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PubMed2000

Application of response surface methodology to the study of methyl glucoside polyester synthesis parameters in a solvent-free system.

Response surface methodology (RSM) and 3-level-3-factor fractional factorial design were used to evaluate the effects of synthesis parameters, including reaction time (4 to 8 h), temperature (110 to 130 degrees C), and substrate molar ratio of fatty acid methyl esters (FAME) from soybean oil to methyl glucoside (4:1 to 6:1) on the percent molar conversion to methyl glucoside polyester (MGPE), utilizing 15 g of methyl glucoside as the reactant in a solvent-free system. All synthesis variables (reaction time, temperature, and substrate molar ratio) exhibited significant effects on percent molar conversion to MPGE in the experimental range. Optimization of the synthesis reaction was suggested by ridge max analysis to compute the estimated ridge of optimum response for increasing radii from the center of the original design. Based on the ridge max analysis, optimum conditions were: reaction time 6.3 h, synthesis temperature 123.8 degrees C, and substrate molar ratio 5.9:1. The predicted molar conversion was 55.68% (i.e., 15 g methyl glucoside yielded 56.5 g MGPE) at the optimum point.

Glucosides↗

PubMed1987

B lymphocyte-specific protein binding near an immunoglobulin kappa-chain gene J segment.

Nuclear extracts from pre-B and B cell lines contain a nuclear DNA binding protein (kappa locus protein, KLP) that specifically recognizes a DNA sequence in the immunoglobulin kappa light chain joining (J) segment gene region. KLP is not observed in mature B cells, T cells, or nonlymphoid cell types. Two tandem binding sites for KLP designated KI and KII have been identified by methylation interference analysis to be immediately proximal to the J kappa 1 nonamer-heptamer recognition sequences and separated by 38 base pairs from each other. Fragments of DNA containing KI and KII sites compete for binding to KLP, and both protein-DNA complexes have the same electrophoretic mobility. Other flanking sequences of immunoglobulin gene fragments do not bind to KLP. The position of KLP-DNA binding and its tissue-specific expression suggest that it may be involved in the regulation of lymphoid gene DNA rearrangements by targeting recombinase to the kappa-chain gene region.

Animals↗

PubMed2004

Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus.

Transfer RNA (m7G46) methyltransferase catalyzes the methyl transfer from S-adenosylmethionine to N7 atom of the guanine 46 residue in tRNA. Analysis of the Aquifex aeolicus genome revealed one candidate open reading frame, aq065, encoding this gene. The aq065 protein was expressed in Escherichia coli and purified to homogeneity on 15% SDS-polyacrylamide gel electrophoresis. Although the overall amino acid sequence of the aq065 protein differs considerably from that of E. coli YggH, the purified aq065 protein possessed a tRNA (m7G46) methyltransferase activity. The modified nucleoside and its location were determined by liquid chromatography-mass spectroscopy. To clarify the RNA recognition mechanism of the enzyme, we investigated the methyl transfer activity to 28 variants of yeast tRNAPhe and E. coli tRNAThr. It was confirmed that 5'-leader and 3'-trailer RNAs of tRNA precursor are not required for the methyl transfer. We found that the enzyme specificity was critically dependent on the size of the variable loop. Experiments using truncated variants showed that the variable loop sequence inserted between two stems is recognized as a substrate, and the most important recognition site is contained within the T stem. These results indicate that the L-shaped tRNA structure is not required for methyl acceptance activity. It was also found that nucleotide substitutions around G46 in three-dimensional core decrease the activity.

Amino Acid Sequence↗

PubMed1984

Prospective study of "fibrocystic breast disease" and caffeine consumption.

Conflicting reports have appeared in the recent literature on a presumed association between coffee intake and "fibrocystic breast disease." The hypothesis suggesting that abstention from coffee and caffeine consumption eliminates breast pain and resolves breast nodules was based on an uncontrolled clinical study. For a condition with a notorious reputation for "waxing and waning," it is essential to document the stability versus instability of clinical findings, keeping the methyl xanthine consumption constant. Seventy-two women, all with palpable breast nodules, were followed over a 6-month period with monthly examinations and questionnaires on intake of coffee, tea, soft drinks, chocolate, candies, and caffeine-containing drugs. The methyl xanthine consumption remained remarkably constant throughout the observation time. The examiner was uninformed as to previous examination findings and pain reports of each woman. In 21 (15%) breasts with nodularity, the nodules had completely disappeared by the termination of the study. A total of 125 (87%) breasts of the sample manifested a change in the number of nodules or a change in position. Forty-eight of the 72 women were menopausal. For a chronic condition with so much variety of subjective sensitivity and great variability of objective clinical palpation, it is difficult to relate the amount of methyl xanthine consumption to "fibrocystic breast disease."

Adult↗

PubMed2006

The epigenetic magic of histone lysine methylation.

Epigenetic mechanisms control eukaryotic development beyond DNA-stored information. There are several pathways, including histone tail modifications, histone variant incorporation, nucleosome remodelling, DNA methylation and noncoding RNAs that together all contribute to the dynamic 'make-up' of chromatin under distinct developmental options. The histone tail modifications are most variable and over 50 marks have by now been mapped. While the majority of these modifications are transient, histone lysine methylation and, in particular, a histone lysine tri-methyl state has been regarded as a more robust signal, consistent with proposed roles to impart long-term epigenetic memory. Based on the paradigm of SET-domain histone lysine methyltransferases (HMTases) and chromo-domain adaptor proteins, and in conjunction with the Sir Hans Krebs Medal 2005, I describe here my personal view on the discovery of the first HMTase in 2000, and the subsequent advances on the biology of histone lysine methylation. This discovery has changed my scientific career and significantly contributed to a better understanding of epigenetic control, with important implications for heterochromatin formation, X inactivation, Polycomb group silencing and novel insights into stem cell research, nuclear reprogramming and cancer.

Animals↗

PubMed2006

Genomic instability in Solanum tuberosum x Solanum kurtzianum interspecific hybrids.

The use of interspecific crosses in breeding is an important strategy in improving the genetic base of the modern cultivated potato, Solanum tuberosum L. Until now, it has normally been interspecific Solanum hybrids that have been morphologically and cytologically characterized. However, little is known about the genomic changes that may occur in the hybrid nucleus owing to the combination of genomes of different origin. We have observed novel AFLP bands in Solanum tuberosum x Solanum kurtzianum diploid hybrids; 40 novel fragments were detected out of 138 AFLP fragments analyzed. No cytological abnormalities were observed in the hybrids; however, we found