransferase. We have used two different programs to predict the secondary structure of M.EcoRV. The resulting consensus prediction was tested by a mutant profiling analysis. 29 neutral mutations of M.EcoRV were generated by five cycles of random mutagenesis and selection for active variants to increase the reliability of the prediction and to get a secondary structure prediction for some ambiguously predicted regions. The predicted consensus secondary structure elements could be aligned to the common topology of the structures of the catalytic domains of M.HhaI and M.TaqI. In a complementary approach we have isolated nine catalytically inactive single mutants. Five of these mutants contain an amino acid exchange within the catalytic domain of M.EcoRV (Val2-Ala, Lys81Arg, Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant binds DNA similarly to wild-type M.EcoRV, but is catalytically inactive. Hence this mutant behaves like a bona fide active site mutant. According to the structure prediction, Trp231 is located in a loop at the putative active site of M.EcoRV. The other inactive mutants were insoluble. They contain amino acid exchanges within the conserved amino acid motifs X, III or IV in M.EcoRV confirming the importance of these regions.

  16. Molecular cloning and functional characterization of a novel isoflavone 3′-O-methyltransferase from Pueraria lobata

    Directory of Open Access Journals (Sweden)

    Jia eLi

    2016-06-01

    Full Text Available Pueraria lobata roots accumulate 3′-, 4′- and 7-O-methylated isoflavones and many of these methylated compounds exhibit various pharmacological activities. Either the 4′- or 7-O-methylation activity has been investigated at molecular levels in several legume species. However, the gene encoding the isoflavone 3′-O-methyltransferase has not yet been isolated from any plant species. In this study, we reported the first cDNA encoding the isoflavone 3′-O-methyltransferase from P. lobata (designated PlOMT4. Heterologous expressions in yeast and Escherichia coli cells showed that the gene product exhibits an enzyme activity to methylate the 3′-hydroxy group of the isoflavone substrate. The transcript abundance of PlOMT4 matches well with its enzymatic product in different organs of P. lobata and in the plant roots in response to methyl jasmonate elicitation. Integration of the biochemical with metabolic and transcript data supported the proposed function of PlOMT4. The identification of PlOMT4 would not only help to understand the isoflavonoid metabolism in P. lobata but also potentially provide an enzyme catalyst for methylating existing drug candidates to improve their hydrophobicity.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. Unusual pseudosubstrate specificity of a novel 3,5-dimethoxyphenol O-methyltransferase cloned from Ruta graveolens L.

    Science.gov (United States)

    Burga, Laura; Wellmann, Frank; Lukacin, Richard; Witte, Simone; Schwab, Wilfried; Schröder, Joachim; Matern, Ulrich

    2005-08-01

    A cDNA was cloned from Ruta graveolens cells encoding a novel O-methyltransferase (OMT) with high similarity to orcinol or chavicol/eugenol OMTs, but containing a serine-rich N-terminus and a 13 amino acid insertion between motifs IV and V. Expression in Escherichia coli revealed S-adenosyl-l-methionine-dependent OMT activity with methoxylated phenols only with an apparent Km of 20.4 for the prime substrate 3,5-dimethoxyphenol. The enzyme forms a homodimer of 84 kDa, and the activity was insignificantly affected by 2.0 mM Ca2+ or Mg2+, whereas Fe2+, Co2+, Zn2+, Cu2+ or Hg2+ were inhibitory (78-100%). Dithiothreitol (DTT) suppressed the OMT activity. This effect was examined further, and, in the presence of Zn2+ as a potential thiol methyltransferase (TMT) cofactor, the recombinant OMT methylated DTT to DTT-monomethylthioether. Sets of kinetic OMT experiments with 3,5-dimethoxyphenol at various Zn2+/DTT concentrations revealed the competitive binding of DTT with an apparent Ki of 52.0 microM. Thus, the OMT exhibited TMT activity with almost equivalent affinity to the thiol pseudosubstrate which is structurally unrelated to methoxyphenols.

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

    Science.gov (United States)

    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.

