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PubMed1993

DNA methylation by N-methyl-N-nitrosourea: methylation pattern changes in single- and double-stranded DNA, and in DNA with mismatched or bulged guanines.

The detection of abnormal DNA base pairing arrangements and conformations is chemically probed in synthetic 32P-end-labeled deoxyribonucleotide oligomers using N-methyl-N-nitrosourea (MNU) and 2,12,-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]heptadeca-1 -[17],2,11,13,15 pentaene-Ni (II) (Ni-complex) with KHSO5. The DNA targets studied are single-stranded (s-s) DNA, double-stranded (d-s) DNA, d-s DNA with G-G, G-A and G-T mismatches, d-s DNA with a single bulged G and d-s DNA with two bulged G's. The effect of the non-Watson--Crick structures on the formation of N7-methylguanine (N7-MeG) by MNU and the oxidation of G by Ni-complex is reported along with the Tm's and circular dichroism spectra of the different duplex oligomers. The results for MNU and Ni-complex show that the qualitative and quantitative character of the cleavage patterns at a G3 run change with the nature of the abnormal base pairing motif. Based on the DNA substrates studied, the results indicate that a combination of reagents which report electronic and steric perturbations can be a useful approach to monitor DNA mismatches and bulges.

Autoradiography↗
PubMed1998

Potential antitumor alpha-methylene-gamma-butyrolactone-bearing nucleic acid base. 3. Synthesis of 5'-methyl-5'-[(6-substituted-9H-purin-9-yl)methyl]-2'-oxo-3'- methylenetetrahydrofurans.

Search for a new alpha-methylene-gamma-butyrolactone-bearing 6-substituted purine as a potential antitumor agent has led to synthesize seven, hitherto unreported, 5'-Methyl-5'-[(6-substituted-9H-purin-9-yl)methyl]-2'-oxo-3'- methylenetetrahydrofurans (H, Cl, I, CH3, NH2, SH, > C=O) (6a-g). These include 5'-Methyl-5'-[(9H-purin-9-yl)methyl]-2'-oxo-3'-methylenetetrahydrofur ans (6a), 5'-Methyl-5'-[(chloro-9H-purin-9-yl)methyl]-2'-oxo-3'- methylenetetrahydrofurans (6b), 5'-Methyl-5'-[(6-iodo-9H-purin-9-yl) methyl]-2'-oxo-3'-methylenetetrahydrofurans (6c), 5'-Methyl-5'-[(6-methyl-9H-purin-9-yl) methyl]-2'-oxo-3'-methylenetetrahydrofurans (6d), 5'-Methyl-5'-[(9H-adenin-9-yl)methyl]-2'-oxo-3-methylenetetrahy drofurans (6e), 5'-Methyl-5'-[(6-mercapto-9H-purin-9-yl) methyl]-2'-oxo-3'-methylenetetrahydrofurans (6f) and 5'-Methyl-5'-[(9H-hypoxanthin-9-yl)methyl]-2'-oxo-3'- methylenetetrahydrofurans (6g) which were made by the Reformatsky-type reaction of ethyl alpha-(bromomethyl) acrylate with the corresponding (6-substituted-9H-purin-9-yl)-2-propanone intermediates (5a-g). These ketone intermediates 5a-g, 1-(9H-purin-9-yl)-2-propanone (5a), 1-(6-chloro-9H-purin-9-yl)-2-propanone (5b), 1-(6-iodo-9H-purin-9-yl)-2-propanone (5c), 1-(6-methyl-9H-purin-9-yl)-2-propanone (5d), 1-(9H-adenin-9-yl)-2-propanone (5e), 1-(6-mercapto-9H-purin-9-yl)-2-propanone (5f), and 1-(9H-hypoxanthin-9-yl)-2-propanone (5g) were directly obtained by the alkylation of the 6-substituted purine bases with the chloroacetone in the presence of K2CO3 (or NaH) under DMF (or DMSO). The preliminary in vitro cytotoxicity assay for the synthetic alpha-methylene-gamma-butyro-lactone compounds (6a-g) were determined against three cell lines (PM-3A, P-388, and K-562) and showed the moderate antitumor activity (IC50 ranged from 1.4 to 4.3 micrograms/ml) with the compound 5'-methyl-5'-[(9H-hypoxanthin-9-yl)methyl]-2'-oxo-3'- methylenetetrahydrofuran (6g) showing the least antitumor activity.

