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PubMed1990

Studies on the permethylation/dephosphorylation of inositol polyphosphates: an approach to a more sensitive assay.

Methods for the permethylation of inositol phosphates (i.e. the formation of the completely substituted C-O-methyl/P-O-methyl derivatives) have been studied as a precursor to preparing C-O-methyl inositols where the remaining inositol hydroxyl groups are at the positions originally occupied by the phosphomonoesters. Classical sodium-driven methylations, diazomethane methylations and methylation with methyl trifluoromethanesulfonate were studied and only the latter was found to produce completely alkylated inositol phosphates. Treatment of the permethylated substrates with methanolic HCl removed the dimethylphosphate groups to produce C-O-methyl inositols which are candidates for negative ion chemical ionization gas chromatographic/mass spectrometric analysis as heptafluorobutyryl C-O-methyl inositols. As an example, gas chromatographic/mass spectrometric analysis of myo-inositol 1,2,6-trisphosphate was carried out by methylation, dephosphorylation and conversion to the tris(heptafluorobutyryl) derivative. Detection at the low-femtomole level was achieved by this means. A limitation of the method may be that the methylation procedure appears to produce a variable degree of phosphate positional isomerization, with resulting loss of specificity. If stable isotope internal standards were available for the inositol polyphosphates of interest, this limitation could be compensated for.

Gas Chromatography-Mass Spectrometry↗
PubMedDate not supplied

Exposure to methyl tertiary-butyl ether from oxygenated gasoline in Stamford, Connecticut.

In 1993, state health officials in Connecticut invited the Centers for Disease Control and Prevention (CDC) to assist in an investigation of exposure to methyl tertiary-butyl ether in oxygenated gasoline in Stamford, Connecticut. Venous blood samples were collected from 14 commuters and from 30 other persons who worked in the vicinity of traffic or automobiles, and the samples were analyzed for methyl tertiary-butyl ether, tertiary-butyl alcohol, benzene, m-/p-xylene, o-xylene, and toluene. The highest levels of methyl tertiary-butyl ether in blood were measured among gasoline service station attendants (median = 15 micrograms/l, range = 7.6-28.9 micrograms/l). Blood levels of methyl tertiary-butyl ether were highly variable among persons who worked in car-repair shops (median = 1.73 micrograms/l, range = 0.17-36.7 micrograms/l) and were generally lowest among commuters (median = 0.11 micrograms/l, range = < 0.05-2.60 micrograms/l). Blood levels of methyl tertiary-butyl ether were correlated strongly with personal-breathing-zone samples of methyl tertiary-butyl ether and blood levels of other volatile organic compounds. This exposure information should prove useful to a future risk analysis of this high-volume chemical.

Air Pollutants↗
PubMed1994

The isolation and maintenance of the human pilosebaceous unit.

We have previously developed methods for the isolation and maintenance of human sebaceous glands and hair follicles. However, in long-term culture the maintenance of both is suboptimal. This may be due to a lack of stem cells, which are thought to be located in the bulge area of the hair follicle, and this region is not present in either model. Isolation of the entire pilosebaceous unit would retain this region, and may lead to improved maintenance of both structures. We describe a method for the isolation of viable, individual, pilosebaceous units by microdissection from human scalp face-lift skin. The viability of isolated pilosebaceous units has been determined by light microscopy, patterns of DNA synthesis by [methyl-3H] thymidine autoradiography, and lipogenesis by [U-14C] acetate uptake into lipids. When maintained for 7 days in supplemented Williams E medium, isolated pilosebaceous units showed a significant increase in length. This was due to the production of a keratinized hair fibre which grew at the in vivo rate of 0.3 mm/day. Light microscopy and [methyl-3H] thymidine autoradiography confirmed that after 7 days maintenance the hair follicle retained apparently normal morphology and patterns of DNA synthesis, However, the morphology of the sebaceous gland on maintenance was more variable, generally showing luminal keratinization. Moreover [methyl-3H] thymidine autoradiography of sebaceous glands showed a marked reduction on maintenance. The rates and patterns of lipogenesis by the whole pilosebaceous unit were, respectively, lower and different from those seen with isolated human sebaceous glands; this indicates that the bulk of pilosebaceous lipogenesis is derived from the hair follicle. Rates of recovery of [14C] from 2 mM-[U-14C] sodium acetate into thin-layer chromatography plates after 7 days maintenance decreased, although this was not statistically significant, indicating that rates of lipogenesis may fall on maintenance. Pilosebaceous units were maintained for 7 days on Gelfoam (an absorbable gelatin sponge) at the media-air interface. Initial results show a marked improvement in sebaceous gland morphology. It is possible, therefore, to obtain viable human pilosebaceous units by microdissection, and to maintain them in vitro for up to 7 days, with apparently full retention of hair follicle function, but only partial retention of sebaceous gland function.

