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At least 19 recordsLinked to original sources
PubMed1979

Attractants and repellents control demethylation of methylated chemotaxis proteins in Escherichia coli.

A group of methylated proteins, the methyl-accepting chemotaxis proteins (MCP), has been shown to play a central role in bacterial chemotaxis. Both methylation and demethylation of MCP occur continuously in the absence of added stimuli; these two processes are in balance such that a basal level of methylation is maintained. Attractants cause the methylation level to increase to a new value, whereas repellents bring about a decrease in level. Therefore, attractants and repellents must somehow perturb the balance between methylation and demethylation of MCP. In this report the effect of attractants on demethylation of MCP was monitored in two ways: (i) by following the disappearance of [methyl-3H]MCP and (ii) by measuring formation of [3H]methanol, the product of MCP demethylation. Both methods showed that addition of attractants causes a transient inhibition of MCP demethylation. Repellent addition has previously been shown to stimulate MCP demethylation. It is therefore concluded that control of demethylation plays a crucial role in changing the level of methylation of MCP in response to attractants and repellents.

Bacterial Proteins↗
PubMedDate not supplied

Rabbit hepatic microsomal O-demethylation of chlorotrianisene.

The in vitro hepatic O-demethylation of the nonsteroidal estrogen chlorotrianisene (CTA) has been studied. The rate of O-demethylation was maximal at 0.4 mM NADPH. Although NADH did not catalyze the reaction alone, it had a synergistic effect in the presence of equimolar amounts of NADPH. Carbon monoxide decreased the rate of O-demethylation. Nicotinamide was found to decrease the O-demethylation rate at a concentration of 40 mM, but had no apparent effect at concentrations of 20 mM or lower. Extracts from incubation mixtures contained one major (mono-O-demethylated) and a minor (bis-O-demethylated) metabolite. Extracts of mixtures containing soluble rather than microsomal enzymes or from mixtures in which microsomal protein had been denatured did not contain these metabolites.

Animals↗
PubMed1978

Demethylation in erythrocytes: a reaction involving hemoglobin.

A reaction is described in erythrocytes of rat and of man whereby O-methylated metabolites of the catecholamines are demethylated to the corresponding catechols. The reaction was studied by incubating aliquots of erythrocyte lysates with radiolabeled O-methylated compounds and isolating the catechol product by alumina adsorption chromatography. The demethylating activity was located in the cytosol of the erythrocytes. Evidence was strong that oxyhemoglobin was responsible for the reaction: the demethylase activity was inseparable from oxyhemoglobin in several chromatographic separations. In addition, although commercially available hemoglobins were inactive in the reactions, after their conversion to oxyhemoglobin and purification, they did effect demethylation. Methemoglobin did not demethylate guaiacols and in fact inhibited demethylation by oxyhemoglobin. The reaction was inhibited by the addition of reduced pyridine nucleotides and of the methyl acceptor tetrahydrofolic acid.

Animals↗
PubMed1978

Pharmacological studies on N-demethylated carbachol.

In attempts to find a drug more active than pilocarpine, the tertiary nitrogen derivative of carbachol, N-demethylated carbachol, was synthetized and tested on several autonomic nervous system preparations. N-Demethylated carbachol was active at muscarinic and nicotinic sites in vivo and in vitro. In superfusion studies, N-demethylated carbachol contracted the smooth muscle of the guinea pig ileum as well as skeletal muscles of frog recus abdominis and chick biventer cervicis. N-Demethylated carbachol decreased blood pressure in the rat, with an ED50 ("/- SEM) of 4.82 +/- 0.78 mg/kg. After close arterial injection to the cat superior cervical ganglion, N-demethylated carbachol elicited contractions of the nictitating membrane (ED50 of 1.68 +/- 0.24 mg/kg) that were not significantly affected by atropine. N-D-methylated carbachol stimulated salivation in dog Wharton duct preparations with an ED50 of 2.55 +/- 0.81 mg/kg. In contrast, pilocarpine had no effects on skeletal muscles in vitro, produced ganglionic effects blocked by atropine, had a prominent effect on salivation, and tended to elevate blood pressure.

