Preparation and separation of methyl ethers of D-arabinose

All possible methyl ethers of methyl β-d-arabinofuranoside and methyl β-d-arabinopyranoside were prepared by partial methylation using Kuhn's method (Ag₂O and MeI in N,N-dimethylformamide) and Haworth's method (Me₂SO₄ and 40% NaOH in water). The ratios of the methyl ethers obtained from the furanoside were found to be considerably more dependent on the method of methylation than those from the pyranoside. All of the methyl ethers of d-arabinofuranose and d-arabinopyranose are separable by thin-layer chromatography on silica gel and paper electrophoresis in borate buffer.     

Chromatography of methyl ethers of D-glucosamine

Identification of methyl ethers obtained by methylation and subsequent hydrolysis is a powerful technique for determination of linkage positions in structure studies of complex carbohydrates. Although methyl ethers of neutral sugars have been separated by various chromatographic methods, separation of methyl ethers of 2-amino-2-deoxy-d-glucose has been more difficult. Only recently, successful procedures for separation of methyl ethers of d-glucosamine based on thin-layer (1), gas (2), and column (3) chromatography have appeared in literature. We wish to report here a method for separation of d-glucosamine methyl ethers using a combination of partition and ion-exchange chromatography.     

The characterization of hemiacetal bonds formed after periodate oxidation of heteroglycans

The location of inter-residue, cyclic hemiacetals formed following the periodate oxidation of four representative heteroglycans has been determined by methylation analysis of the periodate-oxidized glycans. The cyclic hemiacetals led to the protection of hydroxyl groups during methylation in methyl sulfoxide, and their positions were located by analysis of the resulting di- and mono-methyl ethers. Such derivatives were not observed upon methylation analysis of the native and the periodate-oxidized-borohydride-reduced glycans. Inter-residue hemiacetals were thus identified in all oxidized glycans, between aldehydic groups at C-2 or C-3 of oxidized residues and hydroxyl groups at C-3 or C-2 of adjacent, unoxidized residues. Selective removal of 6-O-substituents from oxidized residues resulted in a decreased ability of the latter to form the inter-residue hemiacetals. Analysis of the types and proportions of the methyl ethers resulting from inter-residue hemiacetal formation may also yield structural information on the glycan.     

The MTHFR C677T polymorphism and global DNA methylation in oral epithelial cells

DNA methylation is mediated by DNA methyltransferases (DNMTs) that add a methyl group to the 5′-carbon of cytosine. The enzyme methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate in the rate-limiting step of the cycle involving the methyl donor S-adenosyl-L-methionine (SAM). The MTHFR C677T polymorphism results in a thermolabile enzyme with reduced activity that is predicted to influence the DNA methylation status. In this study, we investigated the impact of the MTHFR C677T polymorphism on the global DNA methylation of oral epithelial cells obtained from 54 healthy subjects. There were no significant differences in global DNA methylation among the MTHFR CC, CT and TT genotypes (p = 0.75; Kruskal-Wallis test).     

The methylation of transfer ribonucleic acid during regeneration of the liver

Transfer ribonucleic acid¹ is methylated after the molecule is synthesized; at least eight enzymes are involved in the transfer of methyl groups (derived from methionine). The time courses of methylation and synthesis of tRNA during rat liver regeneration have been compared in an in vivo radioisotopic study, using 6-orotic acid-¹⁴C and ³H-methyl-L-methionine as precursors in double label pulses. Liver regeneration is a synchronized system in which biochemical events of the cell cycle are separable. Transfer RNA methylation increase precedes by several hours tRNA synthesis during regeneration, although the curves overlap. A ratio of the relative rate of methylation to the relative rate of synthesis has been made; that curve positively correlates with the rise and fall of protein synthesis during regeneration. It is clear that methylation and synthesis of tRNA are only weakly coupled; changing methyl content of the tRNA "pool" resulting from differential tRNA methylase and polymerase activities may regulate the rate of protein synthesis in the cell cycle at the translational level. The "pool sizes" of uridine monophosphate (UMP) and S-adenosylmethionine (SAM) were measured indirectly; UMP and SAM were isolated from perchloric acid supernatants and their specific activities were computed. Differential changes in radioactivity available to tRNA methylases and polymerases are not a source of artifact. That is, the control of both the synthesis and methylation of tRNA is at the enzyme level in vivo, rather than at some enzymatic step prior to those enzymatic reactions.     