Autoradiography↗
PubMed1995

Synergistic effect of intrastriatal co-administration of L-NAME and quinolinic acid.

Chronic dialytic intrastriatal co-administration of quinolinic acid (QUIN) and four concentrations of the nitric oxide synthase (NOS) inhibitor NG nitro-L-arginine methyl ester (L-NAME) produced variable results. Low concentrations of L-NAME (1 microM and 50 microM) co-administered with 15 mM QUIN produced lesions not significantly different from those produced by 15 mM QUIN alone. In contrast, higher concentrations of L-NAME (1 mM and 100 mM) co-administered with 15 mM QUIN produced striatal lesions significantly larger than those produced by 15 mM QUIN alone. Administered by itself, 100 mM L-NAME produced little striatal damage. These findings suggest that low levels of NOS inhibition have little or no effect on NMDA neurotoxicity in the striatum, whereas high levels of NOS inhibition increase NMDA-induced striatal lesion volume.

Analysis of Variance↗
PubMed1995

Interindividual variability in the urinary excretion of inorganic arsenic metabolites by C57 BL/6J mice: possible involvement of a thiol/disulfide exchange mechanism.

The 24-h urine of 75 C57 BL/6J mice injected s.c. with 0.5 mg/kg arsenic as sodium arsenite were examined for creatinine, S-adenosylmethionine (SAM), urea and inorganic arsenic metabolites including inorganic arsenic (ASi), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). There was interindividual variability of about a 10-fold range in the 24-h urinary excretion of creatinine (80-642 micrograms/24 h, variability (cv) of 33%), SAM (7.5-67.2 micrograms/24 h, cv of 38%), urea (9.5-89.7 mg/24 h, cv of 36%), ASi (0.1-1.6 microgram/24 h, cv of 48%), MMA (0.17-2.1 micrograms/24 h, cv of 50%), DMA (0.73-8.13 micrograms/24 h, cv of 32%) and total arsenic metabolites (1.0-10.4 micrograms/24 h, cv of 31%). Interindividual differences, varying by as much as 5-7-fold, were also found in the urinary proportion of ASi (3-23%, cv of 41%) and MMA (5-22%, cv of 37%), but not in the urinary proportion of DMA (64-90%, cv of 7%). The 24-h urinary excretion of all arsenic metabolites correlated with the 24-h urinary excretion of urea (r = 0.81), creatinine (r = 0.88) and SAM (r = 0.83) as did the 24-h urinary excretion of urea with creatinine (r = 0.94) and SAM (r = 0.86), and the 24-h urinary excretion of creatinine with SAM (r = 0.94). Taken together, these results suggest that the overall intracellular glutathione (GSH)-dependent redox state, as reflected by the 24-h urinary excretion of SAM and creatinine, is involved in the interindividual variability in total arsenic metabolite excretion by C57 BL/6J mice. These preliminary results were also discussed with regard to the involvement of intracellular GSH-dependent redox state in the regulation of the reduction and of the methylation steps of arsenic, and to interindividual variability in the urinary excretion of total arsenic metabolites as a possible complicating factor in the biological monitoring of occupational exposure to arsenic.

Animals↗
PubMed1994

Hypomethylation of CCGG sites in the 3' region of H-ras protooncogene is frequent and is associated with H-ras allele loss in non-small cell lung cancer.

The methylation of MspI/HpaII sites flanking a variable tandem repeat in the 3' region of the c-Ha-ras protooncogene was analyzed in 74 DNA samples of non-small cell lung carcinomas and control lung tissues. Of 39 informative samples, 7 allelic deletions (18%) were found at the c-Ha-ras locus and of these, five (71%) showed hypomethylation of the nondeleted allele. Heterozygous DNA samples without allele loss revealed hypomethylation in 37% (12 of 32). Among 35 homozygotes, 9 showed hypomethylation (26%). We also analyzed c-Ha-ras mutations at codons 12, 13, and 61 by polymerase chain reaction and designed restriction fragment length polymorphism and found no mutation. Thus, c-Ha-ras mutations are not associated with the development of the detected abnormalities. We conclude that hypomethylation at specific sites in the 3' region is associated with loss of heterozygosity for the c-Ha-ras gene in non-small cell lung cancer. The finding that, in informative samples, hypomethylation occurs 2-3 times more frequently than allelic loss suggests that it might be a change contributing or predisposing to a genetic instability that can ultimately lead to c-Ha-ras allelic deletions found in tumor DNA.