Animals↗
PubMed1979

Attractants and repellents influence methylation and demethylation of methyl-accepting chemotaxis proteins in an extract of Escherichia coli.

During bacterial chemotaxis, attractants and repellents alter the methylation levels of the methyl-accepting chemotaxis proteins (MCPs). These methylation levels represent a balance between two enzymatic processes: methylation and demethylation. In vivo experiments previously have shown that chemoeffectors influence the demethylation process; effects on the methylation system have not been reported. Here we show that in a cell-free extract of Escherichia coli both methylation and demethylation of the MCPs are affected by attractants and repellents. Attractants enhance methylation and inhibit demethylation. Repellents inhibit methylation and stimulate demethylation. The cell-free system provides an opportunity for further study of the mechanisms by which attractants and repellents influence the levels of methylation of the MCPs.

Bacterial Proteins↗
PubMed1979

The involvement of cytochrome P-488 and P-450 in NADH-dependent O-demethylation of p-nitroanisole in rat liver microsomes.

These studies have shown that addition of p-nitroanisole to a reaction mixture containing rat liver microsomes resulted in an increase the reoxidation rate of NADH-reduced cytochrome b5. Fortification of rat liver microsomes with partially purified cytochrome b5 produces an increase in both NADPH-dependent and NADH-dependent p-nitroanisole O-demethylation activity. Antiserum to cytochrome P-450 isolated from phenobarbital-treated rat liver microsomes inhibited the NADH-dependent O-demethylation activity as well as the NADPH-dependent O-demethylation activity seen in rat liver microsomes. Addition of either purified cytochrome P-450 or cytochrome P-448 to an incubation mixture containing phenobarbital-treated rat liver microsomes enhanced the NADH-dependent p-nitroanisole O-demethylation activity. These results suggest that NADH-dependent and, in part, NADPH-dependent O-demethylations are catalyzed by cytochrome P-448 and cytochrome P-450 receiving electrons from cytochrome b5.

Animals↗
PubMed1976

The binding of the optical isomers of methadone, alpha-methadol, alpha-acetylmethadol and their N-demethylated derivatives to the opiate receptors of rat brain.

The optical isomers of methadone, alpha-methadol, alpha-acetylmethadol and their N-demethylated derivatives have been systematically studied for their effects on the binding of 3H-dihydromorphine (3H-DHM) and 3H-naloxone (3H-NLX) to opiate receptors in rat brain homogenate. The relative affinities of these agents in competing for both 3H-DHM and 3H-NLX binding parallel their analgesic effects. 1-Methadone is about 30 times as effective as d-methadone in competing for both 3H-DHM and 3H-NLX binding sites. The reduction of 1-methadone to alpha-d-methadol and subsequent N-demethylation to alpha-d-normethadol reduce its effectiveness as indicated by the increase in the IC50 values for both 3H-DHM and 3H-NLX binding. The reduction of d-methadone followed by N-demethylation produces a potent derivative, alpha-1-normethadol, which has IC50 values on 3H-DHM and 3H-NLX binding similar to those of 1-methadone. The affinity of alpha-1-acetyl-methadol on the binding of both 3H-ligands falls between those of 1-methadone and d-methadone, and increases as it is N-demethylated. alpha-d-Acetyl-methadol is more effective than alpha-1-acetylmethadol in competing for both 3H-ligands from the opiate receptors, and its affinity, unlike that of alpha-1-acetylmethadol, decreases when it is N-demethylated. The affinities of the methadone isomers and related compounds on the binding of 3H-NLX fall in the presence of Na+. The latter property indicates the agonistic nature of this series of druges.

Animals↗
PubMed1976

Species and phenobarbitone-induced differences in the kinetic constants of liver microsomal harmine O-demethylation.