4-Butyrolactone↗
PubMed1968

Nitrosamine-induced carcinogenesis. The alklylation of nucleic acids of the rat by N-methyl-N-nitrosourea, dimethylnitrosamine, dimethyl sulphate and methyl methanesulphonate.

1. N[(14)C]-Methyl-N-nitrosourea, [(14)C]dimethylnitrosamine, [(14)C]dimethyl sulphate and [(14)C]methyl methanesulphonate were injected into rats, and nucleic acids were isolated from several organs after various time-intervals. Radioactivity was detected in DNA and RNA, partly in major base components and partly as the methylated base, 7-methylguanine. 2. No 7-methylguanine was detected in liver DNA from normal untreated rats. 3. The specific radioactivity of 7-methylguanine isolated from DNA prepared from rats treated with [(14)C]dimethylnitrosamine was virtually the same as that of the dimethylnitrosamine injected. 4. The degree of methylation of RNA and DNA produced in various organs by each compound was determined, and expressed as a percentage of guanine residues converted into 7-methylguanine. With dimethylnitrosamine both nucleic acids were considerably more highly methylated in the liver (RNA, about 1% of guanine residues methylated; DNA, about 0.6% of guanine residues methylated) than in the other organs. Kidney nucleic acids were methylated to about one-tenth of the extent of those in the liver, lung showed slightly lower values and the other organs only very low values. N-Methyl-N-nitrosourea methylated nucleic acids to about the same extent in all the organs studied, the amount being about the same as that in the kidney after treatment with dimethylnitrosamine. In each case the RNA was more highly methylated than the DNA. Methyl methanesulphonate methylated the nucleic acids in several organs to about the same extent as N-methyl-N-nitrosourea, but the DNA was more highly methylated than the RNA. Dimethyl sulphate, even in toxic doses, gave considerably less methylation than N-methyl-N-nitrosourea in all the organs studied, the greatest methylation being in the brain. 5. The rate of removal of 7-methylguanine from DNA of kidneys from rats treated with dimethylnitrosamine was compared with the rate after treatment of rats with methyl methanesulphonate. No striking difference was found. 6. The results are discussed in connexion with the organ distribution of tumours induced by the compounds under study and in relation to the possible importance of alkylation of cellular components for the induction of cancer.

Animals↗
PubMed2006

Epigenetic regulation of metallothionein-i gene expression: differential regulation of methylated and unmethylated promoters by DNA methyltransferases and methyl CpG binding proteins.

Metallothioneins (MTs) are a group of cysteine-rich stress response proteins that scavenge reactive oxygen species and heavy metals. Recently, we have shown that MT-I promoter is methylated and suppressed in some solid and liquid tumors and can be robustly activated following treatment with inhibitors of DNA methyltransferase (DNMT) and histone deacetylase (HDAC). Here, we have analyzed MT-I chromatin structure in active, unmethylated (Hepa cells) and in repressed, methylated state (lymphosarcoma cells). Restriction enzyme accessibility assay showed that the MT-I promoter has an open conformation in unmethylated state as opposed to refractory chromatin structure in methylated state. Positioning of nucleosomal arrays on the methylated promoter further confirmed the closed chromatin structure of the methylated promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated that the unmethylated promoter is associated with K9-acetyl, K4-methyl, and S10-phospho histone H3 whereas the methylated promoter is predominantly associated with K9-methyl H3. HP1alpha that recognizes K9-methyl H3 inhibited methylated MT-1 promoter activity whereas closely related HP1gamma repressed the promoter irrespective of its methylation status. Ubiquitously expressed DNA methyltransferase 1 (DNMT1) suppressed MT-I promoter activity irrespective of its methylation status that does not require its catalytic activity. The DNMT1-mediated repression of MT-I promoter was relieved by trichostatin A, an HDAC inhibitor. Among the methyl CpG binding proteins, MBD2 and MBD4 specifically associated with the methylated promoter and inhibited its activity. In contrast, MBD1 and MeCP2 interacted with both promoters and suppressed the promoter activity irrespective of its methylation status. These results demonstrate that the methylated and unmethylated MT-I promoter are differentially regulated by DNA methyltransferase and methyl-CpG binding proteins, and DNMT1 could suppress MT promoter by a transcriptional mechanism independent of its enzymatic function. These studies suggest that the components of epigenetic machinery differentially regulate methylated and unmethylated MT-I gene expression.

