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PubMed2002

Evaluation of the studies on decline of pesticide residues.

A specifically designed field trial was carried out in an apple orchard by applying Reldan 50 EC (active ingredient, chlorpyrifos-methyl) according to registered uses in Hungary to study the variability of results derived from supervised field trials. Two types of composite samples (A, size 24; and C, size 12) were taken at days 0, 3, 7, 10, and 14 after application to study the uncertainty of estimated residue values derived from supervised trials. In the case of type A the sampling officer selected the fruits from the specified quadrant of the tree, whereas for type C the fruits were taken from the vicinity of the marked position at consecutive sampling times. An evaluation model applying various formulas for the linearization of the decline curves of pesticide residues was applied, which enabled using the statistics of linear regression for calculating the best fit and confidence intervals for the experimental data. The results indicated that the uncertainty of sampling contributed approximately 84-90% of the combined uncertainty of the results (24-30%). In the decline studies performed simultaneously on the same field, the estimated time required to decrease the initial concentration to half ranged from 0.64 to 4.7 days. Despite the fact that the sample size of type C is half that of type A, both sampling methods provided similar results.

Chlorpyrifos↗

PubMed1998

Use of cholesterol precursors to assess changes in cholesterol synthesis under non-steady-state conditions.

BACKGROUND: Quantification of plasma levels of an early and late intermediate on the cholesterol pathway, mevalonic acid (MVA) and lathosterol respectively, provides a useful method of estimating cholesterol synthesis in humans. The aim of this study was to assess further their roles as indices of cholesterol synthesis under non-steady-state conditions. METHODS: The short-term effects of pharmacological inhibition of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase on both variables were determined in four normolipidaemic subjects during and after treatment with simvastatin 20 mg daily. Plasma MVA was measured using gas chromatography-mass spectrometry, and lathosterol using gas chromatography. RESULTS: A single dose of 20 mg of simvastatin decreased plasma MVA after 2 h and decreased the lathosterol-cholesterol (L/C) ratio after 4 h. Treatment with simvastatin 20 mg daily for 9 days decreased both variables by approximately 50%, the nadir of plasma MVA occurring on the second day and of the L/C ratio on the fifth day, and resulted in a 39% reduction in low-density lipoprotein (LDL)-cholesterol. After discontinuing simvastatin, there were rebounds in plasma MVA and the L/C ratio to above basal levels but not in LDL cholesterol or apolipoprotein B (apoB), the latter continuing to decrease for a further 2 days. CONCLUSION: These results suggest that simvastatin rapidly down-regulates cholesterol synthesis, which is then up-regulated when the drug is withdrawn.

Acyl Coenzyme A↗

PubMed2000

Thiopurine methyltransferase polymorphic tandem repeat: genotype-phenotype correlation analysis.

BACKGROUND: Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. Recently, a variable number tandem repeat (VNTR) within the TPMT promoter has been reported to "modulate" levels of this enzyme activity. METHODS: We set out to perform genotype-phenotype correlation analysis for the polymorphic TPMT tandem repeat in 1211 clinical laboratory samples in which red blood cell (RBC) TPMT activity had been measured and to compare those results with data for 279 control DNA samples. RESULTS: TPMT VNTR length varied from three to nine repeats ( *V3 to *V9), but the most common alleles were *V4 and *V5, with frequencies in the control samples of 0.54 and 0.36, respectively. The clinical laboratory samples were then stratified into those with "low," "intermediate," or "high" levels of RBC TPMT activity; that is, samples presumed to be homozygous for open reading frame (ORF)-based variant alleles, heterozygous for those alleles, or homozygous for the "wild-type" ORF sequence, respectively. TPMT VNTR genotype *V4/*V5 was associated with significantly higher RBC TPMT activity than were *V4/*V4 or *V5/*V5. Lowest activity levels were associated with genotypes that included an allele with more than 5 repeat elements. However, all of these effects were quantitatively small. Finally, there was linkage disequilibrium between VNTR allele *V5 and TPMT*3A, the most common ORF-based polymorphism associated with very low TPMT activity in white persons. CONCLUSIONS: These observations suggest that, in addition to the striking effects of ORF-based single nucleotide polymorphisms on TPMT activity, the VNTR within the 5'-flanking region of the TPMT gene also may modulate levels of RBC TPMT activity.