  1. A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading

    Energy Technology Data Exchange (ETDEWEB)

    Shan, Chun-Min; Wang, Jiyong; Xu, Ke; Chen, Huijie; Yue, Jia-Xing; Andrews, Stuart; Moresco, James J.; Yates, John R.; Nagy, Peter L.; Tong, Liang; Jia, Songtao

    2016-09-20

    Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of large heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.

  2. The purification, crystallization and preliminary structural characterization of PhzM, a phenazine-modifying methyltransferase from Pseudomonas aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Gohain, Neelakshi [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany); Thomashow, Linda S. [USDA Agricultural Research Service, Root Disease and Biological Control Research Unit, Pullman, Washington 99164-6430 (United States); Mavrodi, Dmitri V. [Department of Plant Pathology, Washington State University, Pullman, Washington 99164-6430 (United States); Blankenfeldt, Wulf, E-mail: wulf.blankenfeldt@mpi-dortmund.mpg.de [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)

    2006-09-01

    PhzM, an S-adenosylmethionine-dependent methyltransferase enzyme that catalyzes a reaction involved in the biosynthesis of pyocyanin in P. aeruginosa, was cloned, overexpressed and crystallized. Data collection from native and selenomethionine-labelled crystals is reported. Pyocyanin, phenazine-1-carboxylic acid and more than 70 related compounds collectively known as phenazines are produced by various species of Pseudomonas, including the fluorescent pseudomonad P. aeruginosa, a Gram-negative opportunistic pathogen in humans and animals. P. aeruginosa synthesizes a characteristic blue water-soluble compound called pyocyanin (1-hydroxy-5-methyl-phenazine). Two enzymes designated PhzM and PhzS are involved in the terminal steps of its synthesis and very little is known about these enzymes. In this study, PhzM, a dimeric S-adenosylmethionine-dependent methyltransferase, was purified and crystallized from PEG 3350/sodium cacodylate/sodium citrate pH 6.5. The crystals belong to space group P1, with unit-cell parameters a = 46.1, b = 61.8, c = 69.6 Å, α = 96.3, β = 106.6, γ = 106.9°. They contain one dimer in the asymmetric unit and diffract to a resolution of 1.8 Å. Anomalous data to 2.3 Å resolution have been collected from seleno-l-methionine-labelled PhzM.

  3. Two novel temperate bacteriophages co-existing in Aeromonas sp. ARM81 - characterization of their genomes, proteomes and DNA methyltransferases.

    Science.gov (United States)

    Dziewit, Lukasz; Radlinska, Monika

    2016-08-01

    Aeromonas species are causative agents of a wide spectrum of diseases in animals and humans. Although these bacteria are commonly found in various environments, little is known about their phages. Thus far, only one temperate Aeromonas phage has been characterized. Whole-genome sequencing of an Aeromonas sp. strain ARM81 revealed the presence of two prophage clusters. One of them is integrated into the chromosome and the other was maintained as an extrachromosomal, linear plasmid-like prophage encoding a protelomerase. Both prophages were artificially and spontaneously inducible. We separately isolated both phages and compared their genomes with other known viruses. The novel phages show no similarity to the previously characterized Aeromonas phages and might represent new evolutionary lineages of viruses infecting Aeromonadaceae. Apart from the comparative genomic analyses of these phages, complemented with their structural and molecular characterization, a functional analysis of four DNA methyltransferases encoded by these viruses was conducted. One of the investigated N6-adenine-modifying enzymes shares sequence specificity with a Dam-like methyltransferase of its bacterial host, while another one is non-specific, as it catalyzes adenine methylation in various sequence contexts. The presented results shed new light on the diversity of Aeromonas temperate phages.

  4. The inhibition of the mammalian DNA methyltransferase 3a (Dnmt3a by dietary black tea and coffee polyphenols

    Directory of Open Access Journals (Sweden)

    Jeltsch Albert

    2011-04-01

    Full Text Available Abstract Background Black tea is, second only to water, the most consumed beverage globally. Previously, the inhibition of DNA methyltransferase 1 was shown by dietary polyphenols and epi-gallocatechin gallate (EGCG, the main polyphenolic constituent of green tea, and 5-caffeoyl quinic acid, the main phenolic constituent of the green coffee bean. Results We studied the inhibition of DNA methyltransferase 3a by a series of dietary polyphenols from black tea such as theaflavins and thearubigins and chlorogenic acid derivatives from coffee. For theaflavin 3,3 digallate and thearubigins IC50 values in the lower micro molar range were observed, which when compared to pharmacokinetic data available, suggest an effect of physiological relevance. Conclusions Since Dnnmt3a has been associated with development, cancer and brain function, these data suggest a biochemical mechanism for the beneficial health effect of black tea and coffee and a possible molecular mechanism for the improvement of brain performance and mental health by dietary polyphenols.