1. The apparent kinetic constants for the O-demethylation of harmine to harmol by 10 000 g supernatant fractions from livers of mice, rats, guinea-pigs, rabbits, cats and cows have been determined. The Km values were 10-39 muM and Vmax 0-25 and 1-65 nmol/mg protein/min. 2. Optimal conditions of incubation time and NADP requirements differed between species. In all species except cat and cow the rate of O-demethylation of harmine was linear for 5 min, but in the latter species was linear for 15 min. Maximum stimulation of O-demethylation occurred at NADP concn. of between 50 and 375 muM. 3. Phenobarbitone pre-treatment of weanling, young adult and mature adult mice increased the Vmax for O-demethylation by 2.9- to 4.6-fold but did not change Km. Increased Vmax values were greatest in young and least in old mice and these changes were directly correlated with a decrease of hexobarbitone sleeping time.

Animals↗
PubMed1979

Microsomal mediated metabolism of dialkylaryltriazenes. I. Demethylation of ring halogenated 3,3-dimethyl-1-phenyltriazenes.

The oxidative N-demethylation was investigated for a series of 3,3-dimethyl-l-phenyl-triazenes. Triazenes, deactivated with halogene atoms in the phenylring, were expected to be better demethylated. The results do indicate a good trend that substitution of the ring with deactivating atoms and extent of demethylation compare well. The percentages of demethylation were: For 3,3-dimethyl-l-phenyltriazene, 45%; for 3,3-dimethyl-l (4-chlor-phenyl)-triazene, 92%; for 3,3-dimethyl-l(4-bromophenyl)triazene, 89%; for 3,3-dimethyl-l-(2,4,6-trichlorophenyl)triazene, 122%; and for 3,3-dimethyl-l-l-(2,4,6-tribromophenyl)triazene, 85%.

Animals↗
PubMedDate not supplied

Studies on the N-demethylation and O-de-ethylation of ethylmorphine by hepatic microsomes from male rats.

On the basis of inhibition studies of the dealkylation of morphine and norcodeine, George and Tephly concluded that O-dealkylation and N-dealkylation are catalyzed by different enzymes. We have examined the microsomal dealkylation of 3-O-[1'-14Clethylmorphine by measuring HCHO colorimetrically and [1-14C]acetaldehyde radiometrically. We find that the KM for the O-de-ethylation is 57 muM, which is quite close to the KS(71 muM) for the type I binding of ethylmorphine in similar preparations. On the other hand, the KM for N-demethylation was 250 muM. Further, the N-demethylation was stoichiometric with the stimulation of both NADPH-cytochrome P-450 reductase and NADPH oxidase, whereas the sum of the N-demethylation and O-de-ethylation was significantly greater, suggesting that the O-de-ethylase activity does not involve stimulation of either of these two activities. Induction with phenobarbital increaesed N-demethylation 118% but did not affect O-de-ethylation. Finally, D2O inhibited the N-demethylase more than the O-de-ethylase.

Acetaldehyde↗
PubMed1975

The role of cytochrome b5 in mixed function oxidations: effect of microsomal binding of the hemoprotein on hepatic N-demethylations.

Incubation of rat cytochrome b5 (D-b5) with rat liver microsomes resulted in specific binding of the hemoprotein. The bound hemoprotein was rapidly reduced by NADH. The NADH cytochrome c reductase activity in these preparations increased in proportion to the amount of cytochrome. In contrast to D-b5, which inhibited N-demethylation and the NADH synergism, the binding of cytochrome b5 preparations, reconstituted from heme and apocytochrome b5 had no effect on either the NADPH-dependent N-demethylation of aminopyrine or ethylmorphine or the NADH synergism observed with rat liver microsomes. In addition, manganese protoporphyrin-apocytochrome complex, when bound to microsomes in amounts equilvalent to D-b5, showed no effect on N-demethylation activity. These results suggest that homogeneous cytochrome b5 contains contaminating amounts of tightly bound detergent which presumably is removed during the extraction of the heme from the apocytochrome.

Aminopyrine↗
PubMed1978

[Increased demethylation of aminopyrine under long-term treatment with anticonvulsive drugs (author's transl)].