A side-reaction in the methylation of galacturonate derivatives with diazomethane-boron trifluoride etherate

Partial p-nitrobenzoylation of methyl (methyl 2-O-methyl-α-d-galactopyranosid)uronate (1) gave the 3-p-nitrobenzoate 2 in good yield. Treatment of 2 or methyl (methyl 2,3-di-O-benzoyl-α-d-galactopyranosid)uronate (11) with diazomethane-BF₃-etherate gave, in addition to the expected 4-methyl ethers, by-products resulting from lengthening of the carbon chain. The by-products were formulated as derivatives of methyl 4,7-anhydro-α-d-galacto-heptopyranosid-6-ulose dimethy acetal on the basis of p.m.r. and i.r. spectral data, by analysis of their mass-spectral fragmentation pattern, and by chemical transformations.     

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

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 [³H]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 (DM Roberts et al. [1986] J Biol Chem 261: 1491-1494) raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity.     

Studies on the koenigs-knorr reaction : Part IV: The effect of participating groups on the stereochemistry of disaccharide formation

Partial benzylation of methyl 2-O-benzyl-α-L-fucopyranoside afforded a mixture of methyl 2,3-, and 2,4-di-O-benzyl-α-L-fucopyranoside which were separated by means of their monoacetates. Partial benzylation of methyl α-L-fucopyranoside gave the 2,4-, and 3,4-dibenzyl ethers in the ratio of 3:2, and no 2,3-isomer could be detected in the reaction mixture. The structures of the three dibenzyl ethers were established: (a) by analysis of the n.m.r. spectra of their acetates, and (b) by methylation, removal of benzyl groups by hydrogenolysis, and characterization of the methyl ethers of the methyl glycosides. Acid hydrolysis of these compounds gave the monomethyl ethers of L-fucose, two of which were identical with known compounds, whereas the third, 4-O-methyl-L-fucose, was a new compound. Selective p-nitrobenzoylation of 2,3-, 2,4-, and 3,4-di-O-benzyl-L-fucose, followed by acetylation and treatment with hydrogen bromide in dichloromethane, gave the three possible mono-O-acetyl-di-O-benzyl-α-L-fucopyranosyl bromides, which were condensed with benzyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-α-D-glucopyranoside. The disaccharide derived from the 2-O-acetyl substituted bromide was enriched in β-L-fucopyranoside, whereas the other two bromides gave mainly the α-L-linked anomer. The α-directing influence of the 3- and 4-O-acetyl substituents is not less than the β-directing influence of the 2-O-acetyl group in similar bromides; participation of acyl groups and electronic-steric influences are discussed as possible explanations for the steric course of the reaction.     

Avoidance of DNA methylation

Theocr ⁺ gene of bacterial virus T7 codes for the first protein recognized to inhibit a specific group of DNA methylases. The recognition sequences of several other DNA methylases, not susceptible to Ocr inhibition, are significantly suppressed in the virus genome. The bacterial virus T3 encodes an Ado-Met hydrolase, destroying the methyl donor and causing T3 DNA to be totally unmethylated. These observations could stimulate analogous investigations into the regulation of DNA methylation patterns of eukaryotic viruses and cells. For instance, an underrepresentation of methylation sites (5′-CG) is also true for animal DNA viruses. Moreover, we were able to disclose some novel properties of DNA restriction-modification enzymes concerning the protection of DNA recognition sequences in which only one strand can be methylated (e.g., type III enzymeEcoP15) and the primary resistance of (unmethylated) DNA recognition sites towards type II restriction endonucleaseEcoRII.     

Betaine attenuates hepatic steatosis by reducing methylation of the MTTP promoter and elevating genomic methylation in mice fed a high-fat diet

Aberrant DNA methylation contributes to the abnormality of hepatic gene expression, one of the main factors in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Betaine is a methyl donor and has been considered to be a lipotropic agent. However, whether betaine supplementation improves NAFLD via its effect on the DNA methylation of specific genes and the genome has not been explored. Male C57BL/6 mice were fed either a control diet or high-fat diet (HFD) supplemented with 0%, 1% and 2% betaine in water (wt/vol) for 12 weeks. Betaine supplementation ameliorated HFD-induced hepatic steatosis in a dose-dependent manner. HFD up-regulated FAS and ACOX messenger RNA (mRNA) expression and down-regulated PPARα, ApoB and MTTP mRNA expression; however, these alterations were reversed by betaine supplementation, except ApoB. MTTP mRNA expression was negatively correlated with the DNA methylation of its CpG sites at −184, −156, −63 and −60. Methylation of these CpG sites was lower in both the 1% and 2% betaine-supplemented groups than in the HFD group (averages; 25.55% and 14.33% vs. 30.13%). In addition, both 1% and 2% betaine supplementation significantly restored the methylation capacity [S-adenosylmethionine (SAM) concentration and SAM/S-adenosylhomocysteine ratios] and genomic methylation level, which had been decreased by HFD (0.37% and 0.47% vs. 0.25%). These results suggest that the regulation of aberrant DNA methylation by betaine might be a possible mechanism of the improvements in NAFLD upon betaine supplementation.     