  5. Epigenetic Guardian: A Review of the DNA Methyltransferase DNMT3A in Acute Myeloid Leukaemia and Clonal Haematopoiesis

    Science.gov (United States)

    Chaudry, Sabah F.

    2017-01-01

    Acute myeloid leukaemia (AML) is a haematological malignancy characterized by clonal stem cell proliferation and aberrant block in differentiation. Dysfunction of epigenetic modifiers contributes significantly to the pathogenesis of AML. One frequently mutated gene involved in epigenetic modification is DNMT3A (DNA methyltransferase-3-alpha), a DNA methyltransferase that alters gene expression by de novo methylation of cytosine bases at CpG dinucleotides. Approximately 22% of AML and 36% of cytogenetically normal AML cases carry DNMT3A mutations and around 60% of these mutations affect the R882 codon. These mutations have been associated with poor prognosis and adverse survival outcomes for AML patients. Advances in whole-exome sequencing techniques have recently identified a large number of DNMT3A mutations present in clonal cells in normal elderly individuals with no features of haematological malignancy. Categorically distinct from other preleukaemic conditions, this disorder has been termed clonal haematopoiesis of indeterminate potential (CHIP). Further insight into the mutational landscape of CHIP may illustrate the consequence of particular mutations found in DNMT3A and identify specific “founder” mutations responsible for clonal expansion that may contribute to leukaemogenesis. This review will focus on current research and understanding of DNMT3A mutations in both AML and CHIP. PMID:28286768

  6. Reprogramming metabolism by histone methyltransferase NSD2 drives endocrine resistance via coordinated activation of pentose phosphate pathway enzymes.

    Science.gov (United States)

    Wang, Junjian; Duan, Zhijian; Nugent, Zoann; Zou, June X; Borowsky, Alexander D; Zhang, Yanhong; Tepper, Clifford G; Li, Jian Jian; Fiehn, Oliver; Xu, Jianzhen; Kung, Hsing-Jien; Murphy, Leigh C; Chen, Hong-Wu

    2016-08-10

    Metabolic reprogramming such as the aerobic glycolysis or Warburg effect is well recognized as a common feature of tumorigenesis. However, molecular mechanisms underlying metabolic alterations for tumor therapeutic resistance are poorly understood. Through gene expression profiling analysis we found that histone H3K36 methyltransferase NSD2/MMSET/WHSC1 expression was highly elevated in tamoxifen-resistant breast cancer cell lines and clinical tumors. IHC analysis indicated that NSD2 protein overexpression was associated with the disease recurrence and poor survival. Ectopic expression of NSD2 wild type, but not the methylase-defective mutant, drove endocrine resistance in multiple cell models and xenograft tumors. Mechanistically, NSD2 was recruited to and methylated H3K36me2 at the promoters of key glucose metabolic enzyme genes. Its overexpression coordinately up-regulated hexokinase 2 (HK2) and glucose-6-phosphate dehydrogenase (G6PD), two key enzymes of glycolysis and the pentose phosphate pathway (PPP), as well as TP53-induced glycolysis regulatory phosphatase TIGAR. Consequently, NSD2-driven tamoxifen-resistant cells and tumors displayed heightened PPP activity, elevated NADPH production, and reduced ROS level, without significantly altered glycolysis. These results illustrate a coordinated, epigenetic activation of key glucose metabolic enzymes in therapeutic resistance and nominate methyltransferase NSD2 as a potential therapeutic target for endocrine resistant breast cancer.

