Assay method and compositions

International Nuclear Information System (INIS)

1977-01-01

Methods are described for measuring catecholamine levels in human and animal body fluids and tissues using the catechol-O-methyl-transferase (COMT) radioassay. The assay involves incubating the biological sample with COMT and the tritiated methyl donor, S-adenosyl-L-methionine( 3 H)-methyl. The O-methylated ( 3 H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin- 3 H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin- 3 H and raising the pH of the aqueous periodate phase from which O-methylated ( 3 H) dopamine is extracted and counted. The assay may be modified depending on whether measurements of undifferentiated total endogenous catecholamine levels or differential analyses of the catecholamine levels are being performed. The sensitivity of the assay can be as low as 5 picograms for norepinephrine and epinephrine and 12 picograms for dopamine. The assemblance of the essential components of the assay into a kit for use in laboratories is also described. (U.K.)

MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

Science.gov (United States)

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-06-01

Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status.

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Townsend, Todd A; Parrish, Marcus C; Engelward, Bevin P; Manjanatha, Mugimane G

2017-08-01

DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposure risk of pharmacological and biological agents. We previously developed a higher-throughput platform for the single cell gel electrophoresis (comet) assay, CometChip, to assess DNA damage and genotoxic potential. Here, we utilized the methylation-dependent endonuclease, McrBC, to develop a modified alkaline comet assay, "EpiComet," which allows single platform evaluation of genotoxicity and global DNA methylation [5-methylcytosine (5-mC)] status of single-cell populations under user-defined conditions. Further, we leveraged the CometChip platform to create an EpiComet-Chip system capable of performing quantification across simultaneous exposure protocols to enable unprecedented speed and simplicity. This system detected global methylation alterations in response to exposures which included chemotherapeutic and environmental agents. Using EpiComet-Chip on 63 matched samples, we correctly identified single-sample hypermethylation (≥1.5-fold) at 87% (20/23), hypomethylation (≥1.25-fold) at 100% (9/9), with a 4% (2/54) false-negative rate (FNR), and 10% (4/40) false-positive rate (FPR). Using a more stringent threshold to define hypermethylation (≥1.75-fold) allowed us to correctly identify 94% of hypermethylation (17/18), but increased our FPR to 16% (7/45). The successful application of this novel technology will aid hazard identification and risk characterization of FDA-regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, as the assay is amenable to cultured cells or nucleated cells from any tissue. Environ. Mol. Mutagen. 58:508-521, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Antimutagenic components in Glycyrrhiza against N-methyl-N-nitrosourea in the Ames assay.

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Inami, Keiko; Mine, Yusuke; Kojo, Yukiko; Tanaka, Satomi; Ishikawa, Satoko; Mochizuki, Masataka

2017-03-01

Antimutagenesis against N-nitroso compounds contribute to prevention of human cancer. We have found that Glycyrrhiza aspera ethanolic extract exhibits antimutagenic activity against N-methyl-N-nitrosourea (MNU) using the Ames assay with Salmonella typhimurium TA1535. In the present study, eight purified components from Glycyrrhiza, namely glabridin, glycyrrhetinic acid, glycyrrhizin, licochalcone A, licoricesaponin H2, licoricesaponin G2, liquiritigenin and liquiritin were evaluated for their antimutagenicity against MNU in the Ames assay with S. typhimurium TA1535. Glycyrrhetinic acid, glycyrrhizin, licoricesaponin G2, licoricesaponin H2 and liquiritin did not show the antimutagenicity against MNU in S. typhimurium TA1535. Glabridin, licochalcone A and liquiritigenin reduced revertant colonies derived from MNU in S. typhimurium TA1535 without showing cytotoxic effects, indicating that these compounds possess antimutagenic activity against MNU. The inhibitory activity of glabridin and licochalcone A was more effective than that of liquiritigenin. Thus, Glycyrrhiza contains antimutagenic components against DNA alkylating, direct-acting carcinogens.

High throughput analysis of red wine and grape phenolics-adaptation and validation of methyl cellulose precipitable tannin assay and modified Somers color assay to a rapid 96 well plate format.

Science.gov (United States)

Mercurio, Meagan D; Dambergs, Robert G; Herderich, Markus J; Smith, Paul A

2007-06-13

The methyl cellulose precipitable (MCP) tannin assay and a modified version of the Somers and Evans color assay were adapted to high-throughput (HTP) analysis. To improve efficiency of the MCP tannin assay, a miniaturized 1 mL format and a HTP format using 96 well plates were developed. The Somers color assay was modified to allow the standardization of pH and ethanol concentrations of wine samples in a simple one-step dilution with a buffer solution, thus removing inconsistencies between wine matrices prior to analysis and allowing for its adaptation to a HTP format. Validation studies showed that all new formats were efficient, and results were reproducible and analogous to the original formats.

Immunosuppressive potential of bardoxolone methyl using a modified murine local lymph node assay (LLNA).

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Kitsukawa, Mika; Tsuchiyama, Hiromi; Maeda, Akihisa; Oshida, Keiyu; Miyamoto, Yohei

2014-08-01

2-Cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid methyl ester (CDDO-Me; bardoxolone methyl) is one of the synthetic oleanane triterpenoids (SOs). It is known that it is the strongest Nrf2/ARE signaling inducer of SOs and slightly inhibits immune response. Little was known about the immunomodulatory action of CDDO-Me in vivo. We assessed its immunosuppressive potential by using the modified mouse lymph node assay (LLNA) including immunosuppression-related gene expression analysis. In the modified LLNA, CDDO-Me showed a significant decrease in lymph node weight and changes in expressions of the immunosuppression-related genes, Zfp459 and Fmo2. It has been already reported that a decrease in lymph node weight was induced by several types of immunosuppressive chemicals such as calcineurin inhibitors, antimetabolites, steroids, and alkylators. In addition, changes in Zfp459 and Fmo2 expression was reported in response after only treatment of antimetabolites. From these results, CDDO-Me is considered to have an immunosuppressive action and similar mechanism to antimetabolites.

MSP-HTPrimer: a high-throughput primer design tool to improve assay design for DNA methylation analysis in epigenetics.

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Pandey, Ram Vinay; Pulverer, Walter; Kallmeyer, Rainer; Beikircher, Gabriel; Pabinger, Stephan; Kriegner, Albert; Weinhäusel, Andreas

2016-01-01

Bisulfite (BS) conversion-based and methylation-sensitive restriction enzyme (MSRE)-based PCR methods have been the most commonly used techniques for locus-specific DNA methylation analysis. However, both methods have advantages and limitations. Thus, an integrated approach would be extremely useful to quantify the DNA methylation status successfully with great sensitivity and specificity. Designing specific and optimized primers for target regions is the most critical and challenging step in obtaining the adequate DNA methylation results using PCR-based methods. Currently, no integrated, optimized, and high-throughput methylation-specific primer design software methods are available for both BS- and MSRE-based methods. Therefore an integrated, powerful, and easy-to-use methylation-specific primer design pipeline with great accuracy and success rate will be very useful. We have developed a new web-based pipeline, called MSP-HTPrimer, to design primers pairs for MSP, BSP, pyrosequencing, COBRA, and MSRE assays on both genomic strands. First, our pipeline converts all target sequences into bisulfite-treated templates for both forward and reverse strand and designs all possible primer pairs, followed by filtering for single nucleotide polymorphisms (SNPs) and known repeat regions. Next, each primer pairs are annotated with the upstream and downstream RefSeq genes, CpG island, and cut sites (for COBRA and MSRE). Finally, MSP-HTPrimer selects specific primers from both strands based on custom and user-defined hierarchical selection criteria. MSP-HTPrimer produces a primer pair summary output table in TXT and HTML format for display and UCSC custom tracks for resulting primer pairs in GTF format. MSP-HTPrimer is an integrated, web-based, and high-throughput pipeline and has no limitation on the number and size of target sequences and designs MSP, BSP, pyrosequencing, COBRA, and MSRE assays. It is the only pipeline, which automatically designs primers on both genomic

Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay.

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Robert Grützmann

Full Text Available BACKGROUND: Colorectal cancer (CRC is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC. METHODOLOGY/PRINCIPAL FINDINGS: Using a PCR assay for analysis of Septin 9 (SEPT9 hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls. 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48% and 7/102 controls (7%. In the test study 73/126 CRCs (58% and 18/183 control samples (10% were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected while maintaining 90% specificity (19/183 for controls. Positive rates for plasmas from the other cancers (11/96 and non-cancerous conditions (41/315 were low. The rate of polyp detection (>1 cm was approximately 20%. CONCLUSIONS/SIGNIFICANCE: Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.

Genetic and non-genetic influences during pregnancy on infant global and site specific DNA methylation: role for folate gene variants and vitamin B12.

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Jill A McKay

Full Text Available Inter-individual variation in patterns of DNA methylation at birth can be explained by the influence of environmental, genetic and stochastic factors. This study investigates the genetic and non-genetic determinants of variation in DNA methylation in human infants. Given its central role in provision of methyl groups for DNA methylation, this study focuses on aspects of folate metabolism. Global (LUMA and gene specific (IGF2, ZNT5, IGFBP3 DNA methylation were quantified in 430 infants by Pyrosequencing®. Seven polymorphisms in 6 genes (MTHFR, MTRR, FOLH1, CβS, RFC1, SHMT involved in folate absorption and metabolism were analysed in DNA from both infants and mothers. Red blood cell folate and serum vitamin B(12 concentrations were measured as indices of vitamin status. Relationships between DNA methylation patterns and several covariates viz. sex, gestation length, maternal and infant red cell folate, maternal and infant serum vitamin B(12, maternal age, smoking and genotype were tested. Length of gestation correlated positively with IGF2 methylation (rho = 0.11, p = 0.032 and inversely with ZNT5 methylation (rho = -0.13, p = 0.017. Methylation of the IGFBP3 locus correlated inversely with infant vitamin B(12 concentration (rho = -0.16, p = 0.007, whilst global DNA methylation correlated inversely with maternal vitamin B(12 concentrations (rho = 0.18, p = 0.044. Analysis of common genetic variants in folate pathway genes highlighted several associations including infant MTRR 66G>A genotype with DNA methylation (χ(2 = 8.82, p = 0.003 and maternal MTHFR 677C>T genotype with IGF2 methylation (χ(2 = 2.77, p = 0.006. These data support the hypothesis that both environmental and genetic factors involved in one-carbon metabolism influence DNA methylation in infants. Specifically, the findings highlight the importance of vitamin B(12 status, infant MTRR genotype and maternal MTHFR genotype, all of which may influence the supply of methyl groups for

Methylation-Sensitive High Resolution Melting (MS-HRM).

Science.gov (United States)

Hussmann, Dianna; Hansen, Lise Lotte

2018-01-01

Methylation-Sensitive High Resolution Melting (MS-HRM) is an in-tube, PCR-based method to detect methylation levels at specific loci of interest. A unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1-1% methylated alleles in an unmethylated background.Primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays. Bisulfite treatment of the DNA prior to performing MS-HRM ensures a different base composition between methylated and unmethylated DNA, which is used to separate the resulting amplicons by high resolution melting.The high sensitivity of MS-HRM has proven useful for detecting cancer biomarkers in a noninvasive manner in urine from bladder cancer patients, in stool from colorectal cancer patients, and in buccal mucosa from breast cancer patients. MS-HRM is a fast method to diagnose imprinted diseases and to clinically validate results from whole-epigenome studies. The ability to detect few copies of methylated DNA makes MS-HRM a key player in the quest for establishing links between environmental exposure, epigenetic changes, and disease.

MethylMix 2.0: an R package for identifying DNA methylation genes.

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Cedoz, Pierre-Louis; Prunello, Marcos; Brennan, Kevin; Gevaert, Olivier

2018-04-14

DNA methylation is an important mechanism regulating gene transcription, and its role in carcinogenesis has been extensively studied. Hyper and hypomethylation of genes is a major mechanism of gene expression deregulation in a wide range of diseases. At the same time, high-throughput DNA methylation assays have been developed generating vast amounts of genome wide DNA methylation measurements. We developed MethylMix, an algorithm implemented in R to identify disease specific hyper and hypomethylated genes. Here we present a new version of MethylMix that automates the construction of DNA-methylation and gene expression datasets from The Cancer Genome Atlas (TCGA). More precisely, MethylMix 2.0 incorporates two major updates: the automated downloading of DNA methylation and gene expression datasets from TCGA and the automated preprocessing of such datasets: value imputation, batch correction and CpG sites clustering within each gene. The resulting datasets can subsequently be analyzed with MethylMix to identify transcriptionally predictive methylation states. We show that the Differential Methylation Values created by MethylMix can be used for cancer subtyping. olivier.gevaert@stanford.edu. https://bioconductor.org/packages/release/bioc/manuals/MethylMix/man/MethylMix.pdf. MethylMix 2.0 was implemented as an R package and is available in bioconductor.

In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyl-tetrazolium reduction assay

OpenAIRE

Hördegen, P.; Cabaret, J.; Hertzberg, H.; Langhans, W.; Maurer, V.

2006-01-01

Because of the increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against gastrointestinal nematodes. Phytotherapy could be one of the major options to control these pathologies. Extracts or ingredients of six different plant species were tested against exsheathed infective larvae of Haemonchus contortus using a modified methyl-thiazolyltetrazolium (MTT) reduction assay. Pyrantel tartrate was u...

The performance of the SEPT9 gene methylation assay and a comparison with other CRC screening tests: A meta-analysis.

Science.gov (United States)

Song, Lele; Jia, Jia; Peng, Xiumei; Xiao, Wenhua; Li, Yuemin

2017-06-08

The SEPT9 gene methylation assay is the first FDA-approved blood assay for colorectal cancer (CRC) screening. Fecal immunochemical test (FIT), FIT-DNA test and CEA assay are also in vitro diagnostic (IVD) tests used in CRC screening. This meta-analysis aims to review the SEPT9 assay performance and compare it with other IVD CRC screening tests. By searching the Ovid MEDLINE, EMBASE, CBMdisc and CJFD database, 25 out of 180 studies were identified to report the SEPT9 assay performance. 2613 CRC cases and 6030 controls were included, and sensitivity and specificity were used to evaluate its performance at various algorithms. 1/3 algorithm exhibited the best sensitivity while 2/3 and 1/1 algorithm exhibited the best balance between sensitivity and specificity. The performance of the blood SEPT9 assay is superior to that of the serum protein markers and the FIT test in symptomatic population, while appeared to be less potent than FIT and FIT-DNA tests in asymptomatic population. In conclusion, 1/3 algorithm is recommended for CRC screening, and 2/3 or 1/1 algorithms are suitable for early detection for diagnostic purpose. The SEPT9 assay exhibited better performance in symptomatic population than in asymptomatic population.

DNA methylation regulates expression of VEGF-C, and S-adenosylmethionine is effective for VEGF-C methylation and for inhibiting cancer growth

Energy Technology Data Exchange (ETDEWEB)

Da, M.X. [Department of Surgical Oncology, Gansu Provincial Hospital, Lanzhou (China); Zhang, Y.B. [Department of Surgery, Ningxia Medical University, Yinchuan (China); Yao, J.B. [Department of Surgical Oncology, Gansu Provincial Hospital, Lanzhou (China); Duan, Y.X. [Department of Surgery, Ningxia Medical University, Yinchuan (China)

2014-09-30

DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.

Validation of methylation-sensitive high-resolution melting (MS-HRM) for the detection of stool DNA methylation in colorectal neoplasms.

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Xiao, Zhujun; Li, Bingsheng; Wang, Guozhen; Zhu, Weisi; Wang, Zhongqiu; Lin, Jinfeng; Xu, Angao; Wang, Xinying

2014-04-20

Methylation-sensitive high-resolution melting (MS-HRM) is a new technique for assaying DNA methylation, but its feasibility for assaying stool in patients with colorectal cancer (CRC) is unknown. First, the MS-HRM and methylation-specific PCR (MSP) detection limits were tested. Second, the methylation statuses of SFRP2 and VIM were analyzed in stool samples by MS-HRM, and in matching tumor and normal colon tissues via bisulfite sequencing PCR (BSP). Third, a case-control study evaluated the diagnostic sensitivity and specificity of MS-HRM relative to results obtained with MSP and the fecal immunochemical test (FIT). Finally, the linearity and reproducibility of MS-HRM were assessed. The detection limits of MS-HRM and MSP were 1% and 5%, respectively. The diagnostic sensitivities of MS-HRM (87.3%, 55/63) in stool and BSP in matching tumor tissue (92.1%, 58/63) were highly consistent (κ=0.744). The MS-HRM assay detected 92.5% (37/40) methylation in CRCs, 94.4% (34/36) in advanced adenomas, and 8.8% (5/57) in normal controls. The results of MS-HRM analysis were stable and reliable and showed fairly good linearity for both SFRP2 (PHRM shows potential for CRC screening. Copyright © 2014 Elsevier B.V. All rights reserved.

A CpG-methylation-based assay to predict survival in clear cell renal cell carcinoma

Science.gov (United States)

Wei, Jin-Huan; Haddad, Ahmed; Wu, Kai-Jie; Zhao, Hong-Wei; Kapur, Payal; Zhang, Zhi-Ling; Zhao, Liang-Yun; Chen, Zhen-Hua; Zhou, Yun-Yun; Zhou, Jian-Cheng; Wang, Bin; Yu, Yan-Hong; Cai, Mu-Yan; Xie, Dan; Liao, Bing; Li, Cai-Xia; Li, Pei-Xing; Wang, Zong-Ren; Zhou, Fang-Jian; Shi, Lei; Liu, Qing-Zuo; Gao, Zhen-Li; He, Da-Lin; Chen, Wei; Hsieh, Jer-Tsong; Li, Quan-Zhen; Margulis, Vitaly; Luo, Jun-Hang

2015-01-01

Clear cell renal cell carcinomas (ccRCCs) display divergent clinical behaviours. Molecular markers might improve risk stratification of ccRCC. Here we use, based on genome-wide CpG methylation profiling, a LASSO model to develop a five-CpG-based assay for ccRCC prognosis that can be used with formalin-fixed paraffin-embedded specimens. The five-CpG-based classifier was validated in three independent sets from China, United States and the Cancer Genome Atlas data set. The classifier predicts the overall survival of ccRCC patients (hazard ratio=2.96−4.82; P=3.9 × 10−6−2.2 × 10−9), independent of standard clinical prognostic factors. The five-CpG-based classifier successfully categorizes patients into high-risk and low-risk groups, with significant differences of clinical outcome in respective clinical stages and individual ‘stage, size, grade and necrosis' scores. Moreover, methylation at the five CpGs correlates with expression of five genes: PITX1, FOXE3, TWF2, EHBP1L1 and RIN1. Our five-CpG-based classifier is a practical and reliable prognostic tool for ccRCC that can add prognostic value to the staging system. PMID:26515236

Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine Genome

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Chih-Jie Shen

2014-01-01

Full Text Available Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming, especially in some important organ development. Osteoponin (OPN is an extracellular-matrix protein involved in heart and bone development and diseases. In this study, we investigated the correlation between OPN mRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay. Aberrant methylation of porcine OPN was frequently found in different tissues of somatic nuclear transferred cloning pigs, and bisulfite sequence data suggested that the OPN promoter region −2615 to −2239 nucleotides (nt may be a crucial regulation DNA element. In pig ear fibroblast cell culture study, the demethylation of OPN promoter was found in dose-dependent response of 5-aza-dc treatment and followed the OPN mRNA reexpression. In cloned pig study, discrepant expression pattern was identified in several cloned pig tissues, especially in brain, heart, and ear. Promoter assay data revealed that four methylated CpG sites presenting in the −2615 to −2239 nt region cause significant downregulation of OPN promoter activity. These data suggested that methylation in the OPN promoter plays a crucial role in the regulation of OPN expression that we found in cloned pigs genome.

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls

OpenAIRE

Phutikanit, Nawapen; Suwimonteerabutr, Junpen; Harrison, Dion; D'Occhio, Michael; Carroll, Bernie; Techakumphu, Mongkol

2010-01-01

Abstract Background The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls. Methods Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII). The native genomic ...

Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists

DEFF Research Database (Denmark)

Hansen, Kasper Bø; Mullasseril, Praseeda; Dawit, Sara

2010-01-01

N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe...... a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal...

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR technique among various cell types of bulls

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Carroll Bernie

2010-03-01

Full Text Available Abstract Background The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls. Methods Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII. The native genomic and enzyme treated DNA samples were used as templates in an arbitrarily primed-PCR assay with 30 sets of single short oligonucleotide primer. The PCR products were separated on silver stained denaturing polyacrylamide gels. Three types of PCR markers; digestion resistant-, digestion sensitive-, and digestion dependent markers, were analyzed based on the presence/absence polymorphism of the markers between the two templates. Results Approximately 1,000 PCR markers per sample were produced from 27 sets of primer and most of them (>90% were digestion resistant markers. The highest percentage of digestion resistant markers was found in leukocytic DNA (94.8% and the lowest in fibroblastic DNA (92.3%, P ≤ 0.05. Spermatozoa contained a higher number of digestion sensitive markers when compared with the others (3.6% vs. 2.2% and 2.6% in leukocytes and fibroblasts respectively, P ≤ 0.05. Conclusions The powerfulness of the AMP PCR assay was the generation of methylation-associated markers without any prior knowledge of the genomic sequence. The data obtained from different primers provided an overview of genome wide DNA methylation content in different cell types. By using this technique, we found that DNA methylation profile is tissue-specific. Male germ cells were hypomethylated at the HpaII locations when compared with somatic cells, while the chromatin of the well-characterized somatic cells was heavily methylated when compared with that of the versatile somatic

Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique.

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Xiong, L Z; Xu, C G; Saghai Maroof, M A; Zhang, Q

1999-04-01

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation in the rice genome, using the technique of methylation-sensitive amplified polymorphism (MSAP), which is a modification of the amplified fragment length polymorphism (AFLP) method that makes use of the differential sensitivity of a pair of isoschizomers to cytosine methylation. The tissues assayed included seedlings and flag leaves of an elite rice hybrid, Shanyou 63, and the parental lines Zhenshan 97 and Minghui 63. In all, 1076 fragments, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified using 16 pairs of selective primers. A total of 195 sites were found to be methylated at cytosines in one or both parents, and the two parents showed approximately the same overall degree of methylation (16.3%), as revealed by the incidence of differential digestion by the isoschizomers. Four classes of patterns were identified in a comparative assay of cytosine methylation in the parents and hybrid; increased methylation was detected in the hybrid compared to the parents at some of the recognition sites, while decreased methylation in the hybrid was detected at other sites. A small proportion of the sites was found to be differentially methylated in seedlings and flag leaves; DNA from young seedlings was methylated to a greater extent than that from flag leaves. Almost all of the methylation patterns detected by MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrate that the MSAP technique is highly efficient for large-scale detection of cytosine methylation in the rice genome. We believe that the technique can be adapted for use in other plant species.

Methylated genes as new cancer biomarkers.

LENUS (Irish Health Repository)

Duffy, M J

2012-02-01

Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested that measurement of the methylation status of the promoter regions of specific genes can aid early detection of cancer, determine prognosis and predict therapy responses. Promising DNA methylation biomarkers include the use of methylated GSTP1 for aiding the early diagnosis of prostate cancer, methylated PITX2 for predicting outcome in lymph node-negative breast cancer patients and methylated MGMT in predicting benefit from alkylating agents in patients with glioblastomas. However, prior to clinical utilisation, these findings require validation in prospective clinical studies. Furthermore, assays for measuring gene methylation need to be standardised, simplified and evaluated in external quality assurance programmes. It is concluded that methylated genes have the potential to provide a new generation of cancer biomarkers.

Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

Science.gov (United States)

Draht, Muriel X G; Smits, Kim M; Jooste, Valérie; Tournier, Benjamin; Vervoort, Martijn; Ramaekers, Chantal; Chapusot, Caroline; Weijenberg, Matty P; van Engeland, Manon; Melotte, Veerle

2016-01-01

Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested

In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyl-tetrazolium reduction assay.

Science.gov (United States)

Hördegen, P; Cabaret, J; Hertzberg, H; Langhans, W; Maurer, V

2006-11-03

Because of the increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against gastrointestinal nematodes. Phytotherapy could be one of the major options to control these pathologies. Extracts or ingredients of six different plant species were tested against exsheathed infective larvae of Haemonchus contortus using a modified methyl-thiazolyl-tetrazolium (MTT) reduction assay. Pyrantel tartrate was used as reference anthelmintic. Bromelain, the enzyme complex of the stem of Ananas comosus (Bromeliaceae), the ethanolic extracts of seeds of Azadirachta indica (Meliaceae), Caesalpinia crista (Caesalpiniaceae) and Vernonia anthelmintica (Asteraceae), and the ethanolic extracts of the whole plant of Fumaria parviflora (Papaveraceae) and of the fruit of Embelia ribes (Myrsinaceae) showed an anthelmintic efficacy of up to 93%, relative to pyrantel tartrate. Based on these results obtained with larval Haemonchus contortus, the modified MTT reduction assay could be a possible method for testing plant products with anthelmintic properties.

Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro

DEFF Research Database (Denmark)

Andersen, Helle Raun; Vinggaard, Anne; Rasmussen, Thomas Høj

2002-01-01

, chlorpyrifos, deltamethrin, tolclofos-methyl, and tribenuron-methyl induced weak responses in one or both estrogenicity assays. Upon cotreatment with 17beta-estradiol, the response was potentiated by endosulfan in the proliferation assay and by pirimicarb, propamocarb, and daminozid in the ER transactivation...

Combined high-performance liquid chromatography/32P-postlabeling assay of N7-methyldeoxyguanosine

International Nuclear Information System (INIS)

Shields, P.G.; Povey, A.C.; Wilson, V.L.; Weston, A.; Harris, C.C.

1990-01-01

A highly sensitive and specific assay for the detection of N7-methyl-2'-deoxyguanosine (N7methyldG) has been developed by combining high-performance liquid chromatography, 32 P-postlabeling, and nucleotide chromatography. Separation of normal nucleotides and adducts by high-performance liquid chromatography and then combining a portion of 2'-deoxyguanosine to the N7methyldG allows for quantitation using an internal standard. The directly determined molar ratio is not subject to errors in digestion, variable ATP-specific activity, or assumptions in relative adduct-labeling efficiency. The detection limit was one N7methyldG adduct in 10(7) unmodified 2'-deoxyguanosine bases. N7methyldG adducts have been detected in 5 human lung samples in which O6-methyl-2'-deoxyguanosine adducts had been previously determined. The mean ratio of N7methyldG to O6-methyl-2'-deoxyguanosine was determined to be approximately 10. The current assay complements the high-performance liquid chromatography/ 32 P-postlabeling assay for O6-methyl-2'-deoxyguanosine and increases the detection sensitivity of DNA methylated by exogenous alkylating agents

A panel of genes methylated with high frequency in colorectal cancer

International Nuclear Information System (INIS)

Mitchell, Susan M; Beetson, Iain; Rand, Keith N; McEvoy, Aidan; Thomas, Melissa L; Baker, Rohan T; Wattchow, David A; Young, Graeme P; Lockett, Trevor J; Pedersen, Susanne K; LaPointe, Lawrence C; Ross, Jason P; Molloy, Peter L; Drew, Horace R; Ho, Thu; Brown, Glenn S; Saunders, Neil FW; Duesing, Konsta R; Buckley, Michael J; Dunne, Rob

2014-01-01

The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. Combined epigenomic methods – activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment – were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes

Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures.

Science.gov (United States)

Schwalbe, E C; Hicks, D; Rafiee, G; Bashton, M; Gohlke, H; Enshaei, A; Potluri, S; Matthiesen, J; Mather, M; Taleongpong, P; Chaston, R; Silmon, A; Curtis, A; Lindsey, J C; Crosier, S; Smith, A J; Goschzik, T; Doz, F; Rutkowski, S; Lannering, B; Pietsch, T; Bailey, S; Williamson, D; Clifford, S C

2017-10-18

Rapid and reliable detection of disease-associated DNA methylation patterns has major potential to advance molecular diagnostics and underpin research investigations. We describe the development and validation of minimal methylation classifier (MIMIC), combining CpG signature design from genome-wide datasets, multiplex-PCR and detection by single-base extension and MALDI-TOF mass spectrometry, in a novel method to assess multi-locus DNA methylation profiles within routine clinically-applicable assays. We illustrate the application of MIMIC to successfully identify the methylation-dependent diagnostic molecular subgroups of medulloblastoma (the most common malignant childhood brain tumour), using scant/low-quality samples remaining from the most recently completed pan-European medulloblastoma clinical trial, refractory to analysis by conventional genome-wide DNA methylation analysis. Using this approach, we identify critical DNA methylation patterns from previously inaccessible cohorts, and reveal novel survival differences between the medulloblastoma disease subgroups with significant potential for clinical exploitation.

Comparison of methods for quantification of global DNA methylation in human cells and tissues.

Directory of Open Access Journals (Sweden)

Sofia Lisanti

Full Text Available DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.

A robust internal control for high-precision DNA methylation analyses by droplet digital PCR.

Science.gov (United States)

Pharo, Heidi D; Andresen, Kim; Berg, Kaja C G; Lothe, Ragnhild A; Jeanmougin, Marine; Lind, Guro E

2018-01-01

Droplet digital PCR (ddPCR) allows absolute quantification of nucleic acids and has potential for improved non-invasive detection of DNA methylation. For increased precision of the methylation analysis, we aimed to develop a robust internal control for use in methylation-specific ddPCR. Two control design approaches were tested: (a) targeting a genomic region shared across members of a gene family and (b) combining multiple assays targeting different pericentromeric loci on different chromosomes. Through analyses of 34 colorectal cancer cell lines, the performance of the control assay candidates was optimized and evaluated, both individually and in various combinations, using the QX200™ droplet digital PCR platform (Bio-Rad). The best-performing control was tested in combination with assays targeting methylated CDO1 , SEPT9 , and VIM . A 4Plex panel consisting of EPHA3 , KBTBD4 , PLEKHF1 , and SYT10 was identified as the best-performing control. The use of the 4Plex for normalization reduced the variability in methylation values, corrected for differences in template amount, and diminished the effect of chromosomal aberrations. Positive Droplet Calling (PoDCall), an R-based algorithm for standardized threshold determination, was developed, ensuring consistency of the ddPCR results. Implementation of a robust internal control, i.e., the 4Plex, and an algorithm for automated threshold determination, PoDCall, in methylation-specific ddPCR increase the precision of DNA methylation analysis.

Colorimetric determination of DNase I activity with a DNA-methyl green substrate.

Science.gov (United States)

Sinicropi, D; Baker, D L; Prince, W S; Shiffer, K; Shak, S

1994-11-01

A simple, high throughput, and precise assay was developed for quantification of deoxyribonuclease I (DNase; IUB 3.1.21.1) activity. The method was adapted from the procedure devised by Kurnick which employs a substrate comprised of highly polymerized native DNA complexed with methyl green. Hydrolysis of the DNA produced unbound methyl green and a decrease in the absorbance of the solution at 620 nm. By adjusting the time and temperature of the reaction, the assay permits quantification of DNase activity over a wide concentration range (0.4 to 8900 ng/ml). Samples and standards were added to the substrate in microtiter plates and were incubated for 1-24 h at 25-37 degrees C to achieve the desired assay range. The DNase activity of the samples was interpolated from a standard curve generated with Pulmozyme recombinant human deoxyribonuclease I (rhDNase). Interassay precision was less than 12% CV and recovery was within 100 +/- 11%. Activity determination by the DNA-methyl green method correlated well with that determined by the widely used "hyperchromicity" method originated by Kunitz, which is based on the increase in absorbance at 260 nm upon hydrolysis of DNA. The DNA-methyl green assay was simpler and more versatile than the hyperchromicity method and was used to characterize the activity of rhDNase and DNase isolated from human urine.

Enhanced microbial decolorization of methyl red with oxidized carbon fiber as redox mediator

Energy Technology Data Exchange (ETDEWEB)

Emilia Rios-Del Toro, E. [División de Ciencias Ambientales, Instituto Potosino de Investigación Científica y Tecnológica (IPICyT), Camino a la Presa San José 2055, Col. Lomas 4a Sección, San Luis Potosí, SLP 78216 (Mexico); Celis, Lourdes B. [División de Geociencias Aplicadas, Instituto Potosino de Investigación Científica y Tecnológica (IPICyT), Camino a la Presa San José 2055, Col. Lomas 4a Sección, San Luis Potosí, SLP 78216 (Mexico); Cervantes, Francisco J. [División de Ciencias Ambientales, Instituto Potosino de Investigación Científica y Tecnológica (IPICyT), Camino a la Presa San José 2055, Col. Lomas 4a Sección, San Luis Potosí, SLP 78216 (Mexico); Rangel-Mendez, J. Rene, E-mail: rene@ipicyt.edu.mx [División de Ciencias Ambientales, Instituto Potosino de Investigación Científica y Tecnológica (IPICyT), Camino a la Presa San José 2055, Col. Lomas 4a Sección, San Luis Potosí, SLP 78216 (Mexico)

2013-09-15

Highlights: • Activated carbon fibers (ACFs) act as redox mediator. • Electron accepting capacity increased with oxidation time of ACF. •ACFs increased 8-fold the reduction of methyl red in biological assays. •Biofilm formed on the ACFs partly blocked their redox mediator capacity. -- Abstract: The anaerobic degradation of azo dyes under anaerobic conditions is possible but at a slow rate. Redox mediators (quinones, activated carbon) are used to improve the reduction rate. The aim of this work was to use activated carbon fiber (ACF) as a redox mediator for the anaerobic reduction of the azo dye methyl red. ACF was chemically modified with 8 M HNO{sub 3} to increase its redox-mediating capacity and used in chemical and anaerobic biological batch assays for the reduction of methyl red. ACF increased its redox-mediating capacity up to 3-fold in chemical assays; in biological assays ACF increased the reduction rate up to 8-fold compared to controls without ACF. However, since the ACF served as support for biomass, a biofilm formed on the fiber significantly reduced its redox-mediating capacity; substrate consumption suggested that the electron transport from ACF to methyl red was the rate-limiting step in the process. These results are the first evidence of the role of ACF as a redox mediator in the reductive decolorization of methyl red, in addition to the effect of biofilm attached to ACF on methyl red reduction. Due to the versatile characteristics of ACF and its redox-mediating capacity, carbon fibers could be used in biological wastewater treatment systems to accelerate the reductive transformation of pollutants commonly found in industrial effluents.

Enhanced microbial decolorization of methyl red with oxidized carbon fiber as redox mediator

International Nuclear Information System (INIS)

Emilia Rios-Del Toro, E.; Celis, Lourdes B.; Cervantes, Francisco J.; Rangel-Mendez, J. Rene

2013-01-01

Highlights: • Activated carbon fibers (ACFs) act as redox mediator. • Electron accepting capacity increased with oxidation time of ACF. •ACFs increased 8-fold the reduction of methyl red in biological assays. •Biofilm formed on the ACFs partly blocked their redox mediator capacity. -- Abstract: The anaerobic degradation of azo dyes under anaerobic conditions is possible but at a slow rate. Redox mediators (quinones, activated carbon) are used to improve the reduction rate. The aim of this work was to use activated carbon fiber (ACF) as a redox mediator for the anaerobic reduction of the azo dye methyl red. ACF was chemically modified with 8 M HNO 3 to increase its redox-mediating capacity and used in chemical and anaerobic biological batch assays for the reduction of methyl red. ACF increased its redox-mediating capacity up to 3-fold in chemical assays; in biological assays ACF increased the reduction rate up to 8-fold compared to controls without ACF. However, since the ACF served as support for biomass, a biofilm formed on the fiber significantly reduced its redox-mediating capacity; substrate consumption suggested that the electron transport from ACF to methyl red was the rate-limiting step in the process. These results are the first evidence of the role of ACF as a redox mediator in the reductive decolorization of methyl red, in addition to the effect of biofilm attached to ACF on methyl red reduction. Due to the versatile characteristics of ACF and its redox-mediating capacity, carbon fibers could be used in biological wastewater treatment systems to accelerate the reductive transformation of pollutants commonly found in industrial effluents

Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

Science.gov (United States)

Nikolova, Teodora; Marini, Federico; Kaina, Bernd

2017-10-01

Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H 2 O 2 ) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H 2 O 2 , less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier

Methylated genes as new cancer biomarkers

DEFF Research Database (Denmark)

Brunner, Nils; Duffy, M.J; Napieralski, R.

2009-01-01

Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested that meas......Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested...... that measurement of the methylation status of the promoter regions of specific genes can aid early detection of cancer, determine prognosis and predict therapy responses. Promising DNA methylation biomarkers include the use of methylated GSTP1 for aiding the early diagnosis of prostate cancer, methylated PITX2...... for predicting outcome in lymph node-negative breast cancer patients and methylated MGMT in predicting benefit from alkylating agents in patients with glioblastomas. However, prior to clinical utilisation, these findings require validation in prospective clinical studies. Furthermore, assays for measuring gene...

CpG Methylation Analysis—Current Status of Clinical Assays and Potential Applications in Molecular Diagnostics

Science.gov (United States)

Sepulveda, Antonia R.; Jones, Dan; Ogino, Shuji; Samowitz, Wade; Gulley, Margaret L.; Edwards, Robin; Levenson, Victor; Pratt, Victoria M.; Yang, Bin; Nafa, Khedoudja; Yan, Liying; Vitazka, Patrick

2009-01-01

Methylation of CpG islands in gene promoter regions is a major molecular mechanism of gene silencing and underlies both cancer development and progression. In molecular oncology, testing for the CpG methylation of tissue DNA has emerged as a clinically useful tool for tumor detection, outcome prediction, and treatment selection, as well as for assessing the efficacy of treatment with the use of demethylating agents and monitoring for tumor recurrence. In addition, because CpG methylation occurs early in pre-neoplastic tissues, methylation tests may be useful as markers of cancer risk in patients with either infectious or inflammatory conditions. The Methylation Working Group of the Clinical Practice Committee of the Association of Molecular Pathology has reviewed the current state of clinical testing in this area. We report here our summary of both the advantages and disadvantages of various methods, as well as the needs for standardization and reporting. We then conclude by summarizing the most promising areas for future clinical testing in cancer molecular diagnostics. PMID:19541921

Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas

International Nuclear Information System (INIS)

Xiang, Shengyan; Liu, Zhaojun; Zhang, Baozhen; Zhou, Jing; Zhu, Bu-Dong; Ji, Jiafu; Deng, Dajun

2010-01-01

Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation. Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0. From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A ~ Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%). Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively

High-performance liquid chromatographic radioenzymatic assay for plasma catecyholamines

International Nuclear Information System (INIS)

Klaniecki, T.S.; Corder, C.N.; McDonald, R.H. Jr.; Feldman, J.A.

1977-01-01

A new assay method for plasma catecholamimes (CA) requiring only 50 μl has been developed, which uses high performance liquid chromatography (HPLC). The norepinephrine (NE), dopamine (D), and epinephrine (E) compounds found in plasma are radioactively o-methylated with S-[methyl- 3 H]-adenosyl-L-methionine ( 3 H-SAM) 3 H-SAM by the reaction of catechol-o-methyl transferase (COMT). The reaction is terminated and a standard mixture of nonradioactive o-methylated analogues of NE, D, and E is added to act as a carrier. Following separation by HPLC, the D,L-normetanephrine (NMN), 3-methoxy-4-hydroxyphenylethyl-amine or 3-methoxytyramine (3-MOT), and metanephrine (MN) radioactive peaks are collected which represent NE, D, and E, respectively. Then MNM and MN are oxidized to vanillin, and 3-MOT is acetylated. The products are subsequently separated by solvent extraction. This is necessary in order to avoid high radioactive blanks and to allow quantitation of the radioactivity by liquid scintillation spectrometry. The mean supine levels of NE, D, and E in normal subjects were respectively 182, 33, and 87 pg/ml of plasma. Similar assays on patients with pheochromocytoma revealed 797, 80, and 470 pg/ml

Genetic and DNA methylation changes in cotton (Gossypium genotypes and tissues.

Directory of Open Access Journals (Sweden)

Kenji Osabe

Full Text Available In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP assays including a methylation insensitive enzyme (BsiSI, and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC. DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP.

Genetic and DNA methylation changes in cotton (Gossypium) genotypes and tissues.

Science.gov (United States)

Osabe, Kenji; Clement, Jenny D; Bedon, Frank; Pettolino, Filomena A; Ziolkowski, Lisa; Llewellyn, Danny J; Finnegan, E Jean; Wilson, Iain W

2014-01-01

In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP) assays including a methylation insensitive enzyme (BsiSI), and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC). DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation) in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP.

Synthesis of tritium labelled methyl vinyl ketone and its use in copolymer analysis

International Nuclear Information System (INIS)

Burfield, D.R.; Savariar, C.M.

1980-01-01

The synthesis of tritiated methyl vinyl ketone by base catalysed exchange and its use in determining the ketone content of styrene/methyl vinyl ketone copolymers are reported. Methods of assay are described in detail and the general applicability of the method is discussed. (author)

Heme polymerization inhibition activity (HPIA) assay of synthesized xanthone derivative as antimalarial compound

Science.gov (United States)

Fitriastuti, Dhina; Jumina, Priatmoko

2017-03-01

Xanthone is a phenolic secondary metabolite of Garcinia and Calophyllum herbs which has been clinically proven to display anti malaria activity. In the present paper, 2,3,4-trihydroxy-5-methyl xanthone which has been synthesized from gallic acid and o-cresol in Eaton's reagent was tested for its activity as antimalarial. Thus, HPIA assay of the synthesized xanthones was successfully conducted. The HPIA assay was carried out towards the xanthone, chloroquine diphosphate as positive control and distilled water as negative control in various concentration. The samples were reacted with hematin (ferriprotoporphyrin IX hydroxide) and the absorbance of the precipitate was observed by using Elisa reader. The results of HPIA assay showed that 2,3,4-trihydroxy-5-methyl xanthone and chloroquine have IC50 values of 0.755 and 1.462 mg/mL or 2.92 and 4.57 mM, respectively. 2,3,4-Trihydroxy-5-methyl xanthone displayed better antimalarial activity than chloroquine.

Whole-genome DNA methylation status associated with clinical PTSD measures of OIF/OEF veterans

Science.gov (United States)

Hammamieh, R; Chakraborty, N; Gautam, A; Muhie, S; Yang, R; Donohue, D; Kumar, R; Daigle, B J; Zhang, Y; Amara, D A; Miller, S-A; Srinivasan, S; Flory, J; Yehuda, R; Petzold, L; Wolkowitz, O M; Mellon, S H; Hood, L; Doyle, F J; Marmar, C; Jett, M

2017-01-01

Emerging knowledge suggests that post-traumatic stress disorder (PTSD) pathophysiology is linked to the patients’ epigenetic changes, but comprehensive studies examining genome-wide methylation have not been performed. In this study, we examined genome-wide DNA methylation in peripheral whole blood in combat veterans with and without PTSD to ascertain differentially methylated probes. Discovery was initially made in a training sample comprising 48 male Operation Enduring Freedom (OEF)/Operation Iraqi Freedom (OIF) veterans with PTSD and 51 age/ethnicity/gender-matched combat-exposed PTSD-negative controls. Agilent whole-genome array detected ~5600 differentially methylated CpG islands (CpGI) annotated to ~2800 differently methylated genes (DMGs). The majority (84.5%) of these CpGIs were hypermethylated in the PTSD cases. Functional analysis was performed using the DMGs encoding the promoter-bound CpGIs to identify networks related to PTSD. The identified networks were further validated by an independent test set comprising 31 PTSD+/29 PTSD− veterans. Targeted bisulfite sequencing was also used to confirm the methylation status of 20 DMGs shown to be highly perturbed in the training set. To improve the statistical power and mitigate the assay bias and batch effects, a union set combining both training and test set was assayed using a different platform from Illumina. The pathways curated from this analysis confirmed 65% of the pool of pathways mined from training and test sets. The results highlight the importance of assay methodology and use of independent samples for discovery and validation of differentially methylated genes mined from whole blood. Nonetheless, the current study demonstrates that several important epigenetically altered networks may distinguish combat-exposed veterans with and without PTSD. PMID:28696412

Experimental vapor pressures (from 1 Pa to 100 kPa) of six saturated Fatty Acid Methyl Esters (FAMEs): Methyl hexanoate, methyl octanoate, methyl decanoate, methyl dodecanoate, methyl tetradecanoate and methyl hexadecanoate

International Nuclear Information System (INIS)

Sahraoui, Lakhdar; Khimeche, Kamel; Dahmani, Abdallah; Mokbel, Ilham; Jose, Jacques

2016-01-01

Highlight: • Vapor-liquid equilibria, Enthalpy of Vaporization, saturated Fatty Acid Methyl Ester. - Abstract: Vapor pressures of six saturated Fatty Acid Methyl Esters (FAMEs), methyl hexanoate (or methyl caproate), methyl octanoate (or methyl caprylate), Methyl decanoate (or methyl caprate), methyl dodecanoate (or methyl laurate), methyl tetradecanoate (or methyl myristate), and methyl hexadecanoate (or methyl palmitate) were measured from 1 Pa to 100 kPa and at temperature range between 262 and 453 K using a static apparatus. The experimental data (P-T) were compared with the available literature data.

DNA methylation in inflammatory genes among children with obstructive sleep apnea.

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Kim, Jinkwan; Bhattacharjee, Rakesh; Khalyfa, Abdelnaby; Kheirandish-Gozal, Leila; Capdevila, Oscar Sans; Wang, Yang; Gozal, David

2012-02-01

Pediatric obstructive sleep apnea (OSA) leads to multiple end-organ morbidities that are mediated by the cumulative burden of oxidative stress and inflammation. Because not all children with OSA exhibit increased systemic inflammation, genetic and environmental factors may be affecting patterns of DNA methylation in genes subserving inflammatory functions. DNA from matched children with OSA with and without high levels of high-sensitivity C-reactive protein (hsCRP) were assessed for DNA methylation levels of 24 inflammatory-related genes. Primer-based polymerase chain reaction assays in a case-control setting involving 47 OSA cases and 31 control subjects were conducted to confirm the findings; hsCRP and myeloid-related protein (MRP) 8/14 levels were also assayed. Forkhead box P3 (FOXP3) and interferon regulatory factor 1 (IRF1) showed higher methylation in six children with OSA and high hsCRP levels compared with matched children with OSA and low hsCRP levels (P DNA methylation levels compared with children with OSA and low CRP levels and control subjects. IRF1 did not exhibit significant differences. FOXP3 DNA methylation levels correlated with hsCRP and MRP 8/14 levels and with apnea-hypopnea index (AHI), BMI z score, and apolipoprotein B levels. A stepwise multiple regression model showed that AHI was independently associated with FOXP3 DNA methylation levels (P gene, which regulates expression of T regulatory lymphocytes, is more likely to display increased methylation among children with OSA who exhibit increased systemic inflammatory responses. Thus, epigenetic modifications may constitute an important determinant of inflammatory phenotype in OSA, and FOXP3 DNA methylation levels may provide a potential biomarker for end-organ vulnerability.

Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells

International Nuclear Information System (INIS)

Liu, Huaying; Li, Guiyuan; Zhang, Liming; Niu, Zhaoxia; Zhou, Ming; Peng, Cong; Li, Xiayu; Deng, Tan; Shi, Lei; Tan, Yixin

2008-01-01

Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription. The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter. We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals. BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC

A refined, rapid and reproducible high resolution melt (HRM-based method suitable for quantification of global LINE-1 repetitive element methylation

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Tse M Yat

2011-12-01

Full Text Available Abstract Background The methylation of DNA is recognized as a key mechanism in the regulation of genomic stability and evidence for its role in the development of cancer is accumulating. LINE-1 methylation status represents a surrogate measure of genome-wide methylation. Findings Using high resolution melt (HRM curve analysis technology, we have established an in-tube assay that is linear (r > 0.9986 with a high amplification efficiency (90-105%, capable of discriminating between partcipant samples with small differences in methylation, and suitable for quantifying a wide range of LINE-1 methylation levels (0-100%--including the biologically relevant range of 50-90% expected in human DNA. We have optimized this procedure to perform using 2 μg of starting DNA and 2 ng of bisulfite-converted DNA for each PCR reaction. Intra- and inter-assay coefficients of variation were 1.44% and 0.49%, respectively, supporting the high reproducibility and precision of this approach. Conclusions In summary, this is a completely linear, quantitative HRM PCR method developed for the measurement of LINE-1 methylation. This cost-efficient, refined and reproducible assay can be performed using minimal amounts of starting DNA. These features make our assay suitable for high throughput analysis of multiple samples from large population-based studies.

Multiple sporadic colorectal cancers display a unique methylation phenotype.

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Victoria Gonzalo

Full Text Available Epigenetics are thought to play a major role in the carcinogenesis of multiple sporadic colorectal cancers (CRC. Previous studies have suggested concordant DNA hypermethylation between tumor pairs. However, only a few methylation markers have been analyzed. This study was aimed at describing the epigenetic signature of multiple CRC using a genome-scale DNA methylation profiling. We analyzed 12 patients with synchronous CRC and 29 age-, sex-, and tumor location-paired patients with solitary tumors from the EPICOLON II cohort. DNA methylation profiling was performed using the Illumina Infinium HM27 DNA methylation assay. The most significant results were validated by Methylight. Tumors samples were also analyzed for the CpG Island Methylator Phenotype (CIMP; KRAS and BRAF mutations and mismatch repair deficiency status. Functional annotation clustering was performed. We identified 102 CpG sites that showed significant DNA hypermethylation in multiple tumors with respect to the solitary counterparts (difference in β value ≥0.1. Methylight assays validated the results for 4 selected genes (p = 0.0002. Eight out of 12(66.6% multiple tumors were classified as CIMP-high, as compared to 5 out of 29(17.2% solitary tumors (p = 0.004. Interestingly, 76 out of the 102 (74.5% hypermethylated CpG sites found in multiple tumors were also seen in CIMP-high tumors. Functional analysis of hypermethylated genes found in multiple tumors showed enrichment of genes involved in different tumorigenic functions. In conclusion, multiple CRC are associated with a distinct methylation phenotype, with a close association between tumor multiplicity and CIMP-high. Our results may be important to unravel the underlying mechanism of tumor multiplicity.

Radio-methyl vorozole and methods for making and using the same

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Kim, Sung Won; Biegon, Anat; Fowler, Joanna S.

2014-08-12

Radiotracer vorozole compounds for in vivo and in vitro assaying, studying and imaging cytochrome P450 aromatase enzymes in humans, animals, and tissues and methods for making and using the same are provided. [N-radio-methyl] vorozole substantially separated from an N-3 radio-methyl isomer of vorozole is provided. Separation is accomplished through use of chromatography resins providing multiple mechanisms of selectivity.

MIRA: An R package for DNA methylation-based inference of regulatory activity.

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Lawson, John T; Tomazou, Eleni M; Bock, Christoph; Sheffield, Nathan C

2018-03-01

DNA methylation contains information about the regulatory state of the cell. MIRA aggregates genome-scale DNA methylation data into a DNA methylation profile for independent region sets with shared biological annotation. Using this profile, MIRA infers and scores the collective regulatory activity for each region set. MIRA facilitates regulatory analysis in situations where classical regulatory assays would be difficult and allows public sources of open chromatin and protein binding regions to be leveraged for novel insight into the regulatory state of DNA methylation datasets. R package available on Bioconductor: http://bioconductor.org/packages/release/bioc/html/MIRA.html. nsheffield@virginia.edu.

Human Papillomavirus DNA Methylation as a Biomarker for Cervical Precancer: Consistency across 12 Genotypes and Potential Impact on Management of HPV-Positive Women.

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Clarke, Megan A; Gradissimo, Ana; Schiffman, Mark; Lam, Jessica; Sollecito, Christopher C; Fetterman, Barbara; Lorey, Thomas; Poitras, Nancy; Raine-Bennett, Tina R; Castle, Philip E; Wentzensen, Nicolas; Burk, Robert D

2018-05-01

Purpose: Human papillomavirus (HPV) DNA methylation testing is a promising triage option for women testing HPV positive during cervical cancer screening. However, the extent to which methylation indicates precancer for all 12 carcinogenic HPV types has not been evaluated. Experimental Design: In this nested case-control study, we tested up to 30 cases of precancer [cervical intraepithelial neoplasia grade 3 (CIN3)/adenocarcinoma in situ (AIS)] and 30 normal controls for each carcinogenic type (single infections with 16/18/31/33/35/39/45/51/52/56/58/59). Next-generation bisulfite sequencing was performed on CpG sites within the L1 and L2 genes. We calculated differences in methylation, ORs, and AUC. Using a fixed sensitivity of 80%, we evaluated the specificity and the risk of CIN3/AIS for best performing CpG sites, and compared the performance of an explorative multi-type methylation assay with current triage strategies. Results: Methylation was positively associated with CIN3/AIS across all 12 types. AUCs for the top sites ranged from 0.71 (HPV51 and HPV56) to 0.86 (HPV18). A combined 12-type methylation assay had the highest Youden index (0.46), compared with cytology (0.31) and a 5-type methylation assay, including only previously described types (0.26). The 12-type methylation assay had higher sensitivity (80% vs. 76.6%) and lower test positivity compared with cytology (38.5% vs. 48.7%). The risk of CIN3/AIS was highest for methylation positives and lowest for cytology or HPV16/18 positives. Conclusions: HPV DNA methylation is a general phenomenon marking the transition from HPV infection to precancer for all 12 carcinogenic types. Development of a combined multitype methylation assay may serve as a triage test for HPV-positive women. Clin Cancer Res; 24(9); 2194-202. ©2018 AACR . ©2018 American Association for Cancer Research.

Methylation profiling identified novel differentially methylated markers including OPCML and FLRT2 in prostate cancer.

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Wu, Yu; Davison, Jerry; Qu, Xiaoyu; Morrissey, Colm; Storer, Barry; Brown, Lisha; Vessella, Robert; Nelson, Peter; Fang, Min

2016-04-02

To develop new methods to distinguish indolent from aggressive prostate cancers (PCa), we utilized comprehensive high-throughput array-based relative methylation (CHARM) assay to identify differentially methylated regions (DMRs) throughout the genome, including both CpG island (CGI) and non-CGI regions in PCa patients based on Gleason grade. Initially, 26 samples, including 8 each of low [Gleason score (GS) 6] and high (GS ≥7) grade PCa samples and 10 matched normal prostate tissues, were analyzed as a discovery cohort. We identified 3,567 DMRs between normal and cancer tissues, and 913 DMRs distinguishing low from high-grade cancers. Most of these DMRs were located at CGI shores. The top 5 candidate DMRs from the low vs. high Gleason comparison, including OPCML, ELAVL2, EXT1, IRX5, and FLRT2, were validated by pyrosequencing using the discovery cohort. OPCML and FLRT2 were further validated in an independent cohort consisting of 20 low-Gleason and 33 high-Gleason tissues. We then compared patients with biochemical recurrence (n=70) vs. those without (n=86) in a third cohort, and they showed no difference in methylation at these DMR loci. When GS 3+4 cases and GS 4+3 cases were compared, OPCML-DMR methylation showed a trend of lower methylation in the recurrence group (n=30) than in the no-recurrence (n=52) group. We conclude that whole-genome methylation profiling with CHARM revealed distinct patterns of differential DNA methylation between normal prostate and PCa tissues, as well as between different risk groups of PCa as defined by Gleason scores. A panel of selected DMRs may serve as novel surrogate biomarkers for Gleason score in PCa.

Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape

International Nuclear Information System (INIS)

Snellenberg, Suzanne; Strooper, Lise MA De; Hesselink, Albertus T; Meijer, Chris JLM; Snijders, Peter JF; Heideman, Daniëlle AM; Steenbergen, Renske DM

2012-01-01

Quantitative methylation-specific PCR (qMSP) analysis for determining the methylation status of (candidate) tumor suppressor genes has potential as objective and valuable test to triage high-risk human papillomavirus (hrHPV) positive women in cervical screening. Particularly combined methylation analysis of a panel of genes shows most promising clinical performance, with sensitivity levels that equal or exceed that of cytology. However, the wide application of such methylation marker panels is hampered by the lack of effective multiplex assays allowing simultaneous methylation detection of various targets in a single reaction. Here, we designed and analyzed a multiplex qMSP assay for three genes whose methylation was previously found to be informative for cervical (pre)cancer (i.e. CADM1, MAL and hsa-miR-124-2) as well as a reference gene β-actin. Based on our experience, we discuss the optimization of the parameters that provide a practical approach towards multiplex qMSP design. Primers and PCR reagents were optimized for multiplex qMSP purposes and the resulting assay was analytically validated on serial dilutions of methylated DNA in unmethylated DNA, and compared with singleplex counterparts on hrHPV-positive cervical scrapings. Upon optimization, including primer redesign and primer limiting assays, the multiplex qMSP showed the same analytical performance as the singleplex qMSPs. A strong correlation between the obtained normalized ratios of the singleplex and multiplex qMSPs on cervical scrapes was found for all three markers: CADM1 (R 2 =0.985), MAL (R 2 =0.986) and hsa-miR-124-2 (R 2 =0.944). Multiplex qMSP offers a promising approach for high-throughput diagnostic analysis of the methylation status of multiple genes, which after proper design and validation can be equally specific, sensitive and reproducible as its singleplex versions

Region of interest methylation analysis: a comparison of MSP with MS-HRM and direct BSP.

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Akika, Reem; Awada, Zainab; Mogharbil, Nahed; Zgheib, Nathalie K

2017-07-01

The aim of this study was to compare and contrast three DNA methylation methods of a specific region of interest (ROI): methylation-specific PCR (MSP), methylation-sensitive high resolution melting (MS-HRM) and direct bisulfite sequencing (BSP). The methylation of a CpG area in the promoter region of Estrogen receptor alpha (ESR1) was evaluated by these three methods with samples and standards of different methylation percentages. MSP data were neither reproducible nor sensitive, and the assay was not specific due to non-specific binding of primers. MS-HRM was highly reproducible and a step forward into categorizing the methylation status of the samples as percent ranges. Direct BSP was the most informative method regarding methylation percentage of each CpG site. Though not perfect, it was reproducible and sensitive. We recommend the use of either method depending on the research question and target amplicon, and provided that the designed primers and expected amplicons are within recommendations. If the research question targets a limited number of CpG sites and simple yes/no results are enough, MSP may be attempted. For short amplicons that are crowded with CpG sites and of single melting domain, MS-HRM may be the method of choice though it only indicates the overall methylation percentage of the entire amplicon. Although the assay is highly reproducible, being semi-quantitative makes it of lesser interest to study ROI methylation of samples with little methylation differences. Direct BSP is a step forward as it gives information about the methylation percentage at each CpG site.

Quantification of 5-methyl-2'-deoxycytidine in the DNA.

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Giel-Pietraszuk, Małgorzata; Insińska-Rak, Małgorzata; Golczak, Anna; Sikorski, Marek; Barciszewska, Mirosława; Barciszewski, Jan

2015-01-01

Methylation at position 5 of cytosine (Cyt) at the CpG sequences leading to formation of 5-methyl-cytosine (m(5)Cyt) is an important element of epigenetic regulation of gene expression. Modification of the normal methylation pattern, unique to each organism, leads to the development of pathological processes and diseases, including cancer. Therefore, quantification of the DNA methylation and analysis of changes in the methylation pattern is very important from a practical point of view and can be used for diagnostic purposes, as well as monitoring of the treatment progress. In this paper we present a new method for quantification of 5-methyl-2'deoxycytidine (m(5)C) in the DNA. The technique is based on conversion of m(5)C into fluorescent 3,N(4)-etheno-5-methyl-2'deoxycytidine (εm(5)C) and its identification by reversed-phase high-performance liquid chromatography (RP-HPLC). The assay was used to evaluate m(5)C concentration in DNA of calf thymus and peripheral blood of cows bred under different conditions. This approach can be applied for measuring of 5-methylcytosine in cellular DNA from different cells and tissues.

Identifying DNA Methylation Biomarkers for Non-Endoscopic Detection of Barrett’s Esophagus

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Moinova, Helen R.; LaFramboise, Thomas; Lutterbaugh, James D.; Chandar, Apoorva Krishna; Dumot, John; Faulx, Ashley; Brock, Wendy; De la Cruz Cabrera, Omar; Guda, Kishore; Barnholtz-Sloan, Jill S.; Iyer, Prasad G.; Canto, Marcia I.; Wang, Jean S.; Shaheen, Nicholas J.; Thota, Prashanti N.; Willis, Joseph E.; Chak, Amitabh; Markowitz, Sanford D.

2018-01-01

We report a biomarker-based non-endoscopic method for detecting Barrett’s esophagus (BE), based on detecting methylated DNAs retrieved via a swallowable balloon-based esophageal sampling device. BE is the precursor of, and a major recognized risk factor for, developing esophageal adenocarcinoma (EAC). Endoscopy, the current standard for BE detection, is not cost-effective for population screening. We performed genome-wide screening to ascertain regions targeted for recurrent aberrant cytosine methylation in BE, identifying high-frequency methylation within the CCNA1 locus. We tested CCNA1 DNA methylation as a BE biomarker in cytology brushings of the distal esophagus from 173 individuals with or without BE. CCNA1 DNA methylation demonstrated an area under the curve (AUC)=0.95 for discriminating BE-related metaplasia and neoplasia cases versus normal individuals, performing identically to methylation of VIM DNA, an established BE biomarker. When combined, the resulting two biomarker panel was 95% sensitive and 91% specific. These results were replicated in an independent validation cohort of 149 individuals, who were assayed using the same cutoff values for test positivity established in the training population. To progress toward non-endoscopic esophageal screening, we engineered a well-tolerated, swallowable, encapsulated balloon device able to selectively sample the distal esophagus within 5 minutes. In balloon samples from 86 individuals, tests of CCNA1 plus VIM DNA methylation detected BE metaplasia with 90.3% sensitivity and 91.7% specificity. Combining the balloon sampling device with molecular assays of CCNA1 plus VIM DNA methylation enables an efficient, well-tolerated, sensitive, and specific method of screening at-risk populations for BE. PMID:29343623

Analysis of mutation/rearrangement frequencies and methylation patterns at a given DNA locus using restriction fragment length polymorphism.

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Boyko, Alex; Kovalchuk, Igor

2010-01-01

Restriction fragment length polymorphism (RFLP) is a difference in DNA sequences of organisms belonging to the same species. RFLPs are typically detected as DNA fragments of different lengths after digestion with various restriction endonucleases. The comparison of RFLPs allows investigators to analyze the frequency of occurrence of mutations, such as point mutations, deletions, insertions, and gross chromosomal rearrangements, in the progeny of stressed plants. The assay involves restriction enzyme digestion of DNA followed by hybridization of digested DNA using a radioactively or enzymatically labeled probe. Since DNA can be digested with methylation sensitive enzymes, the assay can also be used to analyze a methylation pattern of a particular locus. Here, we describe RFLP analysis using methylation-insensitive and methylation-sensitive enzymes.

CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.

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Ogino, S; Cantor, M; Kawasaki, T; Brahmandam, M; Kirkner, G J; Weisenberger, D J; Campan, M; Laird, P W; Loda, M; Fuchs, C S

2006-07-01

The concept of CpG island methylator phenotype (CIMP) is not universally accepted. Even if specific clinicopathological features have been associated with CIMP, investigators often failed to demonstrate a bimodal distribution of the number of methylated markers, which would suggest CIMP as a distinct subtype of colorectal cancer. Previous studies primarily used methylation specific polymerase chain reaction which might detect biologically insignificant low levels of methylation. To demonstrate a distinct genetic profile of CIMP colorectal cancer using quantitative DNA methylation analysis that can distinguish high from low levels of DNA methylation. We developed quantitative real time polymerase chain reaction (MethyLight) assays and measured DNA methylation (percentage of methylated reference) of five carefully selected loci (promoters of CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1) in 460 colorectal cancers from large prospective cohorts. There was a clear bimodal distribution of 80 microsatellite instability-high (MSI-H) tumours according to the number of methylated promoters, with no tumours showing 3/5 methylated loci. Thus we defined CIMP as having >or=4/5 methylated loci, and 17% (78) of the 460 tumours were classified as CIMP. CIMP was significantly associated with female sex, MSI, BRAF mutations, and wild-type KRAS. Both CIMP MSI-H tumours and CIMP microsatellite stable (MSS) tumours showed much higher frequencies of BRAF mutations (63% and 54%) than non-CIMP counterparts (non-CIMP MSI-H (0%, pCIMP MSS tumours (6.6%, pCIMP is best characterised by quantitative DNA methylation analysis. CIMP is a distinct epigenotype of colorectal cancer and may be less frequent than previously reported.

Practical spectrophotometric assay for the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase, a potential antibiotic target.

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Heath, Tahirah K; Lutz, Marlon R; Reidl, Cory T; Guzman, Estefany R; Herbert, Claire A; Nocek, Boguslaw P; Holz, Richard C; Olsen, Kenneth W; Ballicora, Miguel A; Becker, Daniel P

2018-01-01

A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [± 0.2] μM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] μM, phenylboronic acid (IC50 = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] μM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics.

Practical spectrophotometric assay for the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase, a potential antibiotic target.

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Tahirah K Heath

Full Text Available A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18 is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I(COD(S,S-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE including captopril (IC50 = 3.4 [± 0.2] μM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] μM, phenylboronic acid (IC50 = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] μM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics.

Effect of temperature stress on protein methyl esters

International Nuclear Information System (INIS)

Welch, W.; Kracaw, K.

1986-01-01

Protein methyl esters have been implicated in a number of physiological processes. They have measured the effect of temperature stress on the levels of protein methyl esters in the mesophilic fungus Penicillium chrysogenum (PCPS) and the thermophilic fungus P. duponti (PD). PD and PCPS were incubated with [methyl- 3 H]methionine. The mycelia were collected by filtration, frozen in liquid nitrogen and ground to a fine powder. The nitrogen powder was extracted with either phosphate buffer or with SDS, glycerol, phosphate, 2-mercaptoethanol. Insoluble material was removed by centrifugation. The supernatants were assayed for protein methyl esters. The released [ 3 H]methanol was extracted into toluene:isoamyl alcohol (3:2) and quantitated by liquid scintillation. The production of volatile methanol was confirmed by use of Conway diffusion cells. Soluble proteins accounted for about one-fourth of the total protein methyl ester extracted by SDS. In PCPS, the SDS extracted proteins have about three times the level of esterification of the soluble proteins whereas in PD there is little difference between soluble and SDS extracted protein. The level of protein esterification in PD is about one-tenth that observed in PCPS. Temperature stress caused large changes in the level of protein esterification. The data suggest protein methyl esters may contribute to the adaptation to environmental stress

Development of fluorescent methods for DNA methyltransferase assay

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Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

2017-03-01

DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

High resolution melt curve analysis based on methylation status for human semen identification.

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Fachet, Caitlyn; Quarino, Lawrence; Karnas, K Joy

2017-03-01

A high resolution melt curve assay to differentiate semen from blood, saliva, urine, and vaginal fluid based on methylation status at the Dapper Isoform 1 (DACT1) gene was developed. Stains made from blood, saliva, urine, semen, and vaginal fluid were obtained from volunteers and DNA was isolated using either organic extraction (saliva, urine, and vaginal fluid) or Chelex ® 100 extraction (blood and semen). Extracts were then subjected to bisulfite modification in order to convert unmethylated cytosines to uracil, consequently creating sequences whose amplicons have melt curves that vary depending on their initial methylation status. When primers designed to amplify the promoter region of the DACT1 gene were used, DNA from semen samples was distinguishable from other fluids by a having a statistically significant lower melting temperature. The assay was found to be sperm-significant since semen from a vasectomized man produced a melting temperature similar to the non-semen body fluids. Blood and semen stains stored up to 5 months and tested at various intervals showed little variation in melt temperature indicating the methylation status was stable during the course of the study. The assay is a more viable method for forensic science practice than most molecular-based methods for body fluid stain identification since it is time efficient and utilizes instrumentation common to forensic biology laboratories. In addition, the assay is advantageous over traditional presumptive chemical methods for body fluid identification since results are confirmatory and the assay offers the possibility of multiplexing which may test for multiple body fluids simultaneously.

An easy-to-perform photometric assay for methyltransferase activity measurements.

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Schäberle, Till F; Siba, Christian; Höver, Thomas; König, Gabriele M

2013-01-01

Methyltransferases (MTs) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to a suitable substrate. Such methylations are important modifications in secondary metabolisms, especially on natural products produced by polyketide synthases and nonribosomal peptide synthetases, many of which are of special interest due to their prominent pharmacological activities (e.g., lovastatin, cyclosporin). To gain basic biochemical knowledge on the methylation process, it is of immense relevance to simplify methods concerning experimental problems caused by a large variety in substrates. Here, we present a photometric method to analyze MT activity by measuring SAM consumption in a coupled enzyme assay. Copyright © 2012 Elsevier Inc. All rights reserved.

Protective effects of folic acid on DNA damage and DNA methylation levels induced by N-methyl- N'-nitro- N-nitrosoguanidine in Kazakh esophageal epithelial cells.

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Chen, Y; Feng, H; Chen, D; Abuduwaili, K; Li, X; Zhang, H

2018-01-01

The protective effects of folic acid on DNA damage and DNA methylation induced by N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) in Kazakh esophageal epithelial cells were investigated using a 3 × 3 factorial design trial. The cells were cultured in vitro and exposed to media containing different concentrations of folic acid and MNNG, after which growth indices were detected. DNA damage levels were measured using comet assays, and genome-wide DNA methylation levels (MLs) were measured using high-performance liquid chromatography. The DNA methylation of methylenetetrahydrofolate reductase (MTHFR) and folate receptor- α (FR α) genes was detected by bisulfite sequencing polymerase chain reaction (PCR). The results showed significant increases in tail DNA concentration, tail length, and Olive tail moment ( p methylation frequencies of MTHFR and FR α genes. In particular, significant differences were observed in the promoter regions of both genes ( p methylation in Kazakh esophageal epithelial cells upon MNNG exposure. Thus, sufficient folic acid levels could play a protective role against the damage induced by this compound.

A tissue biopsy-based epigenetic multiplex PCR assay for prostate cancer detection

Directory of Open Access Journals (Sweden)

Van Neste Leander

2012-06-01

Full Text Available Abstract Background PSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1, APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence. Results An initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the GSTP1, APC and RASSF1 genes, using methylation specific PCR (MSP. The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity. Conclusions The developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples.

The effect of methyl-lidocaine on the biosynthesis of phospholipids de novo in the isolated hamster heart.

OpenAIRE

Tardi, P G; Man, R Y; Choy, P C

1992-01-01

Methyl-lidocaine is an amphiphilic agent which has been used as an experimental anti-arrhythmic drug. When hamster hearts were perfused with labelled glycerol, the presence of methyl-lidocaine in the perfusate was found to enhance the labelling in phosphatidylserine, phosphatidylinositol, diacylglycerol and triacylglycerol. However, the labelling of phosphatidylcholine and phosphatidylethanolamine was not significantly changed by methyl-lidocaine treatment. Assays in vitro for the enzymes inv...

Analysis of the state of posttranslational calmodulin methylation in developing pea plants

International Nuclear Information System (INIS)

Oh, Sukheung; Roberts, D.M.

1990-01-01

A specific calmodulin-N-methyltransferase was used in a radiometric assay to analyze the degree of methylation of lysine-115 in pea (Pisum sativum) plants. Calmodulin was isolated from dissected segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by incubation with the calmodulin methyltransferase in the presence of [ 3 H]methyl-S-adenosylmethionine. By this approach, the presence of unmethylated calmodulins were demonstrated in pea tissues, and the levels of methylation varied depending on the developmental state of the tissue tested. Calmodulin methylation levels were lower in apical root segments of both etiolated and green plants, and in the young lateral roots compared with the mature, differentiated root tissues. The incorporation of methyl groups into these calmodulin samples appears to be specific for position 115 since site-directed mutants of calmodulin with substitutions at this position competitively inhibited methyl group incorporation. The present findings, combined with previous data showing differences in the ability of methylated and unmethylated calmodulins to activate pea NAD kinase raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity

Cadmium inhibits repair of UV-, methyl methanesulfonate- and N-methyl-N-nitrosourea-induced DNA damage in Chinese hamster ovary cells

International Nuclear Information System (INIS)

Fatur, Tanja; Lah, Tamara T.; Filipic, Metka

2003-01-01

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl 2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl 2 , the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl 2 the MMS-induced DNA strand breaks accumulated during the first 2 h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl 2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair

Association of postmenopausal endogenous sex hormones with global methylation level of leukocyte DNA among Japanese women

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Iwasaki Motoki

2012-07-01

Full Text Available Abstract Background Although global hypomethylation of leukocyte DNA has been associated with an increased risk of several sites of cancer, including breast cancer, determinants of global methylation level among healthy individuals remain largely unexplored. Here, we examined whether postmenopausal endogenous sex hormones were associated with the global methylation level of leukocyte DNA. Methods A cross-sectional study was conducted using the control group of a breast cancer case–control study in Nagano, Japan. Subjects were postmenopausal women aged 55 years or over who provided blood samples. We measured global methylation level of peripheral blood leukocyte DNA by luminometric methylation assay; estradiol, estrone, androstenedione, dehydroepiandrosterone sulfate, testosterone and free testosterone by radioimmunoassay; bioavailable estradiol by the ammonium sulfate precipitation method; and sex-hormone binding globulin by immunoradiometric assay. A linear trend of association between methylation and hormone levels was evaluated by regression coefficients in a multivariable liner regression model. A total of 185 women were included in the analyses. Results Mean global methylation level (standard deviation was 70.3% (3.1 and range was from 60.3% to 79.2%. Global methylation level decreased 0.27% per quartile category for estradiol and 0.39% per quartile category for estrone while it increased 0.41% per quartile category for bioavailable estradiol. However, we found no statistically significant association of any sex hormone level measured in the present study with global methylation level of leukocyte DNA. Conclusions Our findings suggest that endogenous sex hormones are not major determinants of the global methylation level of leukocyte DNA.

DNA methylation analysis from saliva samples for epidemiological studies.

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Nishitani, Shota; Parets, Sasha E; Haas, Brian W; Smith, Alicia K

2018-06-18

Saliva is a non-invasive, easily accessible tissue, which is regularly collected in large epidemiological studies to examine genetic questions. Recently, it is becoming more common to use saliva to assess DNA methylation. However, DNA extracted from saliva is a mixture of both bacterial and human DNA derived from epithelial and immune cells in the mouth. Thus, there are unique challenges to using salivary DNA in methylation studies that can influence data quality. This study assesses: (1) quantification of human DNA after extraction; (2) delineation of human and bacterial DNA; (3) bisulfite conversion (BSC); (4) quantification of BSC DNA; (5) PCR amplification of BSC DNA from saliva and; (6) quantitation of DNA methylation with a targeted assay. The framework proposed will allow saliva samples to be more widely used in targeted epigenetic studies.

A combined HM-PCR/SNuPE method for high sensitive detection of rare DNA methylation

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Tierling Sascha

2010-06-01

Full Text Available Abstract Background DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation. Results This combined method allows detection of 14 pg (that is, four to five genomic copies of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2 and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer. Conclusion The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.

Methyl-Analyzer--whole genome DNA methylation profiling.

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Xin, Yurong; Ge, Yongchao; Haghighi, Fatemeh G

2011-08-15

Methyl-Analyzer is a python package that analyzes genome-wide DNA methylation data produced by the Methyl-MAPS (methylation mapping analysis by paired-end sequencing) method. Methyl-MAPS is an enzymatic-based method that uses both methylation-sensitive and -dependent enzymes covering >80% of CpG dinucleotides within mammalian genomes. It combines enzymatic-based approaches with high-throughput next-generation sequencing technology to provide whole genome DNA methylation profiles. Methyl-Analyzer processes and integrates sequencing reads from methylated and unmethylated compartments and estimates CpG methylation probabilities at single base resolution. Methyl-Analyzer is available at http://github.com/epigenomics/methylmaps. Sample dataset is available for download at http://epigenomicspub.columbia.edu/methylanalyzer_data.html. fgh3@columbia.edu Supplementary data are available at Bioinformatics online.

Radioenzymatic assay of DOPA (3,4-dihydroxyphenylalanine)

International Nuclear Information System (INIS)

Johnson, G.A.; Gren, J.M.; Kupiecki, R.

1978-01-01

We modified the single-isotope radioenzymatic assay for catecholamines [Life Sci. 21, 625(1977)] to assay 3,4-dihydroxyphenylalanine (DOPA). DOPA decarboxylase is used to convert DOPA to dopamine, which concurrently is converted to [ 3 H]-3-O-methyldopamine in the presence of catechol-O-methyltransferase and [methyl- 3 H]-S-adenosylmethionine and assayed radioenzymatically. For assay of plasma DOPA, 50 μl of untreated plasma is added directly into the incubation mixture. A duplicate mixture containing an internal standard requires a second 50-μl aliquot of plasma. Because the assay measures both DOPA and endogenous dopamine, two additional aliquots of plasma must be assayed for dopamine in the absence of the decarboxylase by the differential assay; DOPA is estimated by difference. The assay is sensitive to 25 pg (500 ng/liter of plasma). Analysis of DOPA (DOPA plus dopamine) and the concurrent differential assay of catecholamines in at least 10 samples can be done in a single working day. Plasma DOPA concentrations for 42 normotensive adults were 1430 +- 19 ng/liter (mean +- SEM). In contrast, dopamine concentrations for these same subjects averaged 23 +- 20 ng/liter. Values for the 24 women subjects (1510 +- 62 ng/liter) significantly (P = 0.04) exceeded those for the men

Utility of MLH1 methylation analysis in the clinical evaluation of Lynch Syndrome in women with endometrial cancer.

Science.gov (United States)

Bruegl, Amanda S; Djordjevic, Bojana; Urbauer, Diana L; Westin, Shannon N; Soliman, Pamela T; Lu, Karen H; Luthra, Rajyalakshmi; Broaddus, Russell R

2014-01-01

Clinical screening criteria, such as young age of endometrial cancer diagnosis and family history of signature cancers, have traditionally been used to identify women with Lynch Syndrome, which is caused by mutation of a DNA mismatch repair gene. Immunohistochemistry and microsatellite instability analysis have evolved as important screening tools to evaluate endometrial cancer patients for Lynch Syndrome. A complicating factor is that 15-20% of sporadic endometrial cancers have immunohistochemical loss of the DNA mismatch repair protein MLH1 and high levels of microsatellite instability due to methylation of MLH1. The PCR-based MLH1 methylation assay potentially resolves this issue, yet many clinical laboratories do not perform this assay. The objective of this study was to determine if clinical and pathologic features help to distinguish sporadic endometrial carcinomas with MLH1 loss secondary to MLH1 methylation from Lynch Syndrome-associated endometrial carcinomas with MLH1 loss and absence of MLH1 methylation. Of 337 endometrial carcinomas examined, 54 had immunohistochemical loss of MLH1. 40/54 had MLH1 methylation and were designated as sporadic, while 14/54 lacked MLH1 methylation and were designated as Lynch Syndrome. Diabetes and deep myometrial invasion were associated with Lynch Syndrome; no other clinical or pathological variable distinguished the 2 groups. Combining Society of Gynecologic Oncology screening criteria with these 2 features accurately captured all Lynch Syndrome cases, but with low specificity. In summary, no single clinical/pathologic feature or screening criteria tool accurately identified all Lynch Syndrome-associated endometrial carcinomas, highlighting the importance of the MLH1 methylation assay in the clinical evaluation of these patients.

Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification

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Ito Takashi

2011-06-01

Full Text Available Abstract Background We previously developed a simple method termed HpaII-McrBC PCR (HM-PCR to discriminate allelic methylation status of the genomic sites of interest, and successfully applied it to a comprehensive analysis of CpG islands (CGIs on human chromosome 21q. However, HM-PCR requires 200 ng of genomic DNA to examine one target site, thereby precluding its application to such samples that are limited in quantity. Findings We developed HpaII-McrBC whole-genome-amplification PCR (HM-WGA-PCR that uses whole-genome-amplified DNA as the template. HM-WGA-PCR uses only 1/100th the genomic template material required for HM-PCR. Indeed, we successfully analyzed 147 CGIs by HM-WGA-PCR using only ~300 ng of DNA, whereas previous HM-PCR study had required ~30 μg. Furthermore, we confirmed that allelic methylation status revealed by HM-WGA-PCR is identical to that by HM-PCR in every case of the 147 CGIs tested, proving high consistency between the two methods. Conclusions HM-WGA-PCR would serve as a reliable alternative to HM-PCR in the analysis of allelic methylation status when the quantity of DNA available is limited.

Correlation between the methylation of APC gene promoter and colon cancer.

Science.gov (United States)

Li, Bing-Qiang; Liu, Peng-Peng; Zhang, Cai-Hua

2017-08-01

The present study was planned to explore the correlation between the methylation of APC (adenomatous polyposis coli) and colon carcinogenesis. Colon cancer tissues and tumor-adjacent normal tissues of 60 colon cancer patients (who received surgical operation in our hospital from January 2012 to December 2014) were collected. SW1116 cells in human colon cancer tissues were selected for culturing. 5-aza-2c-deoxycytidine (5-aza-dC) was utilized as an inhibitor of the methylation for APC gene. Methylation specific PCR (MSP) was utilized for detection of APC methylation in SW1116 cells. The MTT and Transwell assays were performed to detect the effect of the methylation of APC gene on the proliferation and invasive abilities of SW1116 cells. The correlation between the methylation of APC gene and pathological parameters of colon cancer patients was analyzed. MSP results revealed that 41 cases (68.33%) showed methylation of APC gene in colon cancer tissues. No methylation of APC gene was found in tumor-adjacent normal tissues. 5-aza-dC was able to inhibit the methylation of APC gene in SW1116 cells. APC gene methylation was correlated with tumor size, differentiation degree, lymph node metastasis and Dukes staging. In conclusion, the levels of the methylation of APC in colon cancer tissues and SW1116 cells are relatively high. The methylation of APC promoted the proliferation and invasion abilities of SW1116 cells. Furthermore, methylation is correlated with a variety of clinicopathological features of colon cancer patients.

Subsets of microsatellite-unstable colorectal cancers exhibit discordance between the CpG island methylator phenotype and MLH1 methylation status.

Science.gov (United States)

Kim, Jung H; Rhee, Ye-Y; Bae, Jeong-M; Kwon, Hyeong-J; Cho, Nam-Y; Kim, Mi J; Kang, Gyeong H

2013-07-01

Although the presence of MLH1 methylation in microsatellite-unstable colorectal cancer generally indicates involvement of the CpG island methylator phenotype (CIMP) in the development of the tumor, these two conditions do not always correlate. A minority of microsatellite-unstable colorectal cancers exhibit discordance between CIMP and MLH1 methylation statuses. However, the clinicopathological features of such microsatellite-unstable colorectal cancers with discrepant MLH1 methylation and CIMP statuses remain poorly studied. Microsatellite-unstable colorectal cancers (n=220) were analyzed for CIMP and MLH1 methylation statuses using the MethyLight assay. Based on the combinatorial CIMP and MLH1 methylation statuses, the microsatellite-unstable colorectal cancers were grouped into four subtypes (CIMP-high (CIMP-H) MLH1 methylation-positive (MLH1m+), CIMP-H MLH1 methylation-negative, CIMP-low/0 (CIMP-L/0) MLH1m+, and CIMP-L/0 MLH1 methylation-negative), which were compared in terms of their associations with clinicopathological and molecular features. The CIMP-L/0 MLH1 methylation-negative and CIMP-H MLH1m+ subtypes were predominant, comprising 63.6 and 24.1% of total microsatellite-unstable colorectal cancers, respectively. The discordant subtypes, CIMP-H MLH1 methylation-negative and CIMP-L/0 MLH1m+, were found in 5 and 7% of microsatellite-unstable colorectal cancers, respectively. The CIMP-H MLH1 methylation-negative subtype exhibited elevated incidence rates in male patients and was associated with larger tumor size, more frequent loss of MSH2 expression, increased frequency of KRAS mutation, and advanced cancer stage. The CIMP-L/0 MLH1m+ subtype was associated with onset at an earlier age, a predominance of MLH1 loss, and earlier cancer stage. None of the CIMP-L/0 MLH1m+ subtype patients succumbed to death during the follow-up. Our findings suggest that the discordant subtypes of colorectal cancers exhibit distinct clinicopathological and molecular features

Validation of SCT Methylation as a Hallmark Biomarker for Lung Cancers.

Science.gov (United States)

Zhang, Yu-An; Ma, Xiaotu; Sathe, Adwait; Fujimoto, Junya; Wistuba, Ignacio; Lam, Stephen; Yatabe, Yasushi; Wang, Yi-Wei; Stastny, Victor; Gao, Boning; Larsen, Jill E; Girard, Luc; Liu, Xiaoyun; Song, Kai; Behrens, Carmen; Kalhor, Neda; Xie, Yang; Zhang, Michael Q; Minna, John D; Gazdar, Adi F

2016-03-01

The human secretin gene (SCT) encodes secretin, a hormone with limited tissue distribution. Analysis of the 450k methylation array data in The Cancer Genome Atlas (TCGA) indicated that the SCT promoter region is differentially hypermethylated in lung cancer. Our purpose was to validate SCT methylation as a potential biomarker for lung cancer. We analyzed data from TCGA and developed and applied SCT-specific bisulfite DNA sequencing and quantitative methylation-specific polymerase chain reaction assays. The analyses of TCGA 450K data for 801 samples showed that SCT hypermethylation has an area under the curve (AUC) value greater than 0.98 that can be used to distinguish lung adenocarcinomas or squamous cell carcinomas from nonmalignant lung tissue. Bisulfite sequencing of lung cancer cell lines and normal blood cells allowed us to confirm that SCT methylation is highly discriminative. By applying a quantitative methylation-specific polymerase chain reaction assay, we found that SCT hypermethylation is frequently detected in all major subtypes of malignant non-small cell lung cancer (AUC = 0.92, n = 108) and small cell lung cancer (AUC = 0.93, n = 40) but is less frequent in lung carcinoids (AUC = 0.54, n = 20). SCT hypermethylation appeared in samples of lung carcinoma in situ during multistage pathogenesis and increased in invasive samples. Further analyses of TCGA 450k data showed that SCT hypermethylation is highly discriminative in most other types of malignant tumors but less frequent in low-grade malignant tumors. The only normal tissue with a high level of methylation was the placenta. Our findings demonstrated that SCT methylation is a highly discriminative biomarker for lung and other malignant tumors, is less frequent in low-grade malignant tumors (including lung carcinoids), and appears at the carcinoma in situ stage. Copyright © 2015 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination.

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Qin, Weihua; Wolf, Patricia; Liu, Nan; Link, Stephanie; Smets, Martha; La Mastra, Federica; Forné, Ignasi; Pichler, Garwin; Hörl, David; Fellinger, Karin; Spada, Fabio; Bonapace, Ian Marc; Imhof, Axel; Harz, Hartmann; Leonhardt, Heinrich

2015-08-01

DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.

Synthetic methyl hexagalacturonate hapten inhibitors of antihomogalacturonan monoclonal antibodies LM7, JIM5 and JIM7

DEFF Research Database (Denmark)

Clausen, Mads Hartvig; Willats, William George Tycho; Knox, J. Paul

2003-01-01

A range of synthetic methyl hexagalacturonates were used as potential hapten inhibitors in competitive-inhibition enzyme-linked immunosorbent assays (ELISAs) with anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. The selective inhibition of these antibodies by different haptens...... provides insight into the structures of the partially methyl-esterified pectin epitopes of these widely used monoclonal antibodies....

Methyl bromide fumigation and delayed mortality: safe trade of live pests?

Science.gov (United States)

Phillips, C B; Iline, I I; Novoselov, M; McNeill, M R; Richards, N K; van Koten, C; Stephenson, B P

Live organisms intercepted from treated commodities during phytosanitary inspections usually arouse suspicions of treatment failure, sub-standard treatment application, or post-treatment infestation. The additional possibility that some treatments could kill slowly, meaning commodities might be inspected before pests have succumbed, is seldom considered for treatments other than irradiation. We used a novel biochemical viability assay to measure delays between methyl bromide fumigation and mortality of dipteran eggs, and evaluated the correspondence between egg viability and egg morphological features. Our experimental conditions simulated shipping of rock melons from Australia to New Zealand by sea and air. No eggs survived fumigation, but they took 3-20 days to die, whereas phytosanitary inspections of rock melons occur within 2-7 days. Delays were not influenced by methyl bromide concentration, but were significantly lengthened by cooler storage temperatures. Methyl bromide's preservative effects delayed degradation of egg morphology, so the biochemical assay detected mortality long before morphological signs of egg death appeared. The results show that commodities subjected to effective methyl bromide treatments are at risk of being inspected before all pests have either died, or started to exhibit morphological signs of death. This could cause commodities to be unnecessarily rejected by quarantine authorities. Better methods than inspection for live pests are needed to assist authorities to gain assurance that treated commodities have been effectively disinfested. These could be developed by exploiting biochemical responses of pests and commodities to treatments.

msgbsR: An R package for analysing methylation-sensitive restriction enzyme sequencing data.

Science.gov (United States)

Mayne, Benjamin T; Leemaqz, Shalem Y; Buckberry, Sam; Rodriguez Lopez, Carlos M; Roberts, Claire T; Bianco-Miotto, Tina; Breen, James

2018-02-01

Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high diversity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between samples. To fill this computational need, we present msgbsR, an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the individual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package ( https://bioconductor.org/packages/release/bioc/html/msgbsR.html ).

HumMeth27QCReport: an R package for quality control and primary analysis of Illumina Infinium methylation data

Directory of Open Access Journals (Sweden)

Mancuso Francesco M

2011-12-01

Full Text Available Abstract Background The study of the human DNA methylome has gained particular interest in the last few years. Researchers can nowadays investigate the potential role of DNA methylation in common disorders by taking advantage of new high-throughput technologies. Among these, Illumina Infinium assays can interrogate the methylation levels of hundreds of thousands of CpG sites, offering an ideal solution for genome-wide methylation profiling. However, like for other high-throughput technologies, the main bottleneck remains at the stage of data analysis rather than data production. Findings We have developed HumMeth27QCReport, an R package devoted to researchers wanting to quickly analyse their Illumina Infinium methylation arrays. This package automates quality control steps by generating a report including sample-independent and sample-dependent quality plots, and performs primary analysis of raw methylation calls by computing data normalization, statistics, and sample similarities. This package is available at CRAN repository, and can be integrated in any Galaxy instance through the implementation of ad-hoc scripts accessible at Galaxy Tool Shed. Conclusions Our package provides users of the Illumina Infinium Methylation assays with a simplified, automated, open-source quality control and primary analysis of their methylation data. Moreover, to enhance its use by experimental researchers, the tool is being distributed along with the scripts necessary for its implementation in the Galaxy workbench. Finally, although it was originally developed for HumanMethylation27, we proved its compatibility with data generated with the HumanMethylation450 Bead Chip.

Skin sensitization potency of methyl methacrylate in the local lymph node assay: comparisons with guinea-pig data and human experience.

Science.gov (United States)

Betts, Catherine J; Dearman, Rebecca J; Heylings, Jon R; Kimber, Ian; Basketter, David A

2006-09-01

There is compelling evidence that contact allergens differ substantially (by 4 or 5 orders of magnitude) with respect to their inherent skin-sensitizing potency. Relative potency can now be measured effectively using the mouse local lymph node assay (LLNA) and such data form the basis of risk assessment and risk management strategies. Such determinations also facilitate distinctions being drawn between the prevalence of skin sensitization to a particular contact allergen and inherent potency. The distinction is important because chemicals that are implicated as common causes of contact allergy are not necessarily potent sensitizers. One example is provided by nickel that is undoubtedly a common cause of allergic contact dermatitis, but is a comparatively weak sensitizer in predictive tests. In an attempt to explore other examples of contact allergens where there may exist a discrepancy between prevalence and potency, we describe here analyses conducted with methyl methacrylate (MMA). Results of LLNA studies have been interpreted in the context of historical clinical data on occupational allergic contact dermatitis associated with exposure to MMA.

Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR) for the sensitive detection of disease-related hypermethylation.

Science.gov (United States)

Mauger, Florence; Kernaleguen, Magali; Lallemand, Céline; Kristensen, Vessela N; Deleuze, Jean-François; Tost, Jörg

2018-05-01

The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2 ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

Methyl-Donor and Cofactor Nutrient Intakes in the First 2–3 Years and Global DNA Methylation at Age 4: A Prospective Cohort Study

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Rachael M. Taylor

2018-02-01

Full Text Available Background: During the early postnatal period, the impact of nutrition on DNA methylation has not been well studied in humans. The aim was to quantify the relationship between one-carbon metabolism nutrient intake during the first three years of life and global DNA methylation levels at four years. Design: Childhood dietary intake was assessed using infant feeding questionnaires, food frequency questionnaires, 4-day weighed food records and 24-h food records. The dietary records were used to estimate the intake of methionine, folate, vitamins B2, B6 and B12 and choline. The accumulative nutrient intake specific rank from three months to three years of age was used for analysis. Global DNA methylation (%5-methyl cytosines (%5-mC was measured in buccal cells at four years of age, using an enzyme-linked immunosorbent assay (ELISA commercial kit. Linear regression models were used to quantify the statistical relationships. Results: Data were collected from 73 children recruited from the Women and their Children’s Health (WATCH study. No association was found between one-carbon metabolism nutrient intake and global DNA methylation levels (P > 0.05. Global DNA methylation levels in males were significantly higher than in females (median %5-mC: 1.82 vs. 1.03, males and females respectively, (P < 0.05. Conclusion: No association was found between the intake of one-carbon metabolism nutrients during the early postnatal period and global DNA methylation levels at age four years. Higher global DNA methylation levels in males warrants further investigation.

Methyl-Donor and Cofactor Nutrient Intakes in the First 2–3 Years and Global DNA Methylation at Age 4: A Prospective Cohort Study

Science.gov (United States)

Taylor, Rachael M.; Smith, Roger; Collins, Clare E.; Mossman, David; Wong-Brown, Michelle W.; Chan, Eng-Cheng; Evans, Tiffany-Jane; Attia, John R.; Smith, Tenele; Butler, Trent

2018-01-01

Background: During the early postnatal period, the impact of nutrition on DNA methylation has not been well studied in humans. The aim was to quantify the relationship between one-carbon metabolism nutrient intake during the first three years of life and global DNA methylation levels at four years. Design: Childhood dietary intake was assessed using infant feeding questionnaires, food frequency questionnaires, 4-day weighed food records and 24-h food records. The dietary records were used to estimate the intake of methionine, folate, vitamins B2, B6 and B12 and choline. The accumulative nutrient intake specific rank from three months to three years of age was used for analysis. Global DNA methylation (%5-methyl cytosines (%5-mC)) was measured in buccal cells at four years of age, using an enzyme-linked immunosorbent assay (ELISA) commercial kit. Linear regression models were used to quantify the statistical relationships. Results: Data were collected from 73 children recruited from the Women and their Children’s Health (WATCH) study. No association was found between one-carbon metabolism nutrient intake and global DNA methylation levels (P 0.05). Global DNA methylation levels in males were significantly higher than in females (median %5-mC: 1.82 vs. 1.03, males and females respectively, (P 0.05)). Conclusion: No association was found between the intake of one-carbon metabolism nutrients during the early postnatal period and global DNA methylation levels at age four years. Higher global DNA methylation levels in males warrants further investigation. PMID:29495543

A high-throughput and sensitive method to measure Global DNA Methylation: Application in Lung Cancer

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Mamaev Sergey

2008-08-01

Full Text Available Abstract Background Genome-wide changes in DNA methylation are an epigenetic phenomenon that can lead to the development of disease. The study of global DNA methylation utilizes technology that requires both expensive equipment and highly specialized skill sets. Methods We have designed and developed an assay, CpGlobal, which is easy-to-use, does not utilize PCR, radioactivity and expensive equipment. CpGlobal utilizes methyl-sensitive restriction enzymes, HRP Neutravidin to detect the biotinylated nucleotides incorporated in an end-fill reaction and a luminometer to measure the chemiluminescence. The assay shows high accuracy and reproducibility in measuring global DNA methylation. Furthermore, CpGlobal correlates significantly with High Performance Capillary Electrophoresis (HPCE, a gold standard technology. We have applied the technology to understand the role of global DNA methylation in the natural history of lung cancer. World-wide, it is the leading cause of death attributed to any cancer. The survival rate is 15% over 5 years due to the lack of any clinical symptoms until the disease has progressed to a stage where cure is limited. Results Through the use of cell lines and paired normal/tumor samples from patients with non-small cell lung cancer (NSCLC we show that global DNA hypomethylation is highly associated with the progression of the tumor. In addition, the results provide the first indication that the normal part of the lung from a cancer patient has already experienced a loss of methylation compared to a normal individual. Conclusion By detecting these changes in global DNA methylation, CpGlobal may have a role as a barometer for the onset and development of lung cancer.

Diagnostic markers of urothelial cancer based on DNA methylation analysis

International Nuclear Information System (INIS)

Chihara, Yoshitomo; Hirao, Yoshihiko; Kanai, Yae; Fujimoto, Hiroyuki; Sugano, Kokichi; Kawashima, Kiyotaka; Liang, Gangning; Jones, Peter A; Fujimoto, Kiyohide; Kuniyasu, Hiroki

2013-01-01

Early detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring. We aimed to establish diagnostic markers for UC based on DNA methylation. In this multi-center study, three independent sample sets were prepared. First, DNA methylation levels at CpG loci were measured in the training sets (tumor samples from 91 UC patients, corresponding normal-appearing tissue from these patients, and 12 normal tissues from age-matched bladder cancer-free patients) using the Illumina Golden Gate methylation assay to identify differentially methylated loci. Next, these methylated loci were validated by quantitative DNA methylation by pyrosequencing, using another cohort of tissue samples (Tissue validation set). Lastly, methylation of these markers was analyzed in the independent urine samples (Urine validation set). ROC analysis was performed to evaluate the diagnostic accuracy of these 12 selected markers. Of the 1303 CpG sites, 158 were hyper ethylated and 356 were hypo ethylated in tumor tissues compared to normal tissues. In the panel analysis, 12 loci showed remarkable alterations between tumor and normal samples, with 94.3% sensitivity and 97.8% specificity. Similarly, corresponding normal tissue could be distinguished from normal tissues with 76.0% sensitivity and 100% specificity. Furthermore, the diagnostic accuracy for UC of these markers determined in urine samples was high, with 100% sensitivity and 100% specificity. Based on these preliminary findings, diagnostic markers based on differential DNA methylation at specific loci can be useful for non-invasive and reliable detection of UC and epigenetic field defect

Methylation signature of lymph node metastases in breast cancer patients

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Barekati, Zeinab; Radpour, Ramin; Lu, Qing; Bitzer, Johannes; Zheng, Hong; Toniolo, Paolo; Lenner, Per; Zhong, Xiao Yan

2012-01-01

Invasion and metastasis are two important hallmarks of malignant tumors caused by complex genetic and epigenetic alterations. The present study investigated the contribution of aberrant methylation profiles of cancer related genes, APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14 (ARF), P16 (CDKN2A), P21 (CDKN1A), PTEN, and TIMP3, in the matched axillary lymph node metastasis in comparison to the primary tumor tissue and the adjacent normal tissue from the same breast cancer patients to identify the potential of candidate genes methylation as metastatic markers. The quantitative methylation analysis was performed using the SEQUENOM’s EpiTYPER™ assay which relies on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The quantitative DNA methylation analysis of the candidate genes showed higher methylation proportion in the primary tumor tissue than that of the matched normal tissue and the differences were significant for the APC, BIN1, BMP6, BRCA1, CST6, ESR-b, P16, PTEN and TIMP3 promoter regions (P<0.05). Among those candidate methylated genes, APC, BMP6, BRCA1 and P16 displayed higher methylation proportion in the matched lymph node metastasis than that found in the normal tissue (P<0.05). The pathway analysis revealed that BMP6, BRCA1 and P16 have a role in prevention of neoplasm metastasis. The results of the present study showed methylation heterogeneity between primary tumors and metastatic lesion. The contribution of aberrant methylation alterations of BMP6, BRCA1 and P16 genes in lymph node metastasis might provide a further clue to establish useful biomarkers for screening metastasis

[GSTP1, APC and RASSF1 gene methylation in prostate cancer samples: comparative analysis of MS-HRM method and Infinium HumanMethylation450 BeadChip beadchiparray diagnostic value].

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Skorodumova, L O; Babalyan, K A; Sultanov, R; Vasiliev, A O; Govorov, A V; Pushkar, D Y; Prilepskaya, E A; Danilenko, S A; Generozov, E V; Larin, A K; Kostryukova, E S; Sharova, E I

2016-11-01

There is a clear need in molecular markers for prostate cancer (PC) risk stratification. Alteration of DNA methylation is one of processes that occur during ÐÑ progression. Methylation-sensitive PCR with high resolution melting curve analysis (MS-HRM) can be used for gene methylation analysis in routine laboratory practice. This method requires very small amounts of DNA for analysis. Numerous results have been accumulated on DNA methylation in PC samples analyzed by the Infinium HumanMethylation450 BeadChip (HM450). However, the consistency of MS-HRM results with chip hybridization results has not been examined yet. The aim of this study was to assess the consistency of results of GSTP1, APC and RASSF1 gene methylation analysis in ÐÑ biopsy samples obtained by MS-HRM and chip hybridization. The methylation levels of each gene determined by MS-HRM were statistically different in the group of PC tissue samples and the samples without signs of tumor growth. Chip hybridization data analysis confirmed the results obtained with the MS-HRM. Differences in methylation levels between tumor tissue and histologically intact tissue of each sample determined by MS-HRM and chip hybridization, were consistent with each other. Thus, we showed that the assessment of GSTP1, APC and RASSF1 gene methylation analysis using MS-HRM is suitable for the design of laboratory assays that will differentiate the PC tissue from the tissue without signs of tumor growth.

Assays of D-Amino Acid Oxidase Activity

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Elena Rosini

2018-01-01

Full Text Available D-amino acid oxidase (DAAO is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs. Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.

Synthesis of [14α-methyl-3H]-24,25-dihydrolanosterol

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DeKeczer, S.; Kertesz, D.; Parnes, H.

1993-01-01

We describe the first synthesis of isomerically pure title compound (6) at high specific activity. This required the development of a convenient, regiospecific synthesis of the δ 8(9) -15-ketone and subsequent alkylation with methyl-[ 3 H 3 ]iodide. A key step in our procedure was the use of an electrochemical reduction of the intermediate [14α-methyl- 3 H]-15-oxo-dihydrolanosterol. This process was effected cleanly and in high yield to give (6), an important assay tool in the search for cholesterol lowering agents. This approach was found to be significantly superior to the Wolff-Kishner reduction of the corresponding 3-benzoate. (Author)

The Role of Dietary Extra Virgin Olive Oil and Corn Oil on the Alteration of Epigenetic Patterns in the Rat DMBA-Induced Breast Cancer Model.

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Cristina Rodríguez-Miguel

Full Text Available Disruption of epigenetic patterns is a major change occurring in all types of cancers. Such alterations are characterized by global DNA hypomethylation, gene-promoter hypermethylation and aberrant histone modifications, and may be modified by environment. Nutritional factors, and especially dietary lipids, have a role in the etiology of breast cancer. Thus, we aimed to analyze the influence of different high fat diets on DNA methylation and histone modifications in the rat dimethylbenz(aanthracene (DMBA-induced breast cancer model. Female Sprague-Dawley rats were fed a low-fat, a high corn-oil or a high extra-virgin olive oil (EVOO diet from weaning or from induction with DMBA. In mammary glands and tumors we analyzed global and gene specific (RASSF1A, TIMP3 DNA methylation by LUMA and bisulfite pyrosequencing assays, respectively. We also determined gene expression and enzymatic activity of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b and evaluated changes in histone modifications (H3K4me2, H3K27me3, H4K20me3 and H4K16ac by western-blot. Our results showed variations along time in the global DNA methylation of the mammary gland displaying decreases at puberty and with aging. The olive oil-enriched diet, on the one hand, increased the levels of global DNA methylation in mammary gland and tumor, and on the other, changed histone modifications patterns. The corn oil-enriched diet increased DNA methyltransferase activity in both tissues, resulting in an increase in the promoter methylation of the tumor suppressor genes RASSF1A and TIMP3. These results suggest a differential effect of the high fat diets on epigenetic patterns with a relevant role in the neoplastic transformation, which could be one of the mechanisms of their differential promoter effect, clearly stimulating for the high corn-oil diet and with a weaker influence for the high EVOO diet, on breast cancer progression.

Methylation Status of miR-182 Promoter in Lung Cancer Cell Lines

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Yongwen LI

2015-05-01

Full Text Available Background and objective It has been proven that the abnormal expression of miR-182 was related to the occurrence and development of tumors. The aim of this study is to explore the relationship between the methylation of miR-182 promoter and its expression in lung cancer cell lines. Methods Real-time quantitative PCR and methylation-specific PCR were used to detect the expression level of miR-182 and its promoter methylation status in five lung cancer cell lines (A549, L9981, NL9980, 95C and 95D. DNA sequencing was used to confirm the methylation results. Results The level of miR-182 expression significantly differs among these lung cancer cell lines. The highly metastatic human lung cancer cell lines, namely, A549 and L9981, demonstrate a relatively lower expression level of miR-182 compared with the lowly metastatic human lung cancer cell line 95C. Methylation-specific PCR and DNA sequencing assay results indicate that these lung cancer cell lines present different levels of miR-182 promoter methylation, and the highest methylation level is observed in A549 cells. Furthermore, the expression of miR-182 in these cell lines significantly increases when treated with 10 μM 5’-Aza-dC. Conclusion DNA methylation occurs in the miR-182 promoter region in lung cancer cell lines. This methylation can regulate the expression level of miR-182. Further study must be conducted to explore the function of miR-182 promoter methylation in lung cancer occurrence and development.

Whole Genome DNA Methylation Analysis of Obstructive Sleep Apnea: IL1R2, NPR2, AR, SP140 Methylation and Clinical Phenotype.

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Chen, Yung-Che; Chen, Ting-Wen; Su, Mao-Chang; Chen, Chung-Jen; Chen, Kuang-Den; Liou, Chia-Wei; Tang, Petrus; Wang, Ting-Ya; Chang, Jen-Chieh; Wang, Chin-Chou; Lin, Hsin-Ching; Chin, Chien-Hung; Huang, Kuo-Tung; Lin, Meng-Chih; Hsiao, Chang-Chun

2016-04-01

We hypothesized that DNA methylation patterns may contribute to disease severity or the development of hypertension and excessive daytime sleepiness (EDS) in patients with obstructive sleep apnea (OSA). Illumina's (San Diego, CA, USA) DNA methylation 27-K assay was used to identify differentially methylated loci (DML). DNA methylation levels were validated by pyrosequencing. A discovery cohort of 15 patients with OSA and 6 healthy subjects, and a validation cohort of 72 patients with sleep disordered breathing (SDB). Microarray analysis identified 636 DMLs in patients with OSA versus healthy subjects, and 327 DMLs in patients with OSA and hypertension versus those without hypertension. In the validation cohort, no significant difference in DNA methylation levels of six selected genes was found between the primary snoring subjects and OSA patients (primary outcome). However, a secondary outcome analysis showed that interleukin-1 receptor 2 (IL1R2) promoter methylation (-114 cytosine followed by guanine dinucleotide sequence [CpG] site) was decreased and IL1R2 protein levels were increased in the patients with SDB with an oxygen desaturation index > 30. Androgen receptor (AR) promoter methylation (-531 CpG site) and AR protein levels were both increased in the patients with SDB with an oxygen desaturation index > 30. Natriuretic peptide receptor 2 (NPR2) promoter methylation (-608/-618 CpG sites) were decreased, whereas levels of both NPR2 and serum C type natriuretic peptide protein were increased in the SDB patients with EDS. Speckled protein 140 (SP140) promoter methylation (-194 CpG site) was increased, and SP140 protein levels were decreased in the patients with SDB and EDS. IL1R2 hypomethylation and AR hypermethylation may constitute an important determinant of disease severity, whereas NPR2 hypomethylation and SP140 hypermethylation may provide a biomarker for vulnerability to EDS in OSA. A commentary on this article appears in this issue on page 723. © 2016

Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors

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Durr, Megan L.; Mydlarz, Wojciech K.; Shao, Chunbo; Zahurak, Marianna L.; Chuang, Alice Y.; Hoque, Mohammad O.; Westra, William H.; Liegeois, Nanette J.; Califano, Joseph A.; Sidransky, David; Ha, Patrick K.

2010-01-01

Background Methylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected. Methodology DNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results. Principal Findings The average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (ptrend for increasing methylation in APC, Mint 1, PGP9.5, RAR-β, and Timp3. Conclusions/Significance Screening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors. PMID:20520817

DNA damage, repair monitoring and epigenetic DNA methylation changes in seedlings of Chernobyl soybeans.

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Georgieva, Mariyana; Rashydov, Namik M; Hajduch, Martin

2017-02-01

This pilot study was carried out to assess the effect of radio-contaminated Chernobyl environment on plant genome integrity 27 years after the accident. For this purpose, nuclei were isolated from root tips of the soybean seedlings harvested from plants grown in the Chernobyl area for seven generations. Neutral, neutral-alkaline, and methylation-sensitive comet assays were performed to evaluate the induction and repair of primary DNA damage and the epigenetic contribution to stress adaptation mechanisms. An increased level of single and double strand breaks in the radio-contaminated Chernobyl seedlings at the stage of primary root development was detected in comparison to the controls. However, the kinetics of the recovery of DNA breaks of radio-contaminated Chernobyl samples revealed that lesions were efficiently repaired at the stage of cotyledon. Methylation-sensitive comet assay revealed comparable levels in the CCGG methylation pattern between control and radio-contaminated samples with a slight increase of approximately 10% in the latter ones. The obtained preliminary data allow us to speculate about the onset of mechanisms providing an adaptation potential to the accumulated internal irradiation after the Chernobyl accident. Despite the limitations of this study, we showed that comet assay is a sensitive and flexible technique which can be efficiently used for genotoxic screening of plant specimens in natural and human-made radio-contaminated areas, as well as for safety monitoring of agricultural products. Copyright © 2016 Elsevier B.V. All rights reserved.

Sensory rhodopsins I and II modulate a methylation/demethylation system in Halobacterium halobium phototaxis

International Nuclear Information System (INIS)

Spudich, E.N.; Takahashi, T.; Spudich, J.L.

1989-01-01

This work demonstrates that phototaxis stimuli in the archaebacterium Halobacterium halobium control a methylation/demethylation system in vivo through photoactivation of sensory rhodopsin I (SR-I) in either its attractant or repellent signaling form as well as through the repellent receptor sensory rhodopsin II (SR-II, also called phoborhodopsin). The effects of positive stimuli that suppress swimming reversals (i.e., an increase in attractant or decrease in repellent light) and negative stimuli that induce swimming reversals (i.e., a decrease in attractant or increase in repellent light) through each photoreceptor were monitored by assaying release of volatile [3H]methyl groups. This assay has been used to measure [3H]methanol produced during the process of adaptation to chemotactic stimuli in eubacteria. In H. halobium positive photostimuli produce a transient increase in the rate of demethylation followed by a decrease below the unstimulated value, whereas negative photostimuli cause an increase followed by a rate similar to that of the unstimulated value. Photoactivation of the SR-I attractant and simultaneous photoactivation of the SR-II repellent receptors cancel in their effects on demethylation, demonstrating the methylation system is regulated by an integrated signal. Analysis of mutants indicates that the source for the volatile methyl groups is intrinsic membrane proteins distinct from the chromoproteins that share the membrane. A methyl-accepting protein (94 kDa) previously correlated in amount with the SR-I chromoprotein (25 kDa) is shown here to be missing in a recently isolated SR-I-SR-II+ mutant (Flx3b), thus confirming the association of this protein with SR-I. Photoactivated SR-II in mutant Flx3b controls demethylation, predicting the existence of a photomodulated methyl-accepting component distinct from the 94-kDa protein of SR-I

DNA Methylation program in normal and alcohol-induced thinning cortex.