Demethylation of aminopyrine was measured in 25 healthy controls and 19 epileptics on long-term treatment with anticonvulsants by the 14C-aminopyrine breath test. Compared to controls epileptics exhibited increased cumulative 14CO2-exhalation rates (88.7% at 30 min, 62.6% at 2 h and 24.8% at 8h) following ingestion of 2 mu Ci 14C-aminopyrine. The results suggest that long-term treatment with antiepileptic drugs results in increased demethylating function of the liver which can be easily detected by a simple breath analysis technique like decreased demethylation in chronic liver disease.

Aminopyrine↗
PubMed2026

Identification of elements determining KIR gene demethylation at the CD56-bright stage of NK cell development.

The variegated expression of the KIR family of class I MHC receptors generates specialized natural killer (NK) cells capable of allele-specific HLA recognition. Understanding the mechanism of KIR gene activation will lead to improved methods for the generation of fully functional NK cells. A central RUNX-binding site in the KIR proximal promoter is required for gene activation. RUNX proteins recruit ten-eleven translocation (TET) proteins that generate 5-hydroxymethylcytosine (5hmC) and drive DNA demethylation. Assessment of 5-methylcytosine (5mC) and 5hmC residues at four stages of NK cell development reveals deposition of 5hmC primarily in a CREB site next to the RUNX site at the CD56Bright stage but not the subsequent CD56Dim stage representing fully mature NK cells. KIR promoter demethylation is delayed relative to other lineage-associated genes, indicating a high threshold for KIR gene demethylation in developing NK cells, and a window of opportunity for RUNX/TET-dependent KIR gene activation in CD56Bright NK cells.

6-base sequencing↗
PubMed1976

Higher activity of oxidative drug demethylation in the liver microsomes from dystrophic mouse.

The activities of NADPH-dependent oxidative demethylation of aminopyrine and other methyl compounds in the liver microsomes from dystrophic mice were found to be about 30% higher than those of the normal mice. Consumption of reduced pyridine nucleotides during the demethylation reactions was also significantly larger in the dystrophic mouse system than in the normal mouse system. The synergistic effect of further addition of NADH on the oxidative demethylation in the reaction system with NADPH, however, was not significant in either the normal or the dystrophic mouse system. The activities of NADPH-cytochrome c reductase and lipid peroxidation were also higher by about 30% in the dystrophic mouse than in the normal mouse, but the contents of cytochrome P-450 and phospholipids in the liver microsomes from normal and dystrophic mice were not appreciably different. The results suggest the possibility that the progressive muscular dystrophy may involve abnormal features in not only muscle but also liver and other tissues.

Aminopyrine↗
PubMed1979

Stimulation of p-nitroanisole O-demethylation by ethanol in perfused livers from fasted rats.

Low concentrations of ethanol (0.2 mM) stimulated p-nitroanisole O-demethylation in perfused livers from fasted, but not fed, phenobarbital-treated rats. The increase in mixed-function oxidation correlated well with the production of NADH from ethanol metabolism (Ka for both processes = 0.2-0.3 mM). This stimulation by ethanol was blocked by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, and pyruvate, a substrate for lactate dehydrogenase. Under these conditions, the characteristic reduction of NAD+ by ethanol was also abolished. p-Nitroanisole O-demethylation by isolated hepatic microsomes was unaffected by low concentrations of ethanol (up to 2 mM); however, when NADH was added to the microsomes, or was generated from ethanol, alcohol dehydrogenase and NAD+, a synergistic increase in p-nitroanisole metabolism occurred. Sorbitol and xylitol, two carbohydrates which reduced pyridine nucleotides in perfused livers, also stimulated p-nitroanisole O-demethylation in livers from fasted rats. The data indicate that NADH produced from the metabolism of ethanol, sorbitol and xylitol stimulates mixed-function oxidation in livers from fasted animals.

Alcohol Oxidoreductases↗
PubMed2026

Epigenetic priming and locus-specific demethylation enhance cell-death susceptibility in liver cancer.