  7. A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-α

    Science.gov (United States)

    Begley, Ulrike; Sosa, Maria Soledad; Avivar-Valderas, Alvaro; Patil, Ashish; Endres, Lauren; Estrada, Yeriel; Chan, Clement TY; Su, Dan; Dedon, Peter C; Aguirre-Ghiso, Julio A; Begley, Thomas

    2013-01-01

    Emerging evidence points to aberrant regulation of translation as a driver of cell transformation in cancer. Given the direct control of translation by tRNA modifications, tRNA modifying enzymes may function as regulators of cancer progression. Here, we show that a tRNA methyltransferase 9-like (hTRM9L/KIAA1456) mRNA is down-regulated in breast, bladder, colorectal, cervix and testicular carcinomas. In the aggressive SW620 and HCT116 colon carcinoma cell lines, hTRM9L is silenced and its re-expression and methyltransferase activity dramatically suppressed tumour growth in vivo. This growth inhibition was linked to decreased proliferation, senescence-like G0/G1-arrest and up-regulation of the RB interacting protein LIN9. Additionally, SW620 cells re-expressing hTRM9L did not respond to hypoxia via HIF1-α-dependent induction of GLUT1. Importantly, hTRM9L-negative tumours were highly sensitive to aminoglycoside antibiotics and this was associated with altered tRNA modification levels compared to antibiotic resistant hTRM9L-expressing SW620 cells. Our study links hTRM9L and tRNA modifications to inhibition of tumour growth via LIN9 and HIF1-α-dependent mechanisms. It also suggests that aminoglycoside antibiotics may be useful to treat hTRM9L-deficient tumours. PMID:23381944

  8. Crystal structure of Bacillus subtilis TrmB, the tRNA (m7G46) methyltransferase.

    Science.gov (United States)

    Zegers, Ingrid; Gigot, Daniel; van Vliet, Françoise; Tricot, Catherine; Aymerich, Stéphane; Bujnicki, Janusz M; Kosinski, Jan; Droogmans, Louis

    2006-01-01

    The structure of Bacillus subtilis TrmB (BsTrmB), the tRNA (m7G46) methyltransferase, was determined at a resolution of 2.1 A. This is the first structure of a member of the TrmB family to be determined by X-ray crystallography. It reveals a unique variant of the Rossmann-fold methyltransferase (RFM) structure, with the N-terminal helix folded on the opposite site of the catalytic domain. The architecture of the active site and a computational docking model of BsTrmB in complex with the methyl group donor S-adenosyl-L-methionine and the tRNA substrate provide an explanation for results from mutagenesis studies of an orthologous enzyme from Escherichia coli (EcTrmB). However, unlike EcTrmB, BsTrmB is shown here to be dimeric both in the crystal and in solution. The dimer interface has a hydrophobic core and buries a potassium ion and five water molecules. The evolutionary analysis of the putative interface residues in the TrmB family suggests that homodimerization may be a specific feature of TrmBs from Bacilli, which may represent an early stage of evolution to an obligatory dimer.

  9. The hemK gene in Escherichia coli encodes the N5-glutamine methyltransferase that modifies peptide release factors

    Science.gov (United States)

    Heurgué-Hamard, Valérie; Champ, Stéphanie; Engström, Åke; Ehrenberg, Måns; Buckingham, Richard H.

    2002-01-01

    Class 1 peptide release factors (RFs) in Escherichia coli are N5-methylated on the glutamine residue of the universally conserved GGQ motif. One other protein alone has been shown to contain N5-methylglutamine: E.coli ribosomal protein L3. We identify the L3 methyltransferase as YfcB and show that it methylates ribosomes from a yfcB strain in vitro, but not RF1 or RF2. HemK, a close orthologue of YfcB, is shown to methylate RF1 and RF2 in vitro. hemK is immediately downstream of and co-expressed with prfA. Its deletion in E.coli K12 leads to very poor growth on rich media and abolishes methylation of RF1. The activity of unmethylated RF2 from K12 strains is extremely low due to the cumulative effects of threonine at position 246, in place of alanine or serine present in all other bacterial RFs, and the lack of N5-methylation of Gln252. Fast-growing spontaneous revertants in hemK K12 strains contain the mutations Thr246Ala or Thr246Ser in RF2. HemK and YfcB are the first identified methyltransferases modifying glutamine, and are widely distributed in nature. PMID:11847124

  10. DNA methyltransferase inhibitors improve the effect of chemotherapeutic agents in SW48 and HT-29 colorectal cancer cells.