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Öztürk, Nail Can; Resendiz, Marisol; Öztürk, Hakan; Zhou, Feng C

2017-05-01

While cerebral underdevelopment is a hallmark of fetal alcohol spectrum disorders (FASD), the mechanism(s) guiding the broad cortical neurodevelopmental deficits are not clear. DNA methylation is known to regulate early development and tissue specification through gene regulation. Here, we examined DNA methylation in the onset of alcohol-induced cortical thinning in a mouse model of FASD. C57BL/6 (B6) mice were administered a 4% alcohol (v/v) liquid diet from embryonic (E) days 7-16, and their embryos were harvested at E17, along with isocaloric liquid diet and lab chow controls. Cortical neuroanatomy, neural phenotypes, and epigenetic markers of methylation were assessed using immunohistochemistry, Western blot, and methyl-DNA assays. We report that cortical thickness, neuroepithelial proliferation, and neuronal migration and maturity were found to be deterred by alcohol at E17. Simultaneously, DNA methylation, including 5-methylcytosine (5mC) and 5-hydroxcylmethylcytosine (5hmC), which progresses as an intrinsic program guiding normal embryonic cortical development, was severely affected by in utero alcohol exposure. The intricate relationship between cortical thinning and this DNA methylation program disruption is detailed and illustrated. DNA methylation, dynamic across the multiple cortical layers during the late embryonic stage, is highly disrupted by fetal alcohol exposure; this disruption occurs in tandem with characteristic developmental abnormalities, ranging from structural to molecular. Finally, our findings point to a significant question for future exploration: whether epigenetics guides neurodevelopment or whether developmental conditions dictate epigenetic dynamics in the context of alcohol-induced cortical teratogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

An optimized algorithm for detecting and annotating regional differential methylation.

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Li, Sheng; Garrett-Bakelman, Francine E; Akalin, Altuna; Zumbo, Paul; Levine, Ross; To, Bik L; Lewis, Ian D; Brown, Anna L; D'Andrea, Richard J; Melnick, Ari; Mason, Christopher E

2013-01-01

DNA methylation profiling reveals important differentially methylated regions (DMRs) of the genome that are altered during development or that are perturbed by disease. To date, few programs exist for regional analysis of enriched or whole-genome bisulfate conversion sequencing data, even though such data are increasingly common. Here, we describe an open-source, optimized method for determining empirically based DMRs (eDMR) from high-throughput sequence data that is applicable to enriched whole-genome methylation profiling datasets, as well as other globally enriched epigenetic modification data. Here we show that our bimodal distribution model and weighted cost function for optimized regional methylation analysis provides accurate boundaries of regions harboring significant epigenetic modifications. Our algorithm takes the spatial distribution of CpGs into account for the enrichment assay, allowing for optimization of the definition of empirical regions for differential methylation. Combined with the dependent adjustment for regional p-value combination and DMR annotation, we provide a method that may be applied to a variety of datasets for rapid DMR analysis. Our method classifies both the directionality of DMRs and their genome-wide distribution, and we have observed that shows clinical relevance through correct stratification of two Acute Myeloid Leukemia (AML) tumor sub-types. Our weighted optimization algorithm eDMR for calling DMRs extends an established DMR R pipeline (methylKit) and provides a needed resource in epigenomics. Our method enables an accurate and scalable way of finding DMRs in high-throughput methylation sequencing experiments. eDMR is available for download at http://code.google.com/p/edmr/.

Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue

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Woodfine Kathryn

2011-01-01

Full Text Available Abstract Background Genes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes. Results Remarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression. Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ

Global DNA methylation responses to low dose radiation exposure

International Nuclear Information System (INIS)

Newman, M.R.; Ormsby, R.J.; Blyth, B.J.; Sykes, P.J.; Bezak, E.

2011-01-01

Full text: High radiation doses cause breaks in the DNA which are considered the critical lesions in initiation of radiation-induced cancer. However, at very low radiation doses relevant for the general public, the induction of such breaks will be rare, and other changes to the DNA such as DNA methylation which affects gene expression may playa role in radiation responses. We are studying global DNA methylation after low dose radiation exposure to determine if low dose radiation has short- and/or long-term effects on chromatin structure. We developed a sensitive high resolution melt assay to measure the levels of DNA methylation across the mouse genome by analysing a stretch of DNA sequence within Long Interspersed Nuclear Elements-I (LINE I) that comprise a very large proportion of the mouse and human genomes. Our initial results suggest no significant short-term or longterm) changes in global NA methylation after low dose whole-body X-radiation of 10 J1Gyor 10 mGy, with a significant transient increase in NA methylation observed I day after a high dose of I Gy. If the low radiation doses tested are inducing changes in bal DNA methylation, these would appear to be smaller than the variation observed between the sexes and following the general stress of the sham-irradiation procedure itself. This research was funded by the Low Dose Radiation Research Program, Biological and Environmental Research, US DOE, Grant DE-FG02-05ER64104 and MN is the recipient of the FMCF/BHP Dose Radiation Research Scholarship.

A significant association between BDNF promoter methylation and the risk of drug addiction.

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Xu, Xuting; Ji, Huihui; Liu, Guili; Wang, Qinwen; Liu, Huifen; Shen, Wenwen; Li, Longhui; Xie, Xiaohu; Zhou, Wenhua; Duan, Shiwei

2016-06-10

As a member of the neurotrophic factor family, brain derived neurotrophic factor (BDNF) plays an important role in the survival and differentiation of neurons. The aim of our work was to evaluate the role of BDNF promoter methylation in drug addiction. A total of 60 drug abusers (30 heroin and 30 methylamphetamine addicts) and 52 healthy age- and gender-matched controls were recruited for the current case control study. Bisulfite pyrosequencing technology was used to determine the methylation levels of five CpGs (CpG1-5) on the BDNF promoter. Among the five CpGs, CpG5 methylation was significantly lower in drug abusers than controls. Moreover, significant associations were found between CpG5 methylation and addictive phenotypes including tension-anxiety, anger-hostility, fatigue-inertia, and depression-dejection. In addition, luciferase assay showed that the DNA fragment of BDNF promoter played a key role in the regulation of gene expression. Our results suggest that BDNF promoter methylation is associated with drug addiction, although further studies are needed to understand the mechanisms by which BDNF promoter methylation contributes to the pathophysiology of drug addiction. Copyright © 2016. Published by Elsevier B.V.

[Novel Approaches in DNA Methylation Studies - MS-HRM Analysis and Electrochemistry].

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Bartošík, M; Ondroušková, E

Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

Screening of molecular cell targets for carcinogenic heterocyclic aromatic amines by using CALUX® reporter gene assays.

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Steinberg, Pablo; Behnisch, Peter A; Besselink, Harrie; Brouwer, Abraham A

2017-06-01

Heterocyclic aromatic amines (HCAs) are compounds formed when meat or fish are cooked at high temperatures for a long time or over an open fire. To determine which pathways of toxicity are activated by HCAs, nine out of the ten HCAs known to be carcinogenic in rodents (2-amino-9H-pyrido[2,3-b]indole (AαC), 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)) were tested in the estrogen receptor α (ERα), androgen receptor (AR), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor γ2 (PPARγ2), polycyclic aromatic hydrocarbons (PAH), Nrf2, and p53 CALUX® reporter gene assays. Trp-P-1 was the only HCA that led to a positive response in the ERα, PPARγ2, and Nrf2 CALUX® assays. In the PAH CALUX® assay, Trp-P-2, MeAαC, and AαC induced luciferase activity to a greater extent than MeIQ and PhIP. In the p53 CALUX® assay without a coupled metabolic activation, only Trp-P-1 and Trp-P-2 enhanced luciferase expression; when a metabolic activation step was coupled to the p53 CALUX® assay, Trp-P-1, Glu-P-2, MeIQ, MeIQx, and PhIP induced a positive response. No HCA was positive in the AR and GR CALUX® assays. Taken together, the results obtained show that the battery of CALUX® assays performed in the present study can successfully be used to screen for molecular cell targets of carcinogenic compounds such as HCAs.

Identification of Differentially Methylated Sites with Weak Methylation Effects

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Hong Tran

2018-02-01

Full Text Available Deoxyribonucleic acid (DNA methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect differentially methylated cytosines (DMCs among treatments. Most statistical methods for DMC detection do not consider the dependency of methylation patterns across the genome, thus possibly inflating type I error. Furthermore, small sample sizes and weak methylation effects among different phenotype categories make it difficult for these statistical methods to accurately detect DMCs. To address these issues, the wavelet-based functional mixed model (WFMM was introduced to detect DMCs. To further examine the performance of WFMM in detecting weak differential methylation events, we used both simulated and empirical data and compare WFMM performance to a popular DMC detection tool methylKit. Analyses of simulated data that replicated the effects of the herbicide glyphosate on DNA methylation in Arabidopsis thaliana show that WFMM results in higher sensitivity and specificity in detecting DMCs compared to methylKit, especially when the methylation differences among phenotype groups are small. Moreover, the performance of WFMM is robust with respect to small sample sizes, making it particularly attractive considering the current high costs of bisulfite sequencing. Analysis of empirical Arabidopsis thaliana data under varying glyphosate dosages, and the analysis of monozygotic (MZ twins who have different pain sensitivities—both datasets have weak methylation effects of <1%—show that WFMM can identify more relevant DMCs related to the phenotype of interest than methylKit. Differentially methylated regions (DMRs are genomic regions with different DNA methylation status across biological samples. DMRs and DMCs are essentially the same

A coupled photometric assay for characterization of S-adenosyl-l-homocysteine hydrolases in the physiological hydrolytic direction.

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Kailing, Lyn L; Bertinetti, Daniela; Herberg, Friedrich W; Pavlidis, Ioannis V

2017-10-25

S-Adenosyl-l-homocysteine hydrolases (SAHases) are important metabolic enzymes and their dysregulation is associated with some severe diseases. In vivo they catalyze the hydrolysis of S-adenosyl-l-homocysteine (SAH), the by-product of methylation reactions in various organisms. SAH is a potent inhibitor of methyltransferases, thus its removal from the equilibrium is an important requirement for methylation reactions. SAH hydrolysis is also the first step in the cellular regeneration process of the methyl donor S-adenosyl-l-methionine (SAM). However, in vitro the equilibrium lies towards the synthetic direction. To enable characterization of SAHases in the physiologically relevant direction, we have developed a coupled photometric assay that shifts the equilibrium towards hydrolysis by removing the product adenosine, using a high affinity adenosine kinase (AK). This converts adenosine to AMP and thereby forms equimolar amounts of ADP, which is phosphorylated by a pyruvate kinase (PK), in turn releasing pyruvate. The readout of the assay is the consumption of NADH during the lactate dehydrogenase (LDH) catalyzed reduction of pyruvate to lactic acid. The applicability of the assay is showcased for the determination of the kinetic constants of an SAHase from Bradyrhizobium elkanii (K M,SAH 41±5μM, v max,SAH 25±1μM/min with 0.13mg/mL enzyme). This assay is a valuable tool for in vitro characterization of SAHases with biotechnological potential, and for monitoring SAHase activity in diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of methylated genes associated with aggressive clinicopathological features in mantle cell lymphoma.

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Anna Enjuanes

Full Text Available BACKGROUND: Mantle cell lymphoma (MCL is genetically characterized by the t(11;14(q13;q32 translocation and a high number of secondary chromosomal alterations. The contribution of DNA methylation to MCL lymphomagenesis is not well known. We sought to identify epigenetically silenced genes in these tumours that might have clinical relevance. METHODOLOGY/PRINCIPAL FINDINGS: To identify potential methylated genes in MCL we initially investigated seven MCL cell lines treated with epigenetic drugs and gene expression microarray profiling. The methylation status of selected candidate genes was validated by a quantitative assay and subsequently analyzed in a series of primary MCL (n = 38. After pharmacological reversion we identified 252 potentially methylated genes. The methylation analysis of a subset of these genes (n = 25 in the MCL cell lines and normal B lymphocytes confirmed that 80% of them were methylated in the cell lines but not in normal lymphocytes. The subsequent analysis in primary MCL identified five genes (SOX9, HOXA9, AHR, NR2F2, and ROBO1 frequently methylated in these tumours. The gene methylation events tended to occur in the same primary neoplasms and correlated with higher proliferation, increased number of chromosomal abnormalities, and shorter survival of the patients. CONCLUSIONS: We have identified a set of genes whose methylation degree and gene expression levels correlate with aggressive clinicopathological features of MCL. Our findings also suggest that a subset of MCL might show a CpG island methylator phenotype (CIMP that may influence the behaviour of the tumours.

2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

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Dong, Hongping; Chang, David C.; Hua, Maggie Ho Chia; Lim, Siew Pheng; Chionh, Yok Hian; Hia, Fabian; Lee, Yie Hou; Kukkaro, Petra; Lok, Shee-Mei; Dedon, Peter C.; Shi, Pei-Yong

2012-01-01

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 (m7GpppAm) 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

Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation

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Schermelleh, Lothar; Haemmer, Andrea; Spada, Fabio; Rösing, Nicole; Meilinger, Daniela; Rothbauer, Ulrich; Cardoso, M. Cristina; Leonhardt, Heinrich

2007-01-01

Postreplicative maintenance of genomic methylation patterns was proposed to depend largely on the binding of DNA methyltransferase 1 (Dnmt1) to PCNA, a core component of the replication machinery. We investigated how the slow and discontinuous DNA methylation could be mechanistically linked with fast and processive DNA replication. Using photobleaching and quantitative live cell imaging we show that Dnmt1 binding to PCNA is highly dynamic. Activity measurements of a PCNA-binding-deficient mutant with an enzyme-trapping assay in living cells showed that this interaction accounts for a 2-fold increase in methylation efficiency. Expression of this mutant in mouse dnmt1−/− embryonic stem (ES) cells restored CpG island methylation. Thus association of Dnmt1 with the replication machinery enhances methylation efficiency, but is not strictly required for maintaining global methylation. The transient nature of this interaction accommodates the different kinetics of DNA replication and methylation while contributing to faithful propagation of epigenetic information. PMID:17576694

Whole-genome methylation caller designed for methyl- DNA ...

African Journals Online (AJOL)

etchie

2013-02-20

Feb 20, 2013 ... Our method uses a single-CpG-resolution, whole-genome methylation ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, ...... methylation is prevalent in embryonic stem cells andmaybe mediated.

CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer.

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Stefansson, Olafur Andri; Hermanowicz, Stefan; van der Horst, Jasper; Hilmarsdottir, Holmfridur; Staszczak, Zuzanna; Jonasson, Jon Gunnlaugur; Tryggvadottir, Laufey; Gudjonsson, Thorkell; Sigurdsson, Stefan

2017-07-05

DNA repair of alkylation damage is defective in various cancers. This occurs through somatically acquired inactivation of the MGMT gene in various cancer types, including breast cancers. In addition to MGMT, the two E. coli AlkB homologs ALKBH2 and ALKBH3 have also been linked to direct reversal of alkylation damage. However, it is currently unknown whether ALKBH2 or ALKBH3 are found inactivated in cancer. Methylome datasets (GSE52865, GSE20713, GSE69914), available through Omnibus, were used to determine whether ALKBH2 or ALKBH3 are found inactivated by CpG promoter methylation. TCGA dataset enabled us to then assess the impact of CpG promoter methylation on mRNA expression for both ALKBH2 and ALKBH3. DNA methylation analysis for the ALKBH3 promoter region was carried out by pyrosequencing (PyroMark Q24) in 265 primary breast tumours and 30 proximal normal breast tissue samples along with 8 breast-derived cell lines. ALKBH3 mRNA and protein expression were analysed in cell lines using RT-PCR and Western blotting, respectively. DNA alkylation damage assay was carried out in cell lines based on immunofluorescence and confocal imaging. Data on clinical parameters and survival outcomes in patients were obtained and assessed in relation to ALKBH3 promoter methylation. The ALKBH3 gene, but not ALKBH2, undergoes CpG promoter methylation and transcriptional silencing in breast cancer. We developed a quantitative alkylation DNA damage assay based on immunofluorescence and confocal imaging revealing higher levels of alkylation damage in association with epigenetic inactivation of the ALKBH3 gene (P = 0.029). In our cohort of 265 primary breast cancer, we found 72 cases showing aberrantly high CpG promoter methylation over the ALKBH3 promoter (27%; 72 out of 265). We further show that increasingly higher degree of ALKBH3 promoter methylation is associated with reduced breast-cancer specific survival times in patients. In this analysis, ALKBH3 promoter methylation at >20

Evaluation of methyl methanesulfonate, 2,6-diaminotoluene and 5-fluorouracil: Part of the Japanese center for the validation of alternative methods (JaCVAM) international validation study of the in vivo rat alkaline comet assay.

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Plappert-Helbig, Ulla; Junker-Walker, Ursula; Martus, Hans-Joerg

2015-07-01

As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined methyl methanesulfonate, 2,6-diaminotoluene, and 5-fluorouracil under coded test conditions. Rats were treated orally with the maximum tolerated dose (MTD) and two additional descending doses of the respective compounds. In the MMS treated groups liver and stomach showed significantly elevated DNA damage at each dose level and a significant dose-response relationship. 2,6-diaminotoluene induced significantly elevated DNA damage in the liver at each dose and a statistically significant dose-response relationship whereas no DNA damage was obtained in the stomach. 5-fluorouracil did not induce DNA damage in either liver or stomach. Copyright © 2015 Elsevier B.V. All rights reserved.

Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm.

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Sahar Houshdaran

2007-12-01

Full Text Available Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete reprogramming of the male germ line could, in theory, result in both altered sperm DNA methylation and compromised spermatogenesis.We determined concentration, motility and morphology of sperm in semen samples collected by male members of couples attending an infertility clinic. Using MethyLight and Illumina assays we measured methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm.This is the first report of a broad epigenetic defect associated with abnormal semen parameters. Our results suggest that the underlying mechanism for these epigenetic changes may be improper erasure of DNA methylation during epigenetic reprogramming of the male germ line.

Identification of endometrial cancer methylation features using combined methylation analysis methods.

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Michael P Trimarchi

Full Text Available DNA methylation is a stable epigenetic mark that is frequently altered in tumors. DNA methylation features are attractive biomarkers for disease states given the stability of DNA methylation in living cells and in biologic specimens typically available for analysis. Widespread accumulation of methylation in regulatory elements in some cancers (specifically the CpG island methylator phenotype, CIMP can play an important role in tumorigenesis. High resolution assessment of CIMP for the entire genome, however, remains cost prohibitive and requires quantities of DNA not available for many tissue samples of interest. Genome-wide scans of methylation have been undertaken for large numbers of tumors, and higher resolution analyses for a limited number of cancer specimens. Methods for analyzing such large datasets and integrating findings from different studies continue to evolve. An approach for comparison of findings from a genome-wide assessment of the methylated component of tumor DNA and more widely applied methylation scans was developed.Methylomes for 76 primary endometrial cancer and 12 normal endometrial samples were generated using methylated fragment capture and second generation sequencing, MethylCap-seq. Publically available Infinium HumanMethylation 450 data from The Cancer Genome Atlas (TCGA were compared to MethylCap-seq data.Analysis of methylation in promoter CpG islands (CGIs identified a subset of tumors with a methylator phenotype. We used a two-stage approach to develop a 13-region methylation signature associated with a "hypermethylator state." High level methylation for the 13-region methylation signatures was associated with mismatch repair deficiency, high mutation rate, and low somatic copy number alteration in the TCGA test set. In addition, the signature devised showed good agreement with previously described methylation clusters devised by TCGA.We identified a methylation signature for a "hypermethylator phenotype" in

Searsia species with affinity to the N-methyl-d-aspartic acid (NMDA) receptor

DEFF Research Database (Denmark)

Jäger, Anna; Knap, D.M.; Nielsen, Birgitte

2012-01-01

Species of Searsia are used in traditional medicine to treat epilepsy. Previous studies on S. dentata and S. pyroides have shown that this is likely mediated via the N-methyl-d-aspartic acid (NMDA) receptor. Ethanolic extracts of leaves of six Searsia species were tested in a binding assay...

Identification of DNA methylation changes associated with human gastric cancer

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Park Jung-Hoon

2011-12-01

Full Text Available Abstract Background Epigenetic alteration of gene expression is a common event in human cancer. DNA methylation is a well-known epigenetic process, but verifying the exact nature of epigenetic changes associated with cancer remains difficult. Methods We profiled the methylome of human gastric cancer tissue at 50-bp resolution using a methylated DNA enrichment technique (methylated CpG island recovery assay in combination with a genome analyzer and a new normalization algorithm. Results We were able to gain a comprehensive view of promoters with various CpG densities, including CpG Islands (CGIs, transcript bodies, and various repeat classes. We found that gastric cancer was associated with hypermethylation of 5' CGIs and the 5'-end of coding exons as well as hypomethylation of repeat elements, such as short interspersed nuclear elements and the composite element SVA. Hypermethylation of 5' CGIs was significantly correlated with downregulation of associated genes, such as those in the HOX and histone gene families. We also discovered long-range epigenetic silencing (LRES regions in gastric cancer tissue and identified several hypermethylated genes (MDM2, DYRK2, and LYZ within these regions. The methylation status of CGIs and gene annotation elements in metastatic lymph nodes was intermediate between normal and cancerous tissue, indicating that methylation of specific genes is gradually increased in cancerous tissue. Conclusions Our findings will provide valuable data for future analysis of CpG methylation patterns, useful markers for the diagnosis of stomach cancer, as well as a new analysis method for clinical epigenomics investigations.

Methylation-Dependent Activation of CDX1 through NF-κB

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Rau, Tilman T.; Rogler, Anja; Frischauf, Myrjam; Jung, Andreas; Konturek, Peter C.; Dimmler, Arno; Faller, Gerhard; Sehnert, Bettina; El-Rifai, Wael; Hartmann, Arndt; Voll, Reinhard E.; Schneider-Stock, Regine

2013-01-01

The caudal homeobox factor 1 (CDX1) is an essential transcription factor for intestinal differentiation. Its aberrant expression in intestinal metaplasia of the upper gastrointestinal tract is a hallmark within the gastritis-metaplasia-carcinoma sequence. CDX1 expression is influenced by certain pathways, such as Wnt, Ras, or NF-κB signaling; however, these pathways alone cannot explain the transient expression of CDX1 in intestinal metaplasia or the molecular inactivation mechanism of its loss in cases of advanced gastric cancer. In this study, we investigated the epigenetic inactivation of CDX1 by promoter methylation, as well as the functional link of CDX1 promoter methylation to the inflammatory NF-κB signaling pathway. We identified methylation-dependent NF-κB binding to the CDX1 promoter and quantified it using competitive electrophoretic mobility shift assays and chromatin immunoprecipitation. A methylated CDX1 promoter was associated with closed chromatin structure, reduced NF-κB binding, and transcriptional silencing. Along the gastritis-metaplasia-carcinoma sequence, we observed a biphasic pattern of tumor necrosis factor-α (TNF-α) protein expression and an inverse biphasic pattern of CDX1 promoter methylation; both are highly consistent with CDX1 protein expression. The stages of hyper-, hypo-, and hyper-methylation patterns of the CDX1 promoter were inversely correlated with the NF-κB signaling activity along this sequence. In conclusion, these functionally interacting events drive CDX1 expression and contribute to intestinal metaplasia, epithelial dedifferentiation, and carcinogenesis in the human stomach. PMID:22749770

Identification of body fluid-specific DNA methylation markers for use in forensic science.

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Park, Jong-Lyul; Kwon, Oh-Hyung; Kim, Jong Hwan; Yoo, Hyang-Sook; Lee, Han-Chul; Woo, Kwang-Man; Kim, Seon-Young; Lee, Seung-Hwan; Kim, Yong Sung

2014-11-01

DNA methylation, which occurs at the 5'-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers, but DNA methylation differences are sometimes low in saliva and vaginal secretions. Moreover, specific DNA methylation markers in four types of body fluids (blood, saliva, semen, and vaginal secretions) have not been investigated with genome-wide profiling. Here, we investigated novel DNA methylation markers for identification of body fluids for use in forensic science using the Illumina HumanMethylation 450K bead array, which contains over 450,000 CpG sites. Using methylome data from 16 samples of blood, saliva, semen, and vaginal secretions, we first selected 2986 hypermethylated or hypomethylated regions that were specific for each type of body fluid. We then selected eight CpG sites as novel, forensically relevant DNA methylation markers: cg06379435 and cg08792630 for blood, cg26107890 and cg20691722 for saliva, cg23521140 and cg17610929 for semen, and cg01774894 and cg14991487 for vaginal secretions. These eight selected markers were evaluated in 80 body fluid samples using pyrosequencing, and all showed high sensitivity and specificity for identification of the target body fluid. We suggest that these eight DNA methylation markers may be good candidates for developing an effective molecular assay for identification of body fluids in forensic science. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Methylation of food commodities during fumigation with methyl bromide

International Nuclear Information System (INIS)

Starratt, A.N.; Bond, E.J.

1990-01-01

Sites of methylation in several commodities (wheat, oatmeal, peanuts, almonds, apples, oranges, maize, alfalfa and potatoes) during fumigation with 14 C-methyl bromide were studied. Differences were observed in levels of the major volatiles: methanol, dimethyl sulphide and methyl mercaptan, products of O- and S-methylation, resulting from treatment of the fumigated materials with 1N sodium hydroxide. In studies of maize and wheat, histidine was the amino acid which underwent the highest level of N-methylation. (author). 24 refs, 3 tabs

Nucleosomes correlate with in vivo progression pattern of de novo methylation of p16 CpG islands in human gastric carcinogenesis.

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Zhe-Ming Lu

Full Text Available BACKGROUND: The exact relationship between nucleosome positioning and methylation of CpG islands in human pathogenesis is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we characterized the nucleosome position within the p16 CpG island and established a seeding methylation-specific PCR (sMSP assay based on bisulfite modification to enrich the p16 alleles containing methylated-CpG at the methylation "seeding" sites within its intron-1 in gastric carcinogenesis. The sMSP-positive rate in primary gastric carcinoma (GC samples (36/40 was significantly higher than that observed in gastritis (19/45 or normal samples (7/13 (P<0.01. Extensive clone sequencing of these sMSP products showed that the density of methylated-CpGs in p16 CpG islands increased gradually along with the severity of pathological changes in gastric tissues. In gastritis lesions the methylation was frequently observed in the region corresponding to the exon-1 coding-nucleosome and the 5'UTR-nucleosome; the methylation was further extended to the region corresponding to the promoter-nucleosome in GC samples. Only few methylated-CpG sites were randomly detected within p16 CpG islands in normal tissues. The significantly inversed relationship between the p16 exon-1 methylation and its transcription was observed in GC samples. An exact p16 promoter-specific 83 bp-MSP assay confirms the result of sMSP (33/55 vs. 1/6, P<0.01. In addition, p16 methylation in chronic gastritis lesions significantly correlated with H. pylori infection; however, such correlation was not observed in GC specimens. CONCLUSIONS/SIGNIFICANCE: It was determined that de novo methylation was initiated in the coding region of p16 exon-1 in gastritis, then progressed to its 5'UTR, and ultimately to the proximal promoter in GCs. Nucleosomes may function as the basic extension/progression unit of de novo methylation of p16 CpG islands in vivo.

Comparison Study of MS-HRM and Pyrosequencing Techniques for Quantification of APC and CDKN2A Gene Methylation

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Migheli, Francesca; Stoccoro, Andrea; Coppedè, Fabio; Wan Omar, Wan Adnan; Failli, Alessandra; Consolini, Rita; Seccia, Massimo; Spisni, Roberto; Miccoli, Paolo; Mathers, John C.; Migliore, Lucia

2013-01-01

There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing. PMID:23326336

Clinical utility of an epigenetic assay to detect occult prostate cancer in histopathologically negative biopsies: results of the MATLOC study.

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Stewart, Grant D; Van Neste, Leander; Delvenne, Philippe; Delrée, Paul; Delga, Agnès; McNeill, S Alan; O'Donnell, Marie; Clark, James; Van Criekinge, Wim; Bigley, Joseph; Harrison, David J

2013-03-01

Concern about possible false-negative prostate biopsy histopathology findings often leads to rebiopsy. A quantitative methylation specific polymerase chain reaction assay panel, including GSTP1, APC and RASSF1, could increase the sensitivity of detecting cancer over that of pathological review alone, leading to a high negative predictive value and a decrease in unnecessary repeat biopsies. The MATLOC study blindly tested archived prostate biopsy needle core tissue samples of 498 subjects from the United Kingdom and Belgium with histopathologically negative prostate biopsies, followed by positive (cases) or negative (controls) repeat biopsy within 30 months. Clinical performance of the epigenetic marker panel, emphasizing negative predictive value, was assessed and cross-validated. Multivariate logistic regression was used to evaluate all risk factors. The epigenetic assay performed on the first negative biopsies of this retrospective review cohort resulted in a negative predictive value of 90% (95% CI 87-93). In a multivariate model correcting for patient age, prostate specific antigen, digital rectal examination and first biopsy histopathological characteristics the epigenetic assay was a significant independent predictor of patient outcome (OR 3.17, 95% CI 1.81-5.53). A multiplex quantitative methylation specific polymerase chain reaction assay determining the methylation status of GSTP1, APC and RASSF1 was strongly associated with repeat biopsy outcome up to 30 months after initial negative biopsy in men with suspicion of prostate cancer. Adding this epigenetic assay could improve the prostate cancer diagnostic process and decrease unnecessary repeat biopsies. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Cytosine methylation is a conserved epigenetic feature found throughout the phylum Platyhelminthes

Science.gov (United States)

2013-01-01

Background The phylum Platyhelminthes (flatworms) contains an important group of bilaterian organisms responsible for many debilitating and chronic infectious diseases of human and animal populations inhabiting the planet today. In addition to their biomedical and veterinary relevance, some platyhelminths are also frequently used models for understanding tissue regeneration and stem cell biology. Therefore, the molecular (genetic and epigenetic) characteristics that underlie trophic specialism, pathogenicity or developmental maturation are likely to be pivotal in our continued studies of this important metazoan group. Indeed, in contrast to earlier studies that failed to detect evidence of cytosine or adenine methylation in parasitic flatworm taxa, our laboratory has recently defined a critical role for cytosine methylation in Schistosoma mansoni oviposition, egg maturation and ovarian development. Thus, in order to identify whether this epigenetic modification features in other platyhelminth species or is a novelty of S. mansoni, we conducted a study simultaneously surveying for DNA methylation machinery components and DNA methylation marks throughout the phylum using both parasitic and non-parasitic representatives. Results Firstly, using both S. mansoni DNA methyltransferase 2 (SmDNMT2) and methyl-CpG binding domain protein (SmMBD) as query sequences, we illustrate that essential DNA methylation machinery components are well conserved throughout the phylum. Secondly, using both molecular (methylation specific amplification polymorphism, MSAP) and immunological (enzyme-linked immunoabsorbent assay, ELISA) methodologies, we demonstrate that representative species (Echinococcus multilocularis, Protopolystoma xenopodis, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica and Polycelis nigra) within all four platyhelminth classes (Cestoda, Monogenea, Trematoda and ‘Turbellaria’) contain methylated cytosines within their genome compartments

Heterogeneity in white blood cells has potential to confound DNA methylation measurements.

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Bjorn T Adalsteinsson

Full Text Available Epigenetic studies are commonly conducted on DNA from tissue samples. However, tissues are ensembles of cells that may each have their own epigenetic profile, and therefore inter-individual cellular heterogeneity may compromise these studies. Here, we explore the potential for such confounding on DNA methylation measurement outcomes when using DNA from whole blood. DNA methylation was measured using pyrosequencing-based methodology in whole blood (n = 50-179 and in two white blood cell fractions (n = 20, isolated using density gradient centrifugation, in four CGIs (CpG Islands located in genes HHEX (10 CpG sites assayed, KCNJ11 (8 CpGs, KCNQ1 (4 CpGs and PM20D1 (7 CpGs. Cellular heterogeneity (variation in proportional white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils, counted by an automated cell counter explained up to 40% (p<0.0001 of the inter-individual variation in whole blood DNA methylation levels in the HHEX CGI, but not a significant proportion of the variation in the other three CGIs tested. DNA methylation levels in the two cell fractions, polymorphonuclear and mononuclear cells, differed significantly in the HHEX CGI; specifically the average absolute difference ranged between 3.4-15.7 percentage points per CpG site. In the other three CGIs tested, methylation levels in the two fractions did not differ significantly, and/or the difference was more moderate. In the examined CGIs, methylation levels were highly correlated between cell fractions. In summary, our analysis detects region-specific differential DNA methylation between white blood cell subtypes, which can confound the outcome of whole blood DNA methylation measurements. Finally, by demonstrating the high correlation between methylation levels in cell fractions, our results suggest a possibility to use a proportional number of a single white blood cell type to correct for this confounding effect in analyses.

Detection of methylated CDO1 in plasma of colorectal cancer; a PCR study.

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Keishi Yamashita

Full Text Available BACKGROUND: Cysteine biology is important for the chemosensitivity of cancer cells. Our research has focused on the epigenetic silencing of cysteine dioxygenase type 1 (CDO1 in colorectal cancer (CRC. In this study, we describe detection of CDO1 methylation in the plasma of CRC patients using methylation specific PCR (Q-MSP and extensive analysis of the PCR reaction. METHODS: DNA was extracted from plasma, and analysed for methylation of the CDO1 gene using Q-MSP. The detection rate of CDO1 gene methylation was calculated and compared with that of diluted DNA extracted from "positive control" DLD1 cells. CDO1 gene methylation in the plasma of 40 CRC patients that were clinicopathologically analysed was then determined. RESULTS: (1 The cloned sequence analysis detected 93.3% methylation of the promoter CpG islands of the CDO1 gene of positive control DLD1 cells and 4.7% methylation of the negative control HepG2 CDO1 gene. (2 DLD1 CDO1 DNA could not be detected in this assay if the extracted DNA was diluted ∼1000 fold. The more DNA that was used for the PCR reaction, the more effectively it was amplified in Q-MSP. (3 By increasing the amount of DNA used, methylated CDO1 could be clearly detected in the plasma of 8 (20% of the CRC patients. However, the percentage of CRC patients detected by methylated CDO1 in plasma was lower than that detected by CEA (35.9% or CA19-9 (23.1% in preoperative serum. Combination of CEA/CA19-9 plus plasma methylated CDO1 could increase the rate of detection of curable CRC patients (39.3% as compared to CEA/CA19-9 (25%. CONCLUSION: We have described detection of CDO1 methylation in the plasma of CRC patients. Although CDO1 methylation was not detected as frequently as conventional tumor markers, analysis of plasma CDO1 methylation in combination with CEA/CA19-9 levels increases the detection rate of curable CRC patients.

Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP).

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Yaish, Mahmoud W; Peng, Mingsheng; Rothstein, Steven J

2014-01-01

DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic

Epigenetic control of viral life-cycle by a DNA-methylation dependent transcription factor.

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Kirsty Flower

Full Text Available Epstein-Barr virus (EBV encoded transcription factor Zta (BZLF1, ZEBRA, EB1 is the prototype of a class of transcription factor (including C/EBPalpha that interact with CpG-containing DNA response elements in a methylation-dependent manner. The EBV genome undergoes a biphasic methylation cycle; it is extensively methylated during viral latency but is reset to an unmethylated state following viral lytic replication. Zta is expressed transiently following infection and again during the switch between latency and lytic replication. The requirement for CpG-methylation at critical Zta response elements (ZREs has been proposed to regulate EBV replication, specifically it could aid the activation of viral lytic gene expression from silenced promoters on the methylated genome during latency in addition to preventing full lytic reactivation from the non-methylated EBV genome immediately following infection. We developed a computational approach to predict the location of ZREs which we experimentally assessed using in vitro and in vivo DNA association assays. A remarkably different binding motif is apparent for the CpG and non-CpG ZREs. Computational prediction of the location of these binding motifs in EBV revealed that the majority of lytic cycle genes have at least one and many have multiple copies of methylation-dependent CpG ZREs within their promoters. This suggests that the abundance of Zta protein coupled with the methylation status of the EBV genome act together to co-ordinate the expression of lytic cycle genes at the majority of EBV promoters.

Atorvastatin Downregulates In Vitro Methyl Methanesulfonate and Cyclophosphamide Alkylation-Mediated Cellular and DNA Injuries

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Carlos F. Araujo-Lima

2018-01-01

Full Text Available Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA reductase inhibitors, and this class of drugs has been studied as protective agents against DNA damages. Alkylating agents (AAs are able to induce alkylation in macromolecules, causing DNA damage, as DNA methylation. Our objective was to evaluate atorvastatin (AVA antimutagenic, cytoprotective, and antigenotoxic potentials against DNA lesions caused by AA. AVA chemopreventive ability was evaluated using antimutagenicity assays (Salmonella/microsome assay, cytotoxicity, cell cycle, and genotoxicity assays in HepG2 cells. The cells were cotreated with AVA and the AA methyl methanesulfonate (MMS or cyclophosphamide (CPA. Our datum showed that AVA reduces the alkylation-mediated DNA damage in different in vitro experimental models. Cytoprotection of AVA at low doses (0.1–1.0 μM was observed after 24 h of cotreatment with MMS or CPA at their LC50, causing an increase in HepG2 survival rates. After all, AVA at 10 μM and 25 μM had decreased effect in micronucleus formation in HepG2 cells and restored cell cycle alterations induced by MMS and CPA. This study supports the hypothesis that statins can be chemopreventive agents, acting as antimutagenic, antigenotoxic, and cytoprotective components, specifically against alkylating agents of DNA.

Evaluation of an ex vivo murine local lymph node assay: multiple endpoint comparison.

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Piccotti, Joseph R; Knight, Stephanie A; Gillhouse, Kimberly; Lagattuta, Mark S; Bleavins, Michael R

2006-01-01

The local lymph node assay (LLNA) is used to assess the skin sensitization potential of chemicals. In the standard assay, mice are treated topically on the dorsum of both ears with test substance for 3 days. Following 2 days of rest, the initiation of the hypersensitivity response is evaluated by injecting (3)H-thymidine into a tail vein, and then measuring the levels of radioisotope incorporated into the DNA of lymph node cells draining the ears. In the current study, BALB/c mice were treated with the contact sensitizers hexylcinnamic aldehyde (HCA) and oxazolone, and the nonsensitizer methyl salicylate. The proliferative response of lymph node cells was evaluated in an ex vivo assay, in which isolated cells were cultured in vitro with (3)H-thymidine. Treatment of mice with HCA at 5-50% resulted in concentration-related increases in (3)H-thymidine incorporation, with stimulation indices ranging from 3 to 14. Low animal-to-animal variability was seen in three replicate assays testing HCA at 25%. As anticipated, the proliferative response induced by the potent sensitizer oxazolone at 0.25% was greater than HCA at all concentrations tested. Stimulation indices of 1.5 and 3 were seen in two independent experiments with methyl salicylate. These equivocal findings were likely due to the irritancy properties of the compound. Importantly, measuring ex vivo (3)H-thymidine incorporation was more sensitive than evaluating lymph node weight and cellularity, and in vitro bromodeoxyuridine incorporation. Furthermore, the results of the ex vivo LLNA were comparable to the standard assay. This study provided evidence that supports the use of an ex vivo LLNA for hazard assessment of contact hypersensitivity. Copyright 2006 John Wiley & Sons, Ltd.

DNA methylation profiles of ovarian epithelial carcinoma tumors and cell lines.

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Sahar Houshdaran

2010-02-01

Full Text Available Epithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies.We obtained DNA methylation profiles of 1,505 CpG sites (808 genes in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes that exhibited 'subtype-specific' DNA methylation patterns (FDR<1% among the tumors. In ovarian cancer cell lines, we estimated that for at least 27% of analyzed autosomal CpG sites, increases in methylation were accompanied by decreases in transcription of the associated gene.The significant difference in DNA methylation profiles between ovarian cancer cell lines and tumors underscores the need to be cautious in using cell lines as tumor models for molecular studies of ovarian cancer and other cancers. Similarly, the distinct methylation profiles of the different histological types of ovarian tumors reinforces the need to treat the different histologies of ovarian cancer as different diseases, both clinically and in biomarker studies. These data provide a useful resource for future studies, including those of

Systemic effects of chronically administered methyl prednisolonate and methyl 17-deoxyprednisolonate.

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Olejniczak, E; Lee, H J

1984-06-01

The systemic activities of methyl prednisolonate and methyl 17-deoxyprednisolonate (1) were studied in rats. Methyl 17-deoxyprednisolonate produced significant changes in the amount of sodium ion (decreased) and potassium ion (increased) in urine; however, methyl prednisolonate had no effect on electrolyte balance. Both methyl prednisolonate and methyl 17-deoxyprednisolonate had no effect on liver glycogen content, plasma corticosterone level and relative adrenal weight. In contrast, the parent compound prednisolone caused a significant decrease in liver glycogen content, plasma corticosterone level and relative adrenal weight.

Biosynthesis and emission of insect-induced methyl salicylate and methyl benzoate from rice

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Zhao, Nan [University of Tennessee, Knoxville (UTK); Guan, Ju [University of Tennessee, Knoxville (UTK); Ferrer, Jean-Luc [Universite Joseph Fourier, France; Engle, Nancy L [ORNL; Chern, Mawsheng [University of California, Davis; Ronald, Pamela [University of California, Davis; Tschaplinski, Timothy J [ORNL; Chen, Feng [University of Tennessee, Knoxville (UTK)

2010-01-01

Two benzenoid esters, methyl salicylate (MeSA) and methyl benzoate (MeBA), were detected from insect-damaged rice plants. By correlating metabolite production with gene expression analysis, five candidate genes encoding putative carboxyl methyltransferases were identified. Enzymatic assays with Escherichia coli-expressed recombinant proteins demonstrated that only one of the five candidates, OsBSMT1, has salicylic acid (SA) methyltransferase (SAMT) and benzoic acid (BA) methyltransferase (BAMT) activities for producing MeSA and MeBA, respectively. Whereas OsBSMT1 is phylogenetically relatively distant from dicot SAMTs, the three-dimensional structure of OsBSMT1, which was determined using homology-based structural modeling, is highly similar to those of characterized SAMTs. Analyses of OsBSMT1 expression in wild-type rice plants under various stress conditions indicate that the jasmonic acid (JA) signaling pathway plays a critical role in regulating the production and emission of MeSA in rice. Further analysis using transgenic rice plants overexpressing NH1, a key component of the SA signaling pathway in rice, suggests that the SA signaling pathway also plays an important role in governing OsBSMT1 expression and emission of its products, probably through a crosstalk with the JA signaling pathway. The role of the volatile products of OsBSMT1, MeSA and MeBA, in rice defense against insect herbivory is discussed.

Biosynthesis and emission of insect-induced methyl salicylate and methyl benzoate from rice.

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Zhao, Nan; Guan, Ju; Ferrer, Jean-Luc; Engle, Nancy; Chern, Mawsheng; Ronald, Pamela; Tschaplinski, Timothy J; Chen, Feng

2010-04-01

Two benzenoid esters, methyl salicylate (MeSA) and methyl benzoate (MeBA), were detected from insect-damaged rice plants. By correlating metabolite production with gene expression analysis, five candidate genes encoding putative carboxyl methyltransferases were identified. Enzymatic assays with Escherichia coli-expressed recombinant proteins demonstrated that only one of the five candidates, OsBSMT1, has salicylic acid (SA) methyltransferase (SAMT) and benzoic acid (BA) methyltransferase (BAMT) activities for producing MeSA and MeBA, respectively. Whereas OsBSMT1 is phylogenetically relatively distant from dicot SAMTs, the three-dimensional structure of OsBSMT1, which was determined using homology-based structural modeling, is highly similar to those of characterized SAMTs. Analyses of OsBSMT1 expression in wild-type rice plants under various stress conditions indicate that the jasmonic acid (JA) signaling pathway plays a critical role in regulating the production and emission of MeSA in rice. Further analysis using transgenic rice plants overexpressing NH1, a key component of the SA signaling pathway in rice, suggests that the SA signaling pathway also plays an important role in governing OsBSMT1 expression and emission of its products, probably through a crosstalk with the JA signaling pathway. The role of the volatile products of OsBSMT1, MeSA and MeBA, in rice defense against insect herbivory is discussed. Copyright 2010 Elsevier Masson SAS. All rights reserved.

γ-radiation induces cellular sensitivity and aberrant methylation in human tumor cell lines.

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Kumar, Ashok; Rai, Padmalatha S; Upadhya, Raghavendra; Vishwanatha; Prasada, K Shama; Rao, B S Satish; Satyamoorthy, Kapettu

2011-11-01

Ionizing radiation induces cellular damage through both direct and indirect mechanisms, which may include effects from epigenetic changes. The purpose of this study was to determine the effect of ionizing radiation on DNA methylation patterns that may be associated with altered gene expression. Sixteen human tumor cell lines originating from various cancers were initially tested for radiation sensitivity by irradiating them with γ-radiation in vitro and subsequently, radiation sensitive and resistant cell lines were treated with different doses of a demethylating agent, 5-Aza-2'-Deoxycytidine (5-aza-dC) and a chromatin modifier, Trichostatin-A (TSA). Survival of these cell lines was measured using 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium (MTT) and clonogenic assays. The effect of radiation on global DNA methylation was measured using reverse phase high performance liquid chromatography (RP-HPLC). The transcription response of methylated gene promoters, from cyclin-dependent kinase inhibitor 2A (p16(INK4a)) and ataxia telangiectasia mutated (ATM) genes, to radiation was measured using a luciferase reporter assay. γ-radiation resistant (SiHa and MDAMB453) and sensitive (SaOS2 and WM115) tumor cell lines were examined for the relationship between radiation sensitivity and DNA methylation. Treatment of cells with 5-aza-dC and TSA prior to irradiation enhanced DNA strand breaks, G2/M phase arrest, apoptosis and cell death. Exposure to γ-radiation led to global demethylation in a time-dependent manner in tumor cells in relation to resistance and sensitivity to radiation with concomitant activation of p16(INK4a) and ATM gene promoters. These results provide important information on alterations in DNA methylation as one of the determinants of radiation effects, which may be associated with altered gene expression. Our results may help in delineating the mechanisms of radiation resistance in tumor cells, which can influence diagnosis, prognosis and

[Comparative analysis of methylation profiles in tissues of oral leukoplakia and oral squamous cell carcinoma].

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Fu, J; Su, Y; Liu, Y; Zhang, X Y

2018-04-09

Objective: To compare the methylation profiles in tissues of oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC) with healthy tissues of oral mucosa, in order to identify the role of DNA methylation played in tumorigenesis. Methods: DNA samples extracted from tissues of 4 healthy oral mucosa, 4 OSCC and 4 OLK collected from patients of the Department of Oral Medicine, Capital Medical University School of Stomatology were examined and compared using Methylation 450 Bead Chip. The genes associated with differentially methylated CpG sites were selected for gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment. Results: Multiple differentially methylated CpG sites were identified by using the above mentioned assay. Hypermethylation constitutes 86.18% (23 290/27 025) of methylation changes in OLK and hypomethylation accounts for 13.82% (3 734/27 025) of methylation changes. Both hypermethylated and hypomethylated CpG sites were markedly increased in OSCC tissue compared with OLK tissue. The majority of differentially methylated CpG sites were located outside CpG islands, with approximately one-fourth in CpG shores flanking the islands, which were considered highly important for gene regulation and tumorigenesis. Pathway analysis revealed that differentially methylated CpG sites in both OLK and OSCC patients shared the same pathway enrichments, most of which were correlated with carcinogenesis and cancer progression (e.g., DNA repair, cell cycle, and apoptosis). Conclusions: In the present study, methylation-associated alterations affect almost all pathways in the cellular network in both OLK and OSCC. OLK and OSCC shared similar methylation changes whether in pathways or genes, indicating that epigenetically they might have the same molecular basis for disease progression.

Assessment of genotoxicity of methyl-tert-butyl ether, benzene, toluene, ethylbenzene, and xylene to human lymphocytes using comet assay

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Chen, Colin S.; Hseu, You C.; Liang, Shih H.; Kuo, J.-Y.; Chen, Ssu. C.

2008-01-01

Methyl-tert-butyl ether (MTBE) is a gasoline oxygenate and antiknock additive substituting for lead alkyls currently in use worldwide. Benzene, toluene, ethylbenzene, and xylene (BTEX) are volatile monoaromatic hydrocarbons which are commonly found together in crude petroleum and petroleum products such as gasoline. The aim of this study is to evaluate the genotoxic effects of these tested chemicals in human lymphocytes. Using the alkaline comet assay, we showed that all of the tested chemicals induce DNA damage in isolated human lymphocytes. This effect could follow from the induction of DNA strands breaks. The neutral version of the test revealed that MTBE, benzene, and xylenes induce DNA double-strand breaks at 200 μM. Apart from MTBE, the spin traps, 5,5-dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-α-phenylnitrone (PBN) can decrease the level of DNA damage in BTEX at 200 μM. This indicated that DNA damage could result from the participation of free radicals in DNA-damaging effect, which was further supported by the fact that post-treatment of formamidopyrimidine-DNA glycosylase (Fpg), enzyme recognizing oxidized DNA purines, gave rise to a significant increase in the extent of DNA damage in cells treated with benzene, and xylene at 200 μM. The results obtained suggested that MTBE and BTEX could induce a variety type of DNA damage such as single-strand breaks (SSBs), double-strand breaks (DSBs), and oxidative base modification

Variable Methylation Potential in Preterm Placenta: Implication for Epigenetic Programming of the Offspring.

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Khot, Vinita V; Chavan-Gautam, Preeti; Mehendale, Savita; Joshi, Sadhana R

2017-06-01

Children born preterm are reported to be at increased risk of developing noncommunicable diseases in later life. Altered placental DNA methylation patterns are implicated in fetal programming of adult diseases. Our earlier animal studies focus on micronutrients (folic acid, vitamin B 12 ) and long-chain polyunsaturated fatty acids (LCPUFAs) that interact in the 1 carbon cycle, thereby influencing methylation reactions. Our previous studies in women delivering preterm show altered plasma levels of micronutrients and lower plasma LCPUFA levels. We postulate that alterations in the micronutrient metabolism may affect the regulation of enzymes, methionine adenosyltransferase ( MAT2A), and SAH-hydrolase ( AHCY), involved in the production of methyl donor S-adenosylmethionine (SAM), thereby influencing the methylation potential (MP) in the placenta of women delivering preterm. The present study, therefore, examines the mRNA, protein levels of enzymes ( MAT2A and AHCY), SAM, S-adenosylhomocysteine (SAH) levels, and global DNA methylation levels from preterm (n = 73) and term (n = 73) placentae. The enzyme messenger RNA (mRNA) levels were analyzed by real-time quantitative polymerase chain reaction, protein levels by enzyme-linked immunosorbent assay, and SAM-SAH levels by high-performance liquid chromatography. The mRNA levels for MAT2A and AHCY are higher ( P programming of the developing fetus.

Decreased viscosity of rat-liver DNA treated by 3'-methyl-4-dimethylaminoazobenzene, detected with a new viscometric approach.

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Parodi, S; Balbi, C; Taningher, M; Pala, M; Russo, P; Abelmoschi, M L; Santi, L

1982-11-01

DNA damage induced in vivo by 3'-methyl-4-dimethylaminoazobenzene (3'CH3DAB) was investigated with 2 differently sensitive techniques: the alkaline elution assay and the viscometric measurement of DNA damage. 3'CH3DAB appeared to be falsely negative with the alkaline elution assay, whereas with the viscometric approach, which is about 30-50 times more sensitive, it appeared positive, and the DNA damage was dose-dependent.

Efficient molecular screening of Lynch syndrome by specific 3' promoter methylation of the MLH1 or BRAF mutation in colorectal cancer with high-frequency microsatellite instability.

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Nakagawa, Hitoshi; Nagasaka, Takeshi; Cullings, Harry M; Notohara, Kenji; Hoshijima, Naoko; Young, Joanne; Lynch, Henry T; Tanaka, Noriaki; Matsubara, Nagahide

2009-06-01

It is sometimes difficult to diagnose Lynch syndrome by the simple but strict clinical criteria, or even by the definitive genetic testing for causative germline mutation of mismatch repair genes. Thus, some practical and efficient screening strategy to select highly possible Lynch syndrome patients is exceedingly desirable. We performed a comprehensive study to evaluate the methylation status of whole MLH1 promoter region by direct bisulfite sequencing of the entire MLH1 promoter regions on Lynch and non-Lynch colorectal cancers (CRCs). Then, we established a convenient assay to detect methylation in key CpG islands responsible for the silencing of MLH1 expression. We studied the methylation status of MLH1 as well as the CpG island methylator phenotype (CIMP) and immunohistochemical analysis of mismatch repair proteins on 16 cases of Lynch CRC and 19 cases of sporadic CRCs with high-frequency microsatellite instability (MSI-H). Sensitivity to detect Lynch syndrome by MLH1 (CCAAT) methylation was 88% and the specificity was 84%. Positive likelihood ratio (PLR) was 5.5 and negative likelihood ratio (NLR) was 0.15. Sensitivity by mutational analysis of BRAF was 100%, specificity was 84%, PLR was 6.3 and NLR was zero. By CIMP analysis; sensitivity was 88%, specificity was 79%, PLR was 4.2, and NLR was 0.16. BRAF mutation or MLH1 methylation analysis combined with MSI testing could be a good alternative to screen Lynch syndrome patients in a cost effective manner. Although the assay for CIMP status also showed acceptable sensitivity and specificity, it may not be practical because of its rather complicated assay.

The Effects of Fenarimol and Methyl Parathion on Glucose 6-Phosphate Dehydrogenase Enzyme Activity in Rats

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Ferda ARI

2017-10-01

Full Text Available Fenarimol and methyl parathion are pesticides that have been used in agriculture for several years. These pesticides have significant effects on environmental and human health. Therefore, we investigated the effects of methyl parathion and fenarimol on glucose 6-phosphate dehydrogenase (EC 1.1.1.49 enzyme activity in rats. The glucose 6- phosphate dehydrogenase is the first enzyme of the pentose phosphate pathway and it is important in detoxifying reactions by NADPH generated. In this study, wistar albino rats administrated with methyl parathion (7 mg kg–1 and fenarimol (200 mg kg−1 by intraperitoneally for different periods (2, 4, 8, 16, 32, 64, and 72 h. The glucose 6-phosphate dehydrogenase enzyme activity was assayed in liver, kidney, brain, and small intestine in male and female rats. The exposure of fenarimol and methyl parathion caused increase of glucose 6-phosphate dehydrogenase enzyme activity in rat tissues, especially at last periods. We suggest that this increment of enzyme activity may be the reason of toxic effects of fenarimol and methyl parathion.

Inhibiting DNA methylation alters olfactory extinction but not acquisition learning in Apis cerana and Apis mellifera.

