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PubMed2000

Dynamic analysis of proviral induction and De Novo methylation: implications for a histone deacetylase-independent, methylation density-dependent mechanism of transcriptional repression.

Methylation of cytosines in the CpG dinucleotide is generally associated with transcriptional repression in mammalian cells, and recent findings implicate histone deacetylation in methylation-mediated repression. Analyses of histone acetylation in in vitro-methylated transfected plasmids support this model; however, little is known about the relationships among de novo DNA methylation, transcriptional repression, and histone acetylation state. To examine these relationships in vivo, we have developed a novel approach that permits the isolation and expansion of cells harboring expressing or silent retroviruses. MEL cells were infected with a Moloney murine leukemia virus encoding the green fluorescent protein (GFP), and single-copy, silent proviral clones were treated weekly with the histone deacetylase inhibitor trichostatin A or the DNA methylation inhibitor 5-azacytidine. Expression was monitored concurrently by flow cytometry, allowing for repeated phenotypic analysis over time, and proviral methylation was determined by Southern blotting and bisulfite methylation mapping. Shortly after infection, proviral expression was inducible and the reporter gene and proviral enhancer showed a low density of methylation. Over time, the efficacy of drug induction diminished, coincident with the accumulation of methyl-CpGs across the provirus. Bisulfite analysis of cells in which 5-azacytidine treatment induced GFP expression revealed measurable but incomplete demethylation of the provirus. Repression could be overcome in late-passage clones only by pretreatment with 5-azacytidine followed by trichostatin A, suggesting that partial demethylation reestablishes the trichostatin-inducible state. These experiments reveal the presence of a silencing mechanism which acts on densely methylated DNA and appears to function independently of histone deacetylase activity.

Animals↗

PubMed2002

DNA methylation density influences the stability of an epigenetic imprint and Dnmt3a/b-independent de novo methylation.

DNA methylation plays an important role in transcriptional repression. To gain insight into the dynamics of demethylation and de novo methylation, we introduced a proviral reporter, premethylated at different densities, into a defined chromosomal site in murine erythroleukemia cells and monitored the stability of the introduced methylation and reporter gene expression. A high density of methylation was faithfully propagated in vivo. In contrast, a low level of methylation was not stable, with complete demethylation and associated transcriptional activation or maintenance-coupled de novo methylation and associated silencing occurring with equal probability. Deletion of the proviral enhancer increased the probability of maintenance-coupled de novo methylation, suggesting that this enhancer functions in part to antagonize such methylation. The DNA methyltransferases (MTases) Dnmt3a and Dnmt3b are thought to be the sole de novo MTases in the mammalian genome. To determine whether these enzymes are responsible for maintenance-coupled de novo methylation, the unmethylated or premethylated proviral reporter was introduced into DNA MTase-deficient embryonic stem cells. These studies revealed the presence of a Dnmt3a/Dnmt3b-independent de novo methyltransferase activity that is stimulated by the presence of preexisting methylation.

Animals↗

PubMed2006

MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeodomain-containing genes in lung cancer cells.

We present a straightforward and comprehensive approach for DNA methylation analysis in mammalian genomes. The methylated-CpG island recovery assay (MIRA), which is based on the high affinity of the MBD2/MBD3L1 complex for methylated DNA, has been used to detect cell type-dependent differences in DNA methylation on a microarray platform. The procedure has been verified and applied to identify a series of novel candidate lung tumor suppressor genes and potential DNA methylation markers that contain methylated CpG islands. One gene of particular interest was DLEC1, located at a commonly deleted area on chromosome 3p22-p21.3, which was frequently methylated in primary lung cancers and melanomas. Among the identified methylated genes, homeodomain-containing genes were unusually frequent (11 of the top 50 hits) and were targeted on different chromosomes. These genes included LHX2, LHX4, PAX7, HOXB13, LBX1, SIX2, HOXD3, DLX1, HOXD1, ONECUT2, and PAX9. The data show that MIRA-assisted microarray analysis has a low false-positive rate and has the capacity to catalogue methylated CpG islands on a genome-wide basis. The results support the hypothesis that cancer-associated DNA methylation events do not occur randomly throughout the genome but at least some are targeted by specific mechanisms.