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    Sylwia Flis

    Full Text Available DNA methylation is an epigenetic phenomenon known to play an important role in the development and progression of human cancer. Enzyme responsible for this process is DNA methyltransferase 1 (DNMT1 that maintains an altered methylation pattern by copying it from parent to daughter DNA strands after replication. Aberrant methylation of the promoter regions of genes critical for normal cellular functions is potentially reversible. Therefore, inactivation of DNMT1 seems to be a valuable target for the development of cancer therapies. Currently, the most popular DNMT inhibitors (DNMTi are cytidine analogues like 5-azacytidine, 5-aza-2'-deoxycytidine (decitabine and pyrimidin-2-one ribonucleoside (zebularine. In colorectal cancer, epigenetic modifications play an essential role at each step of carcinogenesis. Therefore, we have addressed the hypothesis that DNA methyltransferase inhibitors may potentiate inhibitory effects of classical chemotherapeutic agents, such as oxaliplatin and 5-fluorouracil (5-FU, commonly used in colorectal cancer therapy. Here, our report shows that DNMTi can have positive interactions with standard chemotherapeutics in colorectal cancer treatment. Using pharmacological models for the drug-drug interaction analysis, we have revealed that the combination of decitabine with 5-FU or oxaliplatin shows the most attractive interaction (synergism, whereas the effect of zebularine in combinations with chemotherapeutics is moderate and may be depended on genetic/epigenetic background of a cell line or secondary drug used in combination. Our results suggest that DNMTi administered in combination with standard chemotherapeutics might improve the treatment of patients with colorectal cancers.

  11. Histone H3-K9 methyltransferase ESET is essential for early development.

    Science.gov (United States)

    Dodge, Jonathan E; Kang, Yong-Kook; Beppu, Hideyuki; Lei, Hong; Li, En

    2004-03-01

    Methylation of histone H3 at lysine 9 (H3-K9) mediates heterochromatin formation by forming a binding site for HP1 and also participates in silencing gene expression at euchromatic sites. ESET, G9a, SUV39-h1, SUV39-h2, and Eu-HMTase are histone methyltransferases that catalyze H3-K9 methylation in mammalian cells. Previous studies demonstrate that the SUV39-h proteins are preferentially targeted to the pericentric heterochromatin, and mice lacking both Suv39-h genes show cytogenetic abnormalities and an increased incidence of lymphoma. G9a methylates H3-K9 in euchromatin, and G9a null embryos die at 8.5 days postcoitum (dpc). G9a null embryo stem (ES) cells show altered DNA methylation in the Prader-Willi imprinted region and ectopic expression of the Mage genes. So far, an Eu-HMTase mouse knockout has not been reported. ESET catalyzes methylation of H3-K9 and localizes mainly in euchromatin. To investigate the in vivo function of Eset, we have generated an allele that lacks the entire pre- and post-SET domains and that expresses lacZ under the endogenous regulation of the Eset gene. We found that zygotic Eset expression begins at the blastocyst stage and is ubiquitous during postimplantation mouse development, while the maternal Eset transcripts are present in oocytes and persist throughout preimplantation development. The homozygous mutations of Eset resulted in peri-implantation lethality between 3.5 and 5.5 dpc. Blastocysts null for Eset were recovered but in less than Mendelian ratios. Upon culturing, 18 of 24 Eset(-/-) blastocysts showed defective growth of the inner cell mass and, in contrast to the approximately 65% recovery of wild-type and Eset(+/-) ES cells, no Eset(-/-) ES cell lines were obtained. Global H3-K9 trimethylation and DNA methylation at IAP repeats in Eset(-/-) blastocyst outgrowths were not dramatically altered. Together, these results suggest that Eset is required for peri-implantation development and the survival of ES cells.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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

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

    Science.gov (United States)

    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

  14. In vitro anthelmintic efficacy of inhibitors of phosphoethanolamine Methyltransferases in Haemonchus contortus.

    Science.gov (United States)