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Gong, Zhiwen; Wang, Chao; Nieh, James C; Tan, Ken

2016-07-01

DNA methylation plays a key role in invertebrate acquisition and extinction memory. Honey bees have excellent olfactory learning, but the role of DNA methylation in memory formation has, to date, only been studied in Apis mellifera. We inhibited DNA methylation by inhibiting DNA methyltransferase (DNMT) with zebularine (zeb) and studied the resulting effects upon olfactory acquisition and extinction memory in two honey bee species, Apis cerana and A. mellifera. We used the proboscis extension reflex (PER) assay to measure memory. We provide the first demonstration that DNA methylation is also important in the olfactory extinction learning of A. cerana. DNMT did not reduce acquisition learning in either species. However, zeb bidirectionally and differentially altered extinction learning in both species. In particular, zeb provided 1h before acquisition learning improved extinction memory retention in A. mellifera, but reduced extinction memory retention in A. cerana. The reasons for these differences are unclear, but provide a basis for future studies to explore species-specific differences in the effects of methylation on memory formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.

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Barault, L; Amatu, A; Bleeker, F E; Moutinho, C; Falcomatà, C; Fiano, V; Cassingena, A; Siravegna, G; Milione, M; Cassoni, P; De Braud, F; Rudà, R; Soffietti, R; Venesio, T; Bardelli, A; Wesseling, P; de Witt Hamer, P; Pietrantonio, F; Siena, S; Esteller, M; Sartore-Bianchi, A; Di Nicolantonio, F

2015-09-01

O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P alkylating agents. © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Benzopyrene exposure disrupts DNA methylation and growth dynamics in breast cancer cells

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Sadikovic, Bekim; Rodenhiser, David I.

2006-01-01

Exposures to environmental carcinogens and unhealthy lifestyle choices increase the incidence of breast cancer. One such compound, benzo(a)pyrene (BaP), leads to covalent DNA modifications and the deregulation of gene expression. To date, these mechanisms of BaP-induced carcinogenesis are poorly understood, particularly in the case of breast cancer. We tested the effects of BaP exposure on cellular growth dynamics and DNA methylation in four breast cancer cell lines since disruptions in DNA methylation lead to deregulated gene expression and the loss of genomic integrity. We observed robust time- and concentration-dependent loss of proliferation, S phase and G2M accumulation and apoptosis in p53 positive MCF-7 and T47-D cells. We observed minimal responses in p53 negative HCC-1086 and MDA MB 231 cells. Furthermore, BaP increased p53 levels in both p53 positive cell lines, as well as p21 levels in MCF-7 cells, an effect that was prevented by the p53-specific inhibitor pifithrin-α. No changes in global levels of DNA methylation levels induced by BaP were detected by the methyl acceptor assay (MAA) in any cell line, however, methylation profiling by AIMS (amplification of intermethylated sites) analysis showed dynamic, sequence-specific hypo- and hypermethylation events in all cell lines. We also identified BaP-induced hypomethylation events at a number of genomic repeats. Our data confirm the p53-specific disruption of the cell cycle as well as the disruption of DNA methylation as a consequence of BaP treatment, thus reinforcing the link between environmental exposures, DNA methylation and breast cancer

Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

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Calvello, Mariarosaria; Tabano, Silvia; Colapietro, Patrizia; Maitz, Silvia; Pansa, Alessandra; Augello, Claudia; Lalatta, Faustina; Gentilin, Barbara; Spreafico, Filippo; Calzari, Luciano; Perotti, Daniela; Larizza, Lidia; Russo, Silvia; Selicorni, Angelo; Sirchia, Silvia M; Miozzo, Monica

2013-01-01

Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD. PMID:23917791

Resource base influences genome-wide DNA methylation levels in wild baboons (Papio cynocephalus)

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Lea, Amanda J.; Altmann, Jeanne; Alberts, Susan C.; Tung, Jenny

2015-01-01

Variation in resource availability commonly exerts strong effects on fitness-related traits in wild animals. However, we know little about the molecular mechanisms that mediate these effects, or about their persistence over time. To address these questions, we profiled genome-wide whole blood DNA methylation levels in two sets of wild baboons: (i) ‘wild-feeding’ baboons that foraged naturally in a savanna environment and (ii) ‘Lodge’ baboons that had ready access to spatially concentrated human food scraps, resulting in high feeding efficiency and low daily travel distances. We identified 1,014 sites (0.20% of sites tested) that were differentially methylated between wild-feeding and Lodge baboons, providing the first evidence that resource availability shapes the epigenome in a wild mammal. Differentially methylated sites tended to occur in contiguous stretches (i.e., in differentially methylated regions or DMRs), in promoters and enhancers, and near metabolism-related genes, supporting their functional importance in gene regulation. In agreement, reporter assay experiments confirmed that methylation at the largest identified DMR, located in the promoter of a key glycolysis-related gene, was sufficient to causally drive changes in gene expression. Intriguingly, all dispersing males carried a consistent epigenetic signature of their membership in a wild-feeding group, regardless of whether males dispersed into or out of this group as adults. Together, our findings support a role for DNA methylation in mediating ecological effects on phenotypic traits in the wild, and emphasize the dynamic environmental sensitivity of DNA methylation levels across the life course. PMID:26508127

PAX1 methylation analysis by MS-HRM is useful in triage of high-grade squamous intraepithelial lesions.

Science.gov (United States)

Wang, Zhen-Ming

2014-01-01

This study is aimed to investigate the role of paired boxed gene 1 (PAX1) methylation analysis by methylation- sensitive high-resolution melting (MS-HRM) in the detection of high grade lesions in atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H) and compared its performance with the Hybrid Capture 2 (HC2) human papillomavirus (HPV) test. In our study, 130 cases with a diagnosis of ASC-H from the cervical cytological screening by Thinprep cytologic test (TCT) technique were selected for triage. Their cervical scrapings were collected and evaluated by using PAX1 methylation analysis (MS-HRM) and high-risk HPV DNA test (HC2), followed by colposcopy and cervical biopsy. Chi-square test were used to test the differences of PAX1 methylation or HPV infection between groups. In the detection of CIN2+, the sensitivity, specificity, the PPV, NPV and the accuracy of PAX1 MS-HRM assay and high-risk HPV (HR-HPV) tests were respectively 80.6% vs 67.7%, 94.9% vs 54.5%, 83.3%, vs 31.8%, 94.0% vs 84.4%, and 91.5% vs 57.7%. The PAX1 MS-HRM assay proved superior to HR-HPV testing in the detection of high grade lesions (CIN2+) in ASC-H. This approach could screen out the majority of high grade lesion cases of ASC-H, and thus could reduce the referral rate to colposcopy.

Validations of SCT Methylation as a Hallmark Biomarker for Lung Cancers

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Fujimoto, Junya; Wistuba, Ignacio; Lam, Stephen; Yatabe, Yasushi; Wang, Yi-Wei; Stastny, Victor; Gao, Boning; Larsen, Jill E; Girard, Luc; Liu, Xiaoyun; Song, Kai; Behrens, Carmen; Kalhor, Neda; Xie, Yang; Zhang, Michael Q; Minna, John D; Gazdar, Adi F

2016-01-01

Background The human secretin (SCT) gene encodes secretin, a hormone with limited tissue distribution. Analysis of The Cancer Genome Atlas (TCGA) 450K methylation array data indicated that the SCT promoter region is differentially hypermethylated in lung cancer. Our purpose was to validate SCT methylation as a potential cancer biomarker for lung cancer. Methods We analyzed TCGA data, and developed and applied SCT-specific bisulfite DNA sequencing and quantitative methylation specific PCR (qMSP) assays. Results The analyses of TCGA 450K data of 801 samples showed that SCT hypermethylation has an area under curve (AUC) value >0.98 to distinguish lung adenocarcinomas or squamous cell carcinomas from non-malignant lung. We confirmed the highly discriminative SCT methylation by bisulfite sequencing of lung cancer cell lines and normal blood cells. By applying qMSP, we found that SCT hypermethylation was frequently detected in all major subtypes of malignant NSCLC (AUC=0.92, n=108) and SCLC cancers (AUC=0.93, n=40) but less frequently present in lung carcinoids (AUC=0.54, n=20). SCT hypermethylation appeared in lung carcinoma in situ samples during multistage pathogenesis and increased in invasive samples. Further analyses of TCGA 450K data showed that SCT hypermethylation is highly discriminative in most types of other malignant tumors but less frequently present in low-grade malignant tumors. The only normal tissue with high methylation was the placenta. Conclusions Our findings demonstrated that SCT methylation is a highly discriminative biomarker for lung and other malignant tumors, and less frequently present in low-grade malignant tumors including lung carcinoids, and appears at the carcinoma in situ stage. PMID:26725182

Transcriptional regulation and DNA methylation in plastids during transitional conversion of chloroplasts to chromoplasts.

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Kobayashi, H; Ngernprasirtsiri, J; Akazawa, T

1990-01-01

During transitional conversion of chloroplasts to chromoplasts in ripening tomato (Lycopersicon esculentum) fruits, transcripts for several plastid genes for photosynthesis decreased to undetectable levels. Run-on transcription of plastids indicated that transcriptional regulation operated as a predominant factor. We found that most of the genes in chloroplasts were actively transcribed in vitro by Escherichia coli and soluble plastid RNA polymerases, but some genes in chromoplasts seemed to be silent when assayed by the in vitro systems. The regulatory step, therefore, was ascribed to DNA templates. The analysis of modified base composition revealed the presence of methylated bases in chromoplast DNA, in which 5-methylcytosine was most abundant. The presence of 5-methylcytosine detected by isoschizomeric endonucleases and Southern hybridization was correlated with the undetectable transcription activity of each gene in the run-on assay and in vitro transcription experiments. It is thus concluded that the suppression of transcription mediated by DNA methylation is one of the mechanisms governing gene expression in plastids converting from chloroplasts to chromoplasts. Images Fig. 1 Fig. 2 Fig. 3. Fig. 4. Fig. 5. PMID:2303026

Mapuche herbal medicine inhibits blood platelet aggregation.

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Falkenberg, Susan Skanderup; Tarnow, Inge; Guzman, Alfonso; Mølgaard, Per; Simonsen, Henrik Toft

2012-01-01

12 plant species traditionally used by the Mapuche people in Chile to treat wounds and inflammations have been evaluated for their direct blood platelet inhibition. Seven of the 12 tested plant species showed platelet inhibitory effect in sheep blood, and four of these were also able to inhibit the ADP- (5.0 μM) and collagen- (2.0 μg/mL) induced aggregations in human blood. These four species in respective extracts (in brackets) were Blechnum chilense (MeOH), Luma apiculata (H(2)O), Amomyrtus luma (DCM : MeOH 1 : 1) and Cestrum parqui (DCM : MeOH 1 : 1). The platelet aggregating inhibitory effects of A. luma (DCM : MeOH 1 : 1), and L. apiculata (H(2)O) were substantial and confirmed by inhibition of platelet surface activation markers.

Cytosine methylation dysregulation in neonates following intrauterine growth restriction.

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Francine Einstein

2010-01-01

Full Text Available Perturbations of the intrauterine environment can affect fetal development during critical periods of plasticity, and can increase susceptibility to a number of age-related diseases (e.g., type 2 diabetes mellitus; T2DM, manifesting as late as decades later. We hypothesized that this biological memory is mediated by permanent alterations of the epigenome in stem cell populations, and focused our studies specifically on DNA methylation in CD34+ hematopoietic stem and progenitor cells from cord blood from neonates with intrauterine growth restriction (IUGR and control subjects.Our epigenomic assays utilized a two-stage design involving genome-wide discovery followed by quantitative, single-locus validation. We found that changes in cytosine methylation occur in response to IUGR of moderate degree and involving a restricted number of loci. We also identify specific loci that are targeted for dysregulation of DNA methylation, in particular the hepatocyte nuclear factor 4alpha (HNF4A gene, a well-known diabetes candidate gene not previously associated with growth restriction in utero, and other loci encoding HNF4A-interacting proteins.Our results give insights into the potential contribution of epigenomic dysregulation in mediating the long-term consequences of IUGR, and demonstrate the value of this approach to studies of the fetal origin of adult disease.

Epigenetic mechanism of maternal post-traumatic stress disorder in delayed rat offspring development: dysregulation of methylation and gene expression.

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Zhang, X G; Zhang, H; Liang, X L; Liu, Q; Wang, H Y; Cao, B; Cao, J; Liu, S; Long, Y J; Xie, W Y; Peng, D Z

2016-08-19

Maternal post-traumatic stress disorder (PTSD) increases the risk of adverse neurodevelopmental outcomes in the child. Epigenetic alternations may play an essential role in the negative effects of PTSD. This study was aimed to investigate the possible epigenetic alterations of maternal PTSD, which underpins the developmental and behavioral impact. 24 pregnant Sprague-Dawley (SD) rats were randomly grouped into PTSD and control groups. Open-field tests (OFTs), elevated pull maze (EPM) assays, gene expression profile chip tests, and methylated DNA immunoprecipitation sequencing (MeDIP-Seq) were performed on the offsprings 30 days after birth. The results showed that PTSD offsprings had lower body weights and OFT scores than control offsprings. Enzyme-linked immunosorbent assays showed that serotonin receptor (5-HT) and dopamine levels were significantly lower in PTSD offsprings than in control offsprings. In contrast, corticosterone levels were higher in the PTSD group than in the control group. In a comparison of the PTSD group versus the control group, 4,160 significantly differentially methylated loci containing 30,657 CpGs were identified; 2,487 genes, including 13 dysmethylated genes, were validated by gene expression profiling, showing a negative correlation between methylation and gene expression (R = -0.617, P = 0.043). In conclusion, maternal PTSD could delay the physical and behavioral development of offsprings, and the underlying mechanism could contribute to changes in neurotransmitters and gene expression, owing to dysregulation of whole-genome methylation. These findings could support further clinical research on appropriate interventions for maternal PTSD to prevent methylation dysregulation and developmental retardation.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1).

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Nwaobi, Sinifunanya E; Olsen, Michelle L

2015-09-26

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

DNA methylation analysis reveals distinct methylation signatures in pediatric germ cell tumors

International Nuclear Information System (INIS)

Amatruda, James F; Frazier, A Lindsay; Poynter, Jenny N; Ross, Julie A; Christensen, Brock; Fustino, Nicholas J; Chen, Kenneth S; Hooten, Anthony J; Nelson, Heather; Kuriger, Jacquelyn K; Rakheja, Dinesh

2013-01-01

Aberrant DNA methylation is a prominent feature of many cancers, and may be especially relevant in germ cell tumors (GCTs) due to the extensive epigenetic reprogramming that occurs in the germ line during normal development. We used the Illumina GoldenGate Cancer Methylation Panel to compare DNA methylation in the three main histologic subtypes of pediatric GCTs (germinoma, teratoma and yolk sac tumor (YST); N = 51) and used recursively partitioned mixture models (RPMM) to test associations between methylation pattern and tumor and demographic characteristics. We identified genes and pathways that were differentially methylated using generalized linear models and Ingenuity Pathway Analysis. We also measured global DNA methylation at LINE1 elements and evaluated methylation at selected imprinted loci using pyrosequencing. Methylation patterns differed by tumor histology, with 18/19 YSTs forming a distinct methylation class. Four pathways showed significant enrichment for YSTs, including a human embryonic stem cell pluripotency pathway. We identified 190 CpG loci with significant methylation differences in mature and immature teratomas (q < 0.05), including a number of CpGs in stem cell and pluripotency-related pathways. Both YST and germinoma showed significantly lower methylation at LINE1 elements compared with normal adjacent tissue while there was no difference between teratoma (mature and immature) and normal tissue. DNA methylation at imprinted loci differed significantly by tumor histology and location. Understanding methylation patterns may identify the developmental stage at which the GCT arose and the at-risk period when environmental exposures could be most harmful. Further, identification of relevant genetic pathways could lead to the development of new targets for therapy

Modeling of the oxidation of methyl esters—Validation for methyl hexanoate, methyl heptanoate, and methyl decanoate in a jet-stirred reactor

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Glaude, Pierre Alexandre; Herbinet, Olivier; Bax, Sarah; Biet, Joffrey; Warth, Valérie; Battin-Leclerc, Frédérique

2013-01-01

The modeling of the oxidation of methyl esters was investigated and the specific chemistry, which is due to the presence of the ester group in this class of molecules, is described. New reactions and rate parameters were defined and included in the software EXGAS for the automatic generation of kinetic mechanisms. Models generated with EXGAS were successfully validated against data from the literature (oxidation of methyl hexanoate and methyl heptanoate in a jet-stirred reactor) and a new set of experimental results for methyl decanoate. The oxidation of this last species was investigated in a jet-stirred reactor at temperatures from 500 to 1100 K, including the negative temperature coefficient region, under stoichiometric conditions, at a pressure of 1.06 bar and for a residence time of 1.5 s: more than 30 reaction products, including olefins, unsaturated esters, and cyclic ethers, were quantified and successfully simulated. Flow rate analysis showed that reactions pathways for the oxidation of methyl esters in the low-temperature range are similar to that of alkanes. PMID:23710076

Modeling of the oxidation of methyl esters-Validation for methyl hexanoate, methyl heptanoate, and methyl decanoate in a jet-stirred reactor.

Science.gov (United States)

Glaude, Pierre Alexandre; Herbinet, Olivier; Bax, Sarah; Biet, Joffrey; Warth, Valérie; Battin-Leclerc, Frédérique

2010-11-01

The modeling of the oxidation of methyl esters was investigated and the specific chemistry, which is due to the presence of the ester group in this class of molecules, is described. New reactions and rate parameters were defined and included in the software EXGAS for the automatic generation of kinetic mechanisms. Models generated with EXGAS were successfully validated against data from the literature (oxidation of methyl hexanoate and methyl heptanoate in a jet-stirred reactor) and a new set of experimental results for methyl decanoate. The oxidation of this last species was investigated in a jet-stirred reactor at temperatures from 500 to 1100 K, including the negative temperature coefficient region, under stoichiometric conditions, at a pressure of 1.06 bar and for a residence time of 1.5 s: more than 30 reaction products, including olefins, unsaturated esters, and cyclic ethers, were quantified and successfully simulated. Flow rate analysis showed that reactions pathways for the oxidation of methyl esters in the low-temperature range are similar to that of alkanes.

Lack of oestrogenic effects of food preservatives (parabens) in uterotrophic assays

DEFF Research Database (Denmark)

Hossaini, A.; Larsen, Jens-Jørgen; Larsen, John Christian

2000-01-01

The oestrogenic activity of the parabens, methyl-, ethyl- and propyl p-hydroxybenzoate, widely used as antimicrobials in food, and butyl p-hydroxybenzoate, which is used in cosmetic products, and their shared main metabolite p-hydroxybenzoic acid was investigated in a mouse uterotrophic assay......, Immature B6D2F1 mice were treated with oral or subcutaneous doses of the test compounds for three consecutive days, p-Hydroxgybenzoic acid and butyl p-hydroxybenzoate were also tested by the subcutaneous route in a rat uterotrophic assay. A significant increase in the uterus weight at day 4 was considered...... an oestrogenic effect. In the mouse assay, none of the compounds tested produced any oestrogenic response at dose levels up to 100 mg/kg body weight per day, for ethyl p-hydroxybenzoate even at a dose level of 1000 mg/kg body weight per day. In immature Wistar rats, subcutaneous administration of butyl p...

Association of oxidative stress with arsenic methylation in chronic arsenic-exposed children and adults

International Nuclear Information System (INIS)

Xu Yuanyuan; Wang Yi; Zheng Quanmei; Li Xin; Li Bing; Jin Yaping; Sun Xiance; Sun Guifan

2008-01-01

Though oxidative stress is recognized as an important pathogenic mechanism of arsenic, and arsenic methylation capacity is suggested to be highly involved in arsenic-related diseases, the association of arsenic methylation capacity with arsenic-induced oxidative stress remains unclear. To explore oxidative stress and its association with arsenic methylation, cross-sectional studies were conducted among 208 high and 59 low arsenic-exposed subjects. Levels of urinary arsenic species [inorganic arsenic (iAs), monomethylated arsenic (MMA) and dimethylated arsenic (DMA)] were determined by hydride generation atomic absorption spectrometry. Proportions of urinary arsenic species, the first methylation ratio (FMR) and the secondary methylation ratio (SMR) were used as indicators for arsenic methylation capacity. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentrations were analyzed by enzyme-linked immunosorbent assay kits. Reduced glutathione (GSH) levels and superoxide dismutase (SOD) activity in whole blood were determined to reflect anti-oxidative status. The high arsenic-exposed children and adults were significantly increased in urinary 8-OHdG concentrations but decreased in blood GSH levels compared with the low exposed children and adults. In multiple linear regression models, blood GSH levels and urinary 8-OHdG concentrations of arsenic-exposed children and adults showed strong associations with the levels of urinary arsenic species. Arsenic-exposed subjects in the lower and the upper quartiles of proportions of urinary arsenic species, FMR or SMR were significantly different in urinary 8-OHdG, blood GSH and SOD. The associations of arsenic methylation capacity with 8-OHdG, GSH and SOD were also observed in multivariate regression analyses. These results may provide linkage between arsenic methylation capacity and oxidative stress in humans and suggest that adverse health effects induced by arsenic are related to arsenic methylation through oxidative stress

Methylation and silencing of the retinoic acid receptor-β2 gene in cervical cancer

International Nuclear Information System (INIS)

Ivanova, Tatyana; Petrenko, Anatolii; Gritsko, Tatyana; Vinokourova, Svetlana; Eshilev, Ernest; Kobzeva, Vera; Kisseljov, Fjodor; Kisseljova, Natalia

2002-01-01

Expression of the retinoic acid receptor β2 (RAR-β2), a putative tumor suppressor gene, is reduced in various human cancers, including squamous cell carcinomas (SCC) of the uterine cervix. The mechanism of the inhibition of RAR-β2 expression remains obscure. We examined whether methylation of RAR-β2 gene could be responsible for this silencing in cervical SCC. Expression of RAR-β2 mRNA and methylation status of the 5' region of RAR-β2 gene were examined in 20 matched specimens from patients with cervical SCC and in three cervical cancer cell lines by Northern blot analysis and methylation-specific PCR (MSP) assay or Southern blot analysis respectively. In 8 out 20 cervical SCC (40%) the levels of RAR-β2 mRNA were decreased or undetectable in comparison with non-neoplastic cervix tissues. All 8 tumors with reduced levels of RAR-β2 mRNA expression showed methylation of the promoter and the first exon expressed in the RAR-β2 transcript. The RAR-β2 gene from non-neoplastic cervical tissues was mostly unmethylated and expressed, but methylated alleles of the gene were found in three samples of the morphologically normal tissues adjacent to the tumors. Three cervical cancer cell lines with extremely low level of RAR-β2 mRNA expression, SiHA, HeLA and CaSki, also showed methylation of this region of the RAR-β2 gene. These findings suggest that methylation of the 5' region of RAR-β2 gene may contribute to gene silencing and that methylation of this region may be an important and early event in cervical carcinogenesis. These findings may be useful to make retinoids more effective as preventive and therapeutic agents in combination with inhibitors of DNA methylation

Parvovirus b19 DNA CpG dinucleotide methylation and epigenetic regulation of viral expression.

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Francesca Bonvicini

Full Text Available CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression.The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections.The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19.

Parvovirus B19 DNA CpG Dinucleotide Methylation and Epigenetic Regulation of Viral Expression

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Bonvicini, Francesca; Manaresi, Elisabetta; Di Furio, Francesca; De Falco, Luisa; Gallinella, Giorgio

2012-01-01

CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression. The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections. The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19. PMID:22413013

Gene promoter methylation and DNA repair capacity in monozygotic twins with discordant smoking habits.

Science.gov (United States)

Ottini, Laura; Rizzolo, Piera; Siniscalchi, Ester; Zijno, Andrea; Silvestri, Valentina; Crebelli, Riccardo; Marcon, Francesca

2015-02-01

The influence of DNA repair capacity, plasma nutrients and tobacco smoke exposure on DNA methylation was investigated in blood cells of twenty-one couples of monozygotic twins with discordant smoking habits. All study subjects had previously been characterized for mutagen sensitivity with challenge assays with ionizing radiation in peripheral blood lymphocytes. Plasma levels of folic acid, vitamin B12 and homocysteine were also available from a previous investigation. In this work DNA methylation in the promoter region of a panel of ten genes involved in cell cycle control, differentiation, apoptosis and DNA repair (p16, FHIT, RAR, CDH1, DAPK1, hTERT, RASSF1A, MGMT, BRCA1 and PALB2) was assessed in the same batches of cells isolated for previous studies, using the methylation-sensitive high-resolution melting technique. Fairly similar profiles of gene promoter methylation were observed within co-twins compared to unrelated subjects (p= 1.23 × 10(-7)), with no significant difference related to smoking habits (p = 0.23). In a regression analysis the methylation index of study subjects, used as synthetic descriptor of overall promoter methylation, displayed a significant inverse correlation with radiation-induced micronuclei (p = 0.021) and plasma folic acid level (p = 0.007) both in smokers and in non-smokers. The observed association between repair of radiation-induced DNA damage and promoter methylation suggests the involvement of the DNA repair machinery in DNA modification. Data also highlight the possible modulating effect of folate deficiency on DNA methylation and the strong influence of familiarity on the individual epigenetic profile. Copyright © 2015 Elsevier B.V. All rights reserved.

Pleiotropic effects of a methyl donor diet in a novel animal model.

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Kimberly R Shorter

Full Text Available Folate and other methyl-donor pathway components are widely supplemented due to their ability to prevent prenatal neural tube defects. Several lines of evidence suggest that these supplements act through epigenetic mechanisms (e.g. altering DNA methylation. Primary among these are the experiments on the mouse viable yellow allele of the agouti locus (A(vy. In the Avy allele, an Intracisternal A-particle retroelement has inserted into the genome adjacent to the agouti gene and is preferentially methylated. To further test these effects, we tested the same diet used in the Avy studies on wild-derived Peromyscus maniculatus, a native North American rodent. We collected tissues from neonatal offspring whose parents were fed the high-methyl donor diet as well as controls. In addition, we assayed coat-color of a natural variant (wide-band agouti = A(Nb that overexpresses agouti as a phenotypic biomarker. Our data indicate that these dietary components affected agouti protein production, despite the lack of a retroelement at this locus. Surprisingly, the methyl-donor diet was associated with defects (e.g. ovarian cysts, cataracts and increased mortality. We also assessed the effects of the diet on behavior: We scored animals in open field and social interaction tests. We observed significant increases in female repetitive behaviors. Thus these data add to a growing number of studies that suggest that these ubiquitously added nutrients may be a human health concern.

A quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials.

Science.gov (United States)

Tenenbaum, S; DiNardo, J; Morris, W E; Wolf, B A; Schnetzinger, R W

1984-10-01

A quantitative in vitro method for phototoxic evaluation of chemicals has been developed and validated. The assay uses Saccharomyces cerevisiae, seeded in an agar overlay on top of a plate count agar base. 8-Methoxy psoralen is used as a reference standard against which materials are measured. Activity is quantified by cytotoxicity measured as zones of inhibition. Several known phototoxins (heliotropine, lyral, phantolid, and bergamot oil) and photoallergens (6-methyl coumarin and musk ambrette) are used to validate the assay. An excellent correlation is observed between in vivo studies employing Hartley albino guinea pigs and the in vitro assay for several fragrance raw materials and other chemicals. The in vitro assay exhibits a greater sensitivity from 2-500 fold. For three fragrance oils, the in vitro assay detects low levels of photobiological activity while the in vivo assay is negative. Although the in vitro assay does not discriminate between phototoxins and photoallergens, it can be used for screening of raw materials so that reduction in animal usage can be achieved while maintaining the protection of the consumer.

A comparison of the whole genome approach of MeDIP-seq to the targeted approach of the Infinium HumanMethylation450 BeadChip(® for methylome profiling.

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Christine Clark

Full Text Available DNA methylation is one of the most studied epigenetic marks in the human genome, with the result that the desire to map the human methylome has driven the development of several methods to map DNA methylation on a genomic scale. Our study presents the first comparison of two of these techniques - the targeted approach of the Infinium HumanMethylation450 BeadChip® with the immunoprecipitation and sequencing-based method, MeDIP-seq. Both methods were initially validated with respect to bisulfite sequencing as the gold standard and then assessed in terms of coverage, resolution and accuracy. The regions of the methylome that can be assayed by both methods and those that can only be assayed by one method were determined and the discovery of differentially methylated regions (DMRs by both techniques was examined. Our results show that the Infinium HumanMethylation450 BeadChip® and MeDIP-seq show a good positive correlation (Spearman correlation of 0.68 on a genome-wide scale and can both be used successfully to determine differentially methylated loci in RefSeq genes, CpG islands, shores and shelves. MeDIP-seq however, allows a wider interrogation of methylated regions of the human genome, including thousands of non-RefSeq genes and repetitive elements, all of which may be of importance in disease. In our study MeDIP-seq allowed the detection of 15,709 differentially methylated regions, nearly twice as many as the array-based method (8070, which may result in a more comprehensive study of the methylome.

Epigenetic modulation upon exposure of lung fibroblasts to TiO2 and ZnO nanoparticles: alterations in DNA methylation

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Patil NA

2016-09-01

Full Text Available Nayana A Patil,1,2 WN Gade,2 Deepti D Deobagkar1 1Department of Zoology, Molecular Biology Research Laboratory, Centre of Advanced Studies, 2Department of Biotechnology, Proteomic Research Laboratory, Savitribai Phule Pune University, Pune, India Abstract: Titanium dioxide (TiO2 and zinc oxide (ZnO nanoparticles (NPs are promising candidates for numerous applications in consumer products. This will lead to increased human exposure, thus posing a threat to human health. Both these types of NPs have been studied for their cell toxicity, immunotoxicity, and genotoxicity. However, effects of these NPs on epigenetic modulations have not been studied. Epigenetics is an important link in the genotype and phenotype modulation and misregulation can often lead to lifestyle diseases. In this study, we have evaluated the DNA methylation-based epigenetic changes upon exposure to various concentrations of NPs. The investigation was designed to evaluate global DNA methylation, estimating the corresponding methyltransferase activity and expression of Dnmt gene using lung fibroblast (MRC5 cell line as lungs are the primary route of entry and target of occupational exposure to TiO2 and ZnO NPs. Enzyme-linked immunosorbent assay-based immunochemical assay revealed dose-related decrease in global DNA methylation and DNA methyltransferase activity. We also found direct correlation between the concentration of NPs, global methylation levels, and expression levels of Dnmt1, 3A, and 3B genes upon exposure. This is the first study to investigate effect of exposure to TiO2 and ZnO on DNA methylation levels in MRC5 cells. Epigenetic processes are known to play an important role in reprogramming and adaptation ability of an organism and can have long-term consequences. We suggest that changes in DNA methylation can serve as good biomarkers for early exposure to NPs since they occur at concentrations well below the sublethal levels. Our results demonstrate a clear

Mapuche Herbal Medicine Inhibits Blood Platelet Aggregation

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Susan Skanderup Falkenberg

2012-01-01

Full Text Available 12 plant species traditionally used by the Mapuche people in Chile to treat wounds and inflammations have been evaluated for their direct blood platelet inhibition. Seven of the 12 tested plant species showed platelet inhibitory effect in sheep blood, and four of these were also able to inhibit the ADP- (5.0 μM and collagen- (2.0 μg/mL induced aggregations in human blood. These four species in respective extracts (in brackets were Blechnum chilense (MeOH, Luma apiculata (H2O, Amomyrtus luma (DCM : MeOH 1 : 1 and Cestrum parqui (DCM : MeOH 1 : 1. The platelet aggregating inhibitory effects of A. luma (DCM : MeOH 1 : 1, and L. apiculata (H2O were substantial and confirmed by inhibition of platelet surface activation markers.

Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM.

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Kristensen, Lasse S; Wojdacz, Tomasz K; Thestrup, Britta B; Wiuf, Carsten; Hager, Henrik; Hansen, Lise Lotte

2009-12-21

The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample. CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP. MS-HRM and SMART-MSP can be successfully used for single locus

Correlation of SHOX2 Gene Amplification and DNA Methylation in Lung Cancer Tumors

International Nuclear Information System (INIS)

Schneider, Katja U; Liebenberg, Volker; Kneip, Christoph; Seegebarth, Anke; Erdogan, Fikret; Rappold, Gudrun; Schmidt, Bernd; Dietrich, Dimo; Fleischhacker, Michael; Leschber, Gunda; Merk, Johannes; Schäper, Frank; Stapert, Henk R; Vossenaar, Erik R; Weickmann, Sabine

2011-01-01

DNA methylation in the SHOX2 locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with SHOX2 gene expression and/or copy number alterations. An amplification of the SHOX2 gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples. SHOX2 expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect SHOX2 DNA methylation levels. SHOX2 expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH. A hypermethylation of the SHOX2 locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the SHOX2 gene showed no difference. Frequent gene amplification correlated with hypermethylation of the SHOX2 gene locus. This concerted effect qualifies SHOX2 DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples

DNA methylation analysis as novel tool for quality control in regenerative medicine.

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Rapko, Stephen; Baron, Udo; Hoffmüller, Ulrich; Model, Fabian; Wolfe, Leslie; Olek, Sven

2007-09-01

Cell-based regenerative medicine, including tissue engineering, is a novel approach to reconstituting tissues that do not spontaneously heal, such as damaged cartilage, and to curing diseases caused by malfunctioning cells. Typically, manufacturing processes to generate cartilage for replacement therapies involve isolation and expansion of cells from cartilage biopsies. A challenge in the field is potential contamination by other cell types (e.g., fibroblast-like cells), which can overgrow the desired cells during culturing and may ultimately compromise clinical efficacy. No standard analytical system has been absolutely effective in ensuring the identity of these cell-based products. Therefore, we tested deoxyribonucleic acid methylation analysis as a quality assessment tool, applying it to Genzyme's Carticel product, a chondrocyte implant that the Food and Drug Administration has approved. We identified 7 potent discriminators by assaying candidate genomic regions derived from methylation discovery approaches and literature searches regarding a functional role of genes in chondrocyte biology. Using a support vector machine, we trained an optimal cell type classifier that was absolutely effective in discriminating chondrocytes from synovial membrane derived cells, the major potential contaminant of chondrocyte cultures. The abundant marker availability and high quality of this assay format also suggest it as a potential quality control test for other cell types grown or manipulated in vitro.

Assays for noninvasive imaging of reporter gene expression

International Nuclear Information System (INIS)

Gambhir, S.S.; Barrio, J.R.; Herschman, H.R.; Phelps, M.E.

1999-01-01

Repeated, noninvasive imaging of reporter gene expression is emerging as a valuable tool for monitoring the expression of genes in animals and humans. Monitoring of organ/cell transplantation in living animals and humans, and the assessment of environmental, behavioral, and pharmacologic modulation of gene expression in transgenic animals should soon be possible. The earliest clinical application is likely to be monitoring human gene therapy in tumors transduced with the herpes simplex virus type 1 thymidine kinase (HSV1-tk) suicide gene. Several candidate assays for imaging reporter gene expression have been studied, utilizing cytosine deaminase (CD), HSV1-tk, and dopamine 2 receptor (D2R) as reporter genes. For the HSV1-tk reporter gene, both uracil nucleoside derivatives (e.g., 5-iodo-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil [FIAU] labeled with 124 I, 131 I ) and acycloguanosine derivatives {e.g., 8-[ 18 F]fluoro-9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine (8-[ 18 F]-fluoroganciclovir) ([ 18 F]FGCV), 9-[(3-[ 18 F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([ 18 F]FHPG)} have been investigated as reporter probes. For the D2R reporter gene, a derivative of spiperone {3-(2'-[ 18 F]-Fluoroethyl)spiperone ([ 18 F]FESP)} has been used with positron emission tomography (PET) imaging. In this review, the principles and specific assays for imaging reporter gene expression are presented and discussed. Specific examples utilizing adenoviral-mediated delivery of a reporter gene as well as tumors expressing reporter genes are discussed

Regulation of UGT1A1 and HNF1 transcription factor gene expression by DNA methylation in colon cancer cells

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Harvey Mario

2010-01-01

Full Text Available Abstract Background UDP-glucuronosyltransferase 1A1 (UGT1A1 is a pivotal enzyme involved in metabolism of SN-38, the active metabolite of irinotecan commonly used to treat metastatic colorectal cancer. We previously demonstrated aberrant methylation of specific CpG dinucleotides in UGT1A1-negative cells, and revealed that methylation state of the UGT1A1 5'-flanking sequence is negatively correlated with gene transcription. Interestingly, one of these CpG dinucleotides (CpG -4 is found close to a HNF1 response element (HRE, known to be involved in activation of UGT1A1 gene expression, and within an upstream stimulating factor (USF binding site. Results Gel retardation assays revealed that methylation of CpG-4 directly affect the interaction of USF1/2 with its cognate sequence without altering the binding for HNF1-alpha. Luciferase assays sustained a role for USF1/2 and HNF1-alpha in UGT1A1 regulation in colon cancer cells. Based on the differential expression profiles of HNF1A gene in colon cell lines, we also assessed whether methylation affects its expression. In agreement with the presence of CpG islands in the HNF1A promoter, treatments of UGT1A1-negative HCT116 colon cancer cells with a DNA methyltransferase inhibitor restore HNF1A gene expression, as observed for UGT1A1. Conclusions This study reveals that basal UGT1A1 expression in colon cells is positively regulated by HNF1-alpha and USF, and negatively regulated by DNA methylation. Besides, DNA methylation of HNF1A could also play an important role in regulating additional cellular drug metabolism and transporter pathways. This process may contribute to determine local inactivation of drugs such as the anticancer agent SN-38 by glucuronidation and define tumoral response.

Genome-Wide DNA Methylation Analysis and Epigenetic Variations Associated with Congenital Aortic Valve Stenosis (AVS.

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Uppala Radhakrishna

Full Text Available Congenital heart defect (CHD is the most common cause of death from congenital anomaly. Among several candidate epigenetic mechanisms, DNA methylation may play an important role in the etiology of CHDs. We conducted a genome-wide DNA methylation analysis using an Illumina Infinium 450k human methylation assay in a cohort of 24 newborns who had aortic valve stenosis (AVS, with gestational-age matched controls. The study identified significantly-altered CpG methylation at 59 sites in 52 genes in AVS subjects as compared to controls (either hypermethylated or demethylated. Gene Ontology analysis identified biological processes and functions for these genes including positive regulation of receptor-mediated endocytosis. Consistent with prior clinical data, the molecular function categories as determined using DAVID identified low-density lipoprotein receptor binding, lipoprotein receptor binding and identical protein binding to be over-represented in the AVS group. A significant epigenetic change in the APOA5 and PCSK9 genes known to be involved in AVS was also observed. A large number CpG methylation sites individually demonstrated good to excellent diagnostic accuracy for the prediction of AVS status, thus raising possibility of molecular screening markers for this disorder. Using epigenetic analysis we were able to identify genes significantly involved in the pathogenesis of AVS.

Genome-Wide DNA Methylation Analysis and Epigenetic Variations Associated with Congenital Aortic Valve Stenosis (AVS).

Science.gov (United States)

Radhakrishna, Uppala; Albayrak, Samet; Alpay-Savasan, Zeynep; Zeb, Amna; Turkoglu, Onur; Sobolewski, Paul; Bahado-Singh, Ray O

2016-01-01

Congenital heart defect (CHD) is the most common cause of death from congenital anomaly. Among several candidate epigenetic mechanisms, DNA methylation may play an important role in the etiology of CHDs. We conducted a genome-wide DNA methylation analysis using an Illumina Infinium 450k human methylation assay in a cohort of 24 newborns who had aortic valve stenosis (AVS), with gestational-age matched controls. The study identified significantly-altered CpG methylation at 59 sites in 52 genes in AVS subjects as compared to controls (either hypermethylated or demethylated). Gene Ontology analysis identified biological processes and functions for these genes including positive regulation of receptor-mediated endocytosis. Consistent with prior clinical data, the molecular function categories as determined using DAVID identified low-density lipoprotein receptor binding, lipoprotein receptor binding and identical protein binding to be over-represented in the AVS group. A significant epigenetic change in the APOA5 and PCSK9 genes known to be involved in AVS was also observed. A large number CpG methylation sites individually demonstrated good to excellent diagnostic accuracy for the prediction of AVS status, thus raising possibility of molecular screening markers for this disorder. Using epigenetic analysis we were able to identify genes significantly involved in the pathogenesis of AVS.

The methylation of the C-terminal region of hnRNPQ (NSAP1) is important for its nuclear localization

International Nuclear Information System (INIS)

Passos, Dario O.; Quaresma, Alexandre J.C.; Kobarg, Joerg

2006-01-01

Protein arginine methylation is an irreversible post-translational protein modification catalyzed by a family of at least nine different enzymes entitled PRMTs (protein arginine methyl transferases). Although PRMT1 is responsible for 85% of the protein methylation in human cells, its substrate spectrum has not yet been fully characterized nor are the functional consequences of methylation for the protein substrates well understood. Therefore, we set out to employ the yeast two-hybrid system in order to identify new substrate proteins for human PRMT1. We were able to identify nine different PRMT1 interacting proteins involved in different aspects of RNA metabolism, five of which had been previously described either as substrates for PRMT1 or as functionally associated with PRMT1. Among the four new identified possible protein substrates was hnRNPQ3 (NSAP1), a protein whose function has been implicated in diverse steps of mRNA maturation, including splicing, editing, and degradation. By in vitro methylation assays we were able to show that hnRNPQ3 is a substrate for PRMT1 and that its C-terminal RGG box domain is the sole target for methylation. By further studies with the inhibitor of methylation Adox we provide evidence that hnRNPQ1-3 are methylated in vivo. Finally, we demonstrate by immunofluorescence analysis of HeLa cells that the methylation of hnRNPQ is important for its nuclear localization, since Adox treatment causes its re-distribution from the nucleus to the cytoplasm

DNA damage and methylation induced by glyphosate in human peripheral blood mononuclear cells (in vitro study).

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Kwiatkowska, Marta; Reszka, Edyta; Woźniak, Katarzyna; Jabłońska, Ewa; Michałowicz, Jaromir; Bukowska, Bożena

2017-07-01

Glyphosate is a very important herbicide that is widely used in the agriculture, and thus the exposure of humans to this substance and its metabolites has been noted. The purpose of this study was to assess DNA damage (determination of single and double strand-breaks by the comet assay) as well as to evaluate DNA methylation (global DNA methylation and methylation of p16 (CDKN2A) and p53 (TP53) promoter regions) in human peripheral blood mononuclear cells (PBMCs) exposed to glyphosate. PBMCs were incubated with the compound studied at concentrations ranging from 0.1 to 10 mM for 24 h. The study has shown that glyphosate induced DNA lesions, which were effectively repaired. However, PBMCs were unable to repair completely DNA damage induced by glyphosate. We also observed a decrease in global DNA methylation level at 0.25 mM of glyphosate. Glyphosate at 0.25 mM and 0.5 mM increased p53 promoter methylation, while it did not induce statistically significant changes in methylation of p16 promoter. To sum up, we have shown for the first time that glyphosate (at high concentrations from 0.5 to 10 mM) may induce DNA damage in leucocytes such as PBMCs and cause DNA methylation in human cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl-methyl nuclear overhauser enhancement spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Venditti, Vincenzo; Fawzi, Nicolas L.; Clore, G. Marius, E-mail: mariusc@mail.nih.gov [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Laboratory of Chemical Physics (United States)

2011-11-15

Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to {approx}1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methyl-methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.

Methylation of mercury in isopod Porcellio scaber

International Nuclear Information System (INIS)

Jereb, V.; Horvat, M.; Cerne, I.; Drobne, D.

2002-01-01

Due to some remarkable characteristics, more than a decade ago terrestrial isopods were introduced as biornonitoring organisms for metals in industrially polluted environments. These characteristics are: suitable size, abundance, ease of handling in the laboratory, the ability to accumulate metals (Zn, Cd, Pb) and dose-dependent response to different metals and pesticides (diazinon). The isopod Porcellio scaber is a small terrestrial crab, which colonizes upper soil layers and litter. It lives in a humid environment, mostly under tree-leaves, decaying wood and bigger stones. It is an omnivore, but its most common food is decomposing organic matter. Therefore, isopods are important for decomposition and cycling of mineral matter in nature. Porcellio scaber can be also found on the river banks of river ldrijca a mercury contaminated site, but there is a lack of data on effects of Hg on Porcellio scaber. Therefore, it would be of interest to investigate the biological cycle of mercury in this animal. The objectives of our work were: To assess the magnitudes of biological processes (metal Hg 2+ ) uptake, its retention in the animal, accumulation in glands, excretion of Hg by faeces); To investigate the possibility of mercury transformation in the animal (Hg 2+→ MeHg + ). It is known, that intestine is a possible Hg 2+ methylation site; in the gut of Porcellio scaber are present anaerobes and very likely also sulphate-reducing bacteria, which are known to be responsible for Hg 2+ methylation in nature; To validate an appropriate analytical technique for Hg 2+ methylation assays in the isopod Porcellio scaber

Methylation diet and methyl group genetics in risk for adenomatous polyp occurrence

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Mark Lucock

2015-06-01

Conclusion: A methylation diet influences methyl group synthesis in the regulation of blood homocysteine level, and is modulated by genetic interactions. Methylation-related nutrients also interact with key genes to modify risk of AP, a precursor of colorectal cancer. Independent of diet, two methylation-related genes (A2756G-MS and A66G-MSR were directly associated with AP occurrence.

Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants.

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How-Kit, Alexandre; Daunay, Antoine; Mazaleyrat, Nicolas; Busato, Florence; Daviaud, Christian; Teyssier, Emeline; Deleuze, Jean-François; Gallusci, Philippe; Tost, Jörg

2015-07-01

Pyrosequencing permits accurate quantification of DNA methylation of specific regions where the proportions of the C/T polymorphism induced by sodium bisulfite treatment of DNA reflects the DNA methylation level. The commercially available high-throughput locus-specific pyrosequencing instruments allow for the simultaneous analysis of 96 samples, but restrict the DNA methylation analysis to CpG dinucleotide sites, which can be limiting in many biological systems. In contrast to mammals where DNA methylation occurs nearly exclusively on CpG dinucleotides, plants genomes harbor DNA methylation also in other sequence contexts including CHG and CHH motives, which cannot be evaluated by these pyrosequencing instruments due to software limitations. Here, we present a complete pipeline for accurate CpG and non-CpG cytosine methylation analysis at single base-resolution using high-throughput locus-specific pyrosequencing. The devised approach includes the design and validation of PCR amplification on bisulfite-treated DNA and pyrosequencing assays as well as the quantification of the methylation level at every cytosine from the raw peak intensities of the Pyrograms by two newly developed Visual Basic Applications. Our method presents accurate and reproducible results as exemplified by the cytosine methylation analysis of the promoter regions of two Tomato genes (NOR and CNR) encoding transcription regulators of fruit ripening during different stages of fruit development. Our results confirmed a significant and temporally coordinated loss of DNA methylation on specific cytosines during the early stages of fruit development in both promoters as previously shown by WGBS. The manuscript describes thus the first high-throughput locus-specific DNA methylation analysis in plants using pyrosequencing.

Reactions of guanine with methyl chloride and methyl bromide: O6-methylation versus charge transfer complex formation

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Shukla, P. K.; Mishra, P. C.; Suhai, S.

Density functional theory (DFT) at the B3LYP/6-31+G* and B3LYP/AUG-cc-pVDZ levels was employed to study O6-methylation of guanine due to its reactions with methyl chloride and methyl bromide and to obtain explanation as to why the methyl halides cause genotoxicity and possess mutagenic and carcinogenic properties. Geometries of the various isolated species involved in the reactions, reactant complexes (RCs), and product complexes (PCs) were optimized in gas phase. Transition states connecting the reactant complexes with the product complexes were also optimized in gas phase at the same levels of theory. The reactant complexes, product complexes, and transition states were solvated in aqueous media using the polarizable continuum model (PCM) of the self-consistent reaction field theory. Zero-point energy (ZPE) correction to total energy and the corresponding thermal energy correction to enthalpy were made in each case. The reactant complexes of the keto form of guanine with methyl chloride and methyl bromide in water are appreciably more stable than the corresponding complexes involving the enol form of guanine. The nature of binding in the product complexes was found to be of the charge transfer type (O6mG+ · X-, X dbond Cl, Br). Binding of HCl, HBr, and H2O molecules to the PCs obtained with the keto form of guanine did not alter the positions of the halide anions in the PCs, and the charge transfer character of the PCs was also not modified due to this binding. Further, the complexes obtained due to the binding of HCl, HBr, and H2O molecules to the PCs had greater stability than the isolated PCs. The reaction barriers involved in the formation of PCs were found to be quite high (?50 kcal/mol). Mechanisms of genotoxicity, mutagenesis and carcinogenesis caused by the methyl halides appear to involve charge transfer-type complex formation. Thus the mechanisms of these processes involving the methyl halides appear to be quite different from those that involve the

Trans-methylation reactions in plants: focus on the activated methyl cycle.

Science.gov (United States)

Rahikainen, Moona; Alegre, Sara; Trotta, Andrea; Pascual, Jesús; Kangasjärvi, Saijaliisa

2018-02-01

Trans-methylation reactions are vital in basic metabolism, epigenetic regulation, RNA metabolism, and posttranslational control of protein function and therefore fundamental in determining the physiological processes in all living organisms. The plant kingdom is additionally characterized by the production of secondary metabolites that undergo specific hydroxylation, oxidation and methylation reactions to obtain a wide array of different chemical structures. Increasing research efforts have started to reveal the enzymatic pathways underlying the biosynthesis of complex metabolites in plants. Further engineering of these enzymatic machineries offers significant possibilities in the development of bio-based technologies, but necessitates deep understanding of their potential metabolic and regulatory interactions. Trans-methylation reactions are tightly coupled with the so-called activated methyl cycle (AMC), an essential metabolic circuit that maintains the trans-methylation capacity in all living cells. Tight regulation of the AMC is crucial in ensuring accurate trans-methylation reactions in different subcellular compartments, cell types, developmental stages and environmental conditions. This review addresses the organization and posttranslational regulation of the AMC and elaborates its critical role in determining metabolic regulation through modulation of methyl utilization in stress-exposed plants. © 2017 Scandinavian Plant Physiology Society.

Cytosine Methylation Dysregulation in Neonates Following Intrauterine Growth Restriction

Science.gov (United States)

Bhagat, Tushar D.; Fazzari, Melissa J.; Verma, Amit; Barzilai, Nir; Greally, John M.

2010-01-01

Background Perturbations of the intrauterine environment can affect fetal development during critical periods of plasticity, and can increase susceptibility to a number of age-related diseases (e.g., type 2 diabetes mellitus; T2DM), manifesting as late as decades later. We hypothesized that this biological memory is mediated by permanent alterations of the epigenome in stem cell populations, and focused our studies specifically on DNA methylation in CD34+ hematopoietic stem and progenitor cells from cord blood from neonates with intrauterine growth restriction (IUGR) and control subjects. Methods and Findings Our epigenomic assays utilized a two-stage design involving genome-wide discovery followed by quantitative, single-locus validation. We found that changes in cytosine methylation occur in response to IUGR of moderate degree and involving a restricted number of loci. We also identify specific loci that are targeted for dysregulation of DNA methylation, in particular the hepatocyte nuclear factor 4α (HNF4A) gene, a well-known diabetes candidate gene not previously associated with growth restriction in utero, and other loci encoding HNF4A-interacting proteins. Conclusions Our results give insights into the potential contribution of epigenomic dysregulation in mediating the long-term consequences of IUGR, and demonstrate the value of this approach to studies of the fetal origin of adult disease. PMID:20126273

Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human.

Science.gov (United States)

Wu, Chengchao; Yao, Shixin; Li, Xinghao; Chen, Chujia; Hu, Xuehai

2017-02-16

DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features) and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features) by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation.

21 CFR 177.2000 - Vinylidene chloride/methyl acrylate/methyl methacrylate polymers.

Science.gov (United States)

2010-04-01

... methacrylate polymers. 177.2000 Section 177.2000 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF...: POLYMERS Substances for Use as Basic Components of Single and Repeated Use Food Contact Surfaces § 177.2000 Vinylidene chloride/methyl acrylate/methyl methacrylate polymers. The vinylidene chloride/methyl acrylate...

Investigation of differential HDAC4 methylation patterns in eating disorders.

Science.gov (United States)

Subramanian, Subha; Braun, Patricia R; Han, Shizhong; Potash, James B

2018-02-01

The objective of this study was to investigate the relationship between methylation patterns of the histone deacetylase 4 gene and eating disorders in a site previously associated with anorexia nervosa (AN). Women with AN (N=28) or bulimia nervosa (BN) (N=19) were age-matched and sex-matched to controls (N=45). We obtained saliva-derived DNA and use bisulfite pyrosequencing to examine region-specific methylation differences between cases and controls. The region assayed includes 15 CpGs. We found no significant association between the previously implicated CpG and either AN or BN. We found that three CpGs were nominally associated with AN (P=0.02-0.03); the largest difference was a 9% hypermethylation in AN. One CpG was nominally associated with BN (P=0.04), with 4% hypomethylation. None of these results remained significant after correction for multiple testing. We did not replicate previous findings, though through expanded coverage, we identified additional CpGs that were nominally associated with eating disorders.

Whole-genome methylation caller designed for methyl- DNA ...

African Journals Online (AJOL)

etchie

2013-02-20

Feb 20, 2013 ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, Hidden ... its response to environmental cues. .... have a great potential to become the most cost-effective ... hg18 reference genome (set to 0 if not present in retrieved reads). ..... DNA methylation patterns and epigenetic memory.

Comparative analysis of skin sensitization potency of acrylates (methyl acrylate, ethyl acrylate, butyl acrylate, and ethylhexyl acrylate) using the local lymph node assay.

Science.gov (United States)

Dearman, Rebecca J; Betts, Catherine J; Farr, Craig; McLaughlin, James; Berdasco, Nancy; Wiench, Karin; Kimber, Ian

2007-10-01

There are currently available no systematic experimental data on the skin sensitizing properties of acrylates that are of relevance in occupational settings. Limited information from previous guinea-pig tests or from the local lymph node assay (LLNA) is available; however, these data are incomplete and somewhat contradictory. For those reasons, we have examined in the LLNA 4 acrylates: butyl acrylate (BA), ethyl acrylate (EA), methyl acrylate (MA), and ethylhexyl acrylate (EHA). The LLNA data indicated that all 4 compounds have some potential to cause skin sensitization. In addition, the relative potencies of these acrylates were measured by derivation from LLNA dose-response analyses of EC3 values (the effective concentration of chemical required to induce a threefold increase in proliferation of draining lymph node cells compared with control values). On the basis of 1 scheme for the categorization of skin sensitization potency, BA, EA, and MA were each classified as weak sensitizers. Using the same scheme, EHA was considered a moderate sensitizer. However, it must be emphasized that the EC3 value for this chemical of 9.7% is on the borderline between moderate (10%) categories. Thus, the judicious view is that all 4 chemicals possess relatively weak skin sensitizing potential.

[Mifepristone inhibites the migration of endometrial cancer cells through regulating H19 methylation].

Science.gov (United States)

Lu, Z Z; Yan, L; Zhang, H; Li, M J; Zhang, X H; Zhao, X X

2016-06-23

To investigate the effect and mechanism of mifepristone on the migration of human endometrial carcinoma cells. A human endometrial carcinoma cell line, Ishikawa cells, was cultured in vitro and treated with mifepristone at different concentrations. Wound healing assay was applied to detect the migration of Ishikawa cells. RT-PCR and methylation-specific PCR (MSP) were used to detect the levels of H19 mRNA and its DNA methylation. Western-blot was used to detect the expressions of HMGA2 and epithelial to mesenchymal transition (EMT) related proteins. When treated with different concentrations of mifepristone for 48 hours, the width of scratch of the the control group, the 5 mg/L and the 10 mg/L mifepristone treatment groups were (4.18±0.07)mm, (4.68±0.07)mm, and(4.99±0.07)mm, respectively (Pendometrial carcinoma cells partially through methylation-induced of transcriptional inhibition of H19, which results in the down-regulation of HMGA2 and vimentin and upregulation of E-cadherin.