Electron-capture gas chromatography of plasma sulphonylureas after extractive methylation

Conditions for the extractive alkylation of eight sulphonylurea hypoglycemic drugs have been evaluated. Extractive methylation of the compounds was achieved within 90 min using tetrabutylammonium as counter-ion (0.1 M at pH = 6.9) with 5% methyl iodide in dichloromethane as organic phase. Mass spectral analysis showed derivatives methylated at the sulphonamide nitrogen. A higher pH or use of tetrapentylammonium as counter-ion caused hydrolysis of the sulphonylureas.The derivatives showed a high electron-capture response with minimum concentrations detectable in the range 1–4 × 10^?16 moles sec^?1.Therapeutic plasma concentrations of glipizide and tolbutamide were determined by direct extractive methylation of the compounds from the plasma sample. The glipizide derivative was determined by electron-capture gas chromatography down to about 20 ng/ml in a 0.5-ml plasma sample. The relative standard deviation at the 0.2 μg/ml level of glipizide was 6% (n=6). The corresponding figure in the determination of tolbutamide at the 10 μg/ml level was 3% (n=10).     

Genome-wide DNA methylation analysis reveals phytoestrogen modification of promoter methylation patterns during embryonic stem cell differentiation

Background

Environmental challenges during development affect the fetal epigenome, but the period(s) vulnerable to epigenetic dysregulation is(are) not clear. By employing a soy phytoestrogen, genistein, that is known to alter the epigenetic states of the A^vy allele during embryogenesis, we have explored the sensitive period for epigenetic regulation. The post-implantation period, when de novo DNA methylation actively proceeds, is amenable to in vitro analysis using a mouse embryonic stem (ES) cell differentiation system.

Methods and Findings

Mouse ES cells were differentiated in the presence or absence of genistein, and DNA methylation patterns on day 10 were compared by microarray-based promoter methylation analysis coupled with a methylation-sensitive endonuclease (HpaII/McrBC)-dependent enrichment procedure. Moderate changes in methylation levels were observed in a subset of promoters following genistein treatment. Detailed investigation of the Ucp1 and Sytl1 promoters further revealed that genistein does not affect de novo methylation occurring between day 0 and day 4, but interferes with subsequent regulatory processes and leads to a decrease in methylation level for both promoters.

Conclusion

Genistein perturbed the methylation pattern of differentiated ES cells after de novo methylation. Our observations suggest that, for a subset of genes, regulation after de novo DNA methylation in the early embryo may be sensitive to genistein.     

On the role of steric clashes in methylation control of restriction endonuclease activity

Restriction-modification systems digest non-methylated invading DNA, while protecting host DNA against the endonuclease activity by methylation. It is widely believed that the methylated DNA would not ‘fit’ into the binding site of the endonuclease in the productive orientation, and thus steric clashes should account for most of the protection. We test this concept statistically by grafting methyl groups in silico onto non-methylated DNA in co-crystal structures with restriction endonucleases. Clash scores are significantly higher for protective than non-protective methylation (P < 0.05% according to the Wilcoxon rank sum test). Structural data alone are sufficient to distinguish between protective and non-protective DNA methylation with 90% confidence and decision thresholds of 1.1 Å and 48 ų for the most severe distance-based and cumulative volume-based clash with the protein, respectively (0.1 Å was deducted from each interatomic distance to allow for coordinate errors). The most severe clashes are more pronounced for protective methyl groups attached to the nitrogen atoms (N6-methyladenines and N4-methylcytosines) than for C5-methyl groups on cytosines. Cumulative clashes are comparable for all three types of protective methylation.     

Syntheses of methyl ethers of methyl α-D-mannopyranoside,and hydrogen bonding in diastereoisomers of methyl 4-O-(1-ethoxyethyl)-α-D-mannopyranoside

The synthesis of the 4-methyl, the 2,4-dimethyl, and the 2,3,6-trimethyl ethers of methyl α-D-mannopyranoside has been accomplished by the use of selective, benzoyl protecting groups, the 1-ethoxyethyl protecting group, and methylation with diazomethane. Considerable differences were noted in the i.r.- and n.m.r.-spectroscopic and t.l.c. properties of the diastereoisomers of methyl 4-O-[1-ethoxyethyl]-α-D-mannopyranoside. A structure, analogous to that of trans-decalin, maintained by intramolecular hydrogen-bonding is proposed for these compounds. The differences in physical properties of the two diastereoisomers are interpreted on the basis that the (R) isomer has an axially attached methyl group, and, therefore, the ring involved cannot be so stable as that of the (S) isomer.     