    Witola, William H; Matthews, Kwame; McHugh, Mark

    2016-04-01

    The essential phosphobase methylation pathway for synthesis of phosphocholine is unique to nematodes, protozoa and plants, and thus an attractive antiparasitic molecular target. Herein, we screened compounds from the National Cancer Institute (Developmental Therapeutics Program Open Chemical Repository) for specific inhibitory activity against Haemonchus contortus phosphoethanolamine methyltransferases (HcPMT1 and HcPMT2), and tested candidate compounds for anthelmintic activity against adult and third-stage larvae of H. contortus. We identified compound NSC-641296 with IC50 values of 8.3 ± 1.1 μM and 5.1 ± 1.8 μM for inhibition of the catalytic activity of HcPMT1 alone and HcPMT1/HcPMT2 combination, respectively. Additionally we identified compound NSC-668394 with inhibitory IC50 values of 5.9 ± 0.9 μM and 2.8 ± 0.6 μM for HcPMT1 alone and HcPMT1/HcPMT2 combination, respectively. Of the two compounds, NSC-641296 depicted significant anthelmintic activity against third-stage larvae (IC50 = 15 ± 2.9 μM) and adult stages (IC50 = 7 ± 2.9 μM) of H. contortus, with optimal effective in vitro concentrations being 2-fold and 4-fold, respectively, lower than its cytotoxic IC50 (29 ± 2.1 μM) in a mammalian cell line. Additionally, we identified two compounds, NSC-158011 and NSC-323241, with low inhibitory activity against the combined activity of HcPMT1 and HcPMT2, but both compounds did not show any anthelmintic activity against H. contortus. The identification of NSC-641296 that specifically inhibits a unique biosynthetic pathway in H. contortus and has anthelmintic activity against both larval and adult stages of H. contortus, provides impetus for the development of urgently needed new efficacious anthelmintics to address the prevailing problem of anthelmintic-resistant H. contortus.

  15. Histone deacetylase inhibitors inducing human cervical cancer cell apoptosis by decreasing DNA-methyltransferase 3B

    Institute of Scientific and Technical Information of China (English)

    LIU Ning; ZHAO Li-jun; LI Xiao-ping; WANG Jian-liu; CHAI Guo-lin; WEI Li-hui

    2012-01-01

    Background Histone deacetylase (HDAC) inhibitors are a group of small chemical molecules that inhibit histone deacetylase.At cell level,HDAC inhibitors have multiple biological effects such as cell cycle arrest,apoptosis,cell differentiation and auotophagy.At molecular level,HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression.HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis.However,the mechanisms of apoptosis effect are not fully understood.TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research.In this study,we selected Trichostatin A (TSA) to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells.Methods Cervical cancer cell lines such as Hela,Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA.Crystal violent assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed to determine cell number.PARP cleavage and FITC-AnexinV were performed to determine apoptosis.DNA-methyltransferase (DNMT)1,DNMT3A and DNMT3B were determined by regular PCR,qPCR and Western Blotting.Small interfering RNA (SiRNAi) was used to knock down DNMT3B.Results HDAC inhibitors only induce cervical cancer cell apoptosis.At 1 μmol/L of TSA,86% of Hela cell and 76% of Caski went apoptosis.For normal cells,HDAC inhibitors have no cytotoxic effect at therapeutic dosage,(90.0±8.4)% of normal cell survive after treated with 1 μmol/L of TSA.We compared 1 μmol/L group with untreated control with t-test.There was no significance between 1 μmol/L group and untreated control for normal cell (P >0.05).HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell.Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis.More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B.Conclusions DNMT3B was essential to cervical cancer cell survival

  16. Human intelligence and polymorphisms in the DNA methyltransferase genes involved in epigenetic marking.

    Directory of Open Access Journals (Sweden)