DNA Methylation and Methylation Polymorphism in Genetically Stable In vitro Regenerates of Jatropha curcas L. Using Methylation-Sensitive AFLP Markers.

Science.gov (United States)

Rathore, Mangal S; Jha, Bhavanath

2016-03-01

The present investigation aimed to evaluate the degree and pattern of DNA methylation using methylation-sensitive AFLP (MS-AFLP) markers in genetically stable in vitro regenerates of Jatropha curcas L.. The genetically stable in vitro regenerates were raised through direct organogenesis via enhanced axillary shoot bud proliferation (Protocol-1) and in vitro-derived leaf regeneration (Protocol-2). Ten selective combinations of MS-AFLP primers produced 462 and 477 MS-AFLP bands in Protocol-1 (P-1) and Protocol-2 (P-2) regenerates, respectively. In P-1 regenerates, 15.8-31.17 % DNA was found methylated with an average of 25.24 %. In P-2 regenerates, 15.93-32.7 % DNA was found methylated with an average of 24.11 %. Using MS-AFLP in P-1 and P-2 regenerates, 11.52-25.53 % and 13.33-25.47 % polymorphism in methylated DNA was reported, respectively. Compared to the mother plant, P-1 regenerates showed hyper-methylation while P-2 showed hypo-methylation. The results clearly indicated alternation in degree and pattern of DNA methylation; hence, epigenetic instability in the genetically stable in vitro regenerates of J. curcas, developed so far using two different regeneration systems and explants of two different origins. The homologous nucleotide fragments in genomes of P-1 and P-2 regenerates showing methylation re-patterning might be involved in immediate adaptive responses and developmental processes through differential regulation of transcriptome under in vitro conditions.

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers.

Science.gov (United States)

Cervera, M T; Ruiz-García, L; Martínez-Zapater, J M

2002-12-01

AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/ HpaII and EcoRI/ MspI patterns in different ecotypes revealed that 35-43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma.

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Fumiharu Ohka

Full Text Available Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4 have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ, and even low grade gliomas (LGGs, WHO grade 2 eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6-methylguanine-DNA methyltransferase (MGMT that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1 IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2 LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3 LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4 higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.

Recognition of methylated DNA through methyl-CpG binding domain proteins

DEFF Research Database (Denmark)

Zou, Xueqing; Ma, Wen; Solov'yov, Ilia

2012-01-01

DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although...... the function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines...

A seven-gene CpG-island methylation panel predicts breast cancer progression

International Nuclear Information System (INIS)

Li, Yan; Melnikov, Anatoliy A.; Levenson, Victor; Guerra, Emanuela; Simeone, Pasquale; Alberti, Saverio; Deng, Youping

2015-01-01

DNA methylation regulates gene expression, through the inhibition/activation of gene transcription of methylated/unmethylated genes. Hence, DNA methylation profiling can capture pivotal features of gene expression in cancer tissues from patients at the time of diagnosis. In this work, we analyzed a breast cancer case series, to identify DNA methylation determinants of metastatic versus non-metastatic tumors. CpG-island methylation was evaluated on a 56-gene cancer-specific biomarker microarray in metastatic versus non-metastatic breast cancers in a multi-institutional case series of 123 breast cancer patients. Global statistical modeling and unsupervised hierarchical clustering were applied to identify a multi-gene binary classifier with high sensitivity and specificity. Network analysis was utilized to quantify the connectivity of the identified genes. Seven genes (BRCA1, DAPK1, MSH2, CDKN2A, PGR, PRKCDBP, RANKL) were found informative for prognosis of metastatic diffusion and were used to calculate classifier accuracy versus the entire data-set. Individual-gene performances showed sensitivities of 63–79 %, 53–84 % specificities, positive predictive values of 59–83 % and negative predictive values of 63–80 %. When modelled together, these seven genes reached a sensitivity of 93 %, 100 % specificity, a positive predictive value of 100 % and a negative predictive value of 93 %, with high statistical power. Unsupervised hierarchical clustering independently confirmed these findings, in close agreement with the accuracy measurements. Network analyses indicated tight interrelationship between the identified genes, suggesting this to be a functionally-coordinated module, linked to breast cancer progression. Our findings identify CpG-island methylation profiles with deep impact on clinical outcome, paving the way for use as novel prognostic assays in clinical settings. The online version of this article (doi:10.1186/s12885-015-1412-9) contains supplementary

Recommendations for a nomenclature system for reporting methylation aberrations in imprinted domains

DEFF Research Database (Denmark)

Monk, David; Morales, Joannella; den Dunnen, Johan T

2018-01-01

Disorders we have discussed these issues and designed a nomenclature for naming imprinted DMRs as well as for reporting methylation values. We apply these recommendations for imprinted DMRs that are commonly assayed in clinical laboratories and show how they support standardized database submission....... The recommendations are in line with existing recommendations, most importantly the Human Genome Variation Society nomenclature, and should facilitate accurate reporting and data exchange among laboratories and thereby help to avoid future confusion....

A genome-wide methylation study on obesity Differential variability and differential methylation

NARCIS (Netherlands)

Xu, Xiaojing; Su, Shaoyong; Barnes, Vernon A.; De Miguel, Carmen; Pollock, Jennifer; Ownby, Dennis; Shi, Huidong; Zhu, Haidong; Snieder, Harold; Wang, Xiaoling

2013-01-01

Besides differential methylation, DNA methylation variation has recently been proposed and demonstrated to be a potential contributing factor to cancer risk. Here we aim to examine whether differential variability in methylation is also an important feature of obesity, a typical non-malignant common

Maternal intake of methyl-group donors affects DNA methylation of metabolic genes in infants.

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Pauwels, Sara; Ghosh, Manosij; Duca, Radu Corneliu; Bekaert, Bram; Freson, Kathleen; Huybrechts, Inge; Langie, Sabine A S; Koppen, Gudrun; Devlieger, Roland; Godderis, Lode

2017-01-01

Maternal nutrition during pregnancy and infant nutrition in the early postnatal period (lactation) are critically involved in the development and health of the newborn infant. The Maternal Nutrition and Offspring's Epigenome (MANOE) study was set up to assess the effect of maternal methyl-group donor intake (choline, betaine, folate, methionine) on infant DNA methylation. Maternal intake of dietary methyl-group donors was assessed using a food-frequency questionnaire (FFQ). Before and during pregnancy, we evaluated maternal methyl-group donor intake through diet and supplementation (folic acid) in relation to gene-specific ( IGF2 DMR, DNMT1 , LEP , RXRA ) buccal epithelial cell DNA methylation in 6 months old infants ( n  = 114) via pyrosequencing. In the early postnatal period, we determined the effect of maternal choline intake during lactation (in mothers who breast-fed for at least 3 months) on gene-specific buccal DNA methylation ( n  = 65). Maternal dietary and supplemental intake of methyl-group donors (folate, betaine, folic acid), only in the periconception period, was associated with buccal cell DNA methylation in genes related to growth ( IGF2 DMR), metabolism ( RXRA ), and appetite control ( LEP ). A negative association was found between maternal folate and folic acid intake before pregnancy and infant LEP (slope = -1.233, 95% CI -2.342; -0.125, p  = 0.0298) and IGF2 DMR methylation (slope = -0.706, 95% CI -1.242; -0.107, p  = 0.0101), respectively. Positive associations were observed for maternal betaine (slope = 0.875, 95% CI 0.118; 1.633, p  = 0.0241) and folate (slope = 0.685, 95% CI 0.245; 1.125, p  = 0.0027) intake before pregnancy and RXRA methylation. Buccal DNMT1 methylation in the infant was negatively associated with maternal methyl-group donor intake in the first and second trimester of pregnancy and negatively in the third trimester. We found no clear association between maternal choline intake

Detailed Assessment of the Kinetics of Hg-Cell Association, Hg Methylation, and Methylmercury Degradation in Several Desulfovibrio Species

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Graham, Andrew M.; Bullock, Allyson L.; Maizel, Andrew C.; Elias, Dwayne A.

2012-01-01

The kinetics of inorganic Hg [Hg(II)i] association, methylation, and methylmercury (MeHg) demethylation were examined for a group of Desulfovibrio species with and without MeHg production capability. We employed a detailed method for assessing MeHg production in cultures, including careful control of medium chemistry, cell density, and growth phase, plus mass balance of Hg(II)i and MeHg during the assays. We tested the hypothesis that differences in Hg(II)i sorption and/or uptake rates drive observed differences in methylation rates among Desulfovibrio species. Hg(II)i associated rapidly and with high affinity to both methylating and nonmethylating species. MeHg production by Hg-methylating strains was rapid, plateauing after ∼3 h. All MeHg produced was rapidly exported. We also tested the idea that all Desulfovibrio species are capable of Hg(II)i methylation but that rapid demethylation masks its production, but we found this was not the case. Therefore, the underlying reason why MeHg production capability is not universal in the Desulfovibrio is not differences in Hg affinity for cells nor differences in the ability of strains to degrade MeHg. However, Hg methylation rates varied substantially between Hg-methylating Desulfovibrio species even in these controlled experiments and after normalization to cell density. Thus, biological differences may drive cross-species differences in Hg methylation rates. As part of this study, we identified four new Hg methylators (Desulfovibrio aespoeensis, D. alkalitolerans, D. psychrotolerans, and D. sulfodismutans) and four nonmethylating species (Desulfovibrio alcoholivorans, D. tunisiensis, D. carbinoliphilus, and D. piger) in our ongoing effort to generate a library of strains for Hg methylation genomics. PMID:22885751

Synthesis, Antifungal Activity and Structure-Activity Relationships of Novel 3-(Difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic Acid Amides.

Science.gov (United States)

Du, Shijie; Tian, Zaimin; Yang, Dongyan; Li, Xiuyun; Li, Hong; Jia, Changqing; Che, Chuanliang; Wang, Mian; Qin, Zhaohai

2015-05-08

A series of novel 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid amides were synthesized and their activities were tested against seven phytopathogenic fungi by an in vitro mycelia growth inhibition assay. Most of them displayed moderate to excellent activities. Among them N-(2-(5-bromo-1H-indazol-1-yl)phenyl)-3-(difluoro-methyl)-1-methyl-1H-pyrazole-4-carboxamide (9m) exhibited higher antifungal activity against the seven phytopathogenic fungi than boscalid. Topomer CoMFA was employed to develop a three-dimensional quantitative structure-activity relationship model for the compounds. In molecular docking, the carbonyl oxygen atom of 9m could form hydrogen bonds towards the hydroxyl of TYR58 and TRP173 on SDH.

Synthesis, Antifungal Activity and Structure-Activity Relationships of Novel 3-(Difluoromethyl-1-methyl-1H-pyrazole-4-carboxylic Acid Amides

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Shijie Du

2015-05-01

Full Text Available A series of novel 3-(difluoromethyl-1-methyl-1H-pyrazole-4-carboxylic acid amides were synthesized and their activities were tested against seven phytopathogenic fungi by an in vitro mycelia growth inhibition assay. Most of them displayed moderate to excellent activities. Among them N-(2-(5-bromo-1H-indazol-1-ylphenyl-3-(difluoro-methyl-1-methyl-1H-pyrazole-4-carboxamide (9m exhibited higher antifungal activity against the seven phytopathogenic fungi than boscalid. Topomer CoMFA was employed to develop a three-dimensional quantitative structure-activity relationship model for the compounds. In molecular docking, the carbonyl oxygen atom of 9m could form hydrogen bonds towards the hydroxyl of TYR58 and TRP173 on SDH.

MsLDR-creator: a web service to design msLDR assays.

Science.gov (United States)

Bormann, Felix; Dahl, Andreas; Sers, Christine

2012-03-01

MsLDR-creator is a free web service to design assays for the new DNA methylation detection method msLDR. The service provides the user with all necessary information about the oligonucleotides required for the measurement of a given CpG within a sequence of interest. The parameters are calculated by the nearest neighbour approach to achieve optimal behaviour during the experimental procedure. In addition, to guarantee a good start using msLDR, further information, like protocols and hints and tricks, are provided.

Protein methylation in pea chloroplasts

International Nuclear Information System (INIS)

Niemi, K.J.; Adler, J.; Selman, B.R.

1990-01-01

The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with [ 3 H-methyl]-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. One methylinkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile [ 3 H]methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the [ 3 H]methyl group

Dual DNA methylation patterns in the CNS reveal developmentally poised chromatin and monoallelic expression of critical genes.

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Jinhui Wang

Full Text Available As a first step towards discovery of genes expressed from only one allele in the CNS, we used a tiling array assay for DNA sequences that are both methylated and unmethylated (the MAUD assay. We analyzed regulatory regions of the entire mouse brain transcriptome, and found that approximately 10% of the genes assayed showed dual DNA methylation patterns. They include a large subset of genes that display marks of both active and silent, i.e., poised, chromatin during development, consistent with a link between differential DNA methylation and lineage-specific differentiation within the CNS. Sixty-five of the MAUD hits and 57 other genes whose function is of relevance to CNS development and/or disorders were tested for allele-specific expression in F(1 hybrid clonal neural stem cell (NSC lines. Eight MAUD hits and one additional gene showed such expression. They include Lgi1, which causes a subtype of inherited epilepsy that displays autosomal dominance with incomplete penetrance; Gfra2, a receptor for glial cell line-derived neurotrophic factor GDNF that has been linked to kindling epilepsy; Unc5a, a netrin-1 receptor important in neurodevelopment; and Cspg4, a membrane chondroitin sulfate proteoglycan associated with malignant melanoma and astrocytoma in human. Three of the genes, Camk2a, Kcnc4, and Unc5a, show preferential expression of the same allele in all clonal NSC lines tested. The other six genes show a stochastic pattern of monoallelic expression in some NSC lines and bi-allelic expression in others. These results support the estimate that 1-2% of genes expressed in the CNS may be subject to allelic exclusion, and demonstrate that the group includes genes implicated in major disorders of the CNS as well as neurodevelopment.

Polyacrylic acid-coated cerium oxide nanoparticles: An oxidase mimic applied for colorimetric assay to organophosphorus pesticides.

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Zhang, Shi-Xiang; Xue, Shi-Fan; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

2016-11-15

It is important and urgent to develop reliable and highly sensitive methods that can provide on-site and rapid detection of extensively used organophosphorus pesticides (OPs) for their neurotoxicity. In this study, we developed a novel colorimetric assay for the detection of OPs based on polyacrylic acid-coated cerium oxide nanoparticles (PAA-CeO2) as an oxidase mimic and OPs as inhibitors to suppress the activity of acetylcholinesterase (AChE). Firstly, highly dispersed PAA-CeO2 was prepared in aqueous solution, which could catalyze the oxidation of TMB to produce a color reaction from colorless to blue. And the enzyme of AChE was used to catalyze the substrate of acetylthiocholine (ATCh) to produce thiocholine (TCh). As a thiol-containing compound with reducibility, TCh can decrease the oxidation of TMB catalyzed by PAA-CeO2. Upon incubated with OPs, the enzymatic activity of AChE was inhibited to produce less TCh, resulting in more TMB catalytically oxidized by PAA-CeO2 to show an increasing blue color. The two representative OPs, dichlorvos and methyl-paraoxon, were tested using our proposed assay. The novel assay showed notable color change in a concentration-dependent manner, and as low as 8.62 ppb dichlorvos and 26.73 ppb methyl-paraoxon can be readily detected. Therefore, taking advantage of such oxidase-like activity of PAA-CeO2, our proposed colorimetric assay can potentially be a screening tool for the precise and rapid evaluation of the neurotoxicity of a wealth of OPs. Copyright © 2016 Elsevier B.V. All rights reserved.

Genome-wide methylation analysis identified sexually dimorphic methylated regions in hybrid tilapia

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Wan, Zi Yi; Xia, Jun Hong; Lin, Grace; Wang, Le; Lin, Valerie C. L.; Yue, Gen Hua

2016-01-01

Sexual dimorphism is an interesting biological phenomenon. Previous studies showed that DNA methylation might play a role in sexual dimorphism. However, the overall picture of the genome-wide methylation landscape in sexually dimorphic species remains unclear. We analyzed the DNA methylation landscape and transcriptome in hybrid tilapia (Oreochromis spp.) using whole genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq). We found 4,757 sexually dimorphic differentially methylated regions (DMRs), with significant clusters of DMRs located on chromosomal regions associated with sex determination. CpG methylation in promoter regions was negatively correlated with the gene expression level. MAPK/ERK pathway was upregulated in male tilapia. We also inferred active cis-regulatory regions (ACRs) in skeletal muscle tissues from WGBS datasets, revealing sexually dimorphic cis-regulatory regions. These results suggest that DNA methylation contribute to sex-specific phenotypes and serve as resources for further investigation to analyze the functions of these regions and their contributions towards sexual dimorphisms. PMID:27782217

DNA sequence explains seemingly disordered methylation levels in partially methylated domains of Mammalian genomes.

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Dimos Gaidatzis

2014-02-01

Full Text Available For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs, recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%-40% of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS. Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R(2 of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.

NGX6 gene mediated by promoter methylation as a potential molecular marker in colorectal cancer

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Shen Shourong

2010-04-01

Full Text Available Abstract Background Nasopharyngeal carcinoma associated gene 6 (NGX6 is down-regulated in most colon cancer cell lines and tumor tissues when compared with their normal tissue samples. As a novel suppress tumor gene, it could inhibit colon cancer cell growth and cell cycle progression. However, little is known about the transcriptional mechanisms controlling NGX6 gene expression. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumorigenesis of colorectal carcinoma (CRC. In this study, we explored the role of DNA methylation in regulation of NGX6 transcription. Methods In the present study, we cloned the NGX6 promoter with characteristics of a CpG island by luciferase reporter assay. Then, the CpG methylation status around the NGX6 promoter region in colon cancer cell lines and colorectal tumor tissues was examined by methylation-specific PCR and bisulfite DNA sequencing. Finally, 5-Aza-2'-deoxycytidine (5-Aza-dC treatment was used to confirm the correlation between NGX6 promoter methylation and its gene inactivation. Results The sequence spanning positions -157 to +276 was identified as the NGX6 promoter, in which no canonical TATA boxes were found, while two CAAT boxes and GC boxes were discovered. Methylation status was observed more frequently in 40 colorectal cancer samples than in 40 adjacent normal mucosa samples (18/40 versus 7/40; P Conclusions Down-regulation of NGX6 gene is related to the promoter methylation. DNA methylation of NGX6 promoter might be a potential molecular marker for diagnosis or prognosis, or serve as a therapeutic target.

Aberrant TET1 Methylation Closely Associated with CpG Island Methylator Phenotype in Colorectal Cancer.

Science.gov (United States)

Ichimura, Norihisa; Shinjo, Keiko; An, Byonggu; Shimizu, Yasuhiro; Yamao, Kenji; Ohka, Fumiharu; Katsushima, Keisuke; Hatanaka, Akira; Tojo, Masayuki; Yamamoto, Eiichiro; Suzuki, Hiromu; Ueda, Minoru; Kondo, Yutaka

2015-08-01

Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer. The relationship between TET inactivation and CIMP in colorectal cancers is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) colorectal cancers. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in colorectal cancers, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of colorectal cancers as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P = 0.002), suggesting that TET1 methylation is an early event in CIMP tumorigenesis. TET1 methylation was significantly associated with BRAF mutation but not with hMLH1 methylation in the CIMP-P colorectal cancers. Colorectal cancers with TET1 methylation have a significantly greater number of DNA methylated genes and less pathological metastasis compared to those without TET1 methylation (P = 0.007 and 0.045, respectively). Our data suggest that TET1 methylation may contribute to the establishment of a unique pathway in respect to CIMP-mediated tumorigenesis, which may be incidental to hMLH1 methylation. In addition, our findings provide evidence that TET1 methylation may be a good biomarker for the prediction of metastasis in colorectal cancer. ©2015 American Association for Cancer Research.

Evaluating genome-wide DNA methylation changes in mice by Methylation Specific Digital Karyotyping

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Maruoka Shuichiro

2008-12-01

Full Text Available Abstract Background The study of genome-wide DNA methylation changes has become more accessible with the development of various array-based technologies though when studying species other than human the choice of applications are limited and not always within reach. In this study, we adapted and tested the applicability of Methylation Specific Digital Karyotyping (MSDK, a non-array based method, for the prospective analysis of epigenetic changes after perinatal nutritional modifications in a mouse model of allergic airway disease. MSDK is a sequenced based method that allows a comprehensive and unbiased methylation profiling. The method generates 21 base pairs long sequence tags derived from specific locations in the genome. The resulting tag frequencies determine in a quantitative manner the methylation level of the corresponding loci. Results Genomic DNA from whole lung was isolated and subjected to MSDK analysis using the methylation-sensitive enzyme Not I as the mapping enzyme and Nla III as the fragmenting enzyme. In a pair wise comparison of the generated mouse MSDK libraries we identified 158 loci that are significantly differentially methylated (P-value = 0.05 after perinatal dietary changes in our mouse model. Quantitative methylation specific PCR and sequence analysis of bisulfate modified genomic DNA confirmed changes in methylation at specific loci. Differences in genomic MSDK tag counts for a selected set of genes, correlated well with changes in transcription levels as measured by real-time PCR. Furthermore serial analysis of gene expression profiling demonstrated a dramatic difference in expressed transcripts in mice exposed to perinatal nutritional changes. Conclusion The genome-wide methylation survey applied in this study allowed for an unbiased methylation profiling revealing subtle changes in DNA methylation in mice maternally exposed to dietary changes in methyl-donor content. The MSDK method is applicable for mouse models

Geissoschizine methyl ether N-oxide, a new alkaloid with antiacetylcholinesterase activity from Uncaria rhynchophylla.

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Jiang, Wei-Wei; Su, Jia; Wu, Xing-De; He, Juan; Peng, Li-Yan; Cheng, Xiao; Zhao, Qin-Shi

2015-01-01

Geissoschizine methyl ether N-oxide, a new oxindole alkaloid, along with 14 known alkaloids, was isolated from the aerial part of Uncaria rhynchophylla. Their structures were identified by comprehensive spectral methods, including 2D NMR experiments, and confirmed by comparing with the literature data. In vitro acetylcholinesterase (AChE) inhibitory activity assay showed that the new compound exhibited anti-AChE activity with IC₅₀ value of 23.4 μM.

Quantitation of 4-Methyl-4-sulfanylpentan-2-one (4MSP) in Hops by a Stable Isotope Dilution Assay in Combination with GC×GC-TOFMS: Method Development and Application To Study the Influence of Variety, Provenance, Harvest Year, and Processing on 4MSP Concentrations.

Science.gov (United States)

Reglitz, Klaas; Steinhaus, Martin

2017-03-22

A stable isotope dilution assay was developed for quantitation of 4-methyl-4-sulfanylpentan-2-one (4MSP) in hops. The approach included the use of 4-( 13 C)methyl-4-sulfanyl(1,3,5- 13 C 3 )pentan-2-one as internal standard, selective isolation of hop thiols by mercurated agarose, and GC×GC-TOFMS analysis. Application of the method to 53 different hop samples revealed 4MSP concentrations between <1 and 114 μg/kg. Notably high concentrations were associated with United States varieties such as Citra, Eureka, Simcoe, and Apollo, whereas 4MSP was absent from traditional German and English varieties. Further experiments showed that besides the variety, also harvest year and storage vitally influenced 4MSP concentrations, whereas the impact of provenance was less pronounced. Hop processing such as drying and pelletizing had only a minor impact on 4MSP concentrations. Like the majority of other hop volatiles, 4MSP is predominantly located in the lupulin glands.

Fifty years of herbicide research: comparing the discovery of trifluralin and halauxifen-methyl.

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Epp, Jeffrey B; Schmitzer, Paul R; Crouse, Gary D

2018-01-01

Fifty years separate the commercialization of the herbicides trifluralin and halauxifen-methyl. Despite the vast degree of technological change that occurred over that time frame, some aspects of their discovery stories are remarkably similar. For example, both herbicides were prepared very early in the iterative discovery process and both were developed from known lead compound structures by hypothesis-driven research efforts without the use of in vitro assays or computer-aided molecular design. However, there are aspects of the halauxifen-methyl and trifluralin discovery stories that are substantially different. For example, the chemical technology required for the cost-effective production of halauxifen-methyl simply did not exist just two decades prior to its commercial launch. By contrast, the chemical technology required for the cost-effective production of trifluralin was reported in the chemical literature more than two decades prior to its commercial launch. In addition, changes in regulatory environment since the early 1960s ensured that their respective discovery to commercial launch stories would also differ in substantial ways. Ultimately, the time and cost required to develop and register halauxifen-methyl demanded a global initial business case while the lower registration hurdles that trifluralin cleared enabled a narrow initial business case mainly focused on the USA. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

The effect of royal sun agaricus, agaricus brasiliensis S. Wasser et al., Extract on methyl Methanesulfonate caused genotoxicity in Drosophila melanogaster

NARCIS (Netherlands)

Savic, T.; Patenkovic, A.; Sokovic, M.; Glamoclija, J.; Andjelkovic, M.; Griensven, van L.J.L.D.

2011-01-01

The effect of culinary-medicinal Royal Sun Agaricus (Agaricus brasiliensis) hot water extract on methyl methane sulfonate (MMS) induced mutagenicity/genotoxity in Drosophila melanogaster was studied using a quick and broadly applicable in vivo assay, i.e., the wing somatic mutation and recombination

Microbial Community Response to Carbon Substrate Amendment in Mercury Impacted Sediments: Implications on Microbial Methylation of Mercury.

Science.gov (United States)

Elias, D. A.; Somenahally, A. C.; Moberly, J. G.; Hurt, R. A., Jr.; Brown, S. D.; Podar, M.; Palumbo, A. V.; Gilmour, C. C.

2015-12-01

Methylmercury (MeHg) is a neurotoxic and bio-accumulative product of the microbial methylation of inorganic mercury (Hg(II)). Methylating organisms are now known to exist in almost all anaerobic niches including fermentation, Fe(III)- and sulfate- reduction as well as methanogenesis. The study objective was to determine the effect of different carbon sources on the microbial community and methylating populations in particular along a Hg contaminated creek. Sediment cores from upstream and downstream at the Hg contaminated East Fork Poplar Creek (EFPC), Oak Ridge TN, and a background site were sectioned by depth, and Hg-methylation potential (HgMP) assays were performed using stable isotope spikes. Sediments from the lowest depth possessed the highest in-situ activity. Replicate samples were amended with different carbon substrates (cellulose, acetate, propionate, lactate, ethanol and methanol), spiked with stable isotopes for HgMP assays and incubated for 24hrs. Sequencing of the 16S rRNA gene was performed to determine alterations in Bacterial and Archaeal population dynamics. Additionally, bioinformatics and our new qualitative and quantitative hgcAB primers were utilized to determine microbial community structure alterations and correlate organism and gene abundance with altered MeHg generation. HgMP was significantly reduced in cellulose amended sediments while acetate and propionate slightly decreased HgMP in both sites. Methanol, ethanol and lactate increased the HgMP in EFPC downstream while cellulose amendment significantly decreased the Proteobacteria, and the Firmicutes increased but none are currently known to produce MeHg. Geobacter bemidjiensis in particular significantly decreased in cellulose amended sediments in all three sites from being predominant in-situ. This suggests that in EFPC downstream and background sites, the prevalent Hg-methyaltors might be Deltaprotebacteria, since upstream, cellulose amendment did not reduce HgMP even though

Isocitrate dehydrogenase 1 and 2 genes mutations and MGMT methylation in gliomas

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D. V. Tabakov

2017-01-01

Full Text Available Gliomas are the most common brain tumors. It is difficult to detect them at early stages of disease and there is a few available therapies providing significant improvement in survival. Mutations of isocitrate dehydrogenase 1 and 2 genes (IDH1 and IDH2 play significant role in gliomogenesis, diagnostics and selection of patient therapy. We tested the distribution of IDH1 and IDH2 mutations in gliomas of different histological types and grades of malignancy by DNA melting analysis using our protocol with a sensitivity of 5 %. The results of this assay were confirmed by conventional Sanger sequencing. IDH1/2 mutations were detected in 74 % of lower grade gliomas (II and III, World Health Organization and in 14 % of glioblastomas (IV, World Health Organization. Mutation rate in gliomas with oligodendroglioma component were significantly higher then in other glioma types (р = 0.014. The IDH1 mutations was the most common (79 % of general mutation number. IDH1/2 mutations can induce aberrant gene methylation. Detection of methylation rate of the gene encoding for O6-methylguanine-DNA-methyltransferase (MGMT, predictive biomarker for treatment of gliomas with the alkylating agents, has demonstrated a partial association with IDH1/2 mutations. In 73 % of IDH1/2-mutant tumors MGMT promoter methylation were observed. At the same time IDH1/2 mutations were not revealed in 67 % tumors with MGMT promoter methylation. These results indicate existence of another mechanism of MGMT methylation in gliomas. Our data strong support for necessity of both markers testing when patient therapy is selected.

Essential Oil Composition and Antimicrobial Activity of Methyl cinnamate-Linalool Chemovariant of Ocimum basilicum L. from India

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Rajendra Chandra Padalia

2017-03-01

Full Text Available The essential oils obtained from hydrodistillation of Ocimum basilicum L. harvested at four different growth stages during spring-summer and rain-autumn cropping seasons , were characterized using GC and GC-MS. The e ssential oil yield was found to vary from 0.28–0.32% and 0.40–0.52% during spring-summer and rain-autumn cropping season, respectively with its maximal at full bloom stage. Altogether , forty constituents, comprising 94.9–98.3% were identified represented by ( E -methyl cinnamate (36.6 – 66.4%, linalool (11.2 – 43.8%, and (Z -methyl cinnamate (5.4-7.6% as main constituents. Results showed that growth stages strongly influenced the chemical composition of the essential oil in two cropping seasons, particularly concerning to the content of ( E -methyl cinnamate and linalool. Seed setting stage was optimized for harvesting ( E -methyl cinnamate rich oil (66.4% in rain-winter cropping season. The antimicrobial potential of the essential oil was tested againist eight pathogenic bacteria and three fungal strains. Antimicrobial assay showed that the essential oil possessed good antibacterial activity against Streptococcus mutans, Staphylococcus epidermidis, Escherichia coli , and antifungal activity against Candida kefyr and Candida albicans. Ocimum basilicum , essential oil, ( E - methyl cinnamate, linalool, antibacterial activity, antifungal activity

DAPK1 Promoter Methylation and Cervical Cancer Risk: A Systematic Review and a Meta-Analysis.

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Antonella Agodi

Full Text Available The Death-Associated Protein Kinase 1 (DAPK1 gene has been frequently investigated in cervical cancer (CC. The aim of the present study was to carry out a systematic review and a meta-analysis in order to evaluate DAPK1 promoter methylation as an epigenetic marker for CC risk.A systematic literature search was carried out. The Cochrane software package Review Manager 5.2 was used. The fixed-effects or random-effects models, according to heterogeneity across studies, were used to calculate odds ratios (ORs and 95% Confidence Intervals (CIs. Furthermore, subgroup analyses were conducted by histological type, assays used to evaluate DAPK1 promoter methylation, and control sample source.A total of 20 papers, published between 2001 and 2014, on 1929 samples, were included in the meta-analysis. DAPK1 promoter methylation was associated with an increased CC risk based on the random effects model (OR: 21.20; 95%CI = 11.14-40.35. Omitting the most heterogeneous study, the between study heterogeneity decreased and the association increased (OR: 24.13; 95% CI = 15.83-36.78. The association was also confirmed in all the subgroups analyses.A significant strong association between DAPK1 promoter methylation and CC was shown and confirmed independently by histological tumor type, method used to evaluate methylation and source of control samples. Methylation markers may have value in early detection of CC precursor lesions, provide added reassurances of safety for women who are candidates for less frequent screens, and predict outcomes of women infected with human papilloma virus.

Patterns of DNA methylation in development, division of labor and hybridization in an ant with genetic caste determination.

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Chris R Smith

Full Text Available BACKGROUND: DNA methylation is a common regulator of gene expression, including acting as a regulator of developmental events and behavioral changes in adults. Using the unique system of genetic caste determination in Pogonomyrmex barbatus, we were able to document changes in DNA methylation during development, and also across both ancient and contemporary hybridization events. METHODOLOGY/PRINCIPAL FINDINGS: Sodium bisulfite sequencing demonstrated in vivo methylation of symmetric CG dinucleotides in P. barbatus. We also found methylation of non-CpG sequences. This validated two bioinformatics methods for predicting gene methylation, the bias in observed to expected ratio of CpG dinucleotides and the density of CpG/TpG single nucleotide polymorphisms (SNP. Frequencies of genomic DNA methylation were determined for different developmental stages and castes using ms-AFLP assays. The genetic caste determination system (GCD is probably the product of an ancestral hybridization event between P. barbatus and P. rugosus. Two lineages obligately co-occur within a GCD population, and queens are derived from intra-lineage matings whereas workers are produced from inter-lineage matings. Relative DNA methylation levels of queens and workers from GCD lineages (contemporary hybrids were not significantly different until adulthood. Virgin queens had significantly higher relative levels of DNA methylation compared to workers. Worker DNA methylation did not vary among developmental stages within each lineage, but was significantly different between the currently hybridizing lineages. Finally, workers of the two genetic caste determination lineages had half as many methylated cytosines as workers from the putative parental species, which have environmental caste determination. CONCLUSIONS/SIGNIFICANCE: These results suggest that DNA methylation may be a conserved regulatory mechanism moderating division of labor in both bees and ants. Current and historic

Blood DNA methylation biomarkers predict clinical reactivity in food-sensitized infants.

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Martino, David; Dang, Thanh; Sexton-Oates, Alexandra; Prescott, Susan; Tang, Mimi L K; Dharmage, Shyamali; Gurrin, Lyle; Koplin, Jennifer; Ponsonby, Anne-Louise; Allen, Katrina J; Saffery, Richard

2015-05-01

The diagnosis of food allergy (FA) can be challenging because approximately half of food-sensitized patients are asymptomatic. Current diagnostic tests are excellent makers of sensitization but poor predictors of clinical reactivity. Thus oral food challenges (OFCs) are required to determine a patient's risk of reactivity. We sought to discover genomic biomarkers of clinical FA with utility for predicting food challenge outcomes. Genome-wide DNA methylation (DNAm) profiling was performed on blood mononuclear cells from volunteers who had undergone objective OFCs, concurrent skin prick tests, and specific IgE tests. Fifty-eight food-sensitized patients (aged 11-15 months) were assessed, half of whom were clinically reactive. Thirteen nonallergic control subjects were also assessed. Reproducibility was assessed in an additional 48 samples by using methylation data from an independent population of patients with clinical FA. Using a supervised learning approach, we discovered a DNAm signature of 96 CpG sites that predict clinical outcomes. Diagnostic scores were derived from these 96 methylation sites, and cutoffs were determined in a sensitivity analysis. Methylation biomarkers outperformed allergen-specific IgE and skin prick tests for predicting OFC outcomes. FA status was correctly predicted in the replication cohort with an accuracy of 79.2%. DNAm biomarkers with clinical utility for predicting food challenge outcomes are readily detectable in blood. The development of this technology in detailed follow-up studies will yield highly innovative diagnostic assays. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Association between serum organochlorines and global methylation level of leukocyte DNA among Japanese women: a cross-sectional study

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Itoh, Hiroaki [Department of Epidemiology and Environmental Health, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113–8421 Japan (Japan); Iwasaki, Motoki, E-mail: moiwasak@ncc.go.jp [Epidemiology Division, Research Center for Cancer Prevention and Screening, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104–0045 Japan (Japan); Kasuga, Yoshio [Department of Surgery, Nagano Matsushiro General Hospital, 183 Matsushiro, Matsushiro-cho, Nagano City, Nagano Prefecture 381–1231 Japan (Japan); Yokoyama, Shiro; Onuma, Hiroshi [Department of Breast and Thyroid Surgery, Nagano Red Cross Hospital, 5-22-1 Wakasato, Nagano City, Nagano Prefecture 380–8582 Japan (Japan); Nishimura, Hideki [Department of Respiratory Surgery and Breast Surgery, Nagano Municipal Hospital, 1333–1 Tomitake, Nagano City, Nagano Prefecture 381–8551 Japan (Japan); Kusama, Ritsu [Department of Surgery, Hokushin General Hospital, 1-5-63 Nishi, Nakano City, Nagano Prefecture 383–8505 Japan (Japan); Yoshida, Teruhiko [Division of Genetics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104–0045 Japan (Japan); Yokoyama, Kazuhito [Department of Epidemiology and Environmental Health, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113–8421 Japan (Japan); Tsugane, Shoichiro [Dierctor Research Center for Cancer Prevention and Screening, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104–0045 Japan (Japan)

2014-08-15

While the global methylation level of leukocyte DNA may be a suitable biomarker for cancer risk, the level may be influenced by multiple factors, both environmental and host-related, one of which is exposure to environmental pollutants. To date, three epidemiologic studies have examined associations between serum organochlorine levels and global DNA methylation level, but their findings are not fully consistent, and the associations thus require confirmation in other well-characterized populations. We tested the association between organochlorine exposure and the global DNA methylation level of leukocytes in Japanese women. We conducted a cross-sectional study using the control group of a breast cancer case–control study in Japan. Subjects were 403 Japanese women who provided blood samples. Serum polychlorinated biphenyls (PCBs) and nine pesticide-related organochlorines were measured by gas chromatography isotope-dilution high-resolution mass spectrometry. Further, global methylation level of peripheral leukocyte DNA among 399 women was measured by luminometric methylation assay. Linear trends in the association between methylation and quartile levels of organochlorines were evaluated by regression coefficients in a multivariable linear regression model. We found significant inverse associations between the global methylation level in leukocyte DNA and many of the organochlorine levels measured. Global methylation level was significantly decreased by 0.33–0.83% per quartile category for serum o,p′-dichlorodiphenyltrichloroethane (o,p′-DDT), p,p′-DDT, p,p′-dichlorodiphenyldichloroethylene, trans-nonachlor, oxychlordane, hexachlorobenzene, β-hexachlorocyclohexane, PCB17, PCB52/69, PCB74, PCB114, and PCB183. Serum organochlorine levels were inversely associated with the global methylation level of leukocyte DNA in a relatively large sample of Japanese women. - Highlights: • Many serum organochlorine pesticides were inversely associated with the global

Epigenome-wide analysis links SMAD3 methylation at birth to asthma in children of asthmatic mothers.

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DeVries, Avery; Wlasiuk, Gabriela; Miller, Susan J; Bosco, Anthony; Stern, Debra A; Lohman, I Carla; Rothers, Janet; Jones, Anya C; Nicodemus-Johnson, Jessie; Vasquez, Monica M; Curtin, John A; Simpson, Angela; Custovic, Adnan; Jackson, Daniel J; Gern, James E; Lemanske, Robert F; Guerra, Stefano; Wright, Anne L; Ober, Carole; Halonen, Marilyn; Vercelli, Donata

2017-08-01

The timing and mechanisms of asthma inception remain imprecisely defined. Although epigenetic mechanisms likely contribute to asthma pathogenesis, little is known about their role in asthma inception. We sought to assess whether the trajectory to asthma begins already at birth and whether epigenetic mechanisms, specifically DNA methylation, contribute to asthma inception. We used the Methylated CpG Island Recovery Assay chip to survey DNA methylation in cord blood mononuclear cells from 36 children (18 nonasthmatic and 18 asthmatic subjects by age 9 years) from the Infant Immune Study (IIS), an unselected birth cohort closely monitored for asthma for a decade. SMAD3 methylation in IIS (n = 60) and in 2 replication cohorts (the Manchester Asthma and Allergy Study [n = 30] and the Childhood Origins of Asthma Study [n = 28]) was analyzed by using bisulfite sequencing or Illumina 450K arrays. Cord blood mononuclear cell-derived IL-1β levels were measured by means of ELISA. Neonatal immune cells harbored 589 differentially methylated regions that distinguished IIS children who did and did not have asthma by age 9 years. In all 3 cohorts methylation in SMAD3, the most connected node within the network of asthma-associated, differentially methylated regions, was selectively increased in asthmatic children of asthmatic mothers and was associated with childhood asthma risk. Moreover, SMAD3 methylation in IIS neonates with maternal asthma was strongly and positively associated with neonatal production of IL-1β, an innate inflammatory mediator. The trajectory to childhood asthma begins at birth and involves epigenetic modifications in immunoregulatory and proinflammatory pathways. Maternal asthma influences epigenetic mechanisms that contribute to the inception of this trajectory. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Gamma irradiation does not induce detectable changes in DNA methylation directly following exposure of human cells.

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Christoph Lahtz

Full Text Available Environmental chemicals and radiation have often been implicated in producing alterations of the epigenome thus potentially contributing to cancer and other diseases. Ionizing radiation, released during accidents at nuclear power plants or after atomic bomb explosions, is a potentially serious health threat for the exposed human population. This type of high-energy radiation causes DNA damage including single- and double-strand breaks and induces chromosomal rearrangements and mutations, but it is not known if ionizing radiation directly induces changes in the epigenome of irradiated cells. We treated normal human fibroblasts and normal human bronchial epithelial cells with different doses of γ-radiation emitted from a cesium 137 ((137Cs radiation source. After a seven-day recovery period, we analyzed global DNA methylation patterns in the irradiated and control cells using the methylated-CpG island recovery assay (MIRA in combination with high-resolution microarrays. Bioinformatics analysis revealed only a small number of potential methylation changes with low fold-difference ratios in the irradiated cells. These minor methylation differences seen on the microarrays could not be verified by COBRA (combined bisulfite restriction analysis or bisulfite sequencing of selected target loci. Our study shows that acute γ-radiation treatment of two types of human cells had no appreciable direct effect on DNA cytosine methylation patterns in exposed cells.

Inhibition of pectin methyl esterase activity by green tea catechins.

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Lewis, Kristin C; Selzer, Tzvia; Shahar, Chen; Udi, Yael; Tworowski, Dmitry; Sagi, Irit

2008-10-01

Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here, we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin methyl esterases. In a gel assay for PME activity, EGCG blocked esterase activity of pure PME as well as PME extracts from citrus and from parasitic plants. Fluorometric tests were used to determine the IC50 for a synthetic substrate. Molecular docking analysis of PME and EGCG suggests close interaction of EGCG with the catalytic cleft of PME. Inhibition of PME by the green tea compound, EGCG, provides the means to study the diverse roles of PMEs in cell wall metabolism and plant development. In addition, this study introduces the use of EGCG as natural product to be used in the food industry and agriculture.

CpG island methylator phenotype-low (CIMP-low) colorectal cancer shows not only few methylated CIMP-high-specific CpG islands, but also low-level methylation at individual loci.

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Kawasaki, Takako; Ohnishi, Mutsuko; Nosho, Katsuhiko; Suemoto, Yuko; Kirkner, Gregory J; Meyerhardt, Jeffrey A; Fuchs, Charles S; Ogino, Shuji

2008-03-01

The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct phenotype in colorectal cancer. However, the concept of CIMP-low with less extensive CpG island methylation is still evolving. Our aim is to examine whether density of methylation in individual CpG islands was different between CIMP-low and CIMP-high tumors. Utilizing MethyLight technology and 889 population-based colorectal cancers, we quantified DNA methylation (methylation index, percentage of methylated reference) at 14 CpG islands, including 8 CIMP-high-specific loci (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1). Methylation positivity in each locus was defined as methylation index>4. Low-level methylation (methylation index>0, CIMP-high-specific locus was significantly more common in 340 CIMP-low tumors (1/8-5/8 methylation-positive loci) than 133 CIMP-high tumors (> or =6/8 methylation-positive loci) and 416 CIMP-0 tumors (0/8 methylation-positive loci) (PCIMP-high, low-level methylation, was not persistently more prevalent in CIMP-low tumors. In conclusion, compared to CIMP-high and CIMP-0 tumors, CIMP-low colorectal cancers show not only few methylated CIMP-high-specific CpG islands, but also more frequent low-level methylation at individual loci. Our data may provide supporting evidence for a difference in pathogenesis of DNA methylation between CIMP-low and CIMP-high tumors.

Origin and fate of 4-methyl steroid hydrocarbons. I. Diagenesis of 4-methyl sterenes

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Wolff, G.A.; Lamb, N.A.; Maxwell, J.R.

1986-03-01

Treatment of 4-methylcholest-4-ene under mild acid conditions at low temperatures gives chemical evidence for certain features seen in the distributions of sedimentary 4-methyl steroid hydrocarbons, and further indicates that many low temperature diagenetic reactions of steroids are explicable in terms of acid catalyzed rearrangements. Specifically, the results provide: (i) Indirect evidence that the 4-ene skeleton is a key intermediate in the dehydration of 4-methyl stanols in sediments. (ii) An explanation for the distribution of 4-methyl sterenes and A-nor sterenes in the lacustrine Messel shale (Eocene). (iii) An explanation for the presence of 4..beta..-methyl steranes in relatively immature sedimentary rocks, despite the precursor stanols having the 4..cap alpha..-methyl configuration. With increasing maturity in the Paris Basin shales (Lower Toarcian), the less stable 4..beta..-methyl steranes decrease gradually in abundance relative to their 4..cap alpha..-methyl counterparts, at a rate fairly similar to the change in pristane stereochemistry.

Analysis of DNA Cytosine Methylation Patterns Using Methylation-Sensitive Amplification Polymorphism (MSAP).

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Guevara, María Ángeles; de María, Nuria; Sáez-Laguna, Enrique; Vélez, María Dolores; Cervera, María Teresa; Cabezas, José Antonio

2017-01-01

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is the methylation-sensitive amplified polymorphism technique (MSAP) which is a modification of amplified fragment length polymorphism (AFLP). It has been used to study methylation of anonymous CCGG sequences in different fungi, plants, and animal species. The main variation of this technique resides on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent-cutter restriction enzyme. For each sample, MSAP analysis is performed using both EcoRI/HpaII- and EcoRI/MspI-digested samples. A comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) methylation-insensitive polymorphisms that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples and (2) methylation-sensitive polymorphisms which are associated with the amplified fragments that differ in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses the modifications that can be applied to adjust the technology to different species of interest.

Evidence for methyl group transfer between the methyl-accepting chemotaxis proteins in Bacillus subtilis

International Nuclear Information System (INIS)

Bedale, W.A.; Nettleton, D.O.; Sopata, C.S.; Thoelke, M.S.; Ordal, G.W.

1988-01-01

The authors present evidence for methyl (as methyl or methoxy) transfer from the methyl-accepting chemotaxis proteins H1 and possibly H3 of Bacillus subtilis to the methyl-accepting chemotaxis protein H2. This methyl transfer, which has been observed in vitro was strongly stimulated by the chemoattractant aspartate and thus may plan an important role in the sensory processing system of this organism. Although radiolabeling of H1 and H3 began at once after the addition of [ 3 H] methionine, radiolabeling of H2 showed a lag. Furthermore, the addition of excess nonradioactive methionine caused immediate exponential delabeling of H1 and H3 while labeling of H2 continued to increase. Methylation of H2 required the chemotactic methyltransferase, probably to first methylate H1 and H3. Aspartate caused increased labeling of H2 and strongly decreased labeling of H1 and H3 after the addition of nonradioactive methionine. Without the addition of nonradioactive methionine, aspartate caused demethylation of H1 and to a lesser extent H3, with an approximately equal increase of methylation of H2

Methylation profiles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma

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Wang Yifei

2004-09-01

these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients (22/27, 81%, 0/26, 0%, P IRF7 gene hypermethylation preferentially occurred in the male counterparts (11/26, 42.3% to 3/27, 11%, P Conclusions Of the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma.

[Analysis of genomic DNA methylation level in radish under cadmium stress by methylation-sensitive amplified polymorphism technique].

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Yang, Jin-Lan; Liu, Li-Wang; Gong, Yi-Qin; Huang, Dan-Qiong; Wang, Feng; He, Ling-Li

2007-06-01

The level of cytosine methylation induced by cadmium in radish (Raphanus sativus L.) genome was analysed using the technique of methylation-sensitive amplified polymorphism (MSAP). The MSAP ratios in radish seedling exposed to cadmium chloride at the concentration of 50, 250 and 500 mg/L were 37%, 43% and 51%, respectively, and the control was 34%; the full methylation levels (C(m)CGG in double strands) were at 23%, 25% and 27%, respectively, while the control was 22%. The level of increase in MSAP and full methylation indicated that de novo methylation occurred in some 5'-CCGG sites under Cd stress. There was significant positive correlation between increase of total DNA methylation level and CdCl(2) concentration. Four types of MSAP patterns: de novo methylation, de-methylation, atypical pattern and no changes of methylation pattern were identified among CdCl(2) treatments and the control. DNA methylation alteration in plants treated with CdCl(2) was mainly through de novo methylation.

Reaction products from N-methyl-N-nitrosourea and deoxyribonucleic acid containing thymidine residues. Synthesis and identification of a new methylation product, O4-methyl-thymidine

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Lawley, P. D.; Orr, D. J.; Shah, S. A.; Farmer, P. B.; Jarman, M.

1973-01-01

1. DNA was treated with N-methyl-N-nitrosourea at pH7–8, 37°C, degraded to yield 3- and 7-methylpurines and deoxyribonucleosides and the reaction products were separated by chromatography on ion-exchange resins. The following methods for identification and determination of products were used: with unlabelled N-methyl-N-nitrosourea, u.v. absorption; use of methyl-14C-labelled N-methyl-N-nitrosourea and use of [14C]thymine-labelled DNA. 2. The synthesis of O4-methylthymidine and its identification by u.v. and mass spectroscopy are reported. 3. 3-Methylthymidine and O4-methylthymidine were found as methylation products from N-methyl-N-nitrosourea with thymidine and with DNA, in relatively small yields. Unidentified products containing thymine were found in enzymic digests of N-methyl-N-nitrosourea-treated DNA, which may be phosphotriesters. 4. The possible role of formation of methylthymines in mutagenesis by N-methyl-N-nitrosourea is discussed. PMID:4798180

Validation of DAB2IP methylation and its relative significance in predicting outcome in renal cell carcinoma

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Zhao, Liang-Yun; Kapur, Payal; Wu, Kai-Jie; Wang, Bin; Yu, Yan-Hong; Liao, Bing; He, Da-Lin; Chen, Wei; Margulis, Vitaly; Hsieh, Jer-Tsong; Luo, Jun-Hang

2016-01-01

We have recently reported tumor suppressive role of DAB2IP in RCC development. In this study, We identified one CpG methylation biomarker (DAB2IP CpG1) located UTSS of DAB2IP that was associated with poor overall survival in a cohort of 318 ccRCC patients from the Cancer Genome Atlas (TCGA). We further validated the prognostic accuracy of DAB2IP CpG methylation by pyrosequencing quantitative methylation assay in 224 ccRCC patients from multiple Chinese centers (MCHC set), and 239 patients from University of Texas Southwestern Medical Center at Dallas (UTSW set) by using FFPE samples. DAB2IP CpG1 can predict the overall survival of patients in TCGA, MCHC, and UTSW sets independent of patient age, Fuhrman grade and TNM stage (all p<0.05). DAB2IP CpG1 successfully categorized patients into high-risk and low-risk groups with significant differences of clinical outcome in respective clinical subsets, regardless of age, sex, grade, stage, or race (HR: 1.63-7.83; all p<0.05). The detection of DAB2IP CpG1 methylation was minimally affected by ITH in ccRCC. DAB2IP mRNA expression was regulated by DNA methylation in vitro. DAB2IP CpG1 methylation is a practical and repeatable biomarker for ccRCC, which can provide prognostic value that complements the current staging system. PMID:27129174

Synthesis of [methyl-14C]crotonobetaine from DL-[methyl-14C]carnitine

International Nuclear Information System (INIS)

Loester, H.; Seim, H.

1996-01-01

The causes of carnitine deficiency syndromes are not completely understood, but decomposition of L-carnitine in vivo is likely to be involved. Carnitine is metabolized to γ-butyrobetaine, and crotonobetaine is probably an intermediate in this pathway. To validate experimentally the precursor-product relationship between the three physiologically occuring γ-betaines - L-carnitine, crotonobetaine, γ-butyrobetaine - labelling with stable or radioactive isotopes became necessary. Methyl-labelled carnitine isomers (L(-)-, D(+)- or DL-) or γ-butyrobetaine can be easily synthesized by methylation of 4-amino-3-hydroxybutyric acid isomers or 4-aminobutyric acid, respectively. Because of problems with the 4-aminocrotonic acid, we synthesized labelled crotonbetaine from labelled carnitine. Thus, DL-[methyl- 14 C]carnitine was dehydrated by reaction with concentrated sulfuric acid. After removal of the latter the products were separated and purified by ion exchange chromatography on DOWEX 50 WX8 (200 - 400 mesh) and gradient elution with hydrochloric acid. In addition to the labelled main product [methyl- 14 C]crotonobetaine (yield about 50 %), [methyl- 14 C]glycine betaine and [methyl- 14 C]acetonyl-trimethylammonium (ATMA) were formed. The end products were identified by combined thin layer chromatography/autoradiography and quantified by liquid scintillation counting. (Author)

Cord blood buffy coat DNA methylation is comparable to whole cord blood methylation.

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Dou, John; Schmidt, Rebecca J; Benke, Kelly S; Newschaffer, Craig; Hertz-Picciotto, Irva; Croen, Lisa A; Iosif, Ana-Maria; LaSalle, Janine M; Fallin, M Daniele; Bakulski, Kelly M

2018-01-01

Cord blood DNA methylation is associated with numerous health outcomes and environmental exposures. Whole cord blood DNA reflects all nucleated blood cell types, while centrifuging whole blood separates red blood cells, generating a white blood cell buffy coat. Both sample types are used in DNA methylation studies. Cell types have unique methylation patterns and processing can impact cell distributions, which may influence comparability. We evaluated differences in cell composition and DNA methylation between cord blood buffy coat and whole cord blood samples. Cord blood DNA methylation was measured with the Infinium EPIC BeadChip (Illumina) in eight individuals, each contributing buffy coat and whole blood samples. We analyzed principal components (PC) of methylation, performed hierarchical clustering, and computed correlations of mean-centered methylation between pairs. We conducted moderated t-tests on single sites and estimated cell composition. DNA methylation PCs were associated with individual (P PC1 = 1.4 × 10 -9 ; P PC2 = 2.9 × 10 -5 ; P PC3 = 3.8 × 10 -5 ; P PC4 = 4.2 × 10 -6 ; P PC5 = 9.9 × 10 -13 , P PC6 = 1.3 × 10 -11 ) and not with sample type (P PC1-6 >0.7). Samples hierarchically clustered by individual. Pearson correlations of mean-centered methylation between paired samples ranged from r = 0.66 to r = 0.87. No individual site significantly differed between buffy coat and whole cord blood when adjusting for multiple comparisons (five sites had unadjusted Pcoat and whole cord blood are much lower than inter-individual variation, demonstrating that both sample preparation types can be analytically combined and compared.

Digital quantification of gene methylation in stool DNA by emulsion-PCR coupled with hydrogel immobilized bead-array.

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Liu, Yunlong; Wu, Haiping; Zhou, Qiang; Song, Qinxin; Rui, Jianzhong; Zou, Bingjie; Zhou, Guohua

2017-06-15

Aberrations of gene methylation in stool DNA (sDNA) is an effective biomarker for non-invasive colorectal cancer diagnosis. However, it is challenging to accurately quantitate the gene methylation levels in sDNA due to the low abundance and degradation of sDNA. In this study, a digital quantification strategy was proposed by combining emulsion PCR (emPCR) with hydrogel immobilized bead-array. The assay includes following steps: bisulfite conversion of sDNA, pre-amplification by PCR with specific primers containing 5' universal sequences, emPCR of pre-amplicons with beaded primers to achieve single-molecular amplification and identification of hydrogel embedding beads coated with amplicons. The sensitivity and the specificity of the method are high enough to pick up 0.05% methylated targets from unmethylated DNA background. The successful detection of hypermethylated vimentin gene in clinical stool samples suggests that the proposed method should be a potential tool for non-invasive colorectal cancer screening. Copyright © 2016 Elsevier B.V. All rights reserved.