Chromosome Mapping↗

PubMed2000

Immunohistochemical evaluation of global DNA methylation: comparison with in vitro radiolabeled methyl incorporation assay.

The in vitro radiolabeled methyl incorporation assay, a commonly used technique to evaluate global methylation of DNA, has some disadvantages and limitations. The purpose of the present study was to compare the results of global DNA methylation evaluated by radiolabeled methyl incorporation (CPM/microg of DNA) with immunohistochemical staining of the same tissue sections with a monoclonal antibody developed against 5-methylcytosine used (5-mc). We archival specimens of squamous cell cancer (SCC) of the human lung with a matched uninvolved specimen (n = 18 pairs) and 18 lung specimens from subjects without lung cancer (noncancer specimens) to make this comparison. The immunostaining for 5-mc was reported as a percentage of cells positive for staining as well as a weighted average of the intensity score. The results suggested that both radiolabeled methyl incorporation assay and immunostaining for 5-mc can be used to demonstrate hypomethylation of DNA in SCC tissues compared to matched uninvolved tissues. An advantage of immunostaining, however, is its ability to demonstrate hypomethylation of SCC compared to adjacent bronchial mucosa on the same archival specimen, obviating the need to use sections from both SCC and matched uninvolved tissues. Only by using the immunostaining technique were we able to document a statistically significant difference in DNA methylation between SCC and noncancer tissues. We conclude that the immunostaining technique has advantages over the radiolabeled methyl incorporation assay and may be best suited for evaluation of global DNA methylation when the methylation status of cancer cannot be normalized by methyl incorporation of normal tissues or when the number of samples available for evaluation is small.

5-Methylcytosine↗

PubMed1992

Methyl groups in carcinogenesis: effects on DNA methylation and gene expression.

Lipotrope-deficient (methyl-deficient) diets cause fatty livers and increased liver-cell turnover and promote carcinogenesis in rodents. In rats prolonged intake of methyl-deficient diets results in liver tumor development. The mechanisms responsible for the cancer-promoting and carcinogenic properties of this deficiency remain unclear. The results of the experiments described here lend support to the hypothesis that intake of such a diet, by causing depletion of S-adenosylmethionine pools, results in DNA hypomethylation, which in turn leads to changes in expression of genes that may have key roles in regulation of growth. In livers of rats fed a severely methyl-deficient diet (MDD), lowered pools of S-adenosylmethionine and hypomethylated DNA were observed within 1 week. The extent of DNA hypomethylation increased when MDD was fed for longer periods. The decreases in overall levels of DNA methylation were accompanied by simultaneous alterations in gene expression, yielding patterns that closely resembled those reported to occur in livers of animals exposed to cancer-promoting chemicals and in hepatomas. Northern blot analysis of polyadenylated RNAs from livers of rats fed control or deficient diets showed that, after 1 week of MDD intake, there were large increases in levels of mRNAs for the c-myc and c-fos oncogenes, somewhat smaller increases in c-Ha-ras mRNA, and virtually no change in levels of c-Ki-ras mRNA. In contrast, mRNAs for epidermal growth factor receptor decreased significantly. The elevated levels of expression of the c-myc, c-fos, and c-Ha-ras genes were accompanied by selective changes in patterns of methylation within the sequences specifying these genes. Changes in DNA methylation and in gene expression induced in livers of rats fed MDD for 1 month were gradually reversed after restoration of an adequate diet. In hepatomas induced by prolonged dietary methyl deficiency, methylation patterns of c-Ki-ras and c-Ha-ras were abnormal. Although human diets are unlikely to be as severely methyl deficient as those used in these experiments, in some parts of the world intake of diets that are low in methionine and choline and contaminated with mycotoxins, such as aflatoxin, are common. Even in industrialized nations, deficiencies of folic acid and vitamin B12 are not uncommon and are exacerbated by some therapeutic agents and by substance abuse. Thus, it seems possible that interactions of diet and contaminants or drugs, by inducing changes in DNA methylation and aberrant gene expression, may contribute to cancer causation in humans.