    Paul Haggarty

    Full Text Available Epigenetic mechanisms have been implicated in syndromes associated with mental impairment but little is known about the role of epigenetics in determining the normal variation in human intelligence. We measured polymorphisms in four DNA methyltransferases (DNMT1, DNMT3A, DNMT3B and DNMT3L involved in epigenetic marking and related these to childhood and adult general intelligence in a population (n = 1542 consisting of two Scottish cohorts born in 1936 and residing in Lothian (n = 1075 or Aberdeen (n = 467. All subjects had taken the same test of intelligence at age 11yrs. The Lothian cohort took the test again at age 70yrs. The minor T allele of DNMT3L SNP 11330C>T (rs7354779 allele was associated with a higher standardised childhood intelligence score; greatest effect in the dominant analysis but also significant in the additive model (coefficient = 1.40(additive; 95%CI 0.22,2.59; p = 0.020 and 1.99(dominant; 95%CI 0.55,3.43; p = 0.007. The DNMT3L C allele was associated with an increased risk of being below average intelligence (OR 1.25(additive; 95%CI 1.05,1.51; p = 0.011 and OR 1.37(dominant; 95%CI 1.11,1.68; p = 0.003, and being in the lowest 40(th (p(additive = 0.009; p(dominant = 0.002 and lowest 30(th (p(additive = 0.004; p(dominant = 0.002 centiles for intelligence. After Bonferroni correction for the number variants tested the link between DNMT3L 11330C>T and childhood intelligence remained significant by linear regression and centile analysis; only the additive regression model was borderline significant. Adult intelligence was similarly linked to the DNMT3L variant but this analysis was limited by the numbers studied and nature of the test and the association was not significant after Bonferroni correction. We believe that the role of epigenetics in the normal variation in human intelligence merits further study and that this novel finding should be tested in other cohorts.

  17. Risk-Association of DNA Methyltransferases Polymorphisms with Gastric Cancer in the Southern Chinese Population

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    Yang Gao

    2012-07-01

    Full Text Available DNA hypomethylation and/or hypermethylation are presumed to be early events in carcinogenesis, and one or more DNA methyltransferases (DNMTs have been suggested to play roles in carcinogenesis of gastric cancer (GC. However, there have been no systematic studies regarding the association between DNMT gene polymorphisms and GC risk. Here, we examined the associations of 16 single nucleotide polymorphisms (SNPs from DNMT1 (rs2114724, rs2228611, rs2228612, rs8101866, rs16999593, DNMT2 (rs11695471, rs11254413, DNMT3A (rs1550117, rs11887120, rs13420827, rs13428812, rs6733301, DNMT3B (rs2424908, rs2424913, rs6087990 and DNMT3L (rs113593938 with GC in the Southern Chinese population. We assessed the associations of these 16 SNPs with GC in a case-control study that consisted of 242 GC cases and 294 controls, using the Sequenom MALDI-TOF-MS platform. Association analyses based on the χ2 test and binary logistic regression were performed to determine the odds ratio (OR and 95% confidence interval (95%CI for each SNP. We found that rs16999593 in DNMT1, rs11254413 in DNMT2 and rs13420827 in DNMT3A were significantly associated with GC susceptibility (OR 1.45, 0.15, 0.66, respectively; 95% CI 1.00–2.11, p = 0.047; 0.08–0.27, p < 0.01; 0.45–0.97, p = 0.034, respectively, overdominant model. These results suggested that DNMT1, DNMT2 and DNMT3A may play important roles in GC carcinogenesis. However, further studies are required to elucidate the mechanism.

  18. Human intelligence and polymorphisms in the DNA methyltransferase genes involved in epigenetic marking.

    Science.gov (United States)

    Haggarty, Paul; Hoad, Gwen; Harris, Sarah E; Starr, John M; Fox, Helen C; Deary, Ian J; Whalley, Lawrence J