Methyl methacrylate oligomerically-modified clay and its poly(methyl methacrylate) nanocomposites

International Nuclear Information System (INIS)

Zheng Xiaoxia; Jiang, David D.; Wilkie, Charles A.

2005-01-01

A methyl methacrylate oligomerically-modified clay was used to prepare poly(methyl methacrylate) clay nanocomposites by melt blending and the effect of the clay loading level on the modified clay and corresponding nanocomposite was studied. These nanocomposites were characterized by X-ray diffraction, transmission electron microscopy, thermogravimetric analysis and cone calorimetry. The results show a mixed intercalated/delaminated morphology with good nanodispersion. The compatibility between the methylacrylate-subsituted clay and poly(methyl methacrylate) (PMMA) are greatly improved compared to other oligomerically-modified clays

Usability of human Infinium MethylationEPIC BeadChip for mouse DNA methylation studies.

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Needhamsen, Maria; Ewing, Ewoud; Lund, Harald; Gomez-Cabrero, David; Harris, Robert Adam; Kular, Lara; Jagodic, Maja

2017-11-15

The advent of array-based genome-wide DNA methylation methods has enabled quantitative measurement of single CpG methylation status at relatively low cost and sample input. Whereas the use of Infinium Human Methylation BeadChips has shown great utility in clinical studies, no equivalent tool is available for rodent animal samples. We examined the feasibility of using the new Infinium MethylationEPIC BeadChip for studying DNA methylation in mouse. In silico, we identified 19,420 EPIC probes (referred as mEPIC probes), which align with a unique best alignment score to the bisulfite converted reference mouse genome mm10. Further annotation revealed that 85% of mEPIC probes overlapped with mm10.refSeq genes at different genomic features including promoters (TSS1500 and TSS200), 1st exons, 5'UTRs, 3'UTRs, CpG islands, shores, shelves, open seas and FANTOM5 enhancers. Hybridization of mouse samples to Infinium Human MethylationEPIC BeadChips showed successful measurement of mEPIC probes and reproducibility between inter-array biological replicates. Finally, we demonstrated the utility of mEPIC probes for data exploration such as hierarchical clustering. Given the absence of cost and labor convenient genome-wide technologies in the murine system, our findings show that the Infinium MethylationEPIC BeadChip platform is suitable for investigation of the mouse methylome. Furthermore, we provide the "mEPICmanifest" with genomic features, available to users of Infinium Human MethylationEPIC arrays for mouse samples.

Design, Synthesis and Antitumor Activity of Novel 4-Methyl-(3'S,4'S-cis-khellactone Derivatives

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Taigang Liang

2013-04-01

Full Text Available An asymmetric synthesis of a series of novel 4-methyl-(3'S,4'S-cis-khellactone derivatives 3a–o is reported for the first time. Their structures were confirmed by 1H-NMR, 13C-NMR and MS. Their cytotoxic activity was evaluated by the MTT assay against three selected human cancer cell lines: HEPG-2 (human liver carcinoma, SGC-7901 (human gastric carcinoma, LS174T (human colon carcinoma. Some compounds showed high inhibitory activity against these human cancer cell lines. Among them, compound 3a exhibited strong cytotoxicity, with IC50 values ranging from 8.51 to 29.65 μM. The results showed that 4-methyl-cis-khellactone derivatives with S,S configuration could be a potential antitumor agents.

Integrated genetic and epigenetic analysis identifies haplotype-specific methylation in the FTO type 2 diabetes and obesity susceptibility locus.

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Christopher G Bell

2010-11-01

Full Text Available Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D, focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip and absolute methylation values were estimated using a Bayesian algorithm (BATMAN. Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10(-4, permutation p = 1.0×10(-3. Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10(-7. Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM, encapsulates a Highly Conserved Non-Coding Element (HCNE that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases.

Maternal Methyl-Group Donor Intake and Global DNA (HydroxyMethylation before and during Pregnancy

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Sara Pauwels

2016-08-01

Full Text Available It is still unclear to which extent methyl-group intake during pregnancy can affect maternal global DNA (hydroxylmethylation. Pregnancy methylation profiling and its link with methyl-group intake in a healthy population could enhance our understanding of the development of pregnancy related disorders. One hundred forty-eight women were enrolled in the MANOE (MAternal Nutrition and Offspring’s Epigenome study. Thiry-four women were enrolled before pregnancy and 116 during the first trimester of pregnancy. Global DNA (hydroxymethylation in blood using LC-MS/MS and dietary methyl-group intake (methionine, folate, betaine, and choline using a food-frequency questionnaire were estimated pre-pregnancy, during each trimester, and at delivery. Global DNA (hydroxymethylation levels were highest pre-pregnancy and at weeks 18–22 of pregnancy. We observed a positive relation between folic acid and global DNA methylation (p = 0.04 and hydroxymethylation (p = 0.04. A high intake of methionine pre-pregnancy and in the first trimester showed lower (hydroxymethylation percentage in weeks 11–13 and weeks 18–22, respectively. Choline and betaine intake in the first weeks was negatively associated with hydroxymethylation. Women with a high intake of these three methyl groups in the second and third trimester showed higher hyrdoxymethylation/methylation levels in the third trimester. To conclude, a time trend in DNA (hydroxymethylation was found and women with higher methyl-group intake showed higher methylation in the third trimester, and not in earlier phases of pregnancy.

Early de novo DNA methylation and prolonged demethylation in the muscle lineage.

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Tsumagari, Koji; Baribault, Carl; Terragni, Jolyon; Varley, Katherine E; Gertz, Jason; Pradhan, Sirharsa; Badoo, Melody; Crain, Charlene M; Song, Lingyun; Crawford, Gregory E; Myers, Richard M; Lacey, Michelle; Ehrlich, Melanie

2013-03-01

Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues.

Dietary and supplemental maternal methyl-group donor intake and cord blood DNA methylation.

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Pauwels, Sara; Ghosh, Manosij; Duca, Radu Corneliu; Bekaert, Bram; Freson, Kathleen; Huybrechts, Inge; A S Langie, Sabine; Koppen, Gudrun; Devlieger, Roland; Godderis, Lode

2017-01-02

Maternal nutrition is critically involved in the development and health of the fetus. We evaluated maternal methyl-group donor intake through diet (methionine, betaine, choline, folate) and supplementation (folic acid) before and during pregnancy in relation to global DNA methylation and hydroxymethylation and gene specific (IGF2 DMR, DNMT1, LEP, RXRA) cord blood methylation. A total of 115 mother-infant pairs were enrolled in the MAternal Nutrition and Offspring's Epigenome (MANOE) study. The intake of methyl-group donors was assessed using a food-frequency questionnaire. LC-MS/MS and pyrosequencing were used to measure global and gene specific methylation, respectively. Dietary intake of methyl-groups before and during pregnancy was associated with changes in LEP, DNMT1, and RXRA cord blood methylation. Statistically significant higher cord blood LEP methylation was observed when mothers started folic acid supplementation more than 6 months before conception compared with 3-6 months before conception (34.6 ± 6.3% vs. 30.1 ± 3.6%, P = 0.011, LEP CpG1) or no folic acid used before conception (16.2 ± 4.4% vs. 13.9 ± 3%, P = 0.036 for LEP CpG3 and 24.5 ± 3.5% vs. 22.2 ± 3.5%, P = 0.045 for LEP mean CpG). Taking folic acid supplements during the entire pregnancy resulted in statistically significantly higher cord blood RXRA methylation as compared with stopping supplementation in the second trimester (12.3 ± 1.9% vs. 11.1 ± 2%, P = 0.008 for RXRA mean CpG). To conclude, long-term folic acid use before and during pregnancy was associated with higher LEP and RXRA cord blood methylation, respectively. To date, pregnant women are advised to take a folic acid supplement of 400 µg/day from 4 weeks before until 12 weeks of pregnancy. Our results suggest significant epigenetic modifications when taking a folic acid supplement beyond the current advice.

C-Methylated Flavonoids from Cleistocalyx operculatus and Their Inhibitory Effects on Novel Influenza A (H1N1) Neuraminidase

DEFF Research Database (Denmark)

Dao, Trong-Tuan; Tung, Bui-Thanh; Nguyen, Phi-Hung

2010-01-01

As part of an ongoing study focused on the discovery of anti-influenza agents from plants, four new (1-4) and 10 known (5-14) C-methylated flavonoids were isolated from a methanol extract of Cleistocalyx operculatus buds using an influenza H1N1 neuraminidase inhibition assay. Compounds 4, 7, 8...... mutant) expressed in 293T cells with IC50 values of 8.15 ± 1.05 and 3.31 ± 1.34 μM, respectively. Compounds 4, 7, 8, and 14 behaved as noncompetitive inhibitors in the kinetic studies. These results indicate that C-methylated flavonoids from C. operculatus have the potential to be developed...

A tailored approach to BRAF and MLH1 methylation testing in a universal screening program for Lynch syndrome.

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Adar, Tomer; Rodgers, Linda H; Shannon, Kristen M; Yoshida, Makoto; Ma, Tianle; Mattia, Anthony; Lauwers, Gregory Y; Iafrate, Anthony J; Chung, Daniel C

2017-03-01

To determine the correlation between BRAF genotype and MLH1 promoter methylation in a screening program for Lynch syndrome (LS), a universal screening program for LS was established in two medical centers. Tumors with abnormal MLH1 staining were evaluated for both BRAF V600E genotype and MLH1 promoter methylation. Tumors positive for both were considered sporadic, and genetic testing was recommended for all others. A total 1011 colorectal cancer cases were screened for Lynch syndrome, and 148 (14.6%) exhibited absent MLH1 immunostaining. Both BRAF and MLH1 methylation testing were completed in 126 cases. Concordant results (both positive or both negative) were obtained in 86 (68.3%) and 16 (12.7%) cases, respectively, with 81% concordance overall. The positive and negative predictive values for a BRAF mutation in predicting MLH1 promoter methylation were 98.9% and 41%, respectively, and the negative predictive value fell to 15% in patients ≥70 years old. Using BRAF genotyping as a sole test to evaluate cases with absent MLH1 staining would have increased referral rates for genetic testing by 2.3-fold compared with MLH1 methylation testing alone (31% vs 13.5%, respectively, PMLH1 methylation testing for BRAF wild-type cases only would significantly decrease the number of methylation assays performed and reduce the referral rate for genetic testing to 12.7%. A BRAF mutation has an excellent positive predictive value but poor negative predictive value in predicting MLH1 promoter methylation. A hybrid use of these tests may reduce the number of low-risk patients referred to genetic counseling and facilitate wider implementation of Lynch syndrome screening programs.

Association between DNA methylation in the miR-328 5'-flanking region and inter-individual differences in miR-328 and BCRP expression in human placenta.

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Jumpei Saito

Full Text Available MicroRNA (miRNA are non-coding small RNA that regulate gene expression. MiR-328 is reported to influence breast cancer resistance protein (BCRP expression in cancer cells. As a large inter-individual difference in BCRP levels is observed in various human tissues, the contribution of miR-328 to these differences is of interest. We hypothesized that DNA methylation in the miR-328 promoter region is responsible for the difference in miR-328 levels, leading to inter-individual variability in BCRP levels in human placenta. The association between placental miR-328 and BCRP levels was analyzed, and then DNA methylation in the miR-328 5'-flanking region and regulatory mechanisms causing inter-individual differences in miR-328 and BCRP levels were examined. MiR-328 expression was significantly correlated with BCRP mRNA (Rs = -0.560, P < 0.01 and protein (Rs = -0.730, P < 0.01 levels. It was also up-regulated by the demethylating agent 5-aza-2'-deoxycytidine in BCRP-expressing cells. Luciferase assays with differentially methylated reporter constructs indicated that methylation in the miR-328 5'-flanking region including a predicted CpG island remarkably decreased transcriptional activity compared to that in unmethylated constructs. We selected CCAAT/enhancer binding protein α (C/EBPα, located within the predicted CpG island, by in silico analysis. To elucidate the role of C/EBPα in miR-328 expression, a chromatin immunoprecipitation assay, promoter deletion analysis, and electrophoretic mobility shift assay (EMSA were performed. C/EBPα-binding site-truncated constructs showed significantly decreased promoter activity, and EMSA indicated that the C/EBPα-binding sites were located in the CpG island. Finally, the methylation patterns of several CpG dinucleotides proximal to two C/EBPα-binding sites in the miR-328 5'-flanking region were correlated negatively with miR-328 levels, and positively with BCRP levels in human placental samples. These

Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism (MSAP).

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Peraza-Echeverria, S; Herrera-Valencia, V A.; Kay, A -J.

2001-07-01

The extent of DNA methylation polymorphisms was evaluated in micropropagated banana (Musa AAA cv. 'Grand Naine') derived from either the vegetative apex of the sucker or the floral apex of the male inflorescence using the methylation-sensitive amplification polymorphism (MSAP) technique. In all, 465 fragments, each representing a recognition site cleaved by either or both of the isoschizomers were amplified using eight combinations of primers. A total of 107 sites (23%) were found to be methylated at cytosine in the genome of micropropagated banana plants. In plants micropropagated from the male inflorescence explant 14 (3%) DNA methylation events were polymorphic, while plants micropropagated from the sucker explant produced 8 (1.7%) polymorphisms. No DNA methylation polymorphisms were detected in conventionally propagated banana plants. These results demonstrated the usefulness of MSAP to detect DNA methylation events in micropropagated banana plants and indicate that DNA methylation polymorphisms are associated with micropropagation.

Array-based assay detects genome-wide 5-mC and 5-hmC in the brains of humans, non-human primates, and mice.

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Chopra, Pankaj; Papale, Ligia A; White, Andrew T J; Hatch, Andrea; Brown, Ryan M; Garthwaite, Mark A; Roseboom, Patrick H; Golos, Thaddeus G; Warren, Stephen T; Alisch, Reid S

2014-02-13

Methylation on the fifth position of cytosine (5-mC) is an essential epigenetic mark that is linked to both normal neurodevelopment and neurological diseases. The recent identification of another modified form of cytosine, 5-hydroxymethylcytosine (5-hmC), in both stem cells and post-mitotic neurons, raises new questions as to the role of this base in mediating epigenetic effects. Genomic studies of these marks using model systems are limited, particularly with array-based tools, because the standard method of detecting DNA methylation cannot distinguish between 5-mC and 5-hmC and most methods have been developed to only survey the human genome. We show that non-human data generated using the optimization of a widely used human DNA methylation array, designed only to detect 5-mC, reproducibly distinguishes tissue types within and between chimpanzee, rhesus, and mouse, with correlations near the human DNA level (R(2) > 0.99). Genome-wide methylation analysis, using this approach, reveals 6,102 differentially methylated loci between rhesus placental and fetal tissues with pathways analysis significantly overrepresented for developmental processes. Restricting the analysis to oncogenes and tumor suppressor genes finds 76 differentially methylated loci, suggesting that rhesus placental tissue carries a cancer epigenetic signature. Similarly, adapting the assay to detect 5-hmC finds highly reproducible 5-hmC levels within human, rhesus, and mouse brain tissue that is species-specific with a hierarchical abundance among the three species (human > rhesus > mouse). Annotation of 5-hmC with respect to gene structure reveals a significant prevalence in the 3'UTR and an association with chromatin-related ontological terms, suggesting an epigenetic feedback loop mechanism for 5-hmC. Together, these data show that this array-based methylation assay is generalizable to all mammals for the detection of both 5-mC and 5-hmC, greatly improving the utility of mammalian model systems

A novel double-isotope technique for the enzymatic assay of plasma histamine: application to estimation of mast cell activation assessed by antigen challenge in asthmatics

International Nuclear Information System (INIS)

Brown, M.J.; Ind, P.W.; Causon, R.; Lee, T.H.

1982-01-01

The concentration of plasma histamine may provide an index of mast cell activation (degranulation) and can be measured by a sensitive radioenzymatic assay based on its specific conversion to (/sup 3/H)-methylhistamine in the presence of histamine-N-methyltransferase and (/sup 3/H)-S-adenosyl-L-methionine. In this assay, the separation of excess (/sup 3/H)-S-adenosyl-L-methionine from (/sup 3/H)-methylhistamine requires several steps, for which a correction factors is necessary to maintain precision. In the present modification, duplicate 50-microliters aliquots of each plasma sample were incubated with histamine-N-methyltransferase and (/sup 3/H)-S-adenosyl-L-methionine. A further aliquot, with an added standard of 200 ng/ml histamine, was incubated with histamine-N-methyl-transferase and (/sup 14/C)-S-adenosyl-L-methionine. This standard was converted to (/sup 14/C)-methylhistamine, and its recovery at the end of the assay corrected both for varying efficiency of methylation among plasma samples and for losses during the subsequent extraction and separation stages. The sensitivity of the assay was 25 pg/ml. The intra-assay and interassay coefficients of variation were 7.2% and 11.6%, respectively. In five asthmatics, antigen challenge caused a 28% fall in FEV1, and this was associated with a twofold to threefold rise in plasma histamine concentration. This assay may thus prove a useful method for assessing the role of mast cell release of mediators in vivo

Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter

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Dávalos-Salas, Mercedes; Furlan-Magaril, Mayra; González-Buendía, Edgar; Valdes-Quezada, Christian; Ayala-Ortega, Erandi; Recillas-Targa, Félix

2011-01-01

Long-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human retinoblastoma (Rb) gene promoter in different tumoral cell lines. To assess the DNA methylation status of the Rb promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a GFP reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. Rb gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays. We found that the inability of CTCF to bind to the Rb promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation. This study indicates that CTCF plays an important role in maintaining the Rb promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing

Detection of Turner syndrome using X-chromosome inactivation specific differentially methylated CpG sites: A pilot study.

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Zhang, Qiang; Guo, Xiaohong; Tian, Tian; Wang, Teng; Li, Qiaoli; Wang, Lei; Liu, Yun; Xing, Qinghe; He, Lin; Zhao, Xinzhi

2017-05-01

Early diagnosis of Turner syndrome (TS) may improve preventive measures and treatment. X-chromosome inactivation specific differentially methylated CpG sites (XIDMSs) that are high methylated in inactive X chromosomes (Xi) and unmethylated in active X chromosomes (Xa) may be potential makers for TS detection. The candidate XIDMSs were screened from 9 male and 12 female DNA samples with normal karyotypes using the Illumina 450k array and validated by bisulfite sequencing PCR and pyrosequencing assay. X chromosome dosage was calculated according to the methylation level of multiple XIDMSs. Overall, 108 candidate XIDMSs were screened by the 450k array. Validations indicated that XIDMSs gathered and formed the X-chromosome inactivation specific differentially methylated regions (XIDMRs). Using 3 XIDMRs at SAT1, UXT and UTP14A loci, 36 TS, 22 normal female and 6 male samples were analyzed. Methylation levels of the 20 XIDMSs in the XIDMRs could distinguish between TS and normal female DNA samples, the X chromosome dosage was consistent with karyotyping data. Analyzing samples of 2 triple X syndrome and 3 Klinefelter syndrome patients suggested that this method could be used to detect X chromosome aneuploids other than TS. XIDMSs are widely spread along the X chromosome and might be effective markers for detection of TS and other X chromosome aneuploids. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA methylation

DEFF Research Database (Denmark)

Williams, Kristine; Christensen, Jesper; Helin, Kristian

2012-01-01

DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

The origin and fate of 4-methyl steroid hydrocarbons. I. Diagenesis of 4-methyl sterenes

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Wolff, George A.; Lamb, Neil A.; Maxwell, James R.

1986-03-01

Treatment of 4-methylcholest-4-ene under mild acid conditions at low temperatures gives chemical evidence for certain features seen in the distributions of sedimentary 4-methyl steroid hydrocarbons, and further indicates that many low temperature diagenetic reactions of steroids are explicable in terms of acid catalysed rearrangements. Specifically, the results provide: (i) Indirect evidence that the 4-ene skeleton is a key intermediate in the dehydration of 4-methyl stanols in sediments. (ii) An explanation for the distribution of 4-methyl sterenes and A-nor sterenes in the lacustrine Messel shale (Eocene). (iii) An explanation for the presence of 4β-methyl steranes in relatively immature sedimentary rocks, despite the precursor stanols having the 4α-methyl configuration. With increasing maturity in the Paris Basin shales (Lower Toarcian), the less stable 4β-methyl steranes decrease gradually in abundance relative to their 4α-methyl counterparts, at a rate fairly similar to the change in pristane stereochemistry.

A genome-wide methylation study on obesity: differential variability and differential methylation.

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Xu, Xiaojing; Su, Shaoyong; Barnes, Vernon A; De Miguel, Carmen; Pollock, Jennifer; Ownby, Dennis; Shi, Hidong; Zhu, Haidong; Snieder, Harold; Wang, Xiaoling

2013-05-01

Besides differential methylation, DNA methylation variation has recently been proposed and demonstrated to be a potential contributing factor to cancer risk. Here we aim to examine whether differential variability in methylation is also an important feature of obesity, a typical non-malignant common complex disease. We analyzed genome-wide methylation profiles of over 470,000 CpGs in peripheral blood samples from 48 obese and 48 lean African-American youth aged 14-20 y old. A substantial number of differentially variable CpG sites (DVCs), using statistics based on variances, as well as a substantial number of differentially methylated CpG sites (DMCs), using statistics based on means, were identified. Similar to the findings in cancers, DVCs generally exhibited an outlier structure and were more variable in cases than in controls. By randomly splitting the current sample into a discovery and validation set, we observed that both the DVCs and DMCs identified from the first set could independently predict obesity status in the second set. Furthermore, both the genes harboring DMCs and the genes harboring DVCs showed significant enrichment of genes identified by genome-wide association studies on obesity and related diseases, such as hypertension, dyslipidemia, type 2 diabetes and certain types of cancers, supporting their roles in the etiology and pathogenesis of obesity. We generalized the recent finding on methylation variability in cancer research to obesity and demonstrated that differential variability is also an important feature of obesity-related methylation changes. Future studies on the epigenetics of obesity will benefit from both statistics based on means and statistics based on variances.

Detection and discrimination of maintenance and de novo CpG methylation events using MethylBreak.

Science.gov (United States)

Hsu, William; Mercado, Augustus T; Hsiao, George; Yeh, Jui-Ming; Chen, Chung-Yung

2017-05-15

Understanding the principles governing the establishment and maintenance activities of DNA methyltransferases (DNMTs) can help in the development of predictive biomarkers associated with genetic disorders and diseases. A detection system was developed that distinguishes and quantifies methylation events using methylation-sensitive endonucleases and molecular beacon technology. MethylBreak (MB) is a 22-mer oligonucleotide with one hemimethylated and two unmethylated CpG sites, which are also recognition sites for Sau96I and SacII, and is attached to a fluorophore and a quencher. Maintenance methylation was quantified by fluorescence emission due to the digestion of SacII when the hemimethylated CpG site is methylated, which inhibits Sau96I cleavage. The signal difference between SacII digestion of both MB substrate and maintenance methylated MB corresponds to de novo methylation event. Our technology successfully discriminated and measured both methylation activities at different concentrations of MB and achieved a high correlation coefficient of R 2 =0.997. Additionally, MB was effectively applied to normal and cancer cell lines and in the analysis of enzymatic kinetics and RNA inhibition of recombinant human DNMT1. Copyright © 2017 Elsevier B.V. All rights reserved.

Mobility and molecular ions of dimethyl methyl phosphonate, methyl salicylate and acetone

Science.gov (United States)

Nowak, D. M.

1983-06-01

The mobilities of positive and negative reactant ions are reported for (H2O)nH(+); (H2O)2O2 and (H2O)2CO3(-) ion clusters. The formation of positive DMMP monomer and dimer is reported, and equilbria molecular reactions are reported. Acetone is reported as forming a dimer at 81 ppb with a reduced mobility (K sub o) of 1.82, Methyl salicylate is shown to form a protonated and hydrated positive monomer. Mixtures of DMMP and methyl salicylate with acetone showed a substantial change in DMMP ion clustering and little or no change in the methyl salicylate mobility spectra. Negative ions were not observed for DMMP, methyl salicylate, acetone and the mixtures under the conditions reported.

Increased expression and altered methylation of HERVWE1 in the human placentas of smaller fetuses from monozygotic, dichorionic, discordant twins.

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Yu Gao

Full Text Available BACKGROUND: The human endogenous retroviral family W, Env(C7, member 1 gene (HERVWE1 is thought to participate in trophoblast cell fusion, and its expression is diminished in the placentas of singleton intrauterine growth-retarded pregnancies. However, there is limited information about the role of HERVWE1 in discordant fetal growth in twins. This study was to compare HERVWE1 gene expression between the placentas of discordant monozygotic twins and to identify its regulation by methylation. METHODOLOGY/PRINCIPAL FINDINGS: Fetuses from twenty-one pairs of monozygotic, dichorionic, discordant twins were marked as "smaller" or "larger" according to birth weight. Placental HERVWE1 mRNA and protein expression profiles were analyzed using quantitative RT-PCR and immunohistochemistry (IHC staining. Methylation profiles of the HERVWE1 promoter region were analyzed using a pyrosequencing assay. DNA methyltransferase (DNMT transcript levels were analyzed by RT-PCR. 5-methyl cytosine (5-MC was stained using an immunohistochemical assay. There was a significant negative correlation between HERVWE1 mRNA levels and birth weight in twins (P0.05. The DNMT3b3 mRNA levels in the smaller group were significantly downregulated compared with the larger group in discordant twins(P<0.05, whereas the DNMT3b7 mRNA levels in the smaller group were significantly upregulated compared with the larger group in discordant twins(P<0.05. CONCLUSIONS/SIGNIFICANCE: In discordant, monozygotic, dichorionic twins, HERVWE1 expression was higher in smaller fetuses and lower in larger fetuses. Methylation of the HERVWE1 gene promoter region may participate in the regulation of HERVWE1 gene expression in discordant twin pregnancies.

Increased Expression and Altered Methylation of HERVWE1 in the Human Placentas of Smaller Fetuses from Monozygotic, Dichorionic, Discordant Twins

Science.gov (United States)

Wang, Zilian; Luo, Yanmin; Sun, Hongyu; Zhou, Yi; Huang, Linhuan; Li, Manchao; Fang, Qun; Jiang, Shiwen

2012-01-01

Background The human endogenous retroviral family W, Env(C7), member 1 gene (HERVWE1) is thought to participate in trophoblast cell fusion, and its expression is diminished in the placentas of singleton intrauterine growth-retarded pregnancies. However, there is limited information about the role of HERVWE1 in discordant fetal growth in twins. This study was to compare HERVWE1 gene expression between the placentas of discordant monozygotic twins and to identify its regulation by methylation. Methodology/Principal Findings Fetuses from twenty-one pairs of monozygotic, dichorionic, discordant twins were marked as “smaller” or “larger” according to birth weight. Placental HERVWE1 mRNA and protein expression profiles were analyzed using quantitative RT-PCR and immunohistochemistry (IHC) staining. Methylation profiles of the HERVWE1 promoter region were analyzed using a pyrosequencing assay. DNA methyltransferase (DNMT) transcript levels were analyzed by RT-PCR. 5-methyl cytosine (5-MC) was stained using an immunohistochemical assay. There was a significant negative correlation between HERVWE1 mRNA levels and birth weight in twins (P0.05). The DNMT3b3 mRNA levels in the smaller group were significantly downregulated compared with the larger group in discordant twins(P<0.05), whereas the DNMT3b7 mRNA levels in the smaller group were significantly upregulated compared with the larger group in discordant twins(P<0.05). Conclusions/Significance In discordant, monozygotic, dichorionic twins, HERVWE1 expression was higher in smaller fetuses and lower in larger fetuses. Methylation of the HERVWE1 gene promoter region may participate in the regulation of HERVWE1 gene expression in discordant twin pregnancies. PMID:22457770

ICBP90 Regulation of DNA Methylation, Histone Ubiquitination, and Tumor Suppressor Gene Expression in Breast Cancer Cells

Science.gov (United States)

2013-09-01

accomplishments include creation of relevant plant lines, development of in vitro assays, and profiling of mRNA expression in null mutants. 15. SUBJECT TERMS...DNA methylation, UHRF1, VIM1, ubiquitination, epigenetics, chromatin 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF...Molecular Basis of Human Disease ,” which covered several weeks’ worth of material specifically related to the molecular and epigenetic basis of cancer

DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells

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Spada, Fabio; Haemmer, Andrea; Kuch, David; Rothbauer, Ulrich; Schermelleh, Lothar; Kremmer, Elisabeth; Carell, Thomas; Längst, Gernot; Leonhardt, Heinrich

2007-01-01

DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen–binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference–mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required. PMID:17312023

Evolution of DNA Methylation across Insects.

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Bewick, Adam J; Vogel, Kevin J; Moore, Allen J; Schmitz, Robert J

2017-03-01

DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Relationship between methylation status of vitamin D-related genes, vitamin D levels, and methyl-donor biochemistry

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Emma Louise Beckett

2016-12-01

Full Text Available Vitamin D is known for its role in the regulation of gene expression via the vitamin D receptor, a nuclear transcription factor. More recently, a role for vitamin D in regulating DNA methylation has been identified as an additional mechanism of modulation of gene expression. How methylation status influences vitamin D metabolism and response pathways is not yet clear. Therefore, we aimed to assess the relationship between plasma 25-hydroxycholecalciferol (25(OHD and the methylation status of vitamin D metabolism enzyme genes (CYP2R1, CYP27B1 and CYP24A1 and the vitamin D receptor gene (VDR. This analysis was conducted in the context of dietary vitamin D, and background methyl donor related biochemistry, with adjustment for several dietary and lifestyle variables. Percentage methylation at CpG sites was assessed in peripheral blood cells using methylation sensitive and dependent enzymes and qPCR. Standard analytical techniques were used to determine plasma 25(OHD and homocysteine, and serum folate and B12, with the relationship to methylation status assessed using multi-variable regression analysis. CYP2R1 and VDR methylation were found to be independent predictors of plasma 25(OHD, when adjusted for vitamin D intake and other lifestyle variables. CYP24A1 was related to plasma 25(OHD directly, but not in the context of vitamin D intake. Methyl-group donor biochemistry was associated with the methylation status of some genes, but did not alter the relationship between methylation and plasma 25(OHD. Modulation of methylation status of CYP2R1, CYP24A1 and VDR in response to plasma 25(OHD may be part of feedback loops involved in maintaining vitamin D homeostasis, and may explain a portion of the variance in plasma 25(OHD levels in response to intake and sun exposure. Methyl-group donor biochemistry, while a potential independent modulator, did not alter this effect.

Methylation screening of the TGFBI promoter in human lung and prostate cancer by methylation-specific PCR

International Nuclear Information System (INIS)

Shah, Jinesh N; Shao, Genze; Hei, Tom K; Zhao, Yongliang

2008-01-01

Hypermethylation of the TGFBI promoter has been shown to correlate with decreased expression of this gene in human tumor cell lines. In this study, we optimized a methylation-specific polymerase chain reaction (MSP) method and investigated the methylation status of the TGFBI promoter in human lung and prostate cancer specimens. Methylation-specific primers were designed based on the methylation profiles of the TGFBI promoter in human tumor cell lines, and MSP conditions were optimized for accurate and efficient amplification. Genomic DNA was isolated from lung tumors and prostatectomy tissues of prostate cancer patients, bisulfite-converted, and analyzed by MSP. Among 50 lung cancer samples, 44.0% (22/50) harbored methylated CpG sites in the TGFBI promoter. An analysis correlating gene methylation status with clinicopathological cancer features revealed that dense methylation of the TGFBI promoter was associated with a metastatic phenotype, with 42.9% (6/14) of metastatic lung cancer samples demonstrating dense methylation vs. only 5.6% (2/36) of primary lung cancer samples (p < 0.05). Similar to these lung cancer results, 82.0% (41/50) of prostate cancer samples harbored methylated CpG sites in the TGFBI promoter, and dense methylation of the promoter was present in 38.9% (7/18) of prostate cancer samples with the feature of locoregional invasiveness vs. only 19.4% (6/31) of prostate cancer samples without locoregional invasiveness (p < 0.05). Furthermore, promoter hypermethylation correlated with highly reduced expression of the TGFBI gene in human lung and prostate tumor cell lines. We successfully optimized a MSP method for the precise and efficient screening of TGFBI promoter methylation status. Dense methylation of the TGFBI promoter correlated with the extent of TGFBI gene silencing in tumor cell lines and was related to invasiveness of prostate tumors and metastatic status of lung cancer tumors. Thus, TGFBI promoter methylation can be used as a potential

A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer.

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Schwartzman, Jacob; Mongoue-Tchokote, Solange; Gibbs, Angela; Gao, Lina; Corless, Christopher L; Jin, Jennifer; Zarour, Luai; Higano, Celestia; True, Lawrence D; Vessella, Robert L; Wilmot, Beth; Bottomly, Daniel; McWeeney, Shannon K; Bova, G Steven; Partin, Alan W; Mori, Motomi; Alumkal, Joshi

2011-10-01

DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.

DNA methylation and memory formation.

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Day, Jeremy J; Sweatt, J David

2010-11-01

Memory formation and storage require long-lasting changes in memory-related neuronal circuits. Recent evidence indicates that DNA methylation may serve as a contributing mechanism in memory formation and storage. These emerging findings suggest a role for an epigenetic mechanism in learning and long-term memory maintenance and raise apparent conundrums and questions. For example, it is unclear how DNA methylation might be reversed during the formation of a memory, how changes in DNA methylation alter neuronal function to promote memory formation, and how DNA methylation patterns differ between neuronal structures to enable both consolidation and storage of memories. Here we evaluate the existing evidence supporting a role for DNA methylation in memory, discuss how DNA methylation may affect genetic and neuronal function to contribute to behavior, propose several future directions for the emerging subfield of neuroepigenetics, and begin to address some of the broader implications of this work.

Association of Body Mass Index with DNA Methylation and Gene Expression in Blood Cells and Relations to Cardiometabolic Disease: A Mendelian Randomization Approach.

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Michael M Mendelson

2017-01-01

Full Text Available The link between DNA methylation, obesity, and adiposity-related diseases in the general population remains uncertain.We conducted an association study of body mass index (BMI and differential methylation for over 400,000 CpGs assayed by microarray in whole-blood-derived DNA from 3,743 participants in the Framingham Heart Study and the Lothian Birth Cohorts, with independent replication in three external cohorts of 4,055 participants. We examined variations in whole blood gene expression and conducted Mendelian randomization analyses to investigate the functional and clinical relevance of the findings. We identified novel and previously reported BMI-related differential methylation at 83 CpGs that replicated across cohorts; BMI-related differential methylation was associated with concurrent changes in the expression of genes in lipid metabolism pathways. Genetic instrumental variable analysis of alterations in methylation at one of the 83 replicated CpGs, cg11024682 (intronic to sterol regulatory element binding transcription factor 1 [SREBF1], demonstrated links to BMI, adiposity-related traits, and coronary artery disease. Independent genetic instruments for expression of SREBF1 supported the findings linking methylation to adiposity and cardiometabolic disease. Methylation at a substantial proportion (16 of 83 of the identified loci was found to be secondary to differences in BMI. However, the cross-sectional nature of the data limits definitive causal determination.We present robust associations of BMI with differential DNA methylation at numerous loci in blood cells. BMI-related DNA methylation and gene expression provide mechanistic insights into the relationship between DNA methylation, obesity, and adiposity-related diseases.

Association of Body Mass Index with DNA Methylation and Gene Expression in Blood Cells and Relations to Cardiometabolic Disease: A Mendelian Randomization Approach

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Joehanes, Roby; Liu, Chunyu; Aslibekyan, Stella; Demerath, Ellen W.; Guan, Weihua; Zhi, Degui; Willinger, Christine; Courchesne, Paul; Multhaup, Michael; Irvin, Marguerite R.; Schadt, Eric E.; Bressler, Jan; North, Kari; Sundström, Johan; Gustafsson, Stefan; Shah, Sonia; McRae, Allan F.; Harris, Sarah E.; Gibson, Jude; Redmond, Paul; Corley, Janie; Starr, John M.; Visscher, Peter M.; Wray, Naomi R.; Krauss, Ronald M.; Feinberg, Andrew; Fornage, Myriam; Pankow, James S.; Lind, Lars; Fox, Caroline; Ingelsson, Erik; Arnett, Donna K.; Boerwinkle, Eric; Liang, Liming; Levy, Daniel; Deary, Ian J.

2017-01-01

Background The link between DNA methylation, obesity, and adiposity-related diseases in the general population remains uncertain. Methods and Findings We conducted an association study of body mass index (BMI) and differential methylation for over 400,000 CpGs assayed by microarray in whole-blood-derived DNA from 3,743 participants in the Framingham Heart Study and the Lothian Birth Cohorts, with independent replication in three external cohorts of 4,055 participants. We examined variations in whole blood gene expression and conducted Mendelian randomization analyses to investigate the functional and clinical relevance of the findings. We identified novel and previously reported BMI-related differential methylation at 83 CpGs that replicated across cohorts; BMI-related differential methylation was associated with concurrent changes in the expression of genes in lipid metabolism pathways. Genetic instrumental variable analysis of alterations in methylation at one of the 83 replicated CpGs, cg11024682 (intronic to sterol regulatory element binding transcription factor 1 [SREBF1]), demonstrated links to BMI, adiposity-related traits, and coronary artery disease. Independent genetic instruments for expression of SREBF1 supported the findings linking methylation to adiposity and cardiometabolic disease. Methylation at a substantial proportion (16 of 83) of the identified loci was found to be secondary to differences in BMI. However, the cross-sectional nature of the data limits definitive causal determination. Conclusions We present robust associations of BMI with differential DNA methylation at numerous loci in blood cells. BMI-related DNA methylation and gene expression provide mechanistic insights into the relationship between DNA methylation, obesity, and adiposity-related diseases. PMID:28095459

DNA methylation in metabolic disorders

DEFF Research Database (Denmark)

Barres, Romain; Zierath, Juleen R

2011-01-01

DNA methylation is a major epigenetic modification that controls gene expression in physiologic and pathologic states. Metabolic diseases such as diabetes and obesity are associated with profound alterations in gene expression that are caused by genetic and environmental factors. Recent reports...... have provided evidence that environmental factors at all ages could modify DNA methylation in somatic tissues, which suggests that DNA methylation is a more dynamic process than previously appreciated. Because of the importance of lifestyle factors in metabolic disorders, DNA methylation provides...... a mechanism by which environmental factors, including diet and exercise, can modify genetic predisposition to disease. This article considers the current evidence that defines a role for DNA methylation in metabolic disorders....

Histone Lysine Methylation and Neurodevelopmental Disorders

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Jeong-Hoon Kim

2017-06-01

Full Text Available Methylation of several lysine residues of histones is a crucial mechanism for relatively long-term regulation of genomic activity. Recent molecular biological studies have demonstrated that the function of histone methylation is more diverse and complex than previously thought. Moreover, studies using newly available genomics techniques, such as exome sequencing, have identified an increasing number of histone lysine methylation-related genes as intellectual disability-associated genes, which highlights the importance of accurate control of histone methylation during neurogenesis. However, given the functional diversity and complexity of histone methylation within the cell, the study of the molecular basis of histone methylation-related neurodevelopmental disorders is currently still in its infancy. Here, we review the latest studies that revealed the pathological implications of alterations in histone methylation status in the context of various neurodevelopmental disorders and propose possible therapeutic application of epigenetic compounds regulating histone methylation status for the treatment of these diseases.

Avocado and olive oil methyl esters

International Nuclear Information System (INIS)

Knothe, Gerhard

2013-01-01

Biodiesel, the mono-alkyl esters of vegetable oils, animal fats or other triacylglycerol-containing materials and an alternative to conventional petroleum-based diesel fuel, has been derived from a variety of feedstocks. Numerous feedstocks have been investigated as potential biodiesel sources, including commodity oils, however, the methyl esters of avocado and olive oil would likely be suitable as biodiesel fuel. In order to expand the database and comprehensive evaluation of the properties of vegetable oil esters, in this work the fuel-related properties of avocado and olive oil methyl esters, which exhibit similar fatty acid profiles including high oleic acid content, are determined. The cetane numbers of avocado oil methyl esters and olive oil methyl esters are relatively high, determined as 59.2 and 62.5, respectively, due to their elevated content of methyl oleate. Other properties are well within the ranges specified in biodiesel standards. The cloud points of both esters are slightly above 0 °C due to their content of saturated esters, especially methyl palmitate. Overall, avocado and olive oil yield methyl esters with fuel properties comparable to methyl esters from other commodity vegetable oils. The 1 H and 13 C NMR spectra of avocado and olive oil methyl esters are reported. -- Highlights: • Methyl esters of avocado and olive oil meet biodiesel fuel standards. • Provides comparison for methyl esters of other vegetable oils with high oleic content. • Discusses and compares present results with prior literature

Development of a Novel Fluorescence Assay Based on the Use of the Thrombin-Binding Aptamer for the Detection of O6-Alkylguanine-DNA Alkyltransferase Activity

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Maria Tintoré

2010-01-01

Full Text Available Human O6-alkylguanine-DNA alkyltransferase (hAGT is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O6 position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA. The quadruplex structure of TBA is disrupted when a central guanine is replaced by an O6-methyl-guanine. The sequence also contains a fluorophore (fluorescein and a quencher (dabsyl attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of the O6-methyl group.

Stratified randomization controls better for batch effects in 450K methylation analysis: A cautionary tale

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Olive D. Buhule

2014-10-01

Full Text Available Background: Batch effects in DNA methylation microarray experiments can lead to spurious results if not properly handled during the plating of samples. Methods: Two pilot studies examining the association of DNA methylation patterns across the genome with obesity in Samoan men were investigated for chip- and row-specific batch effects. For each study, the DNA of 46 obese men and 46 lean men were assayed using Illumina's Infinium HumanMethylation450 BeadChip. In the first study (Sample One, samples from obese and lean subjects were examined on separate chips. In the second study (Sample Two, the samples were balanced on the chips by lean/obese status, age group, and census region. We used methylumi, watermelon, and limma R packages, as well as ComBat, to analyze the data. Principal component analysis and linear regression were respectively employed to identify the top principal components and to test for their association with the batches and lean/obese status. To identify differentially methylated positions (DMPs between obese and lean males at each locus, we used a moderated t-test.Results: Chip effects were effectively removed from Sample Two but not Sample One. In addition, dramatic differences were observed between the two sets of DMP results. After removing'' batch effects with ComBat, Sample One had 94,191 probes differentially methylated at a q-value threshold of 0.05 while Sample Two had zero differentially methylated probes. The disparate results from Sample One and Sample Two likely arise due to the confounding of lean/obese status with chip and row batch effects.Conclusion: Even the best possible statistical adjustments for batch effects may not completely remove them. Proper study design is vital for guarding against spurious findings due to such effects.

MicroRNA-219-2-3p functions as a tumor suppressor in gastric cancer and is regulated by DNA methylation.

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Huizi Lei

Full Text Available BACKGROUND AIMS: Gastric cancer is the most frequent gastrointestinal tumor in adults and is the most lethal form of human cancer. Despite of the improvements in treatments, the underlying mechanism of gastric carcinogenesis is not well known. To define novel modulators that regulate susceptibility to tumorgenesis, we focused on miR-219-2-3p. METHODS: Quantitative RT-PCR was employed to investigate the level of miR-219-2-3p in gastric cancer (GC tissues (n = 113 and their matched adjacent normal tissues (n = 113. In vitro cell proliferation, apoptosis assays, cell migration, and invasion assays were performed to elucidate biological effects of miR-219-2-3p. Since silencing of miRNA by promoter CpG island methylation may be an important mechanism in tumorgenesis, GC cells were treated with 5-aza-2'-deoxycytidine and trichostatin A, and expression changes of miR-219-2-3p were subsequently examined by quantitative RT-PCR. Finally, the methylation status of CpG island upstream of miR-219-2-3p was analyzed by methylation-specific PCR in GC tissues (n = 22. RESULTS: miR-219-2-3p was down-regulated in GC and cell lines. In addition, the experiments documented the lower expression of miR-219-2-3p in GC specimens with higher grade and later stage tumors. Meanwhile, miR-219-2-3p exerted antiproliferative, proapoptotic, and antimetastatic roles and reduced levels of p-ERK1/2 in GC cells. Furthermore, 5-aza-2'-deoxycytidine and trichostatin A increased the expression (~2 fold of miR-219-2-3p in GC cells. By methylation-specific PCR, DNA methylation in the upstream region of miR-219-2-3p was detected in both adjacent normal tissues and cancer tissues. As expected, the methylation level was considerably higher in the miR-219-2-3p down-regulated group than up-regulated group. CONCLUSIONS: miR-219-2-3p is potentially involved in gastric cancer progression and metastasis by regulating ERK1/2-related signal pathways, which may provide a novel therapeutic strategy

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

Science.gov (United States)

Aiba, Toshiki; Saito, Toshiyuki; Hayashi, Akiko; Sato, Shinji; Yunokawa, Harunobu; Maruyama, Toru; Fujibuchi, Wataru; Kurita, Hisaka; Tohyama, Chiharu; Ohsako, Seiichiroh

2017-03-09

It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

The multi-domain protein Np95 connects DNA methylation and histone modification.

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Rottach, Andrea; Frauer, Carina; Pichler, Garwin; Bonapace, Ian Marc; Spada, Fabio; Leonhardt, Heinrich

2010-04-01

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ss. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways.

Methylation-sensitive amplified polymorphism-based genome-wide analysis of cytosine methylation profiles in Nicotiana tabacum cultivars.

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Jiao, J; Wu, J; Lv, Z; Sun, C; Gao, L; Yan, X; Cui, L; Tang, Z; Yan, B; Jia, Y

2015-11-26

This study aimed to investigate cytosine methylation profiles in different tobacco (Nicotiana tabacum) cultivars grown in China. Methylation-sensitive amplified polymorphism was used to analyze genome-wide global methylation profiles in four tobacco cultivars (Yunyan 85, NC89, K326, and Yunyan 87). Amplicons with methylated C motifs were cloned by reamplified polymerase chain reaction, sequenced, and analyzed. The results show that geographical location had a greater effect on methylation patterns in the tobacco genome than did sampling time. Analysis of the CG dinucleotide distribution in methylation-sensitive polymorphic restriction fragments suggested that a CpG dinucleotide cluster-enriched area is a possible site of cytosine methylation in the tobacco genome. The sequence alignments of the Nia1 gene (that encodes nitrate reductase) in Yunyan 87 in different regions indicate that a C-T transition might be responsible for the tobacco phenotype. T-C nucleotide replacement might also be responsible for the tobacco phenotype and may be influenced by geographical location.

The dynamic changes of DNA methylation and histone modifications of salt responsive transcription factor genes in soybean.

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Yuguang Song

Full Text Available Epigenetic modification contributes to the regulation of gene expression and plant development under salinity stress. Here we describe the identification of 49 soybean transcription factors by microarray analysis as being inducible by salinity stress. A semi-quantitative RT-PCR-based expression assay confirmed the salinity stress inducibility of 45 of these 49 transcription factors, and showed that ten of them were up-regulated when seedlings were exposed to the demethylation agent 5-aza-2-deoxycytidine. Salinity stress was shown to affect the methylation status of four of these ten transcription factors (one MYB, one b-ZIP and two AP2/DREB family members using a combination of bisulfite sequencing and DNA methylation-sensitive DNA gel blot analysis. ChIP analysis indicated that the activation of three of the four DNA methylated transcription factors was correlated with an increased level of histone H3K4 trimethylation and H3K9 acetylation, and/or a reduced level of H3K9 demethylation in various parts of the promoter or coding regions. Our results suggest a critical role for some transcription factors' activation/repression by DNA methylation and/or histone modifications in soybean tolerance to salinity stress.

DNA methylation in sugarcane somaclonal variants assessed through methylation-sensitive amplified polymorphism.

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Francischini, J H M B; Kemper, E L; Costa, J B; Manechini, J R V; Pinto, L R

2017-05-04

Micropropagation is an important tool for large-scale multiplication of plant superior genotypes. However, somaclonal variation is one of the drawbacks of this process. Changes in DNA methylation have been widely reported as one of the main causes of somaclonal variations in plants. In order to investigate the occurrence of changes in the methylation pattern of sugarcane somaclonal variants, the MSAP (methylation-sensitive amplified polymorphism) technique was applied to micro-propagated plantlets sampled at the third subculture phase. The mother plant, in vitro normal plantlets, and in vitro abnormal plantlets (somaclonal variants) of four sugarcane clones were screened against 16 MSAP selective primers for EcoRI/MspI and EcoRI/HpaII restriction enzymes. A total of 1005 and 1200 MSAP-derived markers with polymorphism percentages of 28.36 and 40.67 were obtained for EcoRI/HpaII and EcoRI/MspI restriction enzyme combinations, respectively. The genetic similarity between the mother plant and the somaclonal variants ranged from 0.877 to 0.911 (EcoRI/MspI) and from 0.928 to 0.955 (EcoRI/HpaII). Most of the MASPs among mother plant and micro-propagated plantlets were derived from EcoRI/MspI restriction enzymes suggesting alteration due to gain or loss of internal cytosine methylation. A higher rate of loss of methylation (hypomethylation) than gain of methylation (hypermethylation) was observed in the abnormal in vitro sugarcane plantlets. Although changes in the methylation pattern were also observed in the in vitro normal plantlets, they were lower than those observed for the in vitro abnormal plantlets. The MASP technique proved to be a promising tool to early assessment of genetic fidelity of micro-propagated sugarcane plants.

Evaluation of markers for CpG island methylator phenotype (CIMP) in colorectal cancer by a large population-based sample.

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Ogino, Shuji; Kawasaki, Takako; Kirkner, Gregory J; Kraft, Peter; Loda, Massimo; Fuchs, Charles S

2007-07-01

The CpG island methylator phenotype (CIMP or CIMP-high) with extensive promoter methylation is a distinct phenotype in colorectal cancer. However, a choice of markers for CIMP has been controversial. A recent extensive investigation has selected five methylation markers (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1) as surrogate markers for epigenomic aberrations in tumor. The use of these markers as a CIMP-specific panel needs to be validated by an independent, large dataset. Using MethyLight assays on 920 colorectal cancers from two large prospective cohort studies, we quantified DNA methylation in eight CIMP-specific markers [the above five plus CDKN2A (p16), CRABP1, and MLH1]. A CIMP-high cutoff was set at > or = 6/8 or > or = 5/8 methylated promoters, based on tumor distribution and BRAF/KRAS mutation frequencies. All but two very specific markers [MLH1 (98% specific) and SOCS1 (93% specific)] demonstrated > or = 85% sensitivity and > or = 80% specificity, indicating overall good concordance in methylation patterns and good performance of these markers. Based on sensitivity, specificity, and false positives and negatives, the eight markers were ranked in order as: RUNX3, CACNA1G, IGF2, MLH1, NEUROG1, CRABP1, SOCS1, and CDKN2A. In conclusion, a panel of markers including at least RUNX3, CACNA1G, IGF2, and MLH1 can serve as a sensitive and specific marker panel for CIMP-high.

Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L). Developmental and chemotypic association of allylphenol O-methyltransferase activities.

Science.gov (United States)

Lewinsohn, E; Ziv-Raz, I; Dudai, N; Tadmor, Y; Lastochkin, E; Larkov, O; Chaimovitsh, D; Ravid, U; Putievsky, E; Pichersky, E; Shoham, Y

2000-12-07

Sweet basil (Ocimum basilicum L., Lamiaceae) is a common herb, used for culinary and medicinal purposes. The essential oils of different sweet basil chemotypes contain various proportions of the allyl phenol derivatives estragole (methyl chavicol), eugenol, and methyl eugenol, as well as the monoterpene alcohol linalool. To monitor the developmental regulation of estragole biosynthesis in sweet basil, an enzymatic assay for S-adenosyl-L-methionine (SAM):chavicol O-methyltransferase activity was developed. Young leaves display high levels of chavicol O-methyltransferase activity, but the activity was negligible in older leaves, indicating that the O-methylation of chavicol primarily occurs early during leaf development. The O-methyltransferase activities detected in different sweet basil genotypes differed in their substrate specificities towards the methyl acceptor substrate. In the high-estragole-containing chemotype R3, the O-methyltransferase activity was highly specific for chavicol, while eugenol was virtually not O-methylated. In contrast, chemotype 147/97, that contains equal levels of estragole and methyl eugenol, displayed O-methyltransferase activities that accepted both chavicol and eugenol as substrates, generating estragole and methyl eugenol, respectively. Chemotype SW that contains high levels of eugenol, but lacks both estragole and methyl eugenol, had apparently no allylphenol dependent O-methyltransferase activities. These results indicate the presence of at least two types of allylphenol-specific O-methyltransferase activities in sweet basil chemotypes, one highly specific for chavicol; and a different one that can accept eugenol as a substrate. The relative availability and substrate specificities of these O-methyltransferase activities biochemically rationalizes the variation in the composition of the essential oils of these chemotypes.

Genome-wide methylation analysis identifies differentially methylated CpG loci associated with severe obesity in childhood.

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Huang, R C; Garratt, E S; Pan, H; Wu, Y; Davis, E A; Barton, S J; Burdge, G C; Godfrey, K M; Holbrook, J D; Lillycrop, K A

2015-01-01

Childhood obesity is a major public health issue. Here we investigated whether differential DNA methylation was associated with childhood obesity. We studied DNA methylation profiles in whole blood from 78 obese children (mean BMI Z-score: 2.6) and 71 age- and sex-matched controls (mean BMI Z-score: 0.1). DNA samples from obese and control groups were pooled and analyzed using the Infinium HumanMethylation450 BeadChip array. Comparison of the methylation profiles between obese and control subjects revealed 129 differentially methylated CpG (DMCpG) loci associated with 80 unique genes that had a greater than 10% difference in methylation (P-value obesity were validated using sodium bisulfite pyrosequencing across loci within the FYN, PIWIL4, and TAOK3 genes in individual subjects. Three CpG loci within FYN were hypermethylated in obese individuals (all P obesity was associated with lower methylation of CpG loci within PIWIL4 (P = 0.003) and TAOK3 (P = 0.001). After building logistic regression models, we determined that a 1% increase in methylation in TAOK3, multiplicatively decreased the odds of being obese by 0.91 (95% CI: 0.86 - 0.97), and an increase of 1% methylation in FYN CpG3, multiplicatively increased the odds of being obese by 1.03 (95% CI: 0.99 - 1.07). In conclusion, these findings provide evidence that childhood obesity is associated with specific DNA methylation changes in whole blood, which may have utility as biomarkers of obesity risk.

Caspase 8 and maspin are downregulated in breast cancer cells due to CpG site promoter methylation

International Nuclear Information System (INIS)

Wu, Yanyuan; Alvarez, Monica; Slamon, Dennis J; Koeffler, Phillip; Vadgama, Jaydutt V

2010-01-01

Epigenetic changes associated with promoter DNA methylation results in silencing of several tumor suppressor genes that lead to increased risk for tumor formation and for progression of the cancer. Methylation specific PCR (MSP) and bisulfite sequencing were used for determination of proapoptotic gene Caspase 8 (CASP8) and the tumor suppressor gene maspin promoter methylation in four breast cancer and two non-tumorigenic breast cell lines. Involvement of histone H3 methylation in those cell lines were examined by CHIP assay. The CpG sites in the promoter region of CASP8 and maspin were methylated in all four breast cancer cell lines but not in two non-tumorigenic breast cell lines. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-dc) selectively inhibits DNA methyltransferases, DNMT3a and DNMT3b, and restored CASP8 and maspin gene expression in breast cancer cells. 5-aza-dc also reduced histone H3k9me2 occupancy on CASP8 promoter in SKBR3cells, but not in MCF-7 cells. Combination of histone deacetylase inhibitor Trichostatin A (TSA) and 5-aza-dc significant decrease in nuclear expression of Di-methyl histone H3-Lys27 and slight increase in acetyl histone H3-Lys9 in MCF-7 cells. CASP8 mRNA and protein level in MCF-7 cells were increased by the 5-aza-dc in combination with TSA. Data from our study also demonstrated that treatment with 5-FU caused a significant increase in unmethylated CASP8 and in CASP8 mRNA in all 3 cancer lines. CASP8 and maspin expression were reduced in breast cancer cells due to promoter methylation. Selective application of demethylating agents could offer novel therapeutic opportunities in breast cancer

Potato tuber pectin structure is influenced by pectin methyl esterase activity and impacts on cooked potato texture.