Alleles↗
PubMed1983

True histiocytic lymphoma. A report of four cases.

Clinical, morphologic, cytochemical, immunologic, and ultrastructural features of four cases of true histiocytic lymphoma are described. The neoplastic cells were large, ranging from 20 to 45 mu in diameter with round, folded, or convoluted nuclei, and abundant eosinophilic cytoplasm. They exhibited diffuse nonspecific esterase activity. Diffuse acid phosphatase activity was present in two cases so tested. Muramidase activity was present in half of the cases. Finely granular PAS-positive material was seen in the cytoplasm. Methyl green-pyronin positivity was variable. An occasional neoplastic cell showed erythropagocytosis in one case. Malignant cells either contained no cytoplasmic immunoglobulins (three cases) or had immunoglobulins of multiple classes (one case). Surface markers were studied in two cases; they were absent in one case, and were of multiple classes in another case. Ultrastructurally the neoplastic cells had lysosomal granules in three cases so examined, and phagolysosomes, phagocytized material and residual bodies in one of three cases so studied. Patients ranged in age from 28 to 60 years. Two patients had extralymphatic tumors. Survival of more than 5 years was seen in one patient.

Adult↗
PubMedDate not supplied

Neurophysiological markers as early signs of organophosphate neurotoxicity.

The central and peripheral nervous system effects of acute and subchronic exposure to three organic phosphoro-acid esters (dimethoate, dichlorvos, parathion-methyl) were studied. CNS-dependent variables included mean EEG amplitude, mean frequency of the EEG, and the activity (power density) of six component frequency bands. Peripheral nervous system evaluations included determinations of conduction velocity, and both relative and absolute refractory periods. Cholinesterase activity was measured in blood, brain and other organs. The results indicate that acute large doses of these agents produce substantial changes in these measures of CNS and PNS function. In subchronic experiments it was found that a six weeks administration of 1/50 LD50 of the chemicals induced early functional changes in both the central and the peripheral nervous systems. It is recommended that when cholinesterase inhibition is detected in humans, functional evaluations of CNS and PNS should follow.

Animals↗
PubMed1985

Simplex optimization of extractive alkylation procedures for organic acids in aqueous samples.

Simplex optimization procedures may be used to optimize the yield from an extractive alkylation procedure. Three variables from the derivatization reaction (pH, methylating agent concentration, and phase transfer agent concentration) were chosen for optimization because they interacted with each other and could not be optimized by conventional means. The yield for methylation of adipic acid was doubled with relatively little effort. This procedure may be easily applied to other systems.

Acids↗
PubMed1981

Uptake and metabolism of noradrenaline by blood vessels of perinephritic hypertensive dogs.

Tissue noradrenaline content as well as uptake and metabolism of tritiated exogenous noradrenaline were studied comparatively, in vitro in mesenteric arteries, and in saphenous veins of normotensive and perinephritic hypertensive dogs. The influence of cocaine, iproniazid and 3'-4'-dihydroxy-2-methyl-propiophenone (U-0521) on these variables was also investigated. The concentration of (-)-7-3H-noradrenaline used was 1.08 microM. No changes were observed in noradrenaline content, uptake and metabolism in saphenous vein strips obtained from normotensive or hypertensive animals. However, in mesenteric artery strips obtained from hypertensive dogs, a marked reduction in endogenous noradrenaline content was observed as well as a reduction in noradrenaline accumulation (20 weeks after surgery). The deamination pattern was also modified in these strips: the formation of DOPEG was markedly diminished and the formation of DOMA was increased. These results agree well with the degeneration of the sympathetic innervation of the mesenteric arteries of hypertensive dogs described by Azevedo et al. (1981).

Animals↗
PubMed2026

Population-scale detection of methylation outliers from long-read genome sequencing.