Adolescent↗

PubMedDate not supplied

Evaluation of passive card monitors for hydrogen sulfide for use in kraft pulp mill workplace atmospheres.

Six different brands of passive card monitors representing three different types of card monitor were evaluated for their effectiveness in testing for hydrogen sulfide in workplace atmospheres. The cards were evaluated both in the laboratory and in a kraft pulp mill according to a protocol that closely follows that recommended by the National Institute for Occupational Safety and Health and other researchers for passive samplers. The performances of the Envirometrics Air-Chem Technologies and the Gilian ChemAir quantitative cards, the Gilian ChromAir and VICI Metronics Colortec semiquantitative cards, and the American Gas & Chemical Co. Leak-Tec and Gilian SafeAir indicator cards were evaluated. The detection limits of the cards were determined, and the cards were exposed to a range of hydrogen sulfide concentrations (1, 5, 10, 20 ppm), face velocities (0, 50 ft/min), and temperature/humidity combinations (95 degrees F/90% humidity, 77 degrees F/50% humidity, 40 degrees F/20% humidity). The cards' responses to short-term and variable concentration exposures as well as to the potential interferents methyl mercaptan, dimethyl sulfide, dimethyl disulfide, sulfur dioxide, and carbon monoxide were assessed. Each of the cards tested demonstrated a usefulness for the detection and/or quantification of hydrogen sulfide at concentrations and under conditions relevant to kraft pulp mill workplace atmospheres. Which card is best depends on the application, sampling conditions, and level of confidence necessary in the results. The Gilian ChemAir quantitative card performed well under most conditions with acceptable accuracy (13% lab, 26% field), and no response to potential interferents or to changes in face velocity, temperature, and humidity. The Envirometrics ACT quantitative cards did not perform as well, with an accuracy level of only 46% in the lab. They also seemed to be more affected by changes in face velocity, temperature, and humidity. Both semiquantitative cards (Gillian ChromAir, VICI Metronics Colortec) provided reasonable approximations of hydrogen sulfide levels in the range of the hydrogen sulfide occupational exposure levels although the VICI card had a much lower detection limit. Neither performed well at high temperature and humidity. The response of VICI card was more affected by changes in face velocity, whereas the Gilian ChromAir showed some response to reduced sulfur gas interferents. Both indicator cards (Leak-Tec, Gilian SafeAir) performed well, responding to 20 ppm hydrogen sulfide within 1 min and to 5 ppm within 5 min. Neither was significantly affected by extremes of temperature and humidity.

Air Movements↗

PubMed2006

Stable CpG hypomethylation of adipogenic promoters in freshly isolated, cultured, and differentiated mesenchymal stem cells from adipose tissue.

Mesenchymal stem cells from adipose tissue can differentiate into mesodermal lineages. Differentiation potential, however, varies between clones of adipose stem cells (ASCs), raising the hypothesis that epigenetic differences account for this variability. We report here a bisulfite sequencing analysis of CpG methylation of adipogenic (leptin [LEP], peroxisome proliferator-activated receptor gamma 2 [PPARG2], fatty acid-binding protein 4 [FABP4], and lipoprotein lipase [LPL]) promoters and of nonadipogenic (myogenin [MYOG], CD31, and GAPDH) loci in freshly isolated human ASCs and in cultured ASCs, in relation to gene expression and differentiation potential. Uncultured ASCs display hypomethylated adipogenic promoters, in contrast to myogenic and endothelial loci, which are methylated. Adipogenic promoters exhibit mosaic CpG methylation, on the basis of heterogeneous methylation between cells and of variation in the extent of methylation of a given CpG between donors, and both between and within clonal cell lines. DNA methylation reflects neither transcriptional status nor potential for gene expression upon differentiation. ASC culture preserves hypomethylation of adipogenic promoters; however, between- and within-clone mosaic methylation is detected. Adipogenic differentiation also maintains the overall CpG hypomethylation of LEP, PPARG2, FABP4, and LPL despite demethylation of specific CpGs and transcriptional induction. Furthermore, enhanced methylation at adipogenic loci in primary differentiated cells unrelated to adipogenesis argues for ASC specificity of the hypomethylated state of these loci. Therefore, mosaic hypomethylation of adipogenic promoters may constitute a molecular signature of ASCs, and DNA methylation does not seem to be a determinant of differentiation potential of these cells.