Origin and formation of 1,7-dimethylguanosine in tRNA chemical and enzymatic methylation

tRNA chemical methylation: 1. 1,7-Dimethylguanosine was found in in vivo methylated tRNA from liver and kidney of rat after exposure to a low dose of dimethylnitrosamine (4 mg/kg body weight). 2. At 4 h after dimethylnitrosamine administration, the 1,7-dimethylguanosine:7-methylguanine ratio (product ratio) for liver and kidney tRNA was 0.017 and 0.091, respectively. At 24 h after dimethylnitrosamine administration, the product ratio was lower in both hepatic and renal tRNA. 3. When dimethylnitrosamine was given in four separate daily injections, the product ratio in hepatic tRNA 4 h after the last dose was the same as for the same total dose given by a single injection, but in renal tRNA it was lower. No dialkyl compound was found in liver and kidney tRNA 24 h after the last multiple injection. tRNA enzymatic methylation: 1. Base analyses of Escherichia coli B tRNA methylated in vitro, by using S-adenosylmethionine as physiological methyl donor and enzyme preparations from liver and kidney of normal rat, indicated that 1,7-dimethylguanosine was also a product of enzymatic methylation. 2. The amount of 1,7-dimethylguanosine formed by kidney enzyme preparation was 3-times that produced by the liver extract. 3. A second type of enzymatic methylation assay where chemically methylated tRNA was used as substrate indicated that the 7-methylguanosine residues in the nucleic acid are not the substrate of the methylase activity forming the 1,7-dimethylguanosine moieties. Analogous data were obtained for the origin of 1,7-dimethylguanosine residues in tRNA chemical methylation by dimethyl sulphate.     

MicroRNA mediates DNA methylation of target genes

Small RNAs represented by microRNA (miRNA) plays important roles in plant development and responds to biotic and abiotic stresses. Previous studies have placed special emphasis on gene-repression mediated by miRNA. In this work, the DNA methylation pattern of microRNA genes (MIRs) was interrogated. Full-length cDNA and EST were used to confirm the entity of pri-miRNA. In parallel, miRNA in 24 nucleotides (nt) was pooled to detect chromatin modification effect by using bisulfite sequencing data. 97 MIRs were supported by full-length cDNA and 30 more were hit by EST. Notably, methylation levels of conserved MIRs were significantly lower than the non-conserved at all contexts (CG, CHG, and CHH). Additionally, a substantial part of 24-nt miRNA was able to induce target site methylation, providing a broader perspective for researchers.     

Protein methylation in pea chloroplasts

The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with [³H-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. One is a polypeptide with a molecular mass of 64 kD, a second has an M_r of 48 kD, and the third has a molecular mass of less than 10 kD. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide, having a molecular mass of 24 kD, is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. One methyl-linkage 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 [³H]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 [³H]methyl group.     

Disruption of choline methyl group donation for phosphatidylethanolamine methylation in hepatocarcinoma cells

Despite being widely hypothesized, the actual contribution of choline as a methyl source for phosphatidylethanolamine (PE) methylation has never been demonstrated, mainly due to the inability of conventional methods to distinguish the products from that of the CDP-choline pathway. Using a novel combination of stable-isotope labeling and tandem mass spectrometry, we demonstrated for the first time that choline contributed to phosphatidylcholine (PC) synthesis both as an intact choline moiety via the CDP-choline pathway and as a methyl donor via PE methylation pathway. When hepatocytes were labeled with d(9)-choline containing three deuterium atoms on each of the three methyl groups, d(3)-PC and d(6)-PC were detected, indicating that newly synthesized PC contained one or more individually mobilized methyl groups from d(9)-choline. The synthesis of d(3)-PC and d(6)-PC was sensitive to the general methylation inhibitor 3-deazaadenosine and were specific products of PE methylation using choline as a one-carbon donor. While the contribution to the CDP-choline pathway remained intact in hepatocarcinoma cells, contribution of choline to PE methylation was completely disrupted. In addition to a previously identified lack of PE methyltransferase, hepatocarcinoma cells were found to lack the abilities to oxidize choline to betaine and to donate the methyl group from betaine to homocysteine, whereas the usage of exogenous methionine as a methyl group donor was normal. The failure to use choline as a methyl source in hepatocarcinoma cells may contribute to methionine dependence, a widely observed aberration of one-carbon metabolism in malignancy.