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Ross, Heather A; Wright, Kathryn M; McDougall, Gordon J; Roberts, Alison G; Chapman, Sean N; Morris, Wayne L; Hancock, Robert D; Stewart, Derek; Tucker, Gregory A; James, Euan K; Taylor, Mark A

2011-01-01

Although cooked potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase activity (PME) as a potential factor impacting on textural properties. In this study, tuber PME isoform and gene expression profiles have been determined in potato germplasm with differing textural properties as assessed using an amended wedge fracture method and a sloughing assay, revealing major differences between the potato types. Differences in pectin structure between potato types with different textural properties were revealed using monoclonal antibodies specific for different pectic epitopes. Chemical analysis of tuber pectin clearly demonstrated that, in tubers containing a higher level of total PME activity, there was a reduced degree of methylation of cell wall pectin and consistently higher peak force and work done values during the fracture of cooked tuber samples, demonstrating the link between PME activity, the degree of methylation of cell wall pectin, and cooked tuber textural properties.

Axin gene methylation status correlates with radiosensitivity of lung cancer cells

International Nuclear Information System (INIS)

Yang, Lian-He; Stoecker, Maggie; Wang, Endi; Xu, Ke; Wang, En-Hua; Han, Yang; Li, Guang; Xu, Hong-Tao; Jiang, Gui-Yang; Miao, Yuan; Zhang, Xiu-Peng; Zhao, Huan-Yu; Xu, Zheng-Fan

2013-01-01

We previously reported that Axin1 (Axin) is down-regulated in many cases of lung cancer, and X-ray irradiation increased Axin expression and inhibited lung cancer cells. The mechanisms, however, were not clear. Four lung cancer cell lines were used to detect the methylation status of Axin with or without X-ray treatment. Real-time PCR was used to quantify the expression of Axin, and western blot analysis was applied to measure protein levels of Axin, β-catenin, Cyclin D1, MMP-7, DNMTS, MeCP2 and acetylated histones. Flow cytometric analysis, colony formation assay, transwell assay and xenograft growth experiment were used to study the biological behavior of the cells with hypermethylated or unmethylated Axin gene after X-ray treatment. Hypermethylated Axin gene was detected in 2 of 4 cell lines, and it correlated inversely with Axin expression. X-ray treatment significantly up-regulated Axin expression in H446 and H157 cells, which possess intrinsic hypermethylation of the Axin gene (P<0.01), but did not show up-regulation in LTE and H460 cells, which have unmethylated Axin gene. 2Gy X-ray significantly reduced colony formation (from 71% to 10.5%) in H157 cells, while the reduction was lower in LTE cells (from 71% to 20%). After X-ray irradiation, xenograft growth was significantly decreased in H157 cells (from 1.15 g to 0.28 g) in comparison with LTE cells (from 1.06 g to 0.65 g). Significantly decreased cell invasiveness and increased apoptosis were also observed in H157 cells treated with X-ray irradiation (P<0.01). Down-regulation of DNMTs and MeCP2 and up-regulation of acetylated histones could be detected in lung cancer cells. X-ray-induced inhibition of lung cancer cells may be mediated by enhanced expression of Axin via genomic DNA demethylation and histone acetylation. Lung cancer cells with a different methylation status of the Axin gene showed different radiosensitivity, suggesting that the methylation status of the Axin gene may be one important factor

Protection against de novo methylation is instrumental in maintaining parent-of-origin methylation inherited from the gametes.

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Proudhon, Charlotte; Duffié, Rachel; Ajjan, Sophie; Cowley, Michael; Iranzo, Julian; Carbajosa, Guillermo; Saadeh, Heba; Holland, Michelle L; Oakey, Rebecca J; Rakyan, Vardhman K; Schulz, Reiner; Bourc'his, Déborah

2012-09-28

Identifying loci with parental differences in DNA methylation is key to unraveling parent-of-origin phenotypes. By conducting a MeDIP-Seq screen in maternal-methylation free postimplantation mouse embryos (Dnmt3L-/+), we demonstrate that maternal-specific methylation exists very scarcely at midgestation. We reveal two forms of oocyte-specific methylation inheritance: limited to preimplantation, or with longer duration, i.e. maternally imprinted loci. Transient and imprinted maternal germline DMRs (gDMRs) are indistinguishable in gametes and preimplantation embryos, however, de novo methylation of paternal alleles at implantation delineates their fates and acts as a major leveling factor of parent-inherited differences. We characterize two new imprinted gDMRs, at the Cdh15 and AK008011 loci, with tissue-specific imprinting loss, again by paternal methylation gain. Protection against demethylation after fertilization has been emphasized as instrumental in maintaining parent-of-origin methylation inherited from the gametes. Here we provide evidence that protection against de novo methylation acts as an equal major pivot, at implantation and throughout life. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of methyl butyrate aroma on the survival and viability of human breast cancer cells in vitro

International Nuclear Information System (INIS)

Khan, M.A.; Rumana Ahmad, R.; Srivastava, A.N.

2016-01-01

Background: Aroma can have far reaching effects on mind, body and soul. Pleasant aromas are known to have a soothing effect on the mind and are known to relieve stress and enhance concentration. Recently, it has been demonstrated that aroma may also have some curative effects as well as benefits and can be used both for prophylaxis and therapy of diseases. Our aim was to test our hypothesis whether aroma can cure or prevent cancer. Methyl butyrate (MB) is the methyl ester of butyric acid having a characteristic sweet and fruity odor like that of apples and pineapples. It occurs in many plant products in minute quantities and in pineapple oil. Methods: In the present study, the effect of aroma of MB has been evaluated on human breast cancer cell line MDA-MB-231 in vitro . The percentage viability of the cell line was determined by using Trypan blue dye exclusion assay. Results: It was found that MB at a concentration of 0.01 M was effective in causing considerable cytotoxicity (40%) in breast cancer cells (without even coming in contact with cells) while at 0.02 M, % cytotoxicity was found to be 50%. Mechanism of action of MB on cancer cells was investigated by acridine orange–ethidium bromide assay using fluorescence microscopy and DNA fragmentation assay. MB aroma appeared to induce necrosis in cancer cells exposed to it. Conclusion: No study involving the effect of aroma/smell on cancer cells has ever been reported before and warrants further investigation on other cancer and normal cell lines.

DNA methylation perturbations in genes involved in polyunsaturated Fatty Acid biosynthesis associated with depression and suicide risk.

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Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-Yu; Cooper, Thomas B; Burke, Ainsley K; Oquendo, Maria A; Mann, J John; Sublette, M Elizabeth

2015-01-01

Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention.

DNA methylation perturbations in genes involved in polyunsaturated fatty acid biosynthesis associated with depression and suicide risk

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Fatemeh eHaghighi

2015-04-01

Full Text Available Polyunsaturated fatty acid (PUFA status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6 and α-linolenic acid (ALA, 18:3n-3. We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD with (n=22 and without (n=39 history of suicide attempt, and age- and sex-matched healthy volunteers (n=59. Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1 and 2 (Fads2, and elongation of very long chain fatty acids protein 5 (Elovl5, were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator (LASSO logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention.

Structure, function and carcinogenicity of metabolites of methylated and non-methylated polycyclic aromatic hydrocarbons: a comprehensive review.

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Flesher, James W; Lehner, Andreas F

2016-01-01

The Unified Theory of PAH Carcinogenicity accommodates the activities of methylated and non-methylated polycyclic aromatic hydrocarbons (PAHs) and states that substitution of methyl groups on meso-methyl substituted PAHs with hydroxy, acetoxy, chloride, bromide or sulfuric acid ester groups imparts potent cancer producing properties. It incorporates specific predictions from past researchers on the mechanism of carcinogenesis by methyl-substituted hydrocarbons, including (1) requirement for metabolism to an ArCH2X type structure where X is a good leaving group and (2) biological substitution of a meso-methyl group at the most reactive center in non-methylated hydrocarbons. The Theory incorporates strong inferences of Fieser: (1) The mechanism of carcinogenesis involves a specific metabolic substitution of a hydrocarbon at its most reactive center and (2) Metabolic elimination of a carcinogen is a detoxifying process competitive with that of carcinogenesis and occurring by a different mechanism. According to this outlook, chemical or biochemical substitution of a methyl group at the reactive meso-position of non-methylated hydrocarbons is the first step in the mechanism of carcinogenesis for most, if not all, PAHs and the most potent metabolites of PAHs are to be found among the meso methyl-substituted hydrocarbons. Some PAHs and their known or potential metabolites and closely related compounds have been tested in rats for production of sarcomas at the site of subcutaneous injection and the results strongly support the specific predictions of the Unified Theory.

Are clinicopathological features of colorectal cancers with methylation in half of CpG island methylator phenotype panel markers different from those of CpG island methylator phenotype-high colorectal cancers?

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Bae, Jeong Mo; Rhee, Ye-Young; Kim, Kyung Ju; Wen, Xianyu; Song, Young Seok; Cho, Nam-Yun; Kim, Jung Ho; Kang, Gyeong Hoon

2016-01-01

CpG island methylator phenotype (CIMP)-high (CIMP-H) colorectal cancer (CRC) is defined when a tumor shows methylation at greater than or equal to 60% of CIMP panel markers. Although CRCs with methylation at 50% of panel markers are classified as CIMP-low/CIMP-0 tumors, little is known regarding the clinicopathological and molecular features of CRCs with methylation at 4/8 panel markers (4/8 methylated markers) and whether they are akin to CIMP-H or CIMP-low/CIMP-0 CRCs in terms of their clinicopathological or molecular features. A total of 1164 cases of surgically resected CRC were analyzed for their methylation status in 8 CIMP panel markers, and the frequencies of various clinicopathological and molecular features were compared between CRCs with 0/8, 1/8 to 3/8, 4/8, and 5/8 to 8/8 methylated markers. CRCs with 4/8 methylated markers were closer to CRCs with 5/8 to 8/8 methylated markers in terms of sex distribution, mucin production, serration, nodal metastasis, CK7 expression, CK20 loss, and CDX2 loss frequencies and overall survival rate. CRCs with methylation at 4/8 markers were closer to CRCs with 1/8 to 3/8 methylated markers in terms of less frequent right colon location and poor differentiation. CRCs with 4/8 methylated markers showed the shortest overall survival time compared with CRCs with 0/8, 1/8 to 3/8, 4/8, or 5/8 to 8/8 methylated markers. In terms of clinicopathological and molecular features, CRCs with 4/8 methylated markers appeared to be closer to CIMP-H than to CIMP-low/CIMP-0 and would thus be better classified as CIMP-H if the CRCs require classification into either CIMP-H or CIMP-low/CIMP-0. Copyright © 2015 Elsevier Inc. All rights reserved.

CDO1 promoter methylation is associated with gene silencing and is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients.

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Meller, Sebastian; Zipfel, Lisa; Gevensleben, Heidrun; Dietrich, Jörn; Ellinger, Jörg; Majores, Michael; Stein, Johannes; Sailer, Verena; Jung, Maria; Kristiansen, Glen; Dietrich, Dimo

2016-12-01

Molecular biomarkers may facilitate the distinction between aggressive and clinically insignificant prostate cancer (PCa), thereby potentially aiding individualized treatment. We analyzed cysteine dioxygenase 1 (CDO1) promoter methylation and mRNA expression in order to evaluate its potential as prognostic biomarker. CDO1 methylation and mRNA expression were determined in cell lines and formalin-fixed paraffin-embedded prostatectomy specimens from a first cohort of 300 PCa patients using methylation-specific qPCR and qRT-PCR. Univariate and multivariate Cox proportional hazards and Kaplan-Meier analyses were performed to evaluate biochemical recurrence (BCR)-free survival. Results were confirmed in an independent second cohort comprising 498 PCa cases. Methylation and mRNA expression data from the second cohort were generated by The Cancer Genome Atlas (TCGA) Research Network by means of Infinium HumanMethylation450 BeadChip and RNASeq. CDO1 was hypermethylated in PCa compared to normal adjacent tissues and benign prostatic hyperplasia (P < 0.001) and was associated with reduced gene expression (ρ = -0.91, P = 0.005). Using two different methodologies for methylation quantification, high CDO1 methylation as continuous variable was associated with BCR in univariate analysis (first cohort: HR = 1.02, P = 0.002, 95% CI [1.01-1.03]; second cohort: HR = 1.02, P = 0.032, 95% CI [1.00-1.03]) but failed to reach statistical significance in multivariate analysis. CDO1 promoter methylation is involved in gene regulation and is a potential prognostic biomarker for BCR-free survival in PCa patients following radical prostatectomy. Further studies are needed to validate CDO1 methylation assays and to evaluate the clinical utility of CDO1 methylation for the management of PCa.

Genome-wide DNA methylation profiling in infants born to gestational diabetes mellitus.

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Weng, Xiaoling; Liu, Fatao; Zhang, Hong; Kan, Mengyuan; Wang, Ting; Dong, Mingyue; Liu, Yun

2018-03-26

Offspring exposed to gestational diabetes mellitus (GDM) are at a high risk for metabolic diseases. The mechanisms behind the association between offspring exposed to GDM in utero and an increased risk of health consequences later in life remain unclear. The aim of this study was to clarify the changes in methylation levels in the foetuses of women with GDM and to explore the possible mechanisms linking maternal GDM with a high risk of metabolic diseases in offspring later in life. A genome-wide comparative methylome analysis on the umbilical cord blood of infants born to 30 women with GDM and 33 women with normal pregnancy was performed using Infinium HumanMethylation 450 BeadChip assays. A quantitative methylation analysis of 18 CpG dinucleotides was verified in the validation umbilical cord blood samples from 102 newborns exposed to GDM and 103 newborns who experienced normal pregnancy by MassARRAY EpiTYPER. A total of 4485 differentially methylated sites (DMSs), including 2150 hypermethylated sites and 2335 hypomethylated sites, with a mean β-value difference of >0.05, were identified by the 450k array. Good agreement was observed between the massarray validation data and the 450k array data (R 2 > 0.99; P 0.15 between the GDM and healthy groups were identified and showed potential as clinical biomarkers for GDM. "hsa04940: Type I diabetes mellitus" was the most significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, with a P-value = 3.20E-07 and 1.36E-02 in the hypermethylated and hypomethylated genepathway enrichment analyses, respectively. In the Gene Ontology (GO) pathway analyses, immune MHC-related pathways and neuron development-related pathways were significantly enriched. Our results suggest that GDM has epigenetic effects on genes that are preferentially involved in the Type I diabetes mellitus pathway, immune MHC (major histocompatibility complex)-related pathways and neuron development-related pathways, with consequences on fetal growth

SPE-GC/FTD determination of N-methyl-2-pyrrolidone and its metabolites in urine.

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Kubota, Ryuichi; Endo, Yoko; Takeuchi, Akito; Inoue, Yoshinori; Ogata, Hiroko; Ogawa, Masanori; Nakagawa, Tomoo; Onda, Nobuhiko; Endo, Ginji

2007-07-01

An analytical method using a combination of solid-phase extraction (SPE) and gas chromatography with a flame thermionic detector (GC/FTD) was developed for determination of N-methyl-2-pyrrolidone (NMP), N-methylsuccinimide (MSI), and 2-hydroxy-N-methylsuccinimide (2-HMSI) in human urine. The SPE cartridge of poly(divinylbenzene/hydroxymethacrylate) used was directly loaded with urine sample, followed by elution with methyl isobutyl ketone (MIBK) and subsequent centrifugation, and the supernatant was injected into the capillary GC using a DB1701. This method allowed efficient separation of NMP, MSI, and 2-HMSI, which were nearly free of interference by other GC peaks arising from urine. Recoveries of NMP, MSI, and 2-HMSI from the SPE cartridge were about 98, 101, and 67%, respectively, with limits of detection of 0.04, 0.02, and 0.06 mg/L, respectively, which met the regulatory requirements. The present method was used for assay in biological monitoring of workers exposed to NMP in their occupational environment.

Determination of the quantity of acetyl CoA carboxylase by [14C]methyl avidin binding

International Nuclear Information System (INIS)

Roman-Lopez, C.R.; Goodson, J.; Allred, J.B.

1987-01-01

Conditions are described under which monomeric [ 14 C]methyl avidin binds to SDS-denatured biotin enzymes and remains bound through polyacrylamide gel electrophoresis. The location of radioactive proteins on the dried gel was determined by fluorography and their identity was established by subunit molecular weight. The relative quantity of bound radioactive avidin, stoichiometrically equivalent to the molar quantity of biotin protein, can be determined by scanning the fluorograph with a soft laser densitometer. To determine the absolute quantity of biotin protein, the radioactive areas of the dried gel were cut out, resolubilized, and assayed for radioactivity. Since the specific radioactivity of the [ 14 C]methyl avidin was known, the quantity of avidin bound and therefore the quantity of biotin enzyme could be calculated. The method is illustrated by the analysis of purified acetyl CoA carboxylase and is applied to the analysis of biotin enzymes in isolated rat liver mitochondria

Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis.

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Xie, Xinqiang; Garg, Ashish; Khosla, Chaitan; Cane, David E

2017-03-01

The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR 0 ) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR 0 domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2- 2 H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP + in the presence of redox-inactive, recombinant NanKR1 0 or NanKR5 0 , from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR7 0 from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR 0 -catalyzed isotope exchange of the reversibly generated, transiently formed oxidation product [2- 2 H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR 0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR 0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.

SPONTANEOUS AND MNNG-INDUCED REVERSION OF AN EGFP CONSTRUCT IN HELA CELLS: AN ASSAY FOR OBSERVING MUTATIONS IN LIVING CELLS BY FLUORESCENT MICROSCOPY

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A HeLa cell line stably expressing the Enhanced Green Fluorescence Protein (EGFP) gene, interrupted by the IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 ?M of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done ...

How does methylation suppress the electron-induced decomposition of 1-methyl-nitroimidazoles?

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Kossoski, F.; Varella, M. T. do N.

2017-10-01

The efficient decomposition of nitroimidazoles (NIs) by low energy electrons is believed to underlie their radiosensitizing properties. Recent dissociative electron attachment (DEA) measurements showed that methylation at the N1 site unexpectedly suppresses the electron-induced reactions in 4(5)-NI. We report theoretical results that provide a clear interpretation of that astounding finding. Around 1.5 eV, DEA reactions into several fragments are initiated by a π* resonance, not considered in previous studies. The autoionization lifetime of this anion state, which limits the predissociation dynamics, is considerably shorter in the methylated species, thereby suppressing the DEA signals. On the other hand, the lifetime of the π* resonance located around 3 eV is less affected by methylation, which explains why DEA is still observed at these energies. Our results demonstrate how even a simple methylation can significantly modify the probabilities for DEA reactions, which may be significant for NI-based cancer therapy.

BROMATOMATRIC ASSAY OF GATIFLOXACIN IN PHARMACEUTICALS

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KALSANG THARPA

2008-09-01

Full Text Available Three new, simple, and cost-effective visible spectrophotometric methods are proposed for determination of gatifloxacin (GTF using bromate-bromide mixture, and three dyes, methyl orange, indigocarmine and thymol blue, as reagents.The methods engross the addition of a known excess of bromate-bromide mixture to GTF in hydrochloric acid medium followed by determination of residual bromine by reacting with a fixed amount of either methyl orange andmeasuring the absorbance at 520 nm (method A or indigo carmine and measuring the absorbance at 610 nm (method B or thymol blue and measuring the absorbance at 550 nm (method C. In all the methods, the amount of brominereacted corresponds to the amount of GTF, and the absorbance is found to increase linearly with the concentration of GTF. Under the optimum conditions, GTF could be assayed in the concentration range 0.25-1.5, 0.5-6.0, and 0.5-10μg/mL by method A, method B and method C, respectively. The apparent molar absorptivities are calculated to be 1.6x105, 4.0x104 and 3.2x104 L mol-1 cm-1 for the method A, method B and method C, respectively, and the corresponding Sandell sensitivity values are 0.0025, 0.010 and 0.012 μg/cm2. The intra-day and inter-day precision, and the accuracy of the methods were evaluated as per the current ICH guidelines. The methods were successfully applied to the determination of GTF in pharmaceutical preparations without the interference from any of the pharmaceutical adjuvants.

In vitro antifungal activity of fatty acid methyl esters of the seeds of Annona cornifolia A.St.-Hil. (Annonaceae) against pathogenic fungus Paracoccidioides brasiliensis.

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Lima, Luciana Alves Rodrigues dos Santos; Johann, Susana; Cisalpino, Patrícia Silva; Pimenta, Lúcia Pinheiro Santos; Boaventura, Maria Amélia Diamantino

2011-01-01

Fatty acids are abundant in vegetable oils. They are known to have antibacterial and antifungal properties. Antifungal susceptibility was evaluated by broth microdilution assay following CLSI (formerly the NCCLS) guidelines against 16 fungal strains of clinical interest. In this work, fatty acid methyl esters (FAME) was able to inhibit 12 clinical strains of the pathogenic fungus Paracoccidioides brasiliensis and were also active in the bioautographic assay against Cladosporium sphaerospermum. FAME was a more potent antifungal than trimethoprim-sulfamethoxazole against P. brasiliensis under the experimental conditions tested.

Dissociation dynamics of methylal

Energy Technology Data Exchange (ETDEWEB)

Beaud, P; Frey, H -M; Gerber, T; Mischler, B; Radi, P P; Tzannis, A -P [Paul Scherrer Inst. (PSI), Villigen (Switzerland)

1999-08-01

The dissociation of methylal is investigated using mass spectrometry, combined with a pyrolytic radical source and femtosecond pump probe experiments. Based on preliminary results two reaction paths of methylal dissociation are proposed and discussed. (author) 4 fig., 3 refs.

Cytokinesis-block micronucleus assay evolves into a 'cytome' assay of chromosomal instability, mitotic dysfunction and cell death

International Nuclear Information System (INIS)

Fenech, Michael

2006-01-01

The cytokinesis-block micronucleus (CBMN) assay was originally developed as an ideal system for measuring micronuclei (MNi) however it can also be used to measure nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (necrosis or apoptosis) and nuclear division rate. Current evidence suggests that (a) NPBs originate from dicentric chromosomes in which the centromeres have been pulled to the opposite poles of the cell at anaphase and are therefore indicative of DNA mis-repair, chromosome rearrangement or telomere end-fusions, (b) NPBs may break to form MNi, (c) the nuclear budding process is the mechanism by which cells remove amplified and/or excess DNA and is therefore a marker of gene amplification and/or altered gene dosage, (d) cell cycle checkpoint defects result in micronucleus formation and (e) hypomethylation of DNA, induced nutritionally or by inhibition of DNA methyl transferase can lead to micronucleus formation either via chromosome loss or chromosome breakage. The strong correlation between micronucleus formation, nuclear budding and NPBs (r = 0.75-0.77, P < 0.001) induced by either folic acid deficiency or exposure to ionising radiation is supportive of the hypothesis that folic acid deficiency and/or ionising radiation cause genomic instability and gene amplification by the initiation of breakage-fusion-bridge cycles. In its comprehensive mode, the CBMN assay measures all cells including necrotic and apoptotic cells as well as number of nuclei per cell to provide a measure of cytotoxicity and mitotic activity. The CBMN assay has in fact evolved into a 'cytome' method for measuring comprehensively chromosomal instability phenotype and altered cellular viability caused by genetic defects and/or nutrional deficiencies and/or exogenous genotoxins thus opening up an exciting future for the use of this methodology in the emerging fields of nutrigenomics and toxicogenomics and their combinations

DNA methylation changes detected by methylation-sensitive amplified polymorphism in two contrasting rice genotypes under salt stress.

Science.gov (United States)

Wang, Wensheng; Zhao, Xiuqin; Pan, Yajiao; Zhu, Linghua; Fu, Binying; Li, Zhikang

2011-09-20

DNA methylation, one of the most important epigenetic phenomena, plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli. In the present study, a methylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress. Consistent with visibly different phenotypes in response to salt stress, epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salt-tolerant FL478 and salt-sensitive IR29. In addition, most tissue-specific DNA methylation loci were conserved, while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes. Strikingly, salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29 than in the FL478. This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage, and helps to further elucidate the implications of DNA methylation in crop growth and development. Copyright © 2011. Published by Elsevier Ltd.

MTHFR methylation moderates the impact of smoking on DNA methylation at AHRR for African American young adults.

Science.gov (United States)

Beach, Steven R H; Lei, Man Kit; Ong, Mei Ling; Brody, Gene H; Dogan, Meeshanthini V; Philibert, Robert A

2017-09-01

Smoking has been shown to have a large, reliable, and rapid effect on demethylation of AHRR, particularly at cg05575921, suggesting that methylation may be used as an index of cigarette consumption. Because the availability of methyl donors may also influence the degree of demethylation in response to smoking, factors that affect the activity of methylene tetrahydrofolate reductase (MTHFR), a key regulator of methyl group availability, may be of interest. In the current investigation, we examined the extent to which individual differences in methylation of MTHFR moderated the association between smoking and demethylation at cg05575921 as well as at other loci on AHRR associated with a main effect of smoking. Using a discovery sample (AIM, N = 293), and a confirmatory sample (SHAPE, N = 368) of young adult African Americans, degree of methylation of loci in the first exon of MTHFR was associated with amplification of the association between smoking and AHRR demethylation at cg05575921. However, genetic variation at a commonly studied MTHFR variant, C677T, did not influence cg05575921 methylation. The significant interaction between MTHFR methylation and the smoking-induced response at cg05575921 suggests a role for individual differences in methyl cycle regulation in understanding the effects of cigarette consumption on genome wide DNA methylation. © 2017 Wiley Periodicals, Inc.

Synthesis of [methyl-{sup 14}C]crotonobetaine from DL-[methyl-{sup 14}C]carnitine

Energy Technology Data Exchange (ETDEWEB)

Loester, H.; Seim, H. [Leipzig Univ. (Germany). Inst. of Clinical Chemistry and Pathobiochemistry

1996-02-01

The causes of carnitine deficiency syndromes are not completely understood, but decomposition of L-carnitine in vivo is likely to be involved. Carnitine is metabolized to {gamma}-butyrobetaine, and crotonobetaine is probably an intermediate in this pathway. To validate experimentally the precursor-product relationship between the three physiologically occuring {gamma}-betaines - L-carnitine, crotonobetaine, {gamma}-butyrobetaine - labelling with stable or radioactive isotopes became necessary. Methyl-labelled carnitine isomers (L(-)-, D(+)- or DL-) or {gamma}-butyrobetaine can be easily synthesized by methylation of 4-amino-3-hydroxybutyric acid isomers or 4-aminobutyric acid, respectively. Because of problems with the 4-aminocrotonic acid, we synthesized labelled crotonbetaine from labelled carnitine. Thus, DL-[methyl-{sup 14}C]carnitine was dehydrated by reaction with concentrated sulfuric acid. After removal of the latter the products were separated and purified by ion exchange chromatography on DOWEX 50 WX8 (200 - 400 mesh) and gradient elution with hydrochloric acid. In addition to the labelled main product [methyl-{sup 14}C]crotonobetaine (yield about 50 %), [methyl-{sup 14}C]glycine betaine and [methyl-{sup 14}C]acetonyl-trimethylammonium (ATMA) were formed. The end products were identified by combined thin layer chromatography/autoradiography and quantified by liquid scintillation counting. (Author).

Synergistic antiviral effect in vitro of azidothymidine and amphotericin B methyl ester in combination on HIV infection

DEFF Research Database (Denmark)

Hansen, J E; Nielsen, C; Svenningsen, A

1992-01-01

The nucleoside analogue azidothymidine (AZT) and the methyl ester of amphotericin B (AME) were assayed for antiviral effect on HIV infection singly and in combination. Both compounds were effective in inhibiting HIV infection of MT-4 cells. At concentrations where either compound alone had no sig...... synergistic antiviral properties. Amphotericin B itself significantly reduced HIV infectivity in vitro and should not be used as an antifungal agent in cultures intended to propagate HIV....

Effects on wildlife of ethyl and methyl parathion applied to California rice fields

Science.gov (United States)

Custer, T.W.; Hill, E.F.; Ohlendorf, H.M.

1985-01-01

Selected rice fields on the Sacramento National Wildlife Refuge Complex were aerially sprayed one time during May or June 1982 with either ethyl (0.11 kg Al/ha) or methyl (0.84 kg AI/ha) parathion for control of tadpole shrimp, Triops longicaudatus. No sick or dead vertebrate wildlife were found or adjacent to the treated rice fields after spraying. Specimens of the following birds and mammals were assayed for brain cholinesterase (ChE) activity to determine exposure to either form of parathion; house mouse, Mus musculus; black-tailed jackrabbit, Lepus californicus; mallard, Anas platyrhynchos; ring-necked pheasant, Phasianus colchicus; American coot, Fulica americana; and red-winged blackbird, Agelaius phoeniceus. Both mice and pheasants from methyl parathion-treated fields had overall mean ChE activities that were significantly (P < 0.05) inhibited compared with controls, and 7, 40, 54 and 57% of individual blackbirds, pheasant, mice, and coots, respectively, had inhibited brain ChE activities (i.e., less than -2 SD of control mean). Although no overall species effect was detected for ethyl parathoid treatment, pheasants (43%), coots (33%), and mice (37%) had significantly inhibited brain ChE activities. Neither of the parathion treatment appeared acutely hazardous to wildlife in or adjacent to rice fields, but sufficient information on potential hazards was obtained to warrant caution in use of these chemicals, especially methyl parathion, in rice fields.

Copper induces expression and methylation changes of early development genes in Crassostrea gigas embryos.

Science.gov (United States)

Sussarellu, Rossana; Lebreton, Morgane; Rouxel, Julien; Akcha, Farida; Rivière, Guillaume

2018-03-01

Copper contamination is widespread along coastal areas and exerts adverse effects on marine organisms such as mollusks. In the Pacific oyster, copper induces severe developmental abnormalities during early life stages; however, the underlying molecular mechanisms are largely unknown. This study aims to better understand whether the embryotoxic effects of copper in Crassostrea gigas could be mediated by alterations in gene expression, and the putative role of DNA methylation, which is known to contribute to gene regulation in early embryo development. For that purpose, oyster embryos were exposed to 4 nominal copper concentrations (0.1, 1, 10 and 20 μg L -1 Cu 2+ ) during early development assays. Embryotoxicity was monitored through the oyster embryo-larval bioassay at the D-larva stage 24 h post fertilization (hpf) and genotoxicity at gastrulation 7 hpf. In parallel, the relative expression of 15 genes encoding putative homeotic, biomineralization and DNA methylation proteins was measured at three developmental stages (3 hpf morula stage, 7 hpf gastrula stage, 24 hpf D-larvae stage) using RT-qPCR. Global DNA content in methylcytosine and hydroxymethylcytosine were measured by HPLC and gene-specific DNA methylation levels were monitored using MeDIP-qPCR. A significant increase in larval abnormalities was observed from copper concentrations of 10 μg L -1 , while significant genotoxic effects were detected at 1 μg L -1 and above. All the selected genes presented a stage-dependent expression pattern, which was impaired for some homeobox and DNA methylation genes (Notochord, HOXA1, HOX2, Lox5, DNMT3b and CXXC-1) after copper exposure. While global DNA methylation (5-methylcytosine) at gastrula stage didn't show significant changes between experimental conditions, 5-hydroxymethylcytosine, its degradation product, decreased upon copper treatment. The DNA methylation of exons and the transcript levels were correlated in control samples for HOXA1 but such

DNA methylation of specific CpG sites in the promoter region regulates the transcription of the mouse oxytocin receptor.

Directory of Open Access Journals (Sweden)

Shimrat Mamrut

Full Text Available Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter.

Process for the production of methyl methacrylate

NARCIS (Netherlands)

Eastham, G.R.; Johnson, D.W.; Straathof, A.J.J.; Fraaije, Marco; Winter, Remko

2015-01-01

A process of producing methyl methacrylate or derivatives thereof is described. The process includes the steps of; (i) converting 2-butanone to methyl propionate using a Baeyer-Villiger monooxygenase, and (ii) treating the methyl propionate produced to obtain methyl methacrylate or derivatives

Methylation patterns in marginal zone lymphoma.

Science.gov (United States)

Arribas, Alberto J; Bertoni, Francesco

Promoter DNA methylation is a major regulator of gene expression and transcription. The identification of methylation changes is important for understanding disease pathogenesis, for identifying prognostic markers and can drive novel therapeutic approaches. In this review we summarize the current knowledge regarding DNA methylation in MALT lymphoma, splenic marginal zone lymphoma, nodal marginal zone lymphoma. Despite important differences in the study design for different publications and the existence of a sole large and genome-wide methylation study for splenic marginal zone lymphoma, it is clear that DNA methylation plays an important role in marginal zone lymphomas, in which it contributes to the inactivation of tumor suppressors but also to the expression of genes sustaining tumor cell survival and proliferation. Existing preclinical data provide the rationale to target the methylation machinery in these disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

DNA methylation-based variation between human populations.

Science.gov (United States)

Kader, Farzeen; Ghai, Meenu

2017-02-01

Several studies have proved that DNA methylation affects regulation of gene expression and development. Epigenome-wide studies have reported variation in methylation patterns between populations, including Caucasians, non-Caucasians (Blacks), Hispanics, Arabs, and numerous populations of the African continent. Not only has DNA methylation differences shown to impact externally visible characteristics, but is also a potential biomarker for underlying racial health disparities between human populations. Ethnicity-related methylation differences set their mark during early embryonic development. Genetic variations, such as single-nucleotide polymorphisms and environmental factors, such as age, dietary folate, socioeconomic status, and smoking, impacts DNA methylation levels, which reciprocally impacts expression of phenotypes. Studies show that it is necessary to address these external influences when attempting to differentiate between populations since the relative impacts of these factors on the human methylome remain uncertain. The present review summarises several reported attempts to establish the contribution of differential DNA methylation to natural human variation, and shows that DNA methylation could represent new opportunities for risk stratification and prevention of several diseases amongst populations world-wide. Variation of methylation patterns between human populations is an exciting prospect which inspires further valuable research to apply the concept in routine medical and forensic casework. However, trans-generational inheritance needs to be quantified to decipher the proportion of variation contributed by DNA methylation. The future holds thorough evaluation of the epigenome to understand quantification, heritability, and the effect of DNA methylation on phenotypes. In addition, methylation profiling of the same ethnic groups across geographical locations will shed light on conserved methylation differences in populations.

The multi-domain protein Np95 connects DNA methylation and histone modification

Science.gov (United States)

Rottach, Andrea; Frauer, Carina; Pichler, Garwin; Bonapace, Ian Marc; Spada, Fabio; Leonhardt, Heinrich

2010-01-01

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ß. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways. PMID:20026581

Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene

DEFF Research Database (Denmark)

Candiloro, Ida Lm; Mikeska, Thomas; Hokland, Peter

2008-01-01

ABSTRACT: BACKGROUND: Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) t......ABSTRACT: BACKGROUND: Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (d......MS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation. We used this approach to study methylation of the CDKN2B (p15) cell cycle progression inhibitor gene which is inactivated by DNA methylation in haematological malignancies...... the methylated alleles and assess the degree of methylation. Direct sequencing of selected dMS-HRM products was used to determine the exact DNA methylation pattern and confirmed the degree of methylation estimated by dMS-HRM. CONCLUSION: dMS-HRM is a powerful technique for the analysis of methylation in CDKN2B...

Inductive effect of methyl group in a series of methylated indoles: A ...

Indian Academy of Sciences (India)

Vol. 125, No. 4, July 2013, pp. 905–912. c Indian Academy of Sciences. Inductive effect of methyl group in a series of methylated indoles: A graph theoretical analysis in the light of density functional theory and correlation with experimental charge transfer transition energies. AMIT S TIWARYa,∗ and ASOK K MUKHERJEEb.

SMYD2 promoter DNA methylation is associated with abdominal aortic aneurysm (AAA) and SMYD2 expression in vascular smooth muscle cells.

Science.gov (United States)

Toghill, Bradley J; Saratzis, Athanasios; Freeman, Peter J; Sylvius, Nicolas; Bown, Matthew J

2018-01-01

Abdominal aortic aneurysm (AAA) is a deadly cardiovascular disease characterised by the gradual, irreversible dilation of the abdominal aorta. AAA is a complex genetic disease but little is known about the role of epigenetics. Our objective was to determine if global DNA methylation and CpG-specific methylation at known AAA risk loci is associated with AAA, and the functional effects of methylation changes. We assessed global methylation in peripheral blood mononuclear cell DNA from 92 individuals with AAA and 93 controls using enzyme-linked immunosorbent assays, identifying hyper-methylation in those with large AAA and a positive linear association with AAA diameter ( P  AAA risk loci identified in genome-wide association studies, using bisulphite next-generation sequencing (NGS) in vascular smooth muscle cells (VSMCs) taken from aortic tissues of 44 individuals (24 AAAs and 20 controls). In IL6R , 2 CpGs were hyper-methylated ( P  = 0.0145); in ERG , 13 CpGs were hyper-methylated ( P  = 0.0005); in SERPINB9 , 6 CpGs were hypo-methylated ( P  = 0.0037) and 1 CpG was hyper-methylated ( P  = 0.0098); and in SMYD2 , 4 CpGs were hypo-methylated ( P  = 0.0012).RT-qPCR was performed for each differentially methylated gene on mRNA from the same VSMCs and compared with methylation. This analysis revealed downregulation of SMYD2 and SERPINB9 in AAA, and a direct linear relationship between SMYD2 promoter methylation and SMYD2 expression ( P  = 0.038). Furthermore, downregulation of SMYD2 at the site of aneurysm in the aortic wall was further corroborated in 6 of the same samples used for methylation and gene expression analysis with immunohistochemistry. This study is the first to assess DNA methylation in VSMCs from individuals with AAA using NGS, and provides further evidence there is an epigenetic basis to AAA. Our study shows that methylation status of the SMYD2 promoter may be linked with decreased SMYD2 expression in disease pathobiology. In

Prenatal polycyclic aromatic hydrocarbon, adiposity, peroxisome proliferator-activated receptor (PPAR γ methylation in offspring, grand-offspring mice.

Directory of Open Access Journals (Sweden)

Zhonghai Yan

Full Text Available Greater levels of prenatal exposure to polycyclic aromatic hydrocarbon (PAH have been associated with childhood obesity in epidemiological studies. However, the underlying mechanisms are unclear.We hypothesized that prenatal PAH over-exposure during gestation would lead to weight gain and increased fat mass in offspring and grand-offspring mice. Further, we hypothesized that altered adipose gene expression and DNA methylation in genes important to adipocyte differentiation would be affected.Pregnant dams were exposed to a nebulized PAH mixture versus negative control aerosol 5 days a week, for 3 weeks. Body weight was recorded from postnatal day (PND 21 through PND60. Body composition, adipose cell size, gene expression of peroxisome proliferator-activated receptor (PPAR γ, CCAAT/enhancer-binding proteins (C/EBP α, cyclooxygenase (Cox-2, fatty acid synthase (FAS and adiponectin, and DNA methylation of PPAR γ, were assayed in both the offspring and grand-offspring adipose tissue.Offspring of dams exposed to greater PAH during gestation had increased weight, fat mass, as well as higher gene expression of PPAR γ, C/EBP α, Cox2, FAS and adiponectin and lower DNA methylation of PPAR γ. Similar differences in phenotype and DNA methylation extended through the grand-offspring mice.Greater prenatal PAH exposure was associated with increased weight, fat mass, adipose gene expression and epigenetic changes in progeny.

Methylation-Specific PCR Unraveled

Directory of Open Access Journals (Sweden)

Sarah Derks

2004-01-01

Full Text Available Methylation‐specific PCR (MSP is a simple, quick and cost‐effective method to analyze the DNA methylation status of virtually any group of CpG sites within a CpG island. The technique comprises two parts: (1 sodium bisulfite conversion of unmethylated cytosine's to uracil under conditions whereby methylated cytosines remains unchanged and (2 detection of the bisulfite induced sequence differences by PCR using specific primer sets for both unmethylated and methylated DNA. This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical applications.

Synthesis of 14C-labelled α-methyl tyrosine

International Nuclear Information System (INIS)

Rajagopal, S.; Venkatachalam, T.K.; Conway, T.; Diksic, M.

1992-01-01

A new route for the preparation of radioactively labelled α-methyl L-tyrosine is described. The labelling at the α position has been successfully achieved with 14 C-, 11 C- (very preliminary, unpublished), and 3 H-labelled methyl iodide. A detailed report on 14 C-labelling at the α position and the hydrolysis of 4-methoxy α-methyl phenylalanine is presented. The alkylation proceeds via the methylation of the carbanion of N-benzylidene 4-methoxy phenylalanine methyl ester 2. Hydrolysis of 4-O methyl tyrosine to tyrosine by HBr and HI were analysed and used in the optimization of the hydrolysis conditions of 4. Enantiomeric purity of the isolated L-isomer has been found to be 99% as judged by HPLC. Pseudo first-order rate constant for the hydrolysis of 14 C-labelled α-methyl 4-methoxy phenyl alanine methyl ester was determined. Preliminary findings of the 3 H- and 11 C-radiolabelled α-methyl tyrosine (methyl labelled) are also mentioned. For the first time it was shown that α-methyl D,L-tyrosine can be separated into enantiomerically pure α-methyl D- and L-tyrosine using a CHIRALPAK WH column. (author)

Liberation of methyl acrylate from metallalactone complexes via M-O ring opening (M = Ni, Pd) with methylation agents

KAUST Repository

Lee, S. Y Tina; Ghani, Amylia Abdul; D'Elia, Valerio; Cokoja, Mirza; Herrmann, Wolfgang A.; Basset, Jean-Marie; Kü hn, Fritz

2013-01-01

Ring opening of various nickela- and palladalactones induced by the cleavage of the M-O bond by methyl trifluoromethanesulfonate (MeOTf) and methyl iodide (MeI) is examined. Experimental evidence supports the mechanism of ring opening by the alkylating agent followed by β-H elimination leading to methyl acrylate and a metal-hydride species. MeOTf shows by far higher efficiency in the lactone ring opening than any other methylating agent including the previously reported methyl iodide. © 2013 The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

Methylation pathways in schizophrenia

International Nuclear Information System (INIS)

Sargent, T.W. III.

1982-01-01

Research on the biochemical causes of human psychosis concentrates on investigating whether schizophremia is linked to abnormalities in the metabolism of methyl carbon groups in the body. The metabolism of C-14 labeled methyl groups in methionine is studied in animals, normal subjects and patient volunteers

Simultaneous Determination of Salicylic Acid, Jasmonic Acid, Methyl Salicylate, and Methyl Jasmonate from Ulmus pumila Leaves by GC-MS

Directory of Open Access Journals (Sweden)

Zhi-hong Huang

2015-01-01

Full Text Available Salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate are important phytohormones and defensive signaling compounds, so it is of great importance to determine their levels rapidly and accurately. The study uses Ulmus pumila leaves infected by Tetraneura akinire Sasaki at different stages as materials; after extraction with 80% methanol and ethyl acetate and purification with primary secondary amine (PSA and graphitized carbon blacks (GCB, the contents of signal compounds salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate were determined by GC-MS. The results showed that the level of salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate increased remarkably in U. pumila once infected by T. akinire Sasaki, but the maximums of these four compounds occurred at different times. Salicylic acid level reached the highest at the early stage, and jasmonic acid level went to the maximum in the middle stage; by contrast, change of content of methyl salicylate and methyl jasmonate was the quite opposite.

Simultaneous Determination of Salicylic Acid, Jasmonic Acid, Methyl Salicylate, and Methyl Jasmonate from Ulmus pumila Leaves by GC-MS.

Science.gov (United States)

Huang, Zhi-Hong; Wang, Zhi-Li; Shi, Bao-Lin; Wei, Dong; Chen, Jian-Xin; Wang, Su-Li; Gao, Bao-Jia

2015-01-01

Salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate are important phytohormones and defensive signaling compounds, so it is of great importance to determine their levels rapidly and accurately. The study uses Ulmus pumila leaves infected by Tetraneura akinire Sasaki at different stages as materials; after extraction with 80% methanol and ethyl acetate and purification with primary secondary amine (PSA) and graphitized carbon blacks (GCB), the contents of signal compounds salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate were determined by GC-MS. The results showed that the level of salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate increased remarkably in U. pumila once infected by T. akinire Sasaki, but the maximums of these four compounds occurred at different times. Salicylic acid level reached the highest at the early stage, and jasmonic acid level went to the maximum in the middle stage; by contrast, change of content of methyl salicylate and methyl jasmonate was the quite opposite.

Synthesis of DL-adrenaline (methyl C{sup 14}) (1961); Synthese de la DL-adrenaline (methyle {sup 14}C) (1961)

Energy Technology Data Exchange (ETDEWEB)

Pichat, L; Audinot, M [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

1961-07-01

The sodium derivative of 5-3-4 dibenzyl oxyphenyl 2-oxazolidinone reacted with methyl iodide {sup 14}C, in stoichiometric quantity, gives rise to the corresponding N-methyl {sup 14}C derivative. The oxazolidinone ring is opened by concentrated hydrochloric acid and the benzyl groups removed by catalytic hydrogenolysis. Adrenaline methyl {sup 14}C is then purified on Dowex 50 X-12 exchange resin. Overall-yield is 45 per cent based upon methyl iodide {sup 14}C. (author) [French] Le derive sode de la (dibenzyloxy-3-4-phenyl)-5 oxazolidinone-2 traite par l'iodure de methyle {sup 14}C, en proportion stoechiometrique, fournit le derive N-methyle {sup 14}C correspondant. Apres ouverture du cycle oxazolidinone par HCL concentre et debenzylation par hydrogenation catalytique, on purifie l'adrenaline (methyle {sup 14}C) par chromatographie sur resine echangeuse Dowex 50 X-12. Le rendement est de 45 pour cent par rapport a l'iodure de methyle {sup 14}C. (auteurs)

Synthesis of DL-adrenaline (methyl C{sup 14}) (1961); Synthese de la DL-adrenaline (methyle {sup 14}C) (1961)

Energy Technology Data Exchange (ETDEWEB)

Pichat, L.; Audinot, M. [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

1961-07-01

The sodium derivative of 5-3-4 dibenzyl oxyphenyl 2-oxazolidinone reacted with methyl iodide {sup 14}C, in stoichiometric quantity, gives rise to the corresponding N-methyl {sup 14}C derivative. The oxazolidinone ring is opened by concentrated hydrochloric acid and the benzyl groups removed by catalytic hydrogenolysis. Adrenaline methyl {sup 14}C is then purified on Dowex 50 X-12 exchange resin. Overall-yield is 45 per cent based upon methyl iodide {sup 14}C. (author) [French] Le derive sode de la (dibenzyloxy-3-4-phenyl)-5 oxazolidinone-2 traite par l'iodure de methyle {sup 14}C, en proportion stoechiometrique, fournit le derive N-methyle {sup 14}C correspondant. Apres ouverture du cycle oxazolidinone par HCL concentre et debenzylation par hydrogenation catalytique, on purifie l'adrenaline (methyle {sup 14}C) par chromatographie sur resine echangeuse Dowex 50 X-12. Le rendement est de 45 pour cent par rapport a l'iodure de methyle {sup 14}C. (auteurs)

Transgenerational epigenetics: Inheritance of global cytosine methylation and methylation-related epigenetic markers in the shrub Lavandula latifolia.

Science.gov (United States)

Herrera, Carlos M; Alonso, Conchita; Medrano, Mónica; Pérez, Ricardo; Bazaga, Pilar

2018-04-01

The ecological and evolutionary significance of natural epigenetic variation (i.e., not based on DNA sequence variants) variation will depend critically on whether epigenetic states are transmitted from parents to offspring, but little is known on epigenetic inheritance in nonmodel plants. We present a quantitative analysis of transgenerational transmission of global DNA cytosine methylation (= proportion of all genomic cytosines that are methylated) and individual epigenetic markers (= methylation status of anonymous MSAP markers) in the shrub Lavandula latifolia. Methods based on parent-offspring correlations and parental variance component estimation were applied to epigenetic features of field-growing plants ('maternal parents') and greenhouse-grown progenies. Transmission of genetic markers (AFLP) was also assessed for reference. Maternal parents differed significantly in global DNA cytosine methylation (range = 21.7-36.7%). Greenhouse-grown maternal families differed significantly in global methylation, and their differences were significantly related to maternal origin. Methylation-sensitive amplified polymorphism (MSAP) markers exhibited significant transgenerational transmission, as denoted by significant maternal variance component of marker scores in greenhouse families and significant mother-offspring correlations of marker scores. Although transmission-related measurements for global methylation and MSAP markers were quantitatively lower than those for AFLP markers taken as reference, this study has revealed extensive transgenerational transmission of genome-wide global cytosine methylation and anonymous epigenetic markers in L. latifolia. Similarity of results for global cytosine methylation and epigenetic markers lends robustness to this conclusion, and stresses the value of considering both types of information in epigenetic studies of nonmodel plants. © 2018 Botanical Society of America.

Analysis of DNA methylation of perennial ryegrass under drought using the methylation-sensitive amplification polymorphism (MSAP) technique.

Science.gov (United States)

Tang, Xiao-Mei; Tao, Xiang; Wang, Yan; Ma, Dong-Wei; Li, Dan; Yang, Hong; Ma, Xin-Rong

2014-12-01

Perennial ryegrass (Lolium perenne), an excellent grass for forage and turf, is widespread in temperate regions. Drought is an important factor that limits its growth, distribution, and yield. DNA methylation affects gene expression and plays an important role in adaptation to adverse environments. In this study, the DNA methylation changes in perennial ryegrass under drought stress were assessed using methylation-sensitive amplified polymorphism (MSAP). After 15 days of drought stress treatment, the plant height was less than half of the control, and the leaves were smaller and darker. Genome-wide, a total of 652 CCGG sites were detected by MSAP. The total methylation level was 57.67 and 47.39 % in the control and drought treatment, respectively, indicating a decrease of 10.28 % due to drought exposure. Fifteen differentially displayed DNA fragments in MSAP profiles were cloned for sequencing analysis. The results showed that most of the genes involved in stress responses. The relative expression levels revealed that three demethylated fragments were up-regulated. The expression of a predicted retrotransposon increased significantly, changing from hypermethylation to non-methylation. Although the extent of methylation in two other genes decreased, the sites of methylation remained, and the expression increased only slightly. All of these results suggested that drought stress decreased the total DNA methylation level in perennial ryegrass and demethylation up-regulated related gene expressions and that the extent of methylation was negatively correlated with expression. Overall, the induced epigenetic changes in genome probably are an important regulatory mechanism for acclimating perennial ryegrass to drought and possibly other environmental stresses.

Development of an ELISA assay for screening inhibitors against divalent metal ion dependent alphavirus capping enzyme.

Science.gov (United States)

Kaur, Ramanjit; Mudgal, Rajat; Narwal, Manju; Tomar, Shailly

2018-06-26

Alphavirus non-structural protein, nsP1 has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. Thus, alphavirus capping enzyme nsP1 is a potential drug target. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine (SAM) to a GTP molecule at its N7 position with the help of nsP1 methyltransferase (MTase) followed by guanylylation (GT) reaction which involves the formation of m 7 GMP-nsP1 covalent complex by nsP1 guanylyltransferase (GTase). In subsequent reactions, m 7 GMP moiety is added to the 5' end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. In the present study, chikungunya virus (CHIKV) nsP1 MTase and GT reactions were confirmed by an indirect non-radioactive colorimetric assay and western blot assay using an antibody specific for the m 7 G cap, respectively. The purified recombinant CHIKV nsP1 has been used for the development of a rapid and sensitive non-radioactive enzyme linked immunosorbent assay (ELISA) to identify the inhibitors of CHIKV nsP1. The MTase reaction is followed by GT reaction and resulted in m 7 GMP-nsP1 covalent complex formation. The developed ELISA nsP1 assay measures this m 7 GMP-nsP1 complex by utilizing anti-m 7 G cap monoclonal antibody. The mutation of a conserved residue Asp63 to Ala revealed its role in nsP1 enzyme reaction. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to determine the presence of magnesium ions (Mg 2+ ) in the purified nsP1 protein. The divalent metal ion selectivity and investigation show preference for Mg 2+ ion by CHIKV nsP1. Additionally, using the developed ELISA nsP1 assay, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin were determined and the IC 50 values were estimated to be 2.69 µM, 5.72 µM and 1.18 mM, respectively. Copyright © 2018. Published by Elsevier B.V.

Microwave-assisted pyrolysis of methyl ricinoleate for continuous production of undecylenic acid methyl ester (UAME).

Science.gov (United States)

Nie, Yong; Duan, Ying; Gong, Ruchao; Yu, Shangzhi; Lu, Meizhen; Yu, Fengwen; Ji, Jianbing

2015-06-01

Undecylenic acid methyl ester (UAME) was continuously produced from methyl ricinoleate using a microwave-assisted pyrolysis system with atomization feeding. The UAME yield of 77 wt.% was obtained at 500°C using SiC as the microwave absorbent and heating medium. The methyl ricinoleate conversion and UAME yield from microwave-assisted pyrolysis process were higher than those from conventional pyrolysis. The effect of temperature on the pyrolysis process was also investigated. The methyl ricinoleate conversion increased but the cracking liquid yield decreased when the temperature increased from 460°C to 560°C. The maximum UAME yield was obtained at the temperature of 500°C. Copyright © 2015 Elsevier Ltd. All rights reserved.

Delayed O-methylation of l-DOPA in MB-COMT-deficient mice after oral administration of l-DOPA and carbidopa.

Science.gov (United States)

Tammimäki, Anne; Aonurm-Helm, Anu; Männistö, Pekka T

2018-04-01

1. Catechol-O-methyltransferase (COMT) is involved in the O-methylation of l-DOPA, dopamine, and other catechols. The enzyme is expressed in two isoforms: soluble (S-COMT), which resides in the cytoplasm, and membrane-bound (MB-COMT), which is anchored to intracellular membranes. 2. To obtain specific information on the functions of COMT isoforms, we studied how a complete MB-COMT deficiency affects the total COMT activity in the body, peripheral l-DOPA levels, and metabolism after l-DOPA (10 mg kg -1 ) plus carbidopa (30 mg kg -1 ) administration by gastric tube in wild-type (WT) and MB-COMT-deficient mice. l-DOPA and 3-O-methyl-l-DOPA (3-OMD) levels were assayed in plasma, duodenum, and liver. 3. We showed that the selective lack of MB-COMT did not alter the total COMT activity, COMT enzyme kinetics, l-DOPA levels, or the total O-methylation of l-DOPA but delayed production of 3-OMD in plasma and peripheral tissues.

Quantitative DNA methylation analyses reveal stage dependent DNA methylation and association to clinico-pathological factors in breast tumors

International Nuclear Information System (INIS)

Klajic, Jovana; Tost, Jörg; Kristensen, Vessela N; Fleischer, Thomas; Dejeux, Emelyne; Edvardsen, Hege; Warnberg, Fredrik; Bukholm, Ida; Lønning, Per Eystein; Solvang, Hiroko; Børresen-Dale, Anne-Lise

2013-01-01

Aberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression. Quantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV. Significant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival. In the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

Science.gov (United States)

Volpi, Chiara; Janni, Michela; Lionetti, Vincenzo; Bellincampi, Daniela; Favaron, Francesco; D'Ovidio, Renato

2011-09-01

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

Androgenic effect of honeybee drone milk in castrated rats: roles of methyl palmitate and methyl oleate.