BACKGROUND: Aberrant DNA methylation can mediate the functional effects of rare genetic variation and contribute to imprinting disorders, repeat expansion diseases, and other pathogenic regulatory mechanisms. Long-read sequencing technologies now enable genome-wide detection of CpG methylation alongside genetic variation from a single assay. However, methods for systematic identification and interpretation of methylation outliers from long-read sequencing data remain limited. METHODS: We developed METAFORA, a computational workflow for detecting methylation outlier regions from PacBio and Oxford Nanopore long-read sequencing data. METAFORA constructs population-level methylation references, segments the genome into correlated CpG blocks, infers technical and biological sources of variation through hidden factor estimation, models uncertainty due to variable depth sequencing, and computes covariate-adjusted methylation outlier scores for individual samples. We applied METAFORA across large long-read sequencing cohorts and integrated methylation outliers with multi-omic data. METAFORA is implemented as a snakemake workflow available at https://github.com/tjense25/METAFORA. RESULTS: METAFORA identified methylation outlier regions associated with rare structural variants, tandem repeat expansions, and imprinting abnormalities. We found outlier regions were enriched for molecular outliers across transcriptomic and chromatin accessibility datasets, supporting their functional relevance in gene regulation. In a representative case, METAFORA identified an imprinting defect affecting the GNAS locus associated with an STX16 deletion. CONCLUSIONS: METAFORA enables scalable detection and interpretation of methylation outliers from long-read sequencing data and provides a framework for integrating epigenetic outliers with genomic and multi-omic analyses. These approaches may improve interpretation of rare regulatory variation and support discovery of clinically relevant epigenetic abnormalities in genomic medicine.

DNA methylation↗
PubMed1991

Carrier detection in Wiskott-Aldrich syndrome: combined use of M27 beta for X-inactivation studies and as a linked probe.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder with no clinical or immunologic abnormalities in carrier females. The defective gene has been localized to proximal Xp. Carrier females have nonrandom use of the X chromosome in granulocytes, lymphocytes, and monocytes. We have used the probe M27 beta, which detects both a variable number tandem repeat polymorphism and methylation differences between the active and inactive X chromosome, in the investigation of families referred for genetic counseling. M27 beta detects the locus DXS255, which is tightly linked to WAS. As the probe that is used for investigation of X-inactivation patterns is also linked to the disease locus, it is possible to assign phase in families where this could not be done by conventional use of linked probes. The mothers of four isolated male cases had nonrandom use of the X chromosome. A new mutation was identified in one family with two affected males.

Alleles↗
PubMed2020

Harsh Parenting Predicts Novel HPA Receptor Gene Methylation and NR3C1 Methylation Predicts Cortisol Daily Slope in Middle Childhood.

Adverse experiences in childhood are associated with altered hypothalamic-pituitary-adrenal (HPA) axis function and negative health outcomes throughout life. It is now commonly accepted that abuse and neglect can alter epigenetic regulation of HPA genes. Accumulated evidence suggests harsh parenting practices such as spanking are also strong predictors of negative health outcomes. We predicted harsh parenting at 2.5&#xa0;years old would predict HPA gene DNA methylation similarly to abuse and neglect, and cortisol output at 8.5&#xa0;years old. Saliva samples were collected three times a day across 3 days to estimate cortisol diurnal slopes. Methylation was quantified using the Illumina Infinium MethylationEPIC array BeadChip (850&#xa0;K) with DNA collected from buccal cells. We used principal components analysis to compute a summary statistic for CpG sites across candidate genes. The first and second components were used as outcome variables in mixed linear regression analyses with harsh parenting as a predictor variable. We found harsh parenting significantly predicted methylation of several HPA axis genes, including novel gene associations with AVPRB1, CRHR1, CRHR2, and MC2R (FDR corrected p&#x2009;<&#x2009;0.05). Further, we found NR3C1 methylation predicted a steeper diurnal cortisol slope. Our results extend the current literature by demonstrating harsh parenting may influence DNA methylation similarly to more extreme early life experiences such as abuse and neglect. Further, we show NR3C1 methylation is associated with diurnal HPA function. Elucidating the molecular consequences of harsh parenting on health can inform best parenting practices and provide potential treatment targets for common complex disorders.

Child↗
PubMed1975

A comparison of four quantitative cytochemical methods directed toward demonstration of DNA.

Populations of nuclei isolated from mouse brain tissue were stained by the following cytochemical methods considered stoichiometric for DNA: (1) the Feulgen reaction; (2) gallocyanin-chromalum after RNase; (3) pH 4.0 methylene blue after RNase; and (4) methyl green used in the presence of 2M magnesium chloride. Replicate preparations to be stained with gallocyanin-chromalum, methylene blue, and methyl green were acetylated prior to staining. All of these groups were examined by high-resolution scanning microspectrophotometry. The results indicated that of the methods examined, the Feulgen reaction, gallocyanin-chromalum used without prior acetylation, and methylene blue used with prior acetylation were the most useful in revealing differences attributable to variability in chromatin organization. The greatest variability in total extinction measurements was observed in acetylated, methylene blue-stained nuclei, while the least variability was observed in nuclei stained with methyl green in the presence of 2 M magnesium chloride. Acetylation produced different effects on dye-binding in different groups. It greatly increased binding in nuclei stained with methylene blue; it reduced binding in the methyl green-2 M magnesium chloride series.

Acetylation↗
PubMed1976

Characterization of microsomal methyl sterol demethylase in two Morris hepatomas.

Previously, we reported that the rate of metabolism of methyl sterol intermediates of cholesterol biosynthesis by broken-cell preparations of Morriss hepatoma 7777 is very slow, whereas the intact tumors are known to synthesize cholesterol quite efficiently. Active preparations have now been obtained by substitution of pyrophosphate for phosphate buffer. Although substitution of pyrophosphate buffer markedly enhances microsomal methyl sterol demethylation rates 3- to 4-fold in hepatoma 7777, other microsomal enzymes and electron carriers in either liver or a more slowly growing hepatoma appear to be unaffected by pyrophosphate. Several properties of the active microsomal methyl sterol demethylase have now been compared for control rat liver, host liver, tumor 7777, and tumor 5123C. Conditions necessary for the assay of initial velocities of enzymic reactions in the tumor microsomes have been established with respect to the amount of protein, time-course, concentrations of cofactors and substrate, pH, and other variables. The K'm and the responses to the variables studied above are very similar for methyl sterol demethylase of microsomes isolated from control liver, host liver, tumor 5123C, and tumor 7777. The multienzymic demethylase in the various preparations has been found to be inhibited similarly by in vitro additions of cyanide, cytochrome c, and bile salts. Thus, the enzymes of the microsomal-bound 4-methyl sterol demethylase of cholesterol biosynthesis appear to be very similar in liver and these 2 Morris hepatomas. When xenobiotic inducers of microsomal oxidases, such as phenobarbital and methylcholanthrene, are administered to normal and tumor-bearing rats, elevated rates of methyl sterol demethylation are observed with isolated liver microsomes obtained from both normal and tumor-bearing rats. Similar increases are not observed in the tumors. Furthermore, daily administration of an intestinal bile acid sequestrant elevates hepatic methyl sterol demethylase, but statistically significant changes were not observed in tumors 7777 and 5123C. Since the enzymes of methyl sterol demethylase appear to be grossly similar in liver and these hepatomas, regulation of the activity of the multienzymic system contained in the tumors may be altered. On the other hand, these agents in vivo simply may not affect liver and the hepatomas similarly, due to a lack of uptake of the foreign substances by the tumor that has been transplanted to the thighs.

Animals↗
PubMed1993

DNA methylation and mutation.

5-Methylcytosine (5mC) in DNA is produced by post-synthetic modification of cytosine residues, and it occurs primarily in CpG doublets in the mammalian genome. 5mC is a mutable site, because it can undergo spontaneous deamination to thymine. There is a repair mechanism which specifically recognises G.T mispairs, and replaces thymine with cytosine. However, this repair is not fully efficient, because the 5mC-->T transition mutation occurs about 10 times as frequently as other transitions. Such mutations are frequently seen in inherited diseases, and mutations in the p53 gene in tumours are also very commonly in 5mCpG doublets. As well as mutations, there can also be heritable changes in DNA methylation, known as epimutations, which may be of particular significance in somatic cells. Whereas the pattern of DNA methylation is very constant for any one cell type, the pattern becomes very variable in tumour cells. The breakdown of the normal controls of DNA methylation in tumorigenesis can lead to increased gene expression or to gene silencing. DNA damage increases not only mutation, but also heritable changes in methylation. At present, little is known about the ability of DNA repair to preserve the normal pattern of methylation in somatic cells.

5-Methylcytosine↗