Adipogenesis↗

PubMedDate not supplied

Excitability changes and glucose metabolism in experimentally induced focal cortical dysplasias.

Malformations of cortical development are increasingly recognized in association with severe epileptic syndromes, neuropsychological disorders and mental retardation. Several clinical and experimental studies suggest that functional consequences of cortical dysplasias are not restricted to the area of the dysplastic lesion but also involve remote brain regions. In the present study cortical malformations were induced in newborn rats at day of birth by intracerebral injection of the glutamatergic agonist ibotenate. The resulting cytoarchitectonic lesion associates neuronal depopulation of deep cortical layers, ectopic neurons in superficial layers and sulcus formation, mimicking human polymicrogyria and migration disorders. Electrophysiological recordings of evoked field potentials in slice preparations of adult animals reveal hyperexcitability in widespread cortical regions surrounding the dysplasia. Low-intensity stimulation induced epileptiform activity consisting of long-lasting, multiphasic and N-methyl-D-aspartate-dependent field responses. They appeared with high variability as all-or-none events. These widespread changes in excitability were not observed in sham-operated animals with small superficial ectopias but intact deep cortical layers, indicating that focal loss of these layers induces extended alterations in cortical connectivity and imbalance of excitation and inhibition. Restricted zones of increased excitability were also found in the forelimb and hindlimb representation cortex in sham-operated and control animals, demonstrating that this activity has to be considered as an intrinsic property of specific cortical areas. Deoxyglucose autoradiography showed that the widespread hyperexcitability in ibotenate-injected animals was not accompanied by alterations in glucose metabolism, although in the area of structural abnormality a typical metabolic pattern was found, revealing an increased glucose uptake in layer I. Hypometabolism as described for many types of human dysplastic lesions was not observed. This difference between the experimental and clinical data may be due to the absence of behavioral seizures in this model. However, it can be hypothesized that in patients with developmental malformations, additional pathogenic factors contribute to the manifestation of seizure disorders.

Animals↗

PubMed2006

Diversity of microcystin genotypes among populations of the filamentous cyanobacteria Planktothrix rubescens and Planktothrix agardhii.

Microcystins (MCs) are toxic heptapeptides that are produced by filamentous cyanobacteria Planktothrix rubescens and Planktothrix agardhii via nonribosomal peptide synthesis. MCs share a common structure cyclo (-D-Alanine(1)-L-X(2)- D-erythro-beta-iso-aspartic acid(3)-L-Z(4)-Adda(5)-D-Glutamate(6)- N-methyl-dehydroalanine(7)) where X(2) and Z(2) are variable L-amino acids in positions 2, 4 of the molecule. Part of the mcyB gene (1,451 bp) that is involved in the activation of the X(2) amino acid during MC synthesis was sequenced in 49 strains containing different proportions of arginine, homotyrosine, and leucine in position 2 of the MC molecule. Twenty-five genotypes were found that consisted of eight genotype groups (A-H, comprising 2-11 strains) and 17 unique genotypes. P. rubescens and P. agardhii partly consisted of the same mcyB genotypes. The occurrence of numerous putative recombination events that affected all of the genotypes can explain the conflict between taxonomy and mcyB genotype distribution. Genotypes B (homotyrosine and leucine in X(2)) and C (arginine in X(2)) showed higher nonsynonymous/synonymous (d(N)/d(S)) substitution ratios implying a relaxation of selective constraints. In contrast, other genotypes (arginine, leucine, homotyrosine) showed lowest d(N)/d(S) ratios implying purifying selection. Restriction fragment length polymorphism (RFLP) revealed the unambiguous identification of mcyB genotypes, which are indicative of variable X(2) amino acids in eight populations of P. rubescens in the Alps (Austria, Germany, and Switzerland). The populations were found to differ significantly in the proportion of specific genotypes and the number of genotypes that occurred over several years. It is concluded that spatial isolation might favour the genetic divergence of microcystin synthesis in Planktothrix spp.