    2010-06-25

    Epigenetic mechanisms have been implicated in syndromes associated with mental impairment but little is known about the role of epigenetics in determining the normal variation in human intelligence. We measured polymorphisms in four DNA methyltransferases (DNMT1, DNMT3A, DNMT3B and DNMT3L) involved in epigenetic marking and related these to childhood and adult general intelligence in a population (n = 1542) consisting of two Scottish cohorts born in 1936 and residing in Lothian (n = 1075) or Aberdeen (n = 467). All subjects had taken the same test of intelligence at age 11yrs. The Lothian cohort took the test again at age 70yrs. The minor T allele of DNMT3L SNP 11330C>T (rs7354779) allele was associated with a higher standardised childhood intelligence score; greatest effect in the dominant analysis but also significant in the additive model (coefficient = 1.40(additive); 95%CI 0.22,2.59; p = 0.020 and 1.99(dominant); 95%CI 0.55,3.43; p = 0.007). The DNMT3L C allele was associated with an increased risk of being below average intelligence (OR 1.25(additive); 95%CI 1.05,1.51; p = 0.011 and OR 1.37(dominant); 95%CI 1.11,1.68; p = 0.003), and being in the lowest 40(th) (p(additive) = 0.009; p(dominant) = 0.002) and lowest 30(th) (p(additive) = 0.004; p(dominant) = 0.002) centiles for intelligence. After Bonferroni correction for the number variants tested the link between DNMT3L 11330C>T and childhood intelligence remained significant by linear regression and centile analysis; only the additive regression model was borderline significant. Adult intelligence was similarly linked to the DNMT3L variant but this analysis was limited by the numbers studied and nature of the test and the association was not significant after Bonferroni correction. We believe that the role of epigenetics in the normal variation in human intelligence merits further study and that this novel finding should be tested in other cohorts.

  19. In silico analysis of methyltransferase domains involved in biosynthesis of secondary metabolites

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    Gokhale Rajesh S

    2008-10-01

    Full Text Available Abstract Background Secondary metabolites biosynthesized by polyketide synthase (PKS and nonribosomal peptide synthetase (NRPS family of enzymes constitute several classes of therapeutically important natural products like erythromycin, rapamycin, cyclosporine etc. In view of their relevance for natural product based drug discovery, identification of novel secondary metabolite natural products by genome mining has been an area of active research. A number of different tailoring enzymes catalyze a variety of chemical modifications to the polyketide or nonribosomal peptide backbone of these secondary metabolites to enhance their structural diversity. Therefore, development of powerful bioinformatics methods for identification of these tailoring enzymes and assignment of their substrate specificity is crucial for deciphering novel secondary metabolites by genome mining. Results In this work, we have carried out a comprehensive bioinformatics analysis of methyltransferase (MT domains present in multi functional type I PKS and NRPS proteins encoded by PKS/NRPS gene clusters having known secondary metabolite products. Based on the results of this analysis, we have developed a novel knowledge based computational approach for detecting MT domains present in PKS and NRPS megasynthases, delineating their correct boundaries and classifying them as N-MT, C-MT and O-MT using profile HMMs. Analysis of proteins in nr database of NCBI using these class specific profiles has revealed several interesting examples, namely, C-MT domains in NRPS modules, N-MT domains with significant homology to C-MT proteins, and presence of NRPS/PKS MTs in association with other catalytic domains. Our analysis of the chemical structures of the secondary metabolites and their site of methylation suggested that a possible evolutionary basis for the presence of a novel class of N-MT domains with significant homology to C-MT proteins could be the close resemblance of the chemical

  20. The importance of thiopurine methyltransferase activity for the use of azathioprine in transplant recipients.

    Science.gov (United States)

    Chocair, P R; Duley, J A; Simmonds, H A; Cameron, J S

    1992-05-01

    The immunosuppressive efficacy of azathioprine is related to its rapid metabolism in vivo to 6-mercaptopurine (6MP), with subsequent conversion to thioguanine nucleotides by an anabolic route involving hypoxanthine-guanine phosphoribosyltransferase. Two alternative catabolic routes exist: oxidation to 6-thiouric acid via xanthine oxidase and methylation to 6-methylmercaptopurine via the enzyme thiopurine methyltransferase (TPMT). Catabolism via either route would restrict formation of the active metabolites. We analyzed TPMT activity in erythrocyte lysates of 25 controls, 25 uremic patients on dialysis, and 68 transplanted patients. Median activity was lower in controls (31.0 pmol/hr/mg Hb, range 16.2-43.0) and transplanted patients receiving only cyclosporine and prednisolone (31.7 pmol/hr/mg Hb, range 12.7-43.5) than in the azathioprine treated group, (36.1 pmol/hr/mg Hb, range 16