Liver cancer treatment with epigenetic drugs remains challenging because demethylating agents such as 5-azacytidine (5-AZA) induce genome-wide toxicity and may activate oncogenes. We hypothesized that a low, nontoxic dose of 5-AZA could prime liver cancer cells by partially relaxing chromatin at selected loci to restore silenced cell-death regulators. HepG2 cells treated with 2 μM 5-AZA underwent ATAC-seq and RNA-seq to identify genes with promoter opening and increased expression. Among ten candidates, BFL-1 and SQOR were prioritized for roles in cell death and redox control. Forced expression of either gene increased sensitivity to TNF-α/cycloheximide (CHX) and sorafenib, both of which elevated mitochondrial reactive oxygen species. To establish causality in a physiological context, we used CRISPR-dCas9-TET1 to demethylate CpG-rich promoter regions of BFL-1 or SQOR. Locus-specific editing sensitized cells to TNF-α/CHX more rapidly than conventional overexpression and reproduced the heightened death response elicited by low-dose 5-AZA without baseline toxicity. Analysis of the cancer cell line encyclopedia and The Cancer Genome Atlas datasets showed consistent BFL-1 downregulation in liver cancer, variable SQOR expression across cancers, and positive correlations of both genes with tumor-suppression markers and immune-cell infiltration. These results indicate that targeted reactivation of BFL-1 and SQOR increases cell-death susceptibility in liver cancer cells. Integrating low-dose pharmacologic priming with precise epigenetic editing may preserve genome-wide methylation while restoring cell-death competence, providing proof-of-concept for locus-specific epigenetic therapy in liver cancer.

Humans↗
PubMed2026

YAP1 induces hepatocellular carcinoma via DNA demethylation rather than by canonical driver gene mutations.

Large-scale genome sequencing analyses have identified driver gene mutations (DGMs) in most cancers as well as their associated tumorigenic mechanisms. However, a small fraction of cancers are not positive for these canonical DGMs, leaving the mechanisms underpinning their formation a mystery. We hypothesized that canonical DGM-negative cancers might be driven by activation of the transcriptional coactivator YAP1 that led to the induction of epigenetic changes. To test this theory, we established a mouse mosaic model of hepatocellular carcinoma (HCC) in which we induced YAP1-TEAD activation in a few hepatocytes. Whole-exome sequencing did not identify canonical DGMs in HCCs, but bisulfite sequencing revealed widespread DNA demethylation leading to the transcriptional activation of multiple oncogenes. Knockdown of the DNA demethylation-promoting gene, Tet1, attenuated HCC formation in these mice. Single-cell spatial transcriptomics identified a Tet1-high subpopulation of HCC cells that interacted with other hepatic cell types. Our mechanistic mouse data align with the observation that YAP1-TEAD-TET1-associated signatures were also elevated in hepatocytes from patients with Fontan-associated liver disease (FALD), a condition associated with the development of HCCs with lower frequencies of canonical DGMs. Our study suggests that the YAP1-TEAD-TET1 axis promotes canonical DGM-negative HCC development, and provides new insights into the molecular processes involved.

Animals↗
PubMed1979

Drug metabolism by the fetal stump-tailed monkey (Macaca arctoides). Hepatic microsomal N-demethylation and glucuronidation as measured by radiometric assays.

Sensitive radiometric assays were adapted to study the development and kinetics of meperidine and methadone N-demethylation and morphine glucuronidation by microsomes isolated from livers of fetal stump-tailed macaques (Macaca arctoides). Times in development selected for study were midterm, three-quarter term, near term and newborn (0.5 h and 14 days). With appropriate attention to keeping blanks low, hepatic drug metabolism was demonstrable as early as midterm. Vmax for the N-demethylation reactions (nmole product/10 min/mg microsomal protein) increased throughout gestation, whereas the apparent Kms remained constant. With respect to morphine glucuronidation, all kinetic parameters remained constant throughout the last half of gestation.

Animals↗