Bacterial Toxins↗

PubMed2003

Toward a marker-dense meiotic map of the potato genome: lessons from linkage group I.

Segregation data were obtained for 1260 potato linkage group I-specific AFLP loci from a heterozygous diploid potato population. Analytical tools that identified potential typing errors and/or inconsistencies in the data and that assembled cosegregating markers into bins were applied. Bins contain multiple-marker data sets with an identical segregation pattern, which is defined as the bin signature. The bin signatures were used to construct a skeleton bin map that was based solely on observed recombination events. Markers that did not match any of the bin signatures exactly (and that were excluded from the calculation of the skeleton bin map) were placed on the map by maximum likelihood. The resulting maternal and paternal maps consisted of 95 and 101 bins, respectively. Markers derived from EcoRI/MseI, PstI/MseI, and SacI/MseI primer combinations showed different genetic distributions. Approximately three-fourths of the markers placed into a bin were considered to fit well on the basis of an estimated residual "error rate" of 0-3%. However, twice as many PstI-based markers fit badly, suggesting that parental PstI-site methylation patterns had changed in the population. Recombination frequencies were highly variable across the map. Inert, presumably centromeric, regions caused extensive marker clustering while recombination hotspots (or regions identical by descent) resulted in empty bins, despite the level of marker saturation.

Chromosome Mapping↗

PubMed2001

Specific site methylation in the 5'-flanking region of CYP1A2 interindividual differences in human livers.

Human cytochrome P4501A2 (CYP1A2) is involved in the metabolism of a large number of common drugs and is responsible for the metabolic activation of numerous promutagens and procarcinogens. Large interindividual differences exist in the expression of this enzyme. This variability has important implications for drug efficacy and cancer susceptibility. In this sudy, the methylation status of the CCGG site (bp -2759) located adjacent to an AP-1 site in the 5'-flanking region of the CYP1A2 gene was assessed in liver samples from a pool of 55 human donors. DNA methylation is an important epigenetic mechanism controlling gene expression and may be one of the molecular mechanisms underlying the interindividual variation. Analysis was conducted using Hpa II digestion and PCR. Results showed that individual samples varied in the methylation status at this site. The site was found to be hypermethylated in approximately 60% of the samples. To compare methylation status with level of CYP1A2 expression, results were analyzed by median test. In 44% of the samples with expression levels above the median the CCGG site was hypermethylated. However, for those samples with levels below the median hypermethylation of the site was found in 73% of the samples. The difference was statistically significant (p<0.05). These findings indicate that CpG methylation may be involved in controlling the expression of CYP1A2, with hypermethylation reducing expression. Moreover, this approach can be useful in assessing the role of site-specific DNA methylation in interindividual variation of CYP1A2. Analysis of other CpG sites in potentially important regulatory elements of the CYP1A2 gene will continue.

Cytochrome P-450 CYP1A2↗

PubMed1999

Methylation of inorganic arsenic in different mammalian species and population groups.