Science.gov (United States)

Seres, A B; Ducza, E; Báthori, M; Hunyadi, A; Béni, Z; Dékány, M; Hajagos-Tóth, J; Verli, J; Gáspár, Róbert

2014-04-28

Numerous honeybee (Apis mellifera) products have been used in traditional medicine to treat infertility and to increase vitality in both men and women. Drone milk (DM) is a relatively little-known honeybee product with a putative sexual hormone effect. The oestrogenic effect of a fraction of DM has recently been reported in rats. However, no information is available on the androgenic effects of DM. The purpose of the present study was to determine the androgen-like effect of DM in male rats and to identify effective compounds. A modified Hershberger assay was used to investigate the androgenic effect of crude DM, and the plasma level of testosterone was measured. The prostatic mRNA and protein expression of Spot14-like androgen-inducible protein (SLAP) were also examined with real-time PCR and Western blot techniques. GC-MS and NMR spectroscopic investigations were performed to identify the active components gained by bioactivity-guided fractionation. The crude DM increased the relative weights of the androgen-dependent organs and the plasma testosterone level in castrated rats and these actions were flutamide-sensitive. DM increased the tissue mRNA and protein level of SLAP, providing further evidence of its androgen-like character. After bioactivity-guided fractionation, two fatty acid esters, methyl palmitate (MP) and methyl oleate (MO), were identified as active compounds. MP alone showed an androgenic effect, whereas MO increased the weight of androgen-sensitive tissues and the plasma testosterone level only in combination. The experimental data of DM and its active compounds (MO and MP) show androgenic activity confirming the traditional usage of DM. DM or MP or/and MO treatments may project a natural mode for the therapy of male infertility. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

DNA methylation and transcriptomic changes in response to different lights and stresses in 7B-1 male-sterile tomato.

Directory of Open Access Journals (Sweden)

Vahid Omidvar

Full Text Available We reported earlier that 7B-1 mutant in tomato (Solanum lycopersicum L., cv. Rutgers, an ABA overproducer, is defective in blue light (B signaling leading to B-specific resistance to abiotic and biotic stresses. Using a methylation-sensitive amplified polymorphism (MSAP assay, a number of genes were identified, which were differentially methylated between 7B-1 and its wild type (WT seedlings in white (W, blue (B, red (R lights and dark (D or in response to exogenous ABA and mannitol-induced stresses. The genomic methylation level was almost similar in different lights between 7B-1 and WT seedlings, while significant differences were observed in response to stresses in D, but not B. Using a cDNA-AFLP assay, several transcripts were identified, which were differentially regulated between 7B-1 and WT by B or D or in response to stresses. Blue light receptors cryptochrome 1 and 2 (CRY1 and CRY2 and phototropin 1 and 2 (PHOT1 and PHOT2 were not affected by the 7B-1 mutation at the transcriptional level, instead the mutation had likely affected downstream components of the light signaling pathway. 5-azacytidine (5-azaC induced DNA hypomethylation, inhibited stem elongation and differentially regulated the expression of a number of genes in 7B-1. In addition, it was shown that mir167 and mir390 were tightly linked to auxin signaling pathway in 5-azaC-treated 7B-1 seedlings via the regulation of auxin-response factor (ARF transcripts. Our data showed that DNA methylation remodeling is an active epigenetic response to different lights and stresses in 7B-1 and WT, and highlighted the differences in epigenetic and transcriptional regulation of light and stress responses between 7B-1 and WT. Furthermore, it shed lights on the crosstalk between DNA hypomethylation and miRNA regulation of ARFs expression. This information could also be used as a benchmark for future studies of male-sterility in other crops.

Effects of wildlife of ethyl and methyl parathion applied to California USA rice fields

Science.gov (United States)

Custer, T.W.; Hill, E.F.; Ohlendorf, H.M.

1985-01-01

Selected rice fields on the Sacramento National Wildlife Refuge Complex were aerially sprayed one time during May or June 1982 with either ethyl (0.11 kg Al/ha) or methyl (0.84 kg AI/ha) parathion for control of tadpole shrimp, Triops longicaudatus. No sick or dead vertebrate wildlife were found or adjacent to the treated rice fields after spraying. Specimens of the following birds and mammals were assayed for brain cholinesterase (ChE) activity to determine exposure to either form of parathion; house mouse, Mus musculus; black-tailed jackrabbit, Lepus californicus; mallard, Anas platyrhynchos; ring-necked pheasant, Phasianus colchicus; American coot, Fulica americana; and red-winged blackbird, Agelaius phoeniceus. Both mice and pheasants from methyl parathion-treated fields had overall mean ChE activities that were significantly (P < 0.05) inhibited compared with controls, and 7, 40, 54 and 57% of individual blackbirds, pheasant, mice, and coots, respectively, had inhibited brain ChE activities (i.e., less than -2 SD of control mean). Although no overall species effect was detected for ethyl parathoid treatment, pheasants (43%), coots (33%), and mice (37%) had significantly inhibited brain ChE activities. Neither of the parathion treatment appeared acutely hazardous to wildlife in or adjacent to rice fields, but sufficient information on potential hazards was obtained to warrant caution in use of these chemicals, especially methyl parathion, in rice fields.

Cytosine methylation at CpCpG sites triggers accumulation of non-CpG methylation in gene bodies

OpenAIRE

Zabet, NR; Catoni, Marco; Prischi, F; Paszkowski, Jerzy Waclaw

2017-01-01

Methylation of cytosine is an epigenetic mark involved in the regulation of transcription, usually associated with transcriptional repression. In mammals, methylated cytosines are found predominantly in CpGs but in plants non-CpG methylation (in the CpHpG or CpHpH contexts, where H is A, C or T) is also present and is associated with the transcriptional silencing of transposable elements. In addition, CpG methylation is found in coding regions of active genes. In the absence of the demethylas...

miRNAting control of DNA methylation

Indian Academy of Sciences (India)

DNA methylation is a type of epigenetic modification where a methyl group is added to the cytosine or adenine residue of a given DNA sequence. It has been observed that DNA methylation is achieved by some collaborative agglomeration of certain proteins and non-coding RNAs. The assembly of IDN2 and its ...

Analysis of DNA Methylation of Gracilariopsis lemaneiformis Under Temperature Stress Using the Methylation Sensitive Amplification Polymorphism (MSAP) Technique

Science.gov (United States)

Peng, Chong; Sui, Zhenghong; Zhou, Wei; Hu, Yiyi; Mi, Ping; Jiang, Minjie; Li, Xiaodong; Ruan, Xudong

2018-06-01

Gracilariopsis lemaneiformis is an economically important agarophyte, which contains high quality gel and shows a high growth rate. Wild population of G. lemaneiformis displayed resident divergence, though with a low genetic diversity as was revealed by amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses. In addition, different strains of G. lemaneiformis are diverse in morphology. The highly inconsistence between genetic background and physiological characteristics recommends strongly to the regulation at epigenetic level. In this study, the DNA methylation change in G. lemaneiformis among different generation branches and under different temperature stresses was assessed using methylation sensitive amplified polymorphism (MSAP) technique. It was shown that DNA methylation level among different generation branches was diverse. The full and total methylated DNA level was the lowest in the second generation branch and the highest in the third generation. The total methylation level was 61.11%, 60.88% and 64.12% at 15°C, 22°C and 26°C, respectively. Compared with the control group (22°C), the fully methylated and totally methylated ratios were increased in both experiment groups (15°C and 26°C). All of the cytosine methylation/demethylation transform (CMDT) was further analyzed. High temperature treatment could induce more CMDT than low temperature treatment did.

Maternal vitamin D deficiency during pregnancy results in insulin resistance in rat offspring, which is associated with inflammation and Iκbα methylation.

Science.gov (United States)

Zhang, Huaqi; Chu, Xia; Huang, Yifan; Li, Gang; Wang, Yuxia; Li, Ying; Sun, Changhao

2014-10-01

We aimed to investigate the impact of maternal vitamin D deficiency during pregnancy on insulin resistance in male offspring and examine its mechanism. Pregnant Sprague-Dawley rats were maintained on a vitamin-D-free diet with ultraviolet-free light during pregnancy (early-VDD group). Insulin resistance in the male offspring was assessed by HOMA-IR, OGTT and euglycaemic clamp. NEFA, oxidative stress and inflammation levels were estimated as risk factors for insulin resistance. DNA methylation was examined by bisulfate sequencing PCR analysis. Luciferase reporter assay was performed to validate the effect of DNA methylation. The offspring in the early-VDD group had significantly higher fasting insulin and HOMA-IR levels, markedly reduced glucose tolerance and significantly lower tissue sensitivity to exogenous insulin at 16 weeks (all p insulin resistance in the offspring, which is associated with persistently increased inflammation. Persistently decreased Iκbα expression, potentially caused by changes in Iκbα methylation, plays an important role in persistent inflammation.

Novel double-isotope technique for enzymatic assay of catecholamines, permitting high precision, sensitivity and plasma sample capacity

International Nuclear Information System (INIS)

Brown, M.J.; Jenner, D.A.

1981-01-01

A novel use of a double-isotope method is described which allows radioenzymatic assays to combine precision and sensitivity. In the catechol O-methyltransferase assay separate portions of each plasma sample are incubated with either S-[ 3 H]- or S-[ 14 C]-adenosyl-L-methionine. Standards of noradrenaline and adrenaline are added to the latter portions and are thus converted into standards of [ 14 C]metadrenalines. These are added to the 3 H-labelled portions after the incubation, where they function as tracers. The final recovery of 14 C radioactivity corrects for (a) the efficiency of methylation in the plasma sample concerned and (b) the recovery of metadrenalines during the extraction procedures. The 3 H/ 14 C ratio is constant in each assay for a given catecholamine concentration and is determined for samples to which standards of noradrenaline and adrenaline are added to the 3 H- (as well as the 14 C-) labelled portions before the initial incubation. The sensitivity of the assay is increased by using high specific radioactivity S-[ 3 H]adenosyl-L-methionine, and low backgrounds are maintained by catecholamine depletion in vivo in the rats used for enzyme preparation. Both catecholamines (1.5 pg/ml; 10 pmol/l) may be detected; the coefficients of variation are 3.0 and 3.2% for noradrenaline and adrenaline respectively (intra-assay) and 4.6 and 5.0% (inter-assay). (author)

Colorectal Cancer "Methylator Phenotype": Fact or Artifact?

Directory of Open Access Journals (Sweden)

Charles Anacleto

2005-04-01

Full Text Available It has been proposed that human colorectal tumors can be classified into two groups: one in which methylation is rare, and another with methylation of several loci associated with a "CpG island methylated phenotype (CIMP," characterized by preferential proximal location in the colon, but otherwise poorly defined. There is considerable overlap between this putative methylator phenotype and the well-known mutator phenotype associated with microsatellite instability (MSI. We have examined hypermethylation of the promoter region of five genes (DAPK, MGMT, hMLH1, p16INK4a, and p14ARF in 106 primary colorectal cancers. A graph depicting the frequency of methylated loci in the series of tumors showed a continuous, monotonically decreasing distribution quite different from the previously claimed discontinuity. We observed a significant association between the presence of three or more methylated loci and the proximal location of the tumors. However, if we remove from analysis the tumors with hMLH1 methylation or those with MSI, the significance vanishes, suggesting that the association between multiple methylations and proximal location was indirect due to the correlation with MSI. Thus, our data do not support the independent existence of the so-called methylator phenotype and suggest that it rather may represent a statistical artifact caused by confounding of associations.

Synthesis of N-methyl and N-11C-methyl spiperone by phase transfer catalysis in anhydrous solvent

International Nuclear Information System (INIS)

Omokawa, Hiroyoshi; Tanaka, Akira; Iio, Mayumi; Nishihara, Yoshiaki; Inoue, Osamu; Yamazaki, Toshio.

1985-01-01

Spiperone, a butyrophenone neuroleptic drug, has been used in binding studies of dopamine receptors. Langstrom et al. developed N- 11 C-methyl spiperone, and, in cooperate with Wagner et al., made it possible to visualize the distribution of dopamine receptors in the human brain in vivo. In this paper, we independently developed another synthetic method of N- 11 C-methyl spiperone using the phase transfer catalyst in an anhydrous solvent. Separation of the product is feasible only by passing the reactant solution through a Millipore filter and injecting it onto high pressure liquid chromatography (HPLC). The time required for the synthesis and purification of N- 11 C-methyl spiperone from 11 C-methyl iodide and spiperone was 20 min. Radiochemical yield exceeded 35 % against 11 C-methyl iodide without correcting decay of the radioactivity. (author)

[Specific features of 2-methyl hydroxybenzene and 3-methyl hydroxybenzene distribution in the organism of warm-blooded animals].

Science.gov (United States)

Shormanov, B K; Grishenko, V K; Astashkina, A P; Elizarova, M K

2013-01-01

The present work was designed to study the specific features of 2-methyl hydroxybezene and 3-methyl hydroxybenzene distribution after intragastric administration of these toxicants to warm-blooded animals (rats). They were detected in the unmetabolized form in the internal organs and blood of the animals. The levels of 2-methyl hydroxybezene were especially high in the stomach and blood whereas the maximum content of 3-methyl hydroxybenzene was found in brain, blood, small intestines of the poisoned rats.

Genome-wide DNA methylation maps in follicular lymphoma cells determined by methylation-enriched bisulfite sequencing.

Directory of Open Access Journals (Sweden)

Jeong-Hyeon Choi

Full Text Available BACKGROUND: Follicular lymphoma (FL is a form of non-Hodgkin's lymphoma (NHL that arises from germinal center (GC B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. METHODOLOGY/PRINCIPAL FINDINGS: We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+ B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+ B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+ B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. CONCLUSIONS/SIGNIFICANCE: This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.

Modeling spatiotemporal dynamics of DNA methylation

DEFF Research Database (Denmark)

Lövkvist, Cecilia Elisabet

into how epigenetic marks are distributed in the human genome. In the first part of the thesis, we investigate DNA methylation and maintenance of methylation patterns throughout cell division. We argue that collaborative models, those where the methylation of CpG sites depends on the methylation status...... into the game more explicitly in another type of model that speaks out the duality of the two aspects. Using statistical analysis of experimental data, this thesis further explores a link between DNA methylation and nucleosome occupancy. By comparing the patterns on promoters to regions with similar Cp...... division. The patterns of epigentic marks depend on enzymes that ensure their maintenance and introduction. Using theoretical models, this thesis proposes new mechanisms for how enzymes operate to maintain patterns of epigenetic marks. Through analysis of experimental data this work gives new insight...

Experimental and chemical kinetic modeling study of small methyl esters oxidation: Methyl (E)-2-butenoate and methyl butanoate

Energy Technology Data Exchange (ETDEWEB)

Gail, S.; Sarathy, S.M.; Thomson, M.J. [Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, ON M5S 3G8 (Canada); Dievart, P.; Dagaut, P. [CNRS, 1C, Ave de la Recherche Scientifique, 45071 Orleans Cedex 2 (France)

2008-12-15

This study examines the effect of unsaturation on the combustion of fatty acid methyl esters (FAME). New experimental results were obtained for the oxidation of methyl (E)-2-butenoate (MC, unsaturated C{sub 4} FAME) and methyl butanoate (MB, saturated C{sub 4} FAME) in a jet-stirred reactor (JSR) at atmospheric pressure under dilute conditions over the temperature range 850-1400 K, and two equivalence ratios ({phi}=0.375,0.75) with a residence time of 0.07 s. The results consist of concentration profiles of the reactants, stable intermediates, and final products, measured by probe sampling followed by on-line and off-line gas chromatography analyses. The oxidation of MC and MB in the JSR and under counterflow diffusion flame conditions was modeled using a new detailed chemical kinetic reaction mechanism (301 species and 1516 reactions) derived from previous schemes proposed in the literature. The laminar counterflow flame and JSR (for {phi}=1.13) experimental results used were from a previous study on the comparison of the combustion of both compounds. Sensitivity analyses and reaction path analyses, based on rates of reaction, were used to interpret the results. The data and the model show that MC has reaction pathways analogous to that of MB under the present conditions. The model of MC oxidation provides a better understanding of the effect of the ester function on combustion, and the effect of unsaturation on the combustion of fatty acid methyl ester compounds typically found in biodiesel. (author)

Methyl and p-Bromobenzyl Esters of Hydrogenated Kaurenoic Acid for Controlling Anthracnose in Common Bean Plants.

Science.gov (United States)

Mota, Suellen F; Oliveira, Denilson F; Heleno, Vladimir C G; Soares, Ana Carolina F; Midiwo, Jacob O; Souza, Elaine A

2017-03-01

Kaurenoic acid derivatives were prepared and submitted to in vitro assays with the fungus Colletotrichum lindemuthianum, which causes anthracnose disease in the common bean. The most active substances were found to be methyl and p-bromobenzylesters, 7 and 9, respectively, of the hydrogenated kaurenoic acid, which presented a minimum inhibitory concentration (MIC) of 0.097 and 0.131 mM, respectively, while the commercial fungicide methyl thiophanate (MT) presented a MIC of 0.143 mM. Substances 7 (1.401 mM) and 9 (1.886 mM) reduced the severity of anthracnose in common bean to values statistically comparable to MT (2.044 mM). According to an in silico study, both compounds 7 and 9 are inhibitors of the ketosteroid isomerase (KSI) enzyme produced by other organisms, the amino acid sequence of which could be detected in fungal genomes. These substances appeared to act against C. lindemuthianum by inhibiting its KSI. Therefore, substances 7 and 9 are promising for the development of new fungicides.

Naturally occurring methyl salicylate glycosides.

Science.gov (United States)

Mao, Ping; Liu, Zizhen; Xie, Meng; Jiang, Rui; Liu, Weirui; Wang, Xiaohong; Meng, Shen; She, Gaimei

2014-01-01

As an important part of non steroids anti-inflammation drug (NSAIDs), salicylate has developed from natural substance salicylic acid to natrium salicylicum, to aspirin. Now, methyl salicylate glycoside, a new derivative of salicylic acid, is modified with a -COOH group integrated one methyl radical into formic ether, and a -OH linked with a monosaccharide, a disaccharide or a trisaccharide unit by glycosidic linkage. It has the similar pharmacological activities, anti-inflammatory, analgesic, antipyretic and antithrombotic as the previous salicylates' without resulting in serious side effects, particularly the gastrointestinal toxicity. Owing to the superiority of those significant bioactivities, methyl salicylate glycosides have became a hot research area in NSAIDs for several years. This paper compiles all 9 naturally occurring methyl salicylate glycosides, their distribution of the resource and pharmacological mechanism, which could contribute to the new drug discovery.

[Association of etheno-DNA adduct and DNA methylation level among workers exposed to diesel engine exhaust].

Science.gov (United States)

Shen, M L; He, Z N; Zhang, X; Duan, H W; Niu, Y; Bin, P; Ye, M; Meng, T; Dai, Y F; Yu, S F; Chen, W; Zheng, Y X

2017-06-06

Objective: To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A (P16), Ras association domain family 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in workers with occupational exposure to diesel engine exhaust (DEE). Methods: We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2'-deoxyadenosine (εdA) and 3,N4-etheno-2'-deoxycytidine(εdC). The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine (5mC) over the sum of cytosines (%5mC). Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT. Results: The median ( P (25)- P (75)) of urinary εdA level was 230.00 (98.04-470.91) pmol/g creatinine in DEE exposure group, and 102.10 (49.95-194.48) creatinine in control group. The level of εdA was higher in DEE exposure group than control group ( P 0.05) . Multiple linear regression confirmed the negative correlation between εdA and DNA methylation levels of P16, RASSF1A, and MGMT in non-smoking group (β (95 %CI ) was -0.068 (-0.132--0.003), -0.082 (-0.159--0.004) and -0.048 (-0.090--0.007), P values were 0.039, 0.039 and 0.024, respectively). Moreover, εdC was negative associated with DNA methylation level of MGMT in non-smoking group (β (95 %CI ) was -0.094 (-0.179--0.008), P= 0.032). Conclusion: DEE exposure could induce the increased of εdA and decreased of DNA methylation levels of P16, RASSF1A

Protein methylation reactions in intact pea chloroplasts

International Nuclear Information System (INIS)

Niemi, K.J.

1989-01-01

Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ( 3 H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented

UVB irradiation does not directly induce detectable changes of DNA methylation in human keratinocytes [v1; ref status: indexed, http://f1000r.es/np

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Christoph Lahtz

2013-02-01

Full Text Available Unprotected exposure to UVB radiation from the sun and the resulting DNA damage are thought to be responsible for physiological changes in the skin and for a variety of skin cancers, including basal cell and squamous cell carcinoma and malignant melanoma. Although the mutagenic effects of UVB have been well documented and studied mechanistically, there is only limited information as to whether UV light may also be responsible for inducing epigenetic changes in the genome of exposed cells. DNA methylation is a stable epigenetic modification involved in gene control. To study the effects of UVB radiation on DNA methylation, we repeatedly exposed normal human keratinocytes to a UVB light source. After a recovery period, we analyzed global DNA methylation patterns in the irradiated and control cells using the methylated-CpG island recovery assay (MIRA method in combination with high-resolution microarrays. Bioinformatics analysis revealed only a limited number of possible differences between UVB-exposed and control cells. However, these minor apparent changes could not be independently confirmed by bisulfite sequencing-based approaches. This study reveals that UVB irradiation of keratinocytes has no recognizable global effect on DNA methylation patterns and suggests that changes in DNA methylation, as observed in skin cancers, are not immediate consequences of human exposure to solar UVB irradiation.

Molecular correlates with MGMT promoter methylation and silencing support CpG island methylator phenotype-low (CIMP-low) in colorectal cancer.

Science.gov (United States)

Ogino, Shuji; Kawasaki, Takako; Kirkner, Gregory J; Suemoto, Yuko; Meyerhardt, Jeffrey A; Fuchs, Charles S

2007-11-01

The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer. In contrast, a phenotype with less widespread promoter methylation (CIMP-low) has not been well characterised. O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and silencing have been associated with G>A mutations and microsatellite instability-low (MSI-low). To examine molecular correlates with MGMT methylation/silencing in colorectal cancer. Utilising MethyLight technology, we quantified DNA methylation in MGMT and eight other markers (a CIMP-diagnostic panel; CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) in 920 population-based colorectal cancers. Tumours with both MGMT methylation and loss were correlated positively with MSI-low (p = 0.02), CIMP-high (>or=6/8 methylated CIMP markers, p = 0.005), CIMP-low (1/8-5/8 methylated CIMP markers, p = 0.002, compared to CIMP-0 with 0/8 methylated markers), KRAS G>A mutation (p = 0.02), and inversely with 18q loss of heterozygosity (p = 0.0002). Tumours were classified into nine MSI/CIMP subtypes. Among the CIMP-low group, tumours with both MGMT methylation and loss were far more frequent in MSI-low tumours (67%, 12/18) than MSI-high tumours (5.6%, 1/18; p = 0.0003) and microsatellite stable (MSS) tumours (33%, 52/160; p = 0.008). However, no such relationship was observed among the CIMP-high or CIMP-0 groups. The relationship between MGMT methylation/silencing and MSI-low is limited to only CIMP-low tumours, supporting the suggestion that CIMP-low in colorectal cancer may be a different molecular phenotype from CIMP-high and CIMP-0. Our data support a molecular difference between MSI-low and MSS in colorectal cancer, and a possible link between CIMP-low, MSI-low, MGMT methylation/loss and KRAS mutation.

Evaluation of Methyl-Binding Domain Based Enrichment Approaches Revisited.

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Karolina A Aberg

Full Text Available Methyl-binding domain (MBD enrichment followed by deep sequencing (MBD-seq, is a robust and cost efficient approach for methylome-wide association studies (MWAS. MBD-seq has been demonstrated to be capable of identifying differentially methylated regions, detecting previously reported robust associations and producing findings that replicate with other technologies such as targeted pyrosequencing of bisulfite converted DNA. There are several kits commercially available that can be used for MBD enrichment. Our previous work has involved MethylMiner (Life Technologies, Foster City, CA, USA that we chose after careful investigation of its properties. However, in a recent evaluation of five commercially available MBD-enrichment kits the performance of the MethylMiner was deemed poor. Given our positive experience with MethylMiner, we were surprised by this report. In an attempt to reproduce these findings we here have performed a direct comparison of MethylMiner with MethylCap (Diagenode Inc, Denville, NJ, USA, the best performing kit in that study. We find that both MethylMiner and MethylCap are two well performing MBD-enrichment kits. However, MethylMiner shows somewhat better enrichment efficiency and lower levels of background "noise". In addition, for the purpose of MWAS where we want to investigate the majority of CpGs, we find MethylMiner to be superior as it allows tailoring the enrichment to the regions where most CpGs are located. Using targeted bisulfite sequencing we confirmed that sites where methylation was detected by either MethylMiner or by MethylCap indeed were methylated.

IGFBP3 Promoter Methylation in Colorectal Cancer: Relationship with Microsatellite Instability, CpG Island Methylator Phenotype, p53

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Takako Kawasaki

2007-12-01

Full Text Available Insulin-like growth factor binding protein 3 (IGFBP3, which is induced by wild-type p53, regulates IGF and interacts with the TGF-β pathway. IGFBP3 promoter methylation may occur in colorectal cancer with or without the CpG island methylator phenotype (CIMP, which is associated with microsatellite instability (MSI and TGFBR2 mutation. We examined the relationship between IGFBP3 methylation, p53 expression, CIMP and MSI in 902 population-based colorectal cancers. Utilizing real-time PCR (MethyLight, we quantified promoter methylation in IGFBP3 and eight other CIMP-high-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1. IGFBP3 methylation was far more frequent in non-MSI-high CIMP-high tumors (85% = 35/41 than in MSI-high CIMPhigh (49% = 44/90, P < .0001, MSI-high non-CIMP-high (17% = 6/36, P < .0001, non-MSI-high non-CIMP-high tumors (22% = 152/680, P < .0001. Among CIMPhigh tumors, the inverse relationship between MSI and IGFBP3 methylation persisted in p53-negative tumors (P < .0001, but not in p53-positive tumors. IGFBP3 methylation was associated inversely with TGFBR2 mutation in MSI-high non-CIMP-high tumors (P = .02. In conclusion, IGFBP3 methylation is inversely associated with MSI in CIMP-high colorectal cancers, this relationship is limited to p53-negative tumors. Our data suggest complex relationship between global genomic/epigenomic phenomena (such as MSI/ CIMP, single molecular events (e.g., IGFBP3 methylation, TP53 mutation, TGFBR2 mutation, the related pathways.

Effects of γ-radiation on cell growth, cell cycle and promoter methylation of 22 cell cycle genes in the 1321NI astrocytoma cell line.

Science.gov (United States)

Alghamian, Yaman; Abou Alchamat, Ghalia; Murad, Hossam; Madania, Ammar

2017-09-01

DNA damage caused by radiation initiates biological responses affecting cell fate. DNA methylation regulates gene expression and modulates DNA damage pathways. Alterations in the methylation profiles of cell cycle regulating genes may control cell response to radiation. In this study we investigated the effect of ionizing radiation on the methylation levels of 22 cell cycle regulating genes in correlation with gene expression in 1321NI astrocytoma cell line. 1321NI cells were irradiated with 2, 5 or 10Gy doses then analyzed after 24, 48 and 72h for cell viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu bromide) assay. Flow cytometry were used to study the effect of 10Gy irradiation on cell cycle. EpiTect Methyl II PCR Array was used to identify differentially methylated genes in irradiated cells. Changes in gene expression was determined by qPCR. Azacytidine treatment was used to determine whether DNA methylation affectes gene expression. Our results showed that irradiation decreased cell viability and caused cell cycle arrest at G2/M. Out of 22 genes tested, only CCNF and RAD9A showed some increase in DNA methylation (3.59% and 3.62%, respectively) after 10Gy irradiation, and this increase coincided with downregulation of both genes (by 4 and 2 fold, respectively). with azacytidine confirmed that expression of CCNF and RAD9A genes was regulated by methylation. 1321NI cell line is highly radioresistant and that irradiation of these cells with a 10Gy dose increases DNA methylation of CCNF and RAD9A genes. This dose down-regulates these genes, favoring G2/M arrest. Copyright © 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

Developmental differences in posttranslational calmodulin methylation in pea plants

International Nuclear Information System (INIS)

Oh, Sukheung; Roberts, D.M.

1990-01-01

A calmodulin-N-methyltransferase was used to analyze the degree of lysine-115 methylation of pea calmodulin. Calmodulin was isolated from segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by the calmodulin methyltransferase in the presence of 3 H-methyl-S-adenosylmethionine. Calmodulin methylation levels were lower in apical root segments and in the young lateral roots compared with the mature, differentiated root tissues. The methylation of these calmodulin samples occurs specifically at lysine 115 since site-directed mutants of calmodulin with substitutions at this position were not methylated and competitively inhibited methylation. The present findings, combined with previous data showing differences in NAD kinase activation by methylated and unmethylated calmodulins, raise the possibility that posttranslational methylation could affect calmodulin action

Methylation sensitive amplified polymorphism (MSAP) reveals that ...

African Journals Online (AJOL)

ajl yemi

2011-12-19

Dec 19, 2011 ... Key words: Salt stress, alkali stress, Gossypium hirsutum L., DNA methylation, methylation sensitive amplified polymorphism (MSAP). INTRODUCTION. DNA methylation is one of the key epigenetic mecha- nisms among eukaryotes that can modulate gene expression without the changes of DNA sequence.

Thermal Decomposition of Potential Ester Biofuels. Part I: Methyl Acetate and Methyl Butanoate

Energy Technology Data Exchange (ETDEWEB)

Porterfield, Jessica P.; Bross, David H.; Ruscic, Branko; Thorpe, James H.; Nguyen, Thanh Lam; Baraban, Joshua H.; Stanton, John F.; Daily, John W.; Ellison, G. Barney

2017-06-09

Two methyl esters have been examined as models for the pyrolysis of biofuels. Dilute samples (0.06 - 0.13%) of methyl acetate (CH₃COOCH₃) and methyl butanoate (CH₃CH₂CH₂COOCH₃) were entrained in (He, Ar) carrier gas and decomposed in a set of flash-pyrolysis micro-reactors. The pyrolysis products resulting from the methyl esters were detected and identified by vacuum ultraviolet photoionization mass spectrometry. Complementary product identification was provided by matrix infrared absorption spectroscopy. Pyrolysis pressures in the pulsed micro-reactor were roughly 20 Torr and residence times through the reactors were approximately 25 - 150 µs. Reactor temperatures of 300 – 1600 K were explored. Decomposition of CH₃COOCH₃ commences at 1000 K and the initial products are (CH₂=C=O and CH₃OH). As the micro-reactor is heated to 1300 K, a mixture of (CH₂=C=O and CH₃OH, CH₃, CH₂=O, H, CO, CO₂) appears. The thermal cracking of CH₃CH₂CH₂COOCH₃ begins at 800 K with the formation of (CH₃CH₂CH=C=O, CH₃OH). By 1300 K, the pyrolysis of methyl butanoate yields a complex mixture of (CH₃CH₂CH=C=O, CH₃OH, CH₃, CH₂=O, CO, CO₂, CH₃CH=CH₂, CH₂CHCH₂, CH₂=C=CH₂, HCCCH₂, CH₂=C=C=O, CH₂=CH₂, HCΞCH, CH₂=C=O). Based on the results from the thermal cracking of methyl acetate and methyl butanoate, we predict several important decomposition channels for the pyrolysis of fatty acid methyl esters, R CH₂-COOCH₃. The lowest energy fragmentation will be a 4-center elimination of methanol to form the ketene, RCH=C=O. At higher temperatures, concerted

Electronic transport in methylated fragments of DNA

Energy Technology Data Exchange (ETDEWEB)

Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L., E-mail: umbertofulco@gmail.com; Albuquerque, E. L. [Departamento de Biofísica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal-RN (Brazil); Freire, V. N. [Departamento de Física, Universidade Federal do Ceará, 60455-760 Fortaleza, CE (Brazil); Caetano, E. W. S. [Instituto Federal de Educação, Ciência e Tecnologia do Ceará, 60040-531 Fortaleza, CE (Brazil); Moura, F. A. B. F. de; Lyra, M. L. [Instituto de Física, Universidade Federal de Alagoas, 57072-900 Maceió-AL (Brazil)

2015-11-16

We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

Electronic transport in methylated fragments of DNA

International Nuclear Information System (INIS)

Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L.; Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; Moura, F. A. B. F. de; Lyra, M. L.

2015-01-01

We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics

DNA Methylation Modulates Nociceptive Sensitization after Incision.

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Yuan Sun

Full Text Available DNA methylation is a key epigenetic mechanism controlling DNA accessibility and gene expression. Blockade of DNA methylation can significantly affect pain behaviors implicated in neuropathic and inflammatory pain. However, the role of DNA methylation with regard to postoperative pain has not yet been explored. In this study we sought to investigate the role of DNA methylation in modulating incisional pain and identify possible targets under DNA methylation and contributing to incisional pain. DNA methyltranferase (DNMT inhibitor 5-Aza-2'-deoxycytidine significantly reduced incision-induced mechanical allodynia and thermal sensitivity. Aza-2'-deoxycytidine also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. Global DNA methylation and DNMT3b expression were increased in skin after incision, but none of DNMT1, DNMT3a or DNMT3b was altered in spinal cord or DRG. The expression of proopiomelanocortin Pomc encoding β-endorphin and Oprm1 encoding the mu-opioid receptor were upregulated peripherally after incision; moreover, Oprm1 expression was further increased under DNMT inhibitor treatment. Finally, local peripheral injection of the opioid receptor antagonist naloxone significantly exacerbated incision-induced mechanical hypersensitivity. These results suggest that DNA methylation is functionally relevant to incisional nociceptive sensitization, and that mu-opioid receptor signaling might be one methylation regulated pathway controlling sensitization after incision.

Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay

Energy Technology Data Exchange (ETDEWEB)

Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin, E-mail: cxcai@njnu.edu.cn

2015-04-15

Highlights: • An approach for sensitive and selective DNA demethylase activity assay is reported. • This assay is based on the fluorescence quenching of GO and site-specific cleavage of endonuclease. • It can determine as low as 0.05 ng mL{sup −1} of MBD2 with a linear range of 0.2–300 ng mL{sup −1}. • It has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. • It can avoid false signals, requiring no bisulfite conversion, PCR amplification, radioisotope-labeling. - Abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL{sup −1} (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL{sup −1} and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no

Methylation analysis of polysaccharides: Technical advice.

Science.gov (United States)

Sims, Ian M; Carnachan, Susan M; Bell, Tracey J; Hinkley, Simon F R

2018-05-15

Glycosyl linkage (methylation) analysis is used widely for the structural determination of oligo- and poly-saccharides. The procedure involves derivatisation of the individual component sugars of a polysaccharide to partially methylated alditol acetates which are analysed and quantified by gas chromatography-mass spectrometry. The linkage positions for each component sugar can be determined by correctly identifying the partially methylated alditol acetates. Although the methods are well established, there are many technical aspects to this procedure and both careful attention to detail and considerable experience are required to achieve a successful methylation analysis and to correctly interpret the data generated. The aim of this article is to provide the technical details and critical procedural steps necessary for a successful methylation analysis and to assist researchers (a) with interpreting data correctly and (b) in providing the comprehensive data required for reviewers to fully assess the work. Copyright © 2018 Elsevier Ltd. All rights reserved.

DNA damage, homology-directed repair, and DNA methylation.

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Concetta Cuozzo

2007-07-01

Full Text Available To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP genes (DR-GFP. A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

Wp specific methylation of highly proliferated LCLs

International Nuclear Information System (INIS)

Park, Jung-Hoon; Jeon, Jae-Pil; Shim, Sung-Mi; Nam, Hye-Young; Kim, Joon-Woo; Han, Bok-Ghee; Lee, Suman

2007-01-01

The epigenetic regulation of viral genes may be important for the life cycle of EBV. We determined the methylation status of three viral promoters (Wp, Cp, Qp) from EBV B-lymphoblastoid cell lines (LCLs) by pyrosequencing. Our pyrosequencing data showed that the CpG region of Wp was methylated, but the others were not. Interestingly, Wp methylation was increased with proliferation of LCLs. Wp methylation was as high as 74.9% in late-passage LCLs, but 25.6% in early-passage LCLs. From two Burkitt's lymphoma cell lines, Wp specific hypermethylation was also found (>80%). Interestingly, the expression of EBNA2 gene which located directly next to Wp was associated with its methylation. Our data suggested that Wp specific methylation may be important for the indicator of the proliferation status of LCLs, and the epigenetic viral gene regulation of EBNA2 gene by Wp should be further defined possibly with other biological processes

DNA Methylation Biomarkers: Cancer and Beyond

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Thomas Mikeska

2014-09-01

Full Text Available Biomarkers are naturally-occurring characteristics by which a particular pathological process or disease can be identified or monitored. They can reflect past environmental exposures, predict disease onset or course, or determine a patient’s response to therapy. Epigenetic changes are such characteristics, with most epigenetic biomarkers discovered to date based on the epigenetic mark of DNA methylation. Many tissue types are suitable for the discovery of DNA methylation biomarkers including cell-based samples such as blood and tumor material and cell-free DNA samples such as plasma. DNA methylation biomarkers with diagnostic, prognostic and predictive power are already in clinical trials or in a clinical setting for cancer. Outside cancer, strong evidence that complex disease originates in early life is opening up exciting new avenues for the detection of DNA methylation biomarkers for adverse early life environment and for estimation of future disease risk. However, there are a number of limitations to overcome before such biomarkers reach the clinic. Nevertheless, DNA methylation biomarkers have great potential to contribute to personalized medicine throughout life. We review the current state of play for DNA methylation biomarkers, discuss the barriers that must be crossed on the way to implementation in a clinical setting, and predict their future use for human disease.

Induction of cell death by chemotherapeutic methylating agents

International Nuclear Information System (INIS)

Quiros Barrantes, Steve

2012-01-01

The mechanism of cell death induced by O 6 MeG has been investigated and inhibition of homologous recombination as a strategy for sensitization of tumor cells against methylating agents S N 1. Dependence of the cell cycle was determined toxic responses triggered by O''6 MeG and evaluated by proliferation assays if apoptotic cells have originated exclusively from the second post-treatment cycle. Dependence of O''6 MeG was found at DSB formation. The activation of the control points of the cell cycle and induction of apoptosis is generated during the second cell cycle. Additionally, a portion of the cells has been determined that triggers apoptosis in subsequent generations in the second cell cycle. Inhibition of homologous recombination has been a reasonable strategy to increase S N 1 alkylating agent effectiveness. Evidence has been provided in NHEJ dependent inhibition of DNA-PK that not significantly sensitizes the glioblastoma cells against temozolomide [es

Investigating Resistance of Wild Mustard (Sinapis arvensis L. ‎Populations to Tribenuron-Methyl Herbicide

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‎ Mehdi Afshari

2017-05-01

Full Text Available Tribenuron-methyl is commonly used for post emergence control of broad leaf weeds in wheat fields. In order to survey suspicious resistant weeds in wheat fields to this herbicide thirty-eight fields of Kermanshah province were investigated during 2012- 2013. Seeds of suspected resistance of wild mustard were gathered and tested in a randomized complete blocks design experiment with three replications. First, for early detection of herbicide resistance, the suspected population was screened using discriminating dose of tribenuron-methyl. Determining of the resistance degree was conducted by whole plant bioassay tests using dose-response curves. The resistance mechanisms were assayed by molecular methods, especially using the ALS gene cloning by PJET1.2/blunt Vector. For susceptible populations, the concentration required for complete control was 10.4 g ai ha-1 tribenuron-methyl. Also, in screening tests 50% of populations as resistant populations were identified. According to the Beckie and Tardif, it was found that 57.8% of these population did have a very high degree of resistance, 31.5% with high resistance and 10/5% with low resistance degree. GR50 of the resistant weeds was also increased as compared to sensitive weed, which indicates resistance in this province, Thus to control the resistant populations Z15, this amount increased to 1309 g ai ha-1.The results of DNA sequencing showed that mutation by replacing proline amino acid at position Ala122 causes resistance based on target-site mutation.

Production of Methyl Laurate from Coconut Cream through Fractionation of Methyl Ester

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Johnner P. Sitompul

2015-10-01

Full Text Available This paper concerns the production of methyl laurate from coconut cream through fractionation of methyl esters. Coconut oil was produced by wet processing of coconut cream. The esters were prepared by reacting coconut oil and methanol using homogeneous catalyst KOH in a batch reactor, followed by fractionation of fatty acid methyl esters (FAME at various reduced pressures applying differential batch vacuum distillation. Experimental data were compared with simulation of a batch distillation employing the simple Raoult’s model and modified Raoult’s model of phase equilibria. Activity coefficients (γi were determined by optimization to refine the models. The modified Rault’s model with activity coefficients gave better agreement with the experimental data, giving the value of γi between 0,56-0,73. For a given boiling temperature, lower operating pressure produced higher purity of C10 and C12 FAME for respective distillates.

Radiation effects on DNA methylation in mice

International Nuclear Information System (INIS)

Komura, J.; Kurishita, A.; Miyamura, Y.; Ono, T.; Tawa, R.; Sakurai, H.

1992-01-01

Effects of ionizing radiation on DNA methylation in liver, brain and spleen were examined by high performance liquid chromatography (HPLC). The total methylated cytosine level in the genome was reduced within 8 hours after 3.8 Gy of irradiation in liver of adult mice. But no appreciable effect was observed in brain and spleen. When mice were irradiated at newborn, liver DNA revealed no change in methylated cytosine level. Even though slight effects of radiation were detected in he methylation of the c-myc and c-fos genes, they were only temporary and no long-term effects were observed. These data suggest that the effect of radiation on DNA methylation in vivo is not prevailing a DNA damage, but rather influenced much through biological parameters. (author)

Enhanced susceptibility of a transposable-element-bearing strain of Drosophila melanogaster to somatic eye-color mutations by ethyl nitrosourea, methyl nitrosourea, and X-rays

International Nuclear Information System (INIS)

Ryo, H.; Kondo, S.; Rasmuson, B.

1983-01-01

A strain of Drosophila with the genes z and w + plus a transposable element (TE) is about 3 times more sensitive than a strain without TE toward somatic eye-color mutations after larval exposure to ethyl nitrosourea, methyl nitrosourea and X-rays. The assay system with TE is simple, reliable, and sensitive for detecting somatic mutations induced in vivo by mutagens. (orig.)

Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels

Science.gov (United States)

Lawley, P. D.; Shah, S. A.

1972-01-01

1. The following methods for hydrolysis of methyl-14C-labelled RNA, and for chromatographic isolation and determination of the products, were investigated: enzymic digestion to nucleosides at pH6 or 8; alkaline hydrolysis and conversion into nucleosides; hydrolysis by acid to pyrimidine nucleotides and purine bases, or completely to bases; chromatography on Dowex 50 (NH4+ form) at pH6 or 8.9, or on Dowex 50 (H+ form), or on Sephadex G-10. 2. The suitability of the various methods for determination of methylation products was assessed. The principal product, 7-methylguanosine, was unstable under the conditions used for determinations of nucleosides. 3- and 7-Methyladenine and 3- and 7-methylguanine are best determined as bases; 1-methyladenine and 3-methylcytosine can be isolated as either nucleosides or bases; O6-methylguanine is unstable under the acid hydrolysis conditions used and can be determined as the nucleoside; 3-methyluracil was detected, but may be derived from methylation of the ionized form of uracil. 3. Differences between the patterns of methylation of RNA and homopolyribonucleotides by the N-methyl-N-nitroso compounds and dimethyl sulphate were found: the nitroso compounds were able to methylate O-6 of guanine, were relatively more reactive at N-7 of adenine and probably at N-3 of guanine, but less reactive at N-1 of adenine, N-3 of cytosine and probably at N-3 of uridine. They probably reacted more with the ribose–phosphate chain, but no products from this were identified. 4. The possible influences of these differences on biological action of the methylating agents is discussed. Nitroso compounds may differ principally in their ability to induce miscoding in the Watson–Crick sense by reaction at O-6 of guanine. Both types of agent may induce miscoding to a lesser extent through methylation at N-3 of guanine; both can methylate N atoms, presumably preventing Watson–Crick hydrogen-bonding. N-Methyl-N-nitrosourea can degrade RNA, possibly

Methyl group turnover on methyl-accepting chemotaxis proteins during chemotaxis by Bacillus subtilis

International Nuclear Information System (INIS)

Thoelke, M.S.; Casper, J.M.; Ordal, G.W.

1990-01-01

The addition of attractant to Bacillus subtilis briefly exposed to radioactive methionine causes an increase of labeling of the methyl-accepting chemotaxis proteins. The addition of attractant to cells radiolabeled for longer times shows no change in the extent of methylation. Therefore, the increase in labeling for the briefly labeled cells is due to an increased turnover of methyl groups caused by attractant. All amino acids gave enhanced turnover. This turnover lasted for a prolonged time, probably spanning the period of smooth swimming caused by the attractant addition. Repellent did not affect the turnover when added alone or simultaneously with attractant. Thus, for amino acid attractants, the turnover is probably the excitatory signal, which is seen to extend long into or throughout the adaptation period, not just at the start of it

Diagnosis of Constitutional Mismatch Repair-Deficiency Syndrome Based on Microsatellite Instability and Lymphocyte Tolerance to Methylating Agents.

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Bodo, Sahra; Colas, Chrystelle; Buhard, Olivier; Collura, Ada; Tinat, Julie; Lavoine, Noémie; Guilloux, Agathe; Chalastanis, Alexandra; Lafitte, Philippe; Coulet, Florence; Buisine, Marie-Pierre; Ilencikova, Denisa; Ruiz-Ponte, Clara; Kinzel, Miriam; Grandjouan, Sophie; Brems, Hilde; Lejeune, Sophie; Blanché, Hélène; Wang, Qing; Caron, Olivier; Cabaret, Odile; Svrcek, Magali; Vidaud, Dominique; Parfait, Béatrice; Verloes, Alain; Knappe, Ulrich J; Soubrier, Florent; Mortemousque, Isabelle; Leis, Alexander; Auclair-Perrossier, Jessie; Frébourg, Thierry; Fléjou, Jean-François; Entz-Werle, Natacha; Leclerc, Julie; Malka, David; Cohen-Haguenauer, Odile; Goldberg, Yael; Gerdes, Anne-Marie; Fedhila, Faten; Mathieu-Dramard, Michèle; Hamelin, Richard; Wafaa, Badre; Gauthier-Villars, Marion; Bourdeaut, Franck; Sheridan, Eamonn; Vasen, Hans; Brugières, Laurence; Wimmer, Katharina; Muleris, Martine; Duval, Alex

2015-10-01

Patients with bi-allelic germline mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2) develop a rare but severe variant of Lynch syndrome called constitutional MMR deficiency (CMMRD). This syndrome is characterized by early-onset colorectal cancers, lymphomas or leukemias, and brain tumors. There is no satisfactory method for diagnosis of CMMRD because screens for mutations in MMR genes are noninformative for 30% of patients. MMR-deficient cancer cells are resistant to genotoxic agents and have microsatellite instability (MSI), due to accumulation of errors in repetitive DNA sequences. We investigated whether these features could be used to identify patients with CMMRD. We examined MSI by PCR analysis and tolerance to methylating or thiopurine agents (functional characteristics of MMR-deficient tumor cells) in lymphoblastoid cells (LCs) from 3 patients with CMMRD and 5 individuals with MMR-proficient LCs (controls). Using these assays, we defined experimental parameters that allowed discrimination of a series of 14 patients with CMMRD from 52 controls (training set). We then used the same parameters to assess 23 patients with clinical but not genetic features of CMMRD. In the training set, we identified parameters, based on MSI and LC tolerance to methylation, that detected patients with CMMRD vs controls with 100% sensitivity and 100% specificity. Among 23 patients suspected of having CMMRD, 6 had MSI and LC tolerance to methylation (CMMRD highly probable), 15 had neither MSI nor LC tolerance to methylation (unlikely to have CMMRD), and 2 were considered doubtful for CMMRD based on having only 1 of the 2 features. The presence of MSI and tolerance to methylation in LCs identified patients with CMMRD with 100% sensitivity and specificity. These features could be used in diagnosis of patients. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

Transcription factors as readers and effectors of DNA methylation.

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Zhu, Heng; Wang, Guohua; Qian, Jiang

2016-08-01

Recent technological advances have made it possible to decode DNA methylomes at single-base-pair resolution under various physiological conditions. Many aberrant or differentially methylated sites have been discovered, but the mechanisms by which changes in DNA methylation lead to observed phenotypes, such as cancer, remain elusive. The classical view of methylation-mediated protein-DNA interactions is that only proteins with a methyl-CpG binding domain (MBD) can interact with methylated DNA. However, evidence is emerging to suggest that transcription factors lacking a MBD can also interact with methylated DNA. The identification of these proteins and the elucidation of their characteristics and the biological consequences of methylation-dependent transcription factor-DNA interactions are important stepping stones towards a mechanistic understanding of methylation-mediated biological processes, which have crucial implications for human development and disease.

RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility.

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Shoji, Tatsuma; Takaya, Akiko; Sato, Yoshiharu; Kimura, Satoshi; Suzuki, Tsutomu; Yamamoto, Tomoko

2015-10-15

Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmA(II) enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmA(II), rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmA(II) in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmA(II) activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmA(II), thereby facilitating TEL binding to the ribosome. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Dynamic Alu Methylation during Normal Development, Aging, and Tumorigenesis

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Yanting Luo

2014-01-01

Full Text Available DNA methylation primarily occurs on CpG dinucleotides and plays an important role in transcriptional regulations during tissue development and cell differentiation. Over 25% of CpG dinucleotides in the human genome reside within Alu elements, the most abundant human repeats. The methylation of Alu elements is an important mechanism to suppress Alu transcription and subsequent retrotransposition. Decades of studies revealed that Alu methylation is highly dynamic during early development and aging. Recently, many environmental factors were shown to have a great impact on Alu methylation. In addition, aberrant Alu methylation has been documented to be an early event in many tumors and Alu methylation levels have been associated with tumor aggressiveness. The assessment of the Alu methylation has become an important approach for early diagnosis and/or prognosis of cancer. This review focuses on the dynamic Alu methylation during development, aging, and tumor genesis. The cause and consequence of Alu methylation changes will be discussed.

Solid-phase radioimmunoassay for vitamin B12 in serum, with use of radioiodinated tyrosine methyl ester of vitamin B12

International Nuclear Information System (INIS)

Endres, D.B.; Painter, K.; Niswender, G.D.

1978-01-01

Although radioassays for vitamin B 12 with use of any of several binding proteins have been available for many years, a radioimmunoassay for B 12 has not been reported. We describe here such a radioimmunoassay, incorporating, for the first time, a radioiodinated tyrosine methyl ester of B 12 as the radioactive tracer. Polypropylene tubes are coated with antiserum raised in a rabbit against B 12 /bovine serum albumin to simplify the separation of bound and free radioactivity. Factors affecting the preparation of coated tubes are described. The assay is accurate, sensitive, precise, and specific for vitamin B 12 . Accuracy of the assay is unaffected by the presence of denatured protein. The advantages of this radioimmunoassay over conventional radioassays are discussed

Mercury net methylation in five tropical flood plain regions of Brazil: high in the root zone of floating macrophyte mats but low in surface sediments and flooded soils.

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Guimarães, J R; Meili, M; Hylander, L D; de Castro e Silva, E; Roulet, M; Mauro, J B; de Lemos, R

2000-10-16

In aquatic systems, bottom sediments have often been considered as the main methylmercury (MeHg) production site. In tropical floodplain areas, however, floating meadows and flooded forests extend over large areas and can be important Hg methylating sites. We present here a cross-system comparison of the Hg net methylation capacity in surface sediments, flooded soils and roots of floating aquatic macrophytes, assayed by in situ incubation with 203Hg and extraction of formed Me203 Hg by acid leaching and toluene. The presence of mono-MeHg was confirmed by thin layer chromatography and other techniques. Study areas included floodplain lakes in the Amazon basin (Tapajós, Negro and Amazon rivers), the Pantanal floodplain (Paraguay river basin), freshwater coastal lagoons in Rio de Janeiro and oxbow lakes in the Mogi-Guaçú river, São Paulo state. Different Hg levels were added in assays performed in 1994-1998, but great care was taken to standardise all other test parameters, to allow data comparisons. Net MeHg production was one order of magnitude higher (mean 13.8%, range 0.28-35) in the living or decomposing roots of floating or rooted macrophyte mats (Eichhornia azurea, E. crassipes, Paspalum sp., Eleocharis sellowiana, Salvinia sp., S. rotundifolia and Scirpus cubensis) than in the surface layer of underlying lake sediments (mean 0.6%, range 0.022-2.5). Methylation in flooded soils presented a wide range and was in some cases similar to the one found in macrophyte roots but usually much lower. In a Tapajós floodplain lake, natural concentrations of MeHg in soil and sediment cores taken along a lake-forest transect agreed well with data on net methylation potentials in the same samples. E. azurea, E. crassipes and Salvinia presented the highest methylation potentials, up to 113 times higher than in sediments. Methylation in E. azurea from six lakes of the Paraguay and Cuiabá rivers, high Pantanal, was determined in the 1998 dry and wet seasons and ranged from

Annotating the genome by DNA methylation.

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Cedar, Howard; Razin, Aharon

2017-01-01

DNA methylation plays a prominent role in setting up and stabilizing the molecular design of gene regulation and by understanding this process one gains profound insight into the underlying biology of mammals. In this article, we trace the discoveries that provided the foundations of this field, starting with the mapping of methyl groups in the genome and the experiments that helped clarify how methylation patterns are maintained through cell division. We then address the basic relationship between methyl groups and gene repression, as well as the molecular rules involved in controlling this process during development in vivo. Finally, we describe ongoing work aimed at defining the role of this modification in disease and deciphering how it may serve as a mechanism for sensing the environment.

A systematic assessment of normalization approaches for the Infinium 450K methylation platform.

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Wu, Michael C; Joubert, Bonnie R; Kuan, Pei-fen; Håberg, Siri E; Nystad, Wenche; Peddada, Shyamal D; London, Stephanie J

2014-02-01

The Illumina Infinium HumanMethylation450 BeadChip has emerged as one of the most popular platforms for genome wide profiling of DNA methylation. While the technology is wide-spread, systematic technical biases are believed to be present in the data. For example, this array incorporates two different chemical assays, i.e., Type I and Type II probes, which exhibit different technical characteristics and potentially complicate the computational and statistical analysis. Several normalization methods have been introduced recently to adjust for possible biases. However, there is considerable debate within the field on which normalization procedure should be used and indeed whether normalization is even necessary. Yet despite the importance of the question, there has been little comprehensive comparison of normalization methods. We sought to systematically compare several popular normalization approaches using the Norwegian Mother and Child Cohort Study (MoBa) methylation data set and the technical replicates analyzed with it as a case study. We assessed both the reproducibility between technical replicates following normalization and the effect of normalization on association analysis. Results indicate that the raw data are already highly reproducible, some normalization approaches can slightly improve reproducibility, but other normalization approaches may introduce more variability into the data. Results also suggest that differences in association analysis after applying different normalizations are not large when the signal is strong, but when the signal is more modest, different normalizations can yield very different numbers of findings that meet a weaker statistical significance threshold. Overall, our work provides useful, objective assessment of the effectiveness of key normalization methods.

Analysis of DNA methylation of maize in response to osmotic and salt stress based on methylation-sensitive amplified polymorphism.

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Tan, Ming-pu