Animals↗
PubMed2002

Interplay between two epigenetic marks. DNA methylation and histone H3 lysine 9 methylation.

BACKGROUND: The heterochromatin of many eukaryotes is marked by both DNA methylation and histone H3 lysine 9 (H3-K9) methylation, though the exact relationship between these epigenetic modifications is unknown. In Neurospora, H3-K9 methylation is required for the maintenance of all known DNA methylation. In Arabidopsis, H3-K9 methylation directs some of the CpNpG and asymmetric methylation. However, it is not known in any organism whether DNA methylation may also direct histone H3 methylation. RESULTS: Using chromatin immunoprecipitation (ChIP) assays, we show that Arabidopsis heterochromatin is associated with H3-K9 methylation. This histone methylation is dependent on the KRYPTONITE and DDM1 genes (SU[VAR]3-9 and SWI2/SNF2 homologs, respectively). We also find that a decrease in DNA methylation does not directly cause a loss of H3-K9 methylation. Instead, a decrease in H3-K9 methylation is only seen at loci where transcription is derepressed. CONCLUSIONS: We conclude that DNA methylation does not control the methylation of histone H3-K9. We propose that loss of H3-K9 methylation is due to transcriptional reactivation, coupled with deposition of unmethylated nucleosomes. These findings are consistent with recent observations of DNA replication-independent deposition of histone H3.3 in Drosophila. Our results also suggest that, in Arabidopsis, DNA methylation is sufficient for gene silencing, but H3-K9 methylation is not.

Arabidopsis↗
PubMed2000

Novel ring A stereoisomers of 2-methyl-1alpha,25-dihydroxyvitamin D(3) and 2-methyl-20-epi-1alpha,25-dihydroxyvitamin D(3): transactivation of target genes and modulation of differentiation in human promyelocytic leukemia (HL-60) cells.

We evaluated the biological activity of two sets of ring A stereoisomers of 2-methyl-1alpha,25-dihydroxyvitamin D(3) (2-methyl-1alpha,25(OH)(2)D(3)) and 2-methyl-20-epi-1alpha, 25-dihydroxyvitamin D(3) (2-methyl-20-epi-1alpha,25(OH)(2)D(3)) in terms of the following: transactivation of a rat 25-hydroxyvitamin D(3)-24-hydroxylase gene promoter including two vitamin D response elements (VDREs) and a human osteocalcin gene promoter including a VDRE in transfected human osteosarcoma (MG-63) cells; a vitamin D receptor (VDR)-mediated response using a VDR-GAL4 one-hybrid luciferase reporter system and a retinoid X receptor alpha (RXRalpha)-mediated response using an expressed VDR/RXRalpha-GAL4 modified two-hybrid luciferase reporter system in transfected human epitheloid carcinoma, cervix (HeLa) cells; and modulation of cell surface CD11b antigen expression in human leukemia (HL-60) cells. All the diastereomers of both analogues exhibited unique biological activity profiles depending upon the configurations of the C-1 and C-3 hydroxyl groups, the C-2 methyl group in ring A, and the C-20 methyl group in the side chain. Of the eight possible diastereomers of the 2-methyl analogues, 2alpha-methyl-1alpha,25(OH)(2)D(3) was the most potent and exhibited comparable or even greater biological potency than 1alpha,25(OH)(2)D(3). Of the eight possible diastereomers of the 2-methyl-20-epi analogues, 2alpha-methyl-20-epi-1alpha,25(OH)(2)D(3) was the most potent and exhibited 100- to 200-fold higher transcriptional potencies than 1alpha,25(OH)(2)D(3) and exceptionally high cell regulatory activities. 2beta-methyl-20-epi-1alpha,25(OH)(2)D(3) was nearly as potent as its 2-epimer, 2alpha-methyl-20-epi-1alpha,25(OH)(2)D(3), whereas its 20-epimer, 2beta-methyl-1alpha,25(OH)(2)D(3), was almost completely biologically inactive. In these respects, it can be postulated that the double modification of 2-methyl substitution and 20-epimerization to 1alpha,25(OH)(2)D(3) induces remarkable changes in a VDR/RXRalpha/VDRE-mediated signaling response and greatly enhances biological activity. The other striking finding was that 2beta-methyl-20-epi-3-epi-1beta,25(OH)(2)D(3) is transcriptionally more active than 1alpha,25(OH)(2)D(3) despite lacking the 1alpha-hydroxyl group, which was believed to be essential for expressing VDR-mediated gene transcription. Since the C-20 natural counterpart, 2beta-methyl-3-epi-1beta,25(OH)(2)D(3), was almost completely biologically inactive, 20-epimerization is probably responsible for activation of gene expression. Although earlier extensive structure-activity studies of vitamin D analogues showed stereochemistry at the C-1, C-3, and C-20 of 1alpha,25(OH)(2)D(3) to be the key structural motif for vitamin D action, our results clearly demonstrated that stereochemistry at the C-2 is also an important structural motif for vitamin D action and imply that 2-methyl substitution possibly induces conformational changes in ring A depending upon the combinations of configurations of the C-1 and C-3 hydroxyl groups with C-20 stereochemistry. Consequently, several of these analogues exhibit exceptionally high or unexpected biological activities at the molecular and cellular levels. These results suggest that 2-methyl substitution together with alterations of stereochemistry in both ring A and the side chain of 1alpha, 25(OH)(2)D(3) will provide useful analogues for structure-activity studies and development of therapeutic agents with unique biological activity profiles.