Thousands of people in different parts of the world are exposed to arsenic via drinking water or contaminated soil or food. The high general toxic of arsenic has been known for centuries, and research during the last decades has shown that arsenic is a potent human carcinogen. However, most experimental cancer studies have failed to demonstrate carcinogenicity in experimental animals, indicating marked variation in sensitivity towards arsenic toxicity between species. It has also been suggested that there is a variation in susceptibility among human individuals. One reason for such variability in toxic response may be variation in metabolism. Inorganic arsenic is methylated in humans as well as animals and micro-organisms, but there are considerable differences between species and individuals. In many, but not all, mammalian species, inorganic arsenic is methylated to methylarsonic acid (MMA) and dimethylarsinic acid (DMA), which are more rapidly excreted in urine than is the inorganic arsenic, especially the trivalent form (AsIII, arsenite) which is highly reactive with tissue components. Absorbed arsenate (AsV) is reduced to trivalent arsenic (AsIII) before the methyl groups are attached. It has been estimated that as much as 50-70% of absorbed AsV is rapidly reduced to AsIII, a reaction which seems to be common for most species. In most experimental animal species, DMA is the main metabolite excreted in urine. Compared to human subjects, very little MMA is produced. However, the rate of methylation varies considerably between species, and several species, e.g. the marmoset monkey and the chimpanzee have been shown not to methylate inorganic arsenic at all. In addition, the marmoset monkey accumulates arsenic in the liver. The rat, on the other hand, has an efficient methylation of arsenic but the formed DMA is to a large extent accumulated in the red blood cells. As a result, the rat shows a low rate of excretion of arsenic. In both human subjects and rodents exposed to DMA, about 5% of the dose is excreted in the urine as trimethylarsine oxide. It is obvious from studies on human volunteers exposed to specified doses of inorganic arsenic that the rate of excretion increases with the methylation efficiency, and there are large inter-individual variations in the methylation of arsenic. Recent studies on people exposed to arsenic via drinking water in northern Argentina have shown unusually low urinary excretion of MMA. Furthermore, children had a lower degree of methylation of arsenic than adults. Some studies indicate a lower degree of arsenic methylation in men than in women, especially during pregnancy. Whether the observed differences in methylation of arsenic are associated with variations in the susceptibility of arsenic remains to be investigated.

Adult↗

PubMed2000

Epigenetic regulation of androgen receptor gene expression in human prostate cancers.

Epigenetic mechanisms including DNA methylation and histone deacetylation are thought to play important roles in gene transcriptional inactivation. Heterogenous expression of androgen receptor (AR), which appears to be related to variable responses to endocrine therapy in prostate cancer (PCa) may also be due to epigenetic factors. The methylation status of the 5' CpG island of the AR in 3 prostate cancer cell lines and 10 primary and 14 hormone-refractory PCa samples was determined using the bisulfite PCR methods. In DU145, CpG-rich regions of the AR were hypermethylated. By an immunohistochemical analysis, only one PCa sample had no AR expression, the others being heterogenous. Bisulfite sequencing and methylation-specific PCR analysis showed aberrant methylation of AR 5'-regulatory region in 20% of 10 primary and 28% of 14 hormone-refractory PCa samples. To clarify the effect of epigenetic regulation on AR expression, we treated three prostate cancer cell lines with a demethylating agent, 5-aza-2'-deoxycytidine (azaC), and a histone deacetylase inhibitor, Trichostatin A (TSA). In DU145, re-expression of AR mRNA was detected after treatment with azaC and/or TSA. Our results suggest that epigenetic regulations including CpG methylation and histone acetylation may play important roles in the regulation of the AR.

Antimetabolites, Antineoplastic↗

PubMed2026

Moebius-Plus Phenotype With Positive RCEM Episignature May Indicate Broader Embryologic Malformation Spectrum Detectable by Methylation Profiling.

Moebius syndrome (OMIM #157900) is a rare congenital cranial dysinnervation disorder characterized by abducens (CN VI) and facial (CN VII) nerve palsies with variable craniofacial and limb anomalies. Despite advances in genomic testing, the majority of patients remain genetically unexplained. Episignature testing, which detects syndrome-specific DNA methylation patterns, has emerged as a complementary diagnostic tool for conditions with shared developmental mechanisms. We describe an 8-month-old male born prematurely with bilateral clubfoot, craniofacial dysmorphism, feeding difficulty requiring gastrostomy tube placement, and respiratory failure requiring tracheostomy. Neuroimaging demonstrated absence of bilateral abducens and facial nerves with pontocerebellar hypoplasia, supporting a clinical diagnosis of Moebius syndrome. Extensive genetic evaluation, including genome sequencing and targeted testing for hypotonia and hypoventilation syndromes, was nondiagnostic. Episignature analysis revealed a moderately positive methylation signature consistent with a recurrent constellation of embryonic malformation (RCEM), concordant with two of three previously validated RCEM classifier models. To our knowledge, this is the first report of a patient with a positive RCEM episignature and Moebius syndrome, suggesting a common embryologic pathway. Episignature testing may represent a valuable diagnostic tool in patients with Moebius syndrome and related craniofacial-limb malformation spectra when conventional genomic testing is unrevealing.

RCEM↗

PubMedDate not supplied

Methylation of the insulin-like growth factor binding protein-3 gene and prognosis of epithelial ovarian cancer.

Insulin-like growth factor binding protein-3 (IGFBP-3) is a member of the IGFBP family, which regulates the mitogenic and antiapoptotic effects of insulin-like growth factors. Hypermethylation of the IGFBP-3 promoter has been found to suppress its expression. To evaluate the role of IGFBP-3 in ovarian cancer progression, we examined the survival of 235 consecutively selected epithelial ovarian cancer patients in association with IGFBP-3 promoter methylation and IGFBP-3 expression in tumor tissue. IGFBP-3 promoter methylation was analyzed using methylation-specific polymerase chain reaction. Cytosol protein was extracted and measured using a bicinchoninic acid assay; IGFBP-3 was measured by enzyme linked immunosorbent assay. Promoter methylation of the IGFBP-3 gene was detected in 44% (104/235) of patients. IGFBP-3 promoter methylation was associated with disease progression and death after adjusting for clinical and pathologic variables. The association was more evident in patients with early-stage disease: RR = 2.87 (95% CI: 0.78-10.63) for disease progression and RR = 3.94 (95% CI: 0.91-15.78) for death. Tissue levels of IGFBP-3 did not differ by methylation status but were inversely associated with disease stage and residual tumor size. These results suggest that IGFBP-3 promoter methylation may be a useful prognostic marker for disease progression and death in early-stage ovarian cancer.

Biomarkers, Tumor↗

PubMed1989

Laundry parameters as factors in lowering methyl parathion residue in cotton/polyester fabrics.

Cotton/polyester fabrics were contaminated with a 1.25% solution of methyl parathion (MeP) emulsifiable concentrate formulation, then laboratory laundered. The laundering variables were detergent type, concentration, water volume, and mechanical agitation. Specimens were unfinished (UN) and soil repellent finished (SR) fabrics. Heavy duty liquid and phosphate-built powdered detergents were equally effective when used in combination with a prewash product. A negative linear relationship between detergent concentration and MeP residue remaining after laundering was established. An interaction between detergent concentration and fabric finish was observed. Soil repellent finished fabrics required detergent concentrations above the recommended amount for more efficient soil removal. A negative linear relationship between water volume and after-laundering residue was observed. Water volume played a more significant role in pesticide removal than agitation during laundering.

Chromatography, Gas↗

PubMed1982

Atypical biogroups of Escherichia coli found in clinical specimens and description of Escherichia hermannii sp. nov.

DNA relatedness was used to define the biochemical boundaries of Escherichia coli. A large number of biochemically atypical strains were shown to belong to biogroups of E. coli. These included strains negative in reactions for indole, all three decarboxylases, D-mannitol, lactose, or methyl red and strains positive in reactions for H2S, urea, citrate, KCN, adonitol, myo-inositol, or phenylalanine deaminase. Frequency and source data are presented for these atypical E. coli biogroups. One group of KCN-positive, cellobiose-positive, yellow-pigmented strains was 84 to 91% interrelated but only 35 to 45% related to E. coli. The name Escherichia hermannii sp. nov. is proposed for this group of organisms that was formerly called Enteric Group 11 by the Enteric Section, Centers for Disease Control, Atlanta, GA. Twenty-nine strains of E. hermannii have been isolated in the United States from a variety of clinical sources, principally wounds, sputum, and stools. Three additional strains were isolated from food. E. hermannii strains are gram-negative, oxidase-negative, fermentative, motile rods. In addition to yellow pigment and positive KCN and cellobiose tests, the biochemical reactions characteristic of 32 strains of E. hermannii were as follows: gas from D-glucose, acid from D-glucose, maltose, D-xylose, L-arabinose, L-rhamnose, and D-mannitol; no acid from adonitol or inositol; variable acid production from lactose and sucrose; positive tests for indole, methyl red, and mucate; negative tests for Voges-Proskauer. Simmons citrate, H2S, urea, phenylalanine deaminase, and gelatin hydrolysis; negative or delayed test for L-lysine decarboxylase and negative test for L-arginine dihydrolase; and positive test for ornithine decarboxylase. E. hermannii strains were resistant to penicillin, ampicillin, and carbenicillin and sensitive to other commonly used antibiotics. Wounds account for almost 50% of human isolates of E. hermannii, followed by sputum or lung isolates (ca. 25%) and stool isolates (20%).

Anti-Bacterial Agents↗

PubMed1998

Expression of CYP2E1 in human lung and kidney during development and in full-term placenta: a differential methylation of the gene is involved in the regulation process.

Methylation of dinucleotide CG residues located in the 5' end of the CYP2E1 gene has been demonstrated to play a role in the control of gene expression in the human developing liver. This study was undertaken to examine the CYP2E1 RNA content of human lung, kidney and full-term placenta and to determine whether the expression of CYP2E1 was controlled by its methylation status in these tissues. CYP2E1 was expressed at a very low level in the lung and kidney at whatever age, and at a variable level in full-term placentas. The restriction profile of genomic DNA was identical in lung and kidney and corresponded to a heavy methylation of HpaII/MspI sites located within the promoter, the first exon and first intron of the CYP2E1 gene. A different pattern of methylation was obtained in full-term placentas, indicating that CpG residues located in the 5' end of the gene were predominantly but not fully demethylated. However, the variable level of CYP2E1 RNA in full-term placentas suggests the involvement of other elements in the regulation process of CYP2E1 in this tissue.

Adult↗

PubMed1996

M.BssHII, a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties.

A new multispecific cytosine-C5-DNA-methyltransferase (C5-MTase), M.BssHII, was identified in Bacillus stearothermophilus H3. The M.BssHII gene was cloned and sequenced. The amino acid sequence deduced shows the characteristic building plan of a C5-MTase. By sequencing bisulfite-treated DNA methylated by M.BssHII and by restriction enzyme analysis, we defined the following methylation targets of M.BssHII: ACGCGT/CCGCGG (MluI/SacII), PuGCGCPy (HaeII), PuCCGGPy (Cfr10I) and GCGCGC (BssHII). The relative location of the specificity determinants in the C5-MTase was derived from the analysis of M.BssHII derivatives carrying deletions within the variable region "V" and chimeric C5-Mtases constructed between M.BssHII and the related monospecific enzyme M.phi3TII. Four of the M.BssHII specificities (MluI, SacII, Cfr10I and BssHII) could be associated with amino acid segments within the variable region "V". The determinant for HaeII activity had to be assigned to sequences defining the enzyme core, the first example of a C5-MTase in which a sequence-specific methylation potential is mediated by structures outside of the variable region. Another intriguing result came from the analysis of one particular chimera made between M.BssHII and M.phi3TII. This construct showed a relaxation of the methylation capacity, both with respect to the target recognized and the targeting of methylation within this sequence.

Amino Acid Sequence↗

PubMed1993

An embryonic-like methylation pattern of classical satellite DNA is observed in ICF syndrome.

ICF syndrome has been described as the association of variable immunodeficiency, facial anomalies and centromeric heterochromatin instability. Since the chromosome rearrangements seen in cells of ICF patients are reminiscent of the chromosomal changes induced by the undermethylating agent 5-azacytidine in the late S-phase, we have analyzed the methylation pattern of satellite sequences in four patients. These sequences are almost completely methylated in normal leukocyte DNA. When ICF DNA was tested with methyl-sensitive enzymes, several classical satellite families, but not alphoid sequences, showed a very low level of methylcytosine in leukocyte DNA, with an abnormal pattern compared to the normal germinal and extraembryonic methylation profile. The methylation deficiency affects classical satellite families built from distinct unit sequences but located in the same chromosomal region. This observation may have important implications for the mechanism of chromosomal rearrangements.

5-Methylcytosine↗