Animals↗

PubMed1997

A kinetic study of the methanolysis of the sulfonylureas bensulfuron methyl and sulfometuron methyl using capillary electrophoresis.

The instability of sulfonylureas in solution in methanol has led us to a kinetic study of methanolysis of two sulfonylureas using capillary electrophoresis. In a preliminary experiment, solutions of the seven compounds, bensulfuron methyl, sulfometuron methyl, nicosulfuron, chlorimuron ethyl, thifensulfuron methyl, metsulfuron methyl, and chlorsulfuron were prepared in methanol and in acetonitrile. After six weeks at room temperature the compounds dissolved in acetonitrile were quite stable as shown by electropherograms obtained with free zone capillary electrophoresis (CE). All seven of the compounds dissolved in methanol had undergone extensive degradation and in some cases the sulfonylurea could not be detected. Bensulfuron methyl and sulfometuron methyl were selected for further detailed study. Solutions of these two compounds in methanol were heated at 5, 40, 57, and 65 degrees C. No decomposition of either compound was observed in similar solutions of acetonitrile incubated at 57 degrees C. The rates of decomposition in methanolic solutions were estimated from the decrease in instrumental response representing the peaks of the two sulfonylureas. The methanolysis of both compounds proceeded with pseudo first-order kinetics as evidenced by the fact that semilogarithmic plots of the concentration of the substrate as a function of time were linear (correlation coefficients of 0.99). Rate constants and half-lives for the reactions at 40, 50, 57, and 65 degrees C were determined. These varied by factors of 18 to 22 depending upon the reaction temperature. The energies of activation for these reactions were estimated from the appropriate Arrhenius plots and found to be 26 kcal per mole for bensulfuron methyl and 24.5 kcal per mole for sulfometuron methyl. The kinetics of appearance of the products of methanolysis were directly related to and accurately predicted by the pseudo first-order rate constants observed for disappearance of the sulfonylureas. Kinetic study of the reaction mixture by negative and positive-ion mass spectrometry indicated that the products of methanolysis of sulfometuron methyl were 2-carboxymethyl (N-carboxymethyl)benzsulfonamide and 2-amino-4,6-di-methyl pyrimidine and suggested a general mechanism for methanolysis of each of the seven compounds studied.

Electrophoresis, Capillary↗

PubMed1990

A comparative investigation of the metabolism of methyl bromide and methyl iodide in human erythrocytes.

Human erythrocyte cytoplasm was incubated in head space vials with either methyl bromide or methyl iodide. The decline in concentration of the two methyl halides was monitored by gas chromatography. Simultaneously, the production of S-methylglutathione was determined by thin layer chromatography. In parallel experiments, boiled erythrocyte cytoplasm was used in order to determine non-enzymatic conjugation. Furthermore, inhibition experiments with sulfobromophthalein were performed. The results were compared with previous findings on the metabolism of methyl chloride. In contrast to methyl chloride, both methyl bromide and methyl iodide showed a significant non-enzymatic conjugation with glutathione. In addition, an enzymatic conjugation could be observed in the erythrocyte cytoplasm of the majority of the population, whereas a minority lacks this enzymatic activity. This is consistent with findings on methyl chloride. Inhibition experiments show that a minor form of the erythrocyte glutathione transferase may be responsible for the enzymatic conjugation. Of the three monohalogenated methanes, methyl bromide is the substrate with the highest affinity for the conjugating enzyme(s). In the case of methyl iodide, non-enzymatic reaction overweighs the enzymatic process. There are possible implications of the results for occupational health and the toxicity of the substances.

Cytoplasm↗

PubMed1978

The synthesis, biological activity and metabolism of 15-[6,7-14C2]- and 15-[21-3H]methyl retinone, 15-methyl retinol and 15-dimethyl retinol in rats.

15-Methyl retinone, 15-methyl retinol and 15-dimethyl retinol were synthesized from all-trans retinoic acid. The absorption maxima and molar absorption coefficients (epsilon) in ethanol are: 15-methyl retinone (372 nm, 47 400), 15-methyl retinol (325 nm, 72 000) and 15-dimethyl retinol (325 nm, 72 200). The latter two compounds show fluorescence at 470 nm when excited around 320 nm, but with intensities 40 and 70%, respectively, that of retinol. All react with trifluoroacetic acid in chloroform, and the alcohols are dehydrated in ethanolic HCl. Relative to all-trans retinyl acetate, the biological activities in rat growth assay (+/-S.E.) are: 15-methyl retinone (4.7 +/- 1.5%), 15-methyl retinol (16.2 +/- 2.3%) and 15-dimethyl retinol (0.34 +/- 0.07%). The monomethyl derivatives actively support testicular development and spermatogenesis, but none of the three analogues prevents degeneration of the retina in retinoate-treated vitamin A-deficient rats. By using [6,7-14C2]-, [11,12-3H2]- and [21-3H]-labeled analogues, the 15-methyl derivatives were shown to be well absorbed in the gut but poorly stored (1-3% of the dose) in the liver. Metabolites are excreted extensively (25-65%) in the bile, however, largely as glucuronides, and to some extent (15%) in the urine. 15-Methyl retinone is reduced to 15-methyl retinol in the intestinal mucosa but not in the liver, and fatty acyl esters of the alcohols are present in liver. Insofar as we could judge, none of these 15-methyl analogues is converted into retinol. Both monomethyl and dimethyl retinols are transported in plasma in vivo in the retinol-binding protein fraction. Interestingly, the dosages required for half saturation and saturation of the plasma transport system are inversely related, in a rough way, to the biological activities in growth.

Animals↗

PubMed1989

Effects of 15(S)-15-methyl prostaglandin E2 methyl ester on phospholipid metabolism in rat gastric mucosa.

The effects of 15(S)-15-methyl prostaglandin E2 (PGE2) methyl ester on gastric mucosal metabolism of phospholipids in intact rats and rats injured by intragastric instillation of acidified taurocholic acid were examined by using radioisotope-labeled precursors. The incorporation of palmitic, oleic and arachidonic acids into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was reduced by treatment with 15(S)-15-methyl PGE2 methyl ester in the intact rats, but the incorporation of glycerol was unaffected or affected only slightly. Instillation of acidified taurocholic acid resulted in decreased incorporation of palmitic acid and glycerol into PC and PE, whereas pretreatment with 15(S)-15-methyl PGE2 methyl ester caused the incorporations of these precursors to be maintained after acidified taurocholic acid treatment. These results suggest that 15(S)-15-methyl PGE2 methyl ester may reduce the incorporation of fatty acids into PC and PE by inhibition of the deacylation-reacylation cycle either directly or indirectly, whereas acidified taurocholic acid decreases de novo synthesis of PC and PE, and probably also the reacylation of fatty acid into phospholipids. Pretreatment with 15(S)-15-methyl PGE2 methyl ester protected the PC- and PE-synthesizing activity against the injury induced by acidified taurocholic acid, and this effect may be involved in the prevention of mucosal damage.

Animals↗

PubMed1975

Chemical synthesis, spectral properties, and chromatography of 4alpha-methyl and 4beta-methyl isomers of (24R)-24-ethyl-5alpha-cholestan-3beta-ol and (24S)-24-ethyl-cholesta-5,22-dien-3beta-ol.

Described herein are chemical syntheses of the following compounds: 4-methyl-(24S)-24-ethyl-cholesta-4,22-dien-3-one, 4,4-dimethyl-(24S)-24-ethyl-cholesta-5,22-dien-3-one, 4beta-methyl-(24R)-24-ethyl-5alpha-cholestan-3beta-ol, 4alpha-methyl-(24R)-24-ethyl-5alpha-cholestan-3beta-ol, 4alpha-methyl-(24S)-24-ethyl-5alpha-cholest-22-en-3beta-ol, 4-methyl-6beta-bromo-(24S)-24-ethyl-cholesta-4,22-dien-3-one, 4alpha-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-ol, 4alpha,5alpha-epoxy-(24S)-24-ethyl-cholesta-4,22-dien-3beta-yl acetate, 4beta-methyl-(24S)-24-ethyl-cholest-22-en-3beta,5alpha-diol, 4beta-methyl-5alpha-hydroxy-(24S)-24-ethyl-cholest-22-en-3beta-yl acetate, 4beta-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-yl acetate and 4beta-methyl-(24S)-24-ethyl-cholesta-5,22-dien-3beta-ol. Chromatographic, nuclear magnetic resonance, and mass spectral data are presented for the compounds under consideration.

Cholestadienols↗

PubMed1981

The methyl folate trap. A physiological response in man to prevent methyl group deficiency in kwashiorkor (methionine deficiency) and an explanation for folic-acid induced exacerbation of subacute combined degeneration in pernicious anaemia.

It is suggested that in man the methyl folate trap is a normal physiological response to impending methyl group deficiency resulting from a very low supply of methionine. This decreases cellular S-adenosyl-methionine (SAM), which puts at risk important methylation reactions, including those required to maintain myelin. In order to protect these methylation reactions, the cell has evolved two mechanisms to maintain supplies of methionine and SAM as a first priority. (a) Decreased SAM causes the folate co-factors to be directed through the cycle involving 5-methyl-tetrahydrofolate (5-methyl-THF) and methionine synthetase and away from the cycles that produce purines and pyrimidines for DNA synthesis. This enhances the remethylation of homocysteine to methionine and SAM. In addition, by restricting DNA biosynthesis and with it cell, division, competition for methionine for protein synthesis is reduced. Thus, whatever methionine is available is conserved for the vital methylation reactions in the nerves, brain, and elsewhere. (b) 5-methyl-THF, the form in which almost all folate is transported in human plasma, must react with intracellular homocysteine before it can be retained by the cell as a polyglutamate. Since homocysteine is derived entirely from methionine, methionine deficiency will cause intracellular folate deficiency, and the rate of mitosis of rapidly dividing cells will be reduced. although these two processes have evolved as a response to methionine deficiency, they also occur in B12 deficiency, which the cell mistakenly interprets as lack of methionine. the resulting response is inappropriate and gives rise to a potentially lethal anaemia. In these circumstances the methylation reactions are also partly protected by the reduced rate of cell division. This explains why administration of folic acid, which induces cell division and use of methionine in protein synthesis, impairs methylation of myelin and precipitates or exacerbates subacute combined degeneration (SCD). During folate deficiency methionine biosynthesis is also diminished. As in methionine deficiency, the body responds to decreasing availability of SAM by diverting folate away from DNA biosynthesis towards the remethylation of homocysteine to methionine and SAM. The selective use pf available folate to conserve methionine, together with the ability of nerve tissue to concentrate folate form the plasma, explains the absence of SCD in folate deficiency.

Acute Disease↗

PubMed2000

Enantioselective inclusion of methyl phenyl sulfoxides and benzyl methyl sulfoxides by (R)-phenylglycyl-(R)-phenylglycine and the crystal structures of the inclusion cavities

Crystalline (R)-phenylglycyl-(R)-phenylglycine [(R,R)-1] includes methyl phenyl sulfoxides (2 and 3) and benzyl methyl sulfoxides (4) with high enantioselectivity. The dipeptide exhibited different stereoselectivity depending on four structural isomers of methyl tolyl sulfoxide (C(8)H(10)OS): R for methyl 2-tolyl sulfoxide, S for methyl 3-tolyl sulfoxide, and racemic for methyl 4-tolyl sulfoxide. A structural isomer, benzyl methyl sulfoxide, was included in racemic form. Chlorophenyl methyl sulfoxides 3 (C(7)H(7)ClOS) with a similar volume showed the same enantioselectivity for their recognition. By single-crystal X-ray analyses of these inclusion compounds, it was elucidated that (R,R)-1 molecules self-assembled to form layer structures and included the sulfoxides between these layers and that the origin of the enantioselectivity based on chiral cavities was induced by conformation of the C-terminal phenyl group of the dipeptide. The relative position between the ammonio proton and the C-terminal phenyl group in one molecule of the dipeptide determined the stereochemistry of the methyl sulfinyl groups to be recognized. Various positional isomers of methyl xylyl sulfoxide having the formula of C(9)H(12)OS were subjected to the enantioselective inclusion by (R,R)-1 crystals and these results are also discussed.

Journal Article↗

PubMed1977

RNA methylation and control of eukaryotic RNA biosynthesis. Effects of cycloleucine, a specific inhibitor of methylation, on ribosomal RNA maturation.

The role of RNA methylation in the control of ribosome production in mammalian cells has been reinvestigated through a study of the effects in vivo of cycloleucine, a specific and reversible inhibitor of nucleic acid methylations. No close coupling is observed between methylation and transcription. During an extensive blocking of methylation, the synthesis of preribosomal 45-S RNA continues, although at a slightly reduced rate. Transcription and methylation can be temporarily uncoupled in vivo without impairing significantly the efficiency of the subsequent maturation of the transcript which takes place when the methylation is resumed. At the post-transcriptional level, two main observations are made. First, the cleavage pattern of ribosomal RNA is not qualitatively modified by the drug treatment. Preribosomal 45-S RNA which is synthesized during an extensive blocking of methylations (95%) is cleaved in vivo in a stepwise fashion into molecules resembling the species of the normal processing. Undermethylation does not result in the appearance of new size products and no extensive or rapid degredation of rRNA precursors can be detected. Second, the global efficiency of the process of ribosomal RNA maturation is severely affected, in quantitative terms; the undermethylation partially inhibits several stages in the maturation pathway and does not block selectively at a definite step of processing, as previously reported by others. Two main modifications are observed. (a) In the nucleus, the life times of the various undermethylated intermediates of ribosomal RNA maturation are markedly increased and a significant accumulation of these forms is detected by long-term labelling studies. (b) The rate of appearance of ribosomal 28-S RNA into the cytoplasm is severely affected (85-90% inhibition), in much higher proportions than the formation of its immediate precursor, nuclear 28-S RNA, thus suggesting a particular sensitivity to a lack of methylation of the last stage of preribosome maturation (possibly the release to cytoplasm). Finally, if a normal level of methylation does not appear to be stringently required for the completion of ribosomal RNA maturation, our results indicate that the degree of RNA methylation can modulate the general efficiency of the maturation processess.

Amino Acids↗

PubMed1995

Studies of the bound conformations of methyl alpha-lactoside and methyl beta-allolactoside to ricin B chain using transferred NOE experiments in the laboratory and rotating frames, assisted by molecular mechanics and dynamics calculations.

The conformation in solution of methyl beta-galactopyranosyl-(1-->4)-alpha-glucopyranoside (methyl alpha-lactoside) and methyl beta-galactopyranosyl-(1-->6)-beta-glucopyranoside (methyl beta-allolactoside) has been studied through NMR spectroscopy and molecular mechanics calculations. NOE measurements both in the laboratory and rotating frames, have been interpreted in terms of an ensemble average distribution of conformers. Molecular mechanics calculations have been performed to estimate the probability distribution of conformers from the steric energy maps. The experimental results indicate that methyl alpha-lactoside spends about 90% of its time in a broad low-energy region close to the global minimum, while methyl beta-allolactoside presents much higher flexibility. The conformational changes that occur when both disaccharides are bound to the ricin B chain in aqueous solution have been studied using transferred NOE experiments at several protein/ligand ratios. The observed data indicate that the protein causes a conformational variation in the torsion angles of methyl alpha-lactoside changing towards smaller angle values (phi/psi approximately -20/-20), although the recognized conformer is still within the lowest energy region. In particular, the torsional changes separate Gal H1 from Glc H3 and Glc H6 protons, with a noticeable decrease in the intensities of the corresponding NOE cross-peaks, which were clearly observed for the free disaccharide. On the other hand, different conformations around the phi, psi, and omega glycosidic bonds of methyl beta-allolactoside are recognized by the lectin. In fact, for the methyl-beta-allolactoside-ricin-B complex, only the NOESY cross-peaks corresponding to the protons of the galactose residue are negative, as expected for a molecule in the slow motion regime. In contrast, the corresponding cross peaks for the glucose residue were about zero, as expected for a molecule whose motion is practically independent of the protein. However, for the methyl-alpha-lactoside-ricin-B complex, all the NOESY cross-peaks for both the galactose and glucose moieties were clearly negative. From the NMR experimental point of view, it is demonstrated that the comparison of longitudinal and transversal transferred NOEs allows one to clearly differentiate direct enhancements from spin diffusion effects, which are of major concern when analysing NOE spectra of macromolecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Magnetic Resonance Spectroscopy↗

PubMed1976

Pyrogalloloestrogens: enzymic methylation of pyrogalloloestrogens and interaction of pyrogalloloestrogens with the enzymic methylation of catecholamines in rat liver in vitro.

During incubation of 2,4-dihydroxyoestrone with the 105000 X g supernatant of rat liver in the presence of S-adenosyl-[Me-14C]methionine, the formation of radioactive mono- as well as dimethyl ether derivatives was demonstrated. The products were identified as: 2,4-dihydroxyoestrone 2-methyl ether, 2,4-dihydroxyoestrone 3-methyl ether, 2,4-dihydroxyoestrone 4-methyl ether, 2,4-dihydroxyoestrone 2,3-dimethyl ether, 2,4-dihydroxyoestrone 2,4-dimethyl ether and 2,4-dihydroxyoestrone 3,4-dimethyl ether. The monomethyl ethers were the main products; within this group the 3-methyl ether of 2,4-dihydroxyoestrone was the main metabolite. Among the dimethyl ether derivatives, the 2,4-dihydroxyoestrone 2,3-dimethyl ether represented the quantitatively most important product. When 2,4-dihydroxyoestrone 2-methyl ether was incubated under the same conditions, 2,4-dihydroxyoestrone 2,3- as well as 2,4-dimethyl ether was formed. The 2,3-dimethyl ether was again the main metabolite. The incubation of 2,4-dihydroxyoestrone 4-methyl ether yielded the 2,4- and 3,4-dimethyl ethers, the first being the main product. In contrast, the 3-methyl ether of 2,4-dihydroxyoestrone was not further methylated by the catechol methyltransferase preparation. In further experiments, the effect of the pyrogalloloestrogen and its monomethyl ether derivatives on the enzymatic methylation of catecholamines was investigated. It was demonstrated that the methylation of adrenalin and dopamine was competitively inhibited by 2,4-dihydroxyoestrone and the 2,4-dihydroxyoestrone monomethyl ethers. Only a weak inhibitory effect was observed with the 3- and 4-monomenthyl ethers (Ki values 200 and 160muM). The unsubstituted pyrogalloloestrogen produced a marked inhibition (Ki value 50muM), but the strongest inhibition was found with the 2-monomethyl ether of 2,4-dihydroxyoestrone (Ki value 14muM). The extent of inhibition caused by the addition of the 2-monomethyl ether of 2,4-dihydroxyoestrone was thereby in the same range as the inhibition caused by pyrogallol and the catecholoestrogens.

Animals↗

PubMed2005

The role of phospholipid methylation in 1-methyl-4-phenyl-pyridinium ion (MPP+)-induced neurotoxicity in PC12 cells.

Excessive methylation has been proposed to be involved in the pathogenesis of Parkinson's disease (PD), via mechanisms that involve phospholipid methylation. Meanwhile, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to stimulate phospholipid methylation via the oxidized metabolite, 1-methyl-4-phenyl-pyridinium (MPP+), in the rat brain and liver tissues. In the present study, we investigated the effect of MPP+ on phosphatidylethanolamine N-methyltransferases (PENMT) and the potential role of this pathway in MPP(+)-induced neurotoxicity using PC12 cells. The results obtained indicate that MPP+ stimulated phosphatidylethanolamine (PTE) methylation to phosphatidylcholine (PTC) and correspondingly increased the formation of lysophosphatidylcholine (lyso-PTC). Moreover, the addition of S-adenosylmethionine (SAM) to the cell culture medium increases MPP(+)-induced cytotoxicity. The incubation of 1mM MPP+ and various concentrations of SAM (0-4 mM) decreased the viability of PC12 cells from 80% with MPP+ alone to 38% viability with 4 mM SAM for 4 days incubation. The data also revealed that the addition of S-adenosylhomocysteine (SAH), a methylation inhibitor, offered significant protection against MPP(+)-induced cytotoxicity, indicating that methylation plays a role in MPP(+)-induced cytotoxicity. Interestingly, lyso-PTC showed similar actions to MPP+ in causing many cytotoxic changes with at least 10 times higher potency. Lyso-PTC induced dopamine release and inhibited dopamine uptake in PC12 cells. Lyso-PTC also caused the inhibition of mitochondrial potential and increased the formation of reactive oxygen species in PC12 cells. These results indicate that phospholipid methylation pathway might be involved in MPP+ neurotoxicity and lyso-PTC might play a role in MPP(+)-induced neurotoxicity.

1-Methyl-4-phenylpyridinium↗

PubMed2001

Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation.

Silencing of tumor-suppressor genes by hypermethylation of promoter CpG islands is well documented in human cancer and may be mediated by methyl-CpG-binding proteins, like MeCP2, that are associated in vivo with chromatin modifiers and transcriptional repressors. However, the exact dynamic between methylation and chromatin structure in the regulation of gene expression is not well understood. In this study, we have analyzed the methylation status and chromatin structure of three CpG islands in the p14(ARF)/p16(INK4A) locus in a series of normal and cancer cell lines using methylation-sensitive digestion, MspI accessibility in intact nuclei and chromatin immunoprecipitation (ChIP) assays. We demonstrate the existence of an altered chromatin structure associated with the silencing of tumor-suppressor genes in human cancer cell lines involving CpG island methylation, chromatin condensation, histone deacetylation and MeCP2 binding. The data showed that MeCP2 could bind to methylated CpG islands in both promoters and exons; MeCP2 does not interfere with transcription when bound at an exon, suggesting a more generalized role for the protein beyond transcriptional repression. In the absence of methylation, it is demonstrated that CpG islands located in promoters versus exons display marked differences in the levels of acetylation of associated histone H3, suggesting that chromatin remodeling can be achieved by methylation-independent processes and perhaps explaining why non-promoter CpG islands are more susceptible to de novo methylation than promoter islands.

Acetylation↗

PubMed1981

An in vitro study of the methylation of methyl-accepting chemotaxis protein of Escherichia coli. Construction of the system and effect of mutant proteins on the system.

An in vitro system for the methylation of methyl-accepting chemotaxis proteins (MCP's), which have been shown to be membrane integral proteins, was constructed. The system, consisting of the membrane, the cytoplasm, and labeled S-adenosyl methionine, showed the following characteristics. 1. The methylation of MCP in the membrane required the cytoplasm. The rate of incorporation of the labeled methyl group into MCP was dependent on the amount of the cytoplasm. 2. Incorporation of the labeled methyl moiety into MCP reached a steady state, and the level of the steady state incorporation was dependent on the concentration of the cytoplasm when the concentration of the membrane protein was constant. 3. The methyl moiety which had been incorporated into MCP before the steady state could be exchanged. It was suggested that the amount of methyl group introduced into MCP was equal to that of taken from MCP. 4. The methylated MCP was demethylated faster in the presence of a methyl donor than in its absence. 5. The membranes obtained from cheX-, cheB-, and cheZ mutants were inactive in the present in vitro system even when they were mixed with the wild type cytoplasm.

Bacterial Proteins↗