Animals↗
PubMed2003

Methyl-group dietary intake and risk of breast cancer among African-American women: a case-control study by methylation status of the estrogen receptor alpha genes.

OBJECTIVES: Recent molecular studies show that the absence of estrogen receptor (ER) alpha gene expression in breast cancer is associated with methylation of the CpG island located in the 5' region and the first exon of the ER alpha gene. Because CpG island methylation is an early event in carcinogenesis and because a methyl-deficient diet may lead to abnormal DNA methylation including CpG island methylation, we hypothesized that a methyl-deficient diet is more likely to be associated with breast cancer with methylated ER alpha gene CpG islands. This study aimed to test this hypothesis in African-American women using a case-control design. METHODS: Cases were 304 African-American women pathologically diagnosed with breast cancer during 1995-1998 who lived in three Tennessee counties. Controls were 305 African-American women without breast cancer, who were selected through random-digit dialing and frequency matched to cases by 5-year age range and county of residence. Information on dietary intake and other risk factors was collected through telephone interviews. Dietary methyl-components were defined based on folate and methionine intakes and alcohol consumption. Tumor tissue samples were collected for measuring methylation status of the ER alpha gene. RESULTS: Our results showed that the odds ratio (OR) estimates for lower dietary folate intake were 2.0 (95% confidence interval, CI: 0.8-4.8) for cases with a methylated ER alpha gene, 0.6 (95% CI: 0.3-1.5) for cases with an unmethylated ER alpha gene, and 1.6 (95% CI: 0.7-3.8) for cases with unknown methylation status (presumably including cases with both methylated and un-methylated genes). However, low methionine intake appeared more likely to be associated with tumors with unknown methylation status and high level of alcohol consumption seemed more likely to be related to tumors with un-methylated genes. CONCLUSIONS: These results did not show a pattern consistent with the study hypothesis that methyl-deficient diets are more likely related to breast cancer with a methylated ER gene.

Adult↗
PubMed2002

Diet, methyl donors and DNA methylation: interactions between dietary folate, methionine and choline.

DNA methylation influences the expression of some genes and depends upon the availability of methyl groups from S-adenosylmethionine (SAM). Dietary methyl groups derive from foods that contain methionine, one-carbon units and choline (or the choline metabolite betaine). Humans ingest approximately 50 mmol of methyl groups per day; 60% of them are derived from choline. Transmethylation metabolic pathways closely interconnect choline, methionine, methyltetrahydrofolate (methyl-THF) and vitamins B-6 and B-12. The pathways intersect at the formation of methionine from homocysteine. Perturbing the metabolism of one of these pathways results in compensatory changes in the others. For example, methionine can be formed from homocysteine using methyl groups from methyl-THF, or using methyl groups from betaine that are derived from choline. Similarly, methyl-THF can be formed from one-carbon units derived from serine or from the methyl groups of choline via dimethylglycine, and choline can be synthesized de novo using methyl groups derived from methionine (via SAM). When animals and humans are deprived of choline, they use more methyl-THF to remethylate homocysteine in the liver and increase dietary folate requirements. Conversely, when they are deprived of folate, they use more methyl groups from choline, increasing the dietary requirement for choline. The availability of transgenic and knockout mice has made possible additional studies that demonstrate the interrelationship of these methyl sources. In summary, as we consider dietary requirements and possible effects on DNA methylation, it is important to realize that methionine, methyl-THF and choline can be fungible sources of methyl groups, and the design of our studies should reflect this.

Animals↗
PubMed1988

The role of glutathione in the detoxification of the insecticides methyl parathion and azinphos-methyl in the mouse.

The dimethyl-substituted organothiophosphate insecticides methyl parathion and azinphos-methyl are thought to undergo glutathione-mediated detoxification in mammals. In the present study, depletion of hepatic glutathione in the mouse by pretreatment with diethyl maleate potentiated the acute toxicities of methyl parathion and azinphos-methyl, whereas depletion of hepatic glutathione by pretreatment with buthionine sulfoximine did not. Furthermore incubation of 50 microM methyl parathion with mouse hepatic microsomes for 5 min in the presence of 1 mM diethyl maleate led to significantly greater (p less than 0.05) production of methyl paraoxon, compared to incubations in the absence of diethyl maleate. Conversely, 1 mM diethyl maleate had no effect on metabolic activation of azinphos-methyl by mouse hepatic microsomes, while 10 mM inhibited slightly production of azinphos-methyl oxon from azinphos-methyl. These results suggest normal levels of hepatic glutathione are not required for detoxification of methyl parathion or azinphos-methyl in the mouse. Moreover the potentiation of the acute toxicity of methyl parathion following diethyl maleate pretreatment could result, at least in part, from enhanced production of methyl paraoxon. However, diethyl maleate likely acts through another mechanism(s) as well since it did not enhance the metabolic activation of azinphos-methyl in vitro. These data raise serious doubts about the participation of glutathione in the detoxification of methyl parathion and azinphos-methyl in vivo in the mouse.

Animals↗
PubMed1990

Protein carboxyl methylation and methyl ester turnover in density-fractionated human erythrocytes.

A widely distributed methyltransferase modifies protein D-aspartyl and L-isoaspartyl residues which arise spontaneously as proteins age. Protein carboxyl methylation reactions were analyzed in human erythrocytes which had been separated on density gradients, a procedure which provides fractions enriched in older cells in the denser areas of the gradient. The total flux of methyl groups through the carboxyl methylation pathway was monitored by incubating cells from each fraction with L-[methyl-3H]methionine and measuring the formation of both protein [3H]methyl esters and [3H]methanol, derived from the hydrolysis of protein [3H]methyl esters in vivo. Cells isolated from denser areas of the gradient showed progressively higher rates of both protein carboxyl methylation and methanol production. In all cases, only 10-20% of the total methyl groups transferred were still present as intact protein [3H]methyl esters, consistent with the rapid hydrolysis of protein methyl esters in erythrocytes of all ages. The total flux of methyl groups through the carboxyl methylation pathway was approximately 3-fold higher in cells isolated from densest areas of the gradient compared to cells isolated from least dense areas of the gradient. Increases of a similar magnitude were observed in the numbers of both membrane protein carboxyl methyl esters and cytosolic protein carboxyl methyl esters. The only protein whose methylation was unchanged in denser cells was a 35,000 Da cytosolic protein. It has been proposed that protein carboxyl methyl esters are intermediates in either the repair or metabolism of structurally damaged proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

S1 SINE retroposons are methylated at symmetrical and non-symmetrical positions in Brassica napus: identification of a preferred target site for asymmetrical methylation.

DNA methylation has been often proposed to operate as a genome defence system against parasitic mobile elements. To test this possibility, the methylation status of a class of plant mobile elements, the S1Bn SINEs, was analysed in detail using the bisulfite modification method. We observed that S1Bn SINE retroposons are methylated at symmetrical and asymmetrical positions. Methylated cytosines are not limited to transcriptionally important regions but are well distributed along the sequence. S1Bn SINE retroposons are two-fold more methylated than the average methylation level of the Brassica napus nuclear DNA. By in situ hybridization, we showed that this high level of methylation does not result from the association of S1Bn elements to genomic regions known to be highly methylated suggesting that S1Bn elements were specifically methylated. A detailed analysis of the methylation context showed that S1Bn cytosines in symmetrical CpG and CpNpG sites are methylated at a level of 87% and 44% respectively. We observed that 5.3% of S1Bn cytosines in non-symmetrical positions were also methylated. Of this asymmetrical methylation, 57% occurred at a precise motif (Cp(A/T)pA) that only represented 12% of the asymmetrical sites in S1Bn sequences suggesting that it represents a preferred asymmetrical methylation site. This motif is methylated in S1Bn elements at only half the level observed for the Cp(A/T)pG sites. We show that non-S1Bn CpTpA sites can also be methylated in DNA from B. napus and from other plant species.

5-Methylcytosine↗
PubMed2004

Methylation of the retinoid response gene TIG1 in prostate cancer correlates with methylation of the retinoic acid receptor beta gene.

Methylation of CpG islands and associated gene silencing may lead to malignant progression, but the mechanisms of CpG island methylation in cancer are unknown. The tazarotene-induced gene 1 (TIG1), also known as retinoid acid (RA) receptor-responsive 1 gene was first identified as an RA-responsive gene and was shown to be downregulated in prostate cancer. Here, we show that this downregulation is caused by the methylation of the promoter and CpG island of TIG1. TIG1 was methylated in 26 of 50 (52%) primary prostate cancers, but was not methylated in normal tissues or benign hyperplasias. Three of four tumors that metastasized, five of six that were poorly differentiated and all that were assigned a Gleason score higher than 8 (7/7) were methylated in the promoter of TIG1. The samples with peripheral invasion were more frequently methylated (21/32, 66%) than tissues without peripheral invasion (5/18, 28%). In addition, Gleason 7-10 cancers (21/30, 70%) were significantly more frequently methylated compared with Gleason 4-6 cancers (4/18, 22%) (P<0.01). The retinoic acid receptor beta (RAR-beta) gene was frequently methylated as well (42/50, 84%). When TIG1 showed methylation, RAR-beta was also methylated (25/26 samples). In almost all samples where RAR-beta was not methylated, TIG1 was also in an unmethylated state (14/15 samples). The methylation of TIG1 and RAR-beta was positively correlated (r=0.35; P=0.017). It is possible that the methylation of the retinoid response gene TIG1 occurred in response to the methylation and inactivation of RAR-beta. These observations may contribute to our understanding of mechanistic events leading to CpG island methylation in cancer.

Carcinoma↗
PubMed2004

Electrophysiological characterization of the SK channel blockers methyl-laudanosine and methyl-noscapine in cell lines and rat brain slices.

We have recently shown that the alkaloid methyl-laudanosine blocks SK channel-mediated afterhyperpolarizations (AHPs) in midbrain dopaminergic neurones. However, the relative potency of the compound on the SK channel subtypes and its ability to block AHPs of other neurones were unknown. Using whole-cell patch-clamp experiments in transfected cell lines, we found that the compound blocks SK1, SK2 and SK3 currents with equal potency: its mean IC(50)s were 1.2, 0.8 and 1.8 microM, respectively. IK currents were unaffected. In rat brain slices, methyl-laudanosine blocked apamin-sensitive AHPs in serotonergic neurones of the dorsal raphe and noradrenergic neurones of the locus coeruleus with IC(50)s of 21 and 19 microM, as compared to 15 microM in dopaminergic neurones. However, at 100 microM, methyl-laudanosine elicited a constant hyperpolarization of serotonergic neurones of about 9 mV, which was inconsistently (i.e. not in a reproducible manner) antagonized by atropine and hence partly due to the activation of muscarinic receptors. While exploring the pharmacology of related compounds, we found that methyl-noscapine also blocked SK channels. In cell lines, methyl-noscapine blocked SK1, SK2 and SK3 currents with mean IC(50)s of 5.9, 5.6 and 3.9 microM, respectively. It also did not block IK currents. Methyl-noscapine was slightly less potent than methyl-laudanosine in blocking AHPs in brain slices, its IC(50)s being 42, 37 and 29 microM in dopaminergic, serotonergic and noradrenergic neurones, respectively. Interestingly, no significant non-SK effects were observed with methyl-noscapine in slices. At a concentration of 300 microM, methyl-noscapine elicited the same changes in excitability in the three neuronal types than did a supramaximal concentration of apamin (300 nM). Methyl-laudanosine and methyl-noscapine produced a rapidly reversible blockade of SK channels as compared with apamin. The difference between the IC(50)s of apamin (0.45 nM) and methyl-laudanosine (1.8 microM) in SK3 cells was essentially due to a major difference in their k(-1) (0.028 s(-1) for apamin and >or=20 s(-1) for methyl-laudanosine). These experiments demonstrate that both methyl-laudanosine and methyl-noscapine are medium potency, quickly dissociating, SK channel blockers with a similar potency on the three SK subtypes. Methyl-noscapine may be superior in terms of specificity for the SK channels.

Animals↗
PubMed2006

Bis(1-methyl-1H-tetrazol-5-yl)diazene and two its copper(I) chloride complexes poly[[[mu-1,2-bis(1-methyl-1H-tetrazol-5-yl)diazene-kappa4N',N4:N,N4']dicopper(I)]-di-mu-chloro] and catena-poly[[chlorocopper(I)]-mu-1,2-bis(1-methyl-1H-tetrazol-5-yl)diazene-kappa4N',N4:N,N4'].

While bis(1-methyl-1H-tetrazol-5-yl)diazene, C4H6N10, (I), has no crystallographically imposed symmetry, in the two title chlorocopper(I) complexes, [Cu2Cl2(C4H6N10)]n, (II), and [CuCl(C4H6N10)]n, (III), the organic ligands lie across centres of inversion; in (III), the Cu and Cl atoms additionally lie about a twofold rotation axis in the space group P2/c. Complex (II) forms a two-dimensional coordination polymer containing tetrahedrally coordinated Cu(I) atoms, and complex (III) forms a one-dimensional coordination polymer containing five-coordinate square-pyramidal Cu(I) atoms.

Journal Article↗
PubMed2001

Studies of molecular structure parameters of 20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol and its N-methyl derivative: two inhibitors of Delta24(25) sterol methyl transferase and Delta24(24') sterol methyl reductase of Trypanosoma cruzi.

Molecular structural parameters of two potential drugs against Trypanosoma cruzi epimastigotes, 20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol (1) and 20-N-methylpiperidin-2-yl-5alpha-pregnan-3beta, 20-diol (2) were studied using a combination of a stereoselective synthetic route, spectroscopic characterization and single-crystal X-ray analysis. Both compounds were synthesized with an R configuration at C20. This chirality is a consequence of the stereoselectivity observed during the formation of the intermediate 20-pyridin-2-yl-5alpha-pregnan-3beta,20R-diol (4). NMR data indicated that the six-membered aza ring of (2) is conformationally more restrained, in CDCl3 solution, than (1). X-ray studies showed that maximum deviations among structural molecular parameters of (1) and (2) correspond to torsion angles along the C20-C22 bonds, leading to a different relative orientation of the N atom; a critical structural parameter for the binding properties of aza-sterols to Delta(24(25)) sterol methyl transferase. Cremer-Pople parameters of the five-membered rings of (1) and (2) lie in the observed range for a family of tetracyclic fused ring systems retrieved from the CSD. The phi2 parameter of (1) lies just on the mean of the family, while phi2 of (2) deviates significantly towards the lower limit.

Animals↗
PubMed2001

Identification of methyl halide-utilizing genes in the methyl bromide-utilizing bacterial strain IMB-1 suggests a high degree of conservation of methyl halide-specific genes in gram-negative bacteria.

Strain IMB-1, an aerobic methylotrophic member of the alpha subgroup of the Proteobacteria, can grow with methyl bromide as a sole carbon and energy source. A single cmu gene cluster was identified in IMB-1 that contained six open reading frames: cmuC, cmuA, orf146, paaE, hutI, and partial metF. CmuA from IMB-1 has high sequence homology to the methyltransferase CmuA from Methylobacterium chloromethanicum and Hyphomicrobium chloromethanicum and contains a C-terminal corrinoid-binding motif and an N-terminal methyltransferase motif. However, cmuB, identified in M. chloromethanicum and H. chloromethanicum, was not detected in IMB-1.

Alphaproteobacteria↗
PubMed2000

N-acetyltransferase-dependent activation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine: formation of 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo [4,5-b]pyridine, a possible biomarker for the reactive dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking. PhIP is metabolically activated to the ultimate mutagenic metabolite by CYP P450-mediated N-hydroxylation followed by phase II esterification. Incubation of N-hydroxy-PhIP (N-OH-PhIP) with cytosol, acetyl coenzyme A (AcCoA) and 2'-deoxyguanosine for 24 h resulted in the formation of three different adducts:N(2)-(deoxyguanosin-8-yl)-PhIP, N(2)-(guanosin-8-yl)-PhIP and PhIP-xanthine. One additional product, 5-hydroxy-PhIP (5-OH-PhIP), was also identified in the incubation mixtures. 5-hydroxy-PhIP is formed as a degradation product of conjugates formed from N-acetoxy-PhIP and protein, glutathione or buffer constituents. A similar spectrum of products was obtained using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) instead of acetyl CoA. Addition of glutathione (3 mM) to the incubation mixture resulted in a 50% reduction in both adducts and 5-hydroxy-PhIP formation in liver cytosol. The main product detected was PhIP, suggesting glutathione-dependent reduction of the N-acetoxy-PhIP. Addition of glutathione to incubation mixtures from the other cytosolic preparations had less dramatic effects. In addition, increasing the amount of N-OH-PhIP in the incubation mixture resulted in proportional increased amounts of total adducts and 5-OH-PhIP. Incubation of rat and human S9 with PhIP resulted in the formation of only traces of 5-OH-PhIP. Fortification with AcCoA clearly increased the formation of 5-OH-PhIP. Addition of the CYP 450 1A2 inhibitor, furafylline, completely inhibited the formation of 5-OH-PhIP in incubations with human S9. These results indicate that both PhIP adducts and 5-OH-PhIP are formed by similar routes of activation of N-OH-PhIP. 5-OH-PhIP may therefore serve as a biomarker for the formation of the ultimate mutagenic metabolite of PhIP. A rat dosed orally with PhIP excreted 1% of the dose as 5-OH-PhIP in the urine at 24 h and 0.05 and 0.01% at 48 and 72 h, respectively. This shows that 5-OH-PhIP is also formed in vivo and indicates the possible use of 5-OH-PhIP as a urinary biomarker.

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PubMed1995

[Modulation of the antitumor activity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosoure a by O(6)-methyl-2'-deoxyguanosine--a new inhibitor of O(6)-alkylguanine-DNA-alkyltransferase].

O6-Methyl-2'-deoxyguanosine (O6-MedG), a novel inhibitor of O6-alkylguanine-DNA alkyltransferase (O6-AGT), has been synthesized. The ability of O6-MedG to deplete the O6-AGT activity in leukemia L1210 and melanoma B16 cells in vivo has been studied. After intraperitoneal administration of O6-MedG to mice bearing leukemia L1210 or melanoma B16, the activity of O6-AGT in tumour cells decreased by 50%. Pretreatment of leukemia L1210 bearing mice with O6-MedG (200 mg/kg) 24 hours prior to ACNU (15 mg/kg) administration resulted in six out of seven 60-day survivors. Treatment of mice with ACNU (15 mg/kg) alone increased the life span by 200%. Treatment of melanoma B16 bearing mice with O6-MedG and 3 hours thereafter with ACNU resulted in a 50% inhibition of tumour growth, whereas the inhibiting effect of ACNU alone was 16%. There was no difference in leukemia growth when L1210/BCNU bearing mice were treated with O6-MedG followed by ACNU treatment. In vivo ACNU (15 mg/kg) produced a deep and prolonged inhibition of DNA, RNA and protein synthesis in leukemia L1210 cells. The DNA synthesis in leukemia L1210/BCNU cells was shown to recover more rapidly than in L1210 cells. The activities of DNA-polymerases alpha and beta and, especially, of O6-AGT were elevated in ACNU-resistant leukemia cells as compared with ACNU-sensitive cells. The activation of some repairing enzymes, such as O6-AGT, DNA-polymerases alpha and beta as well as increased levels of GSH may play a role in the development of drug resistance to ACNU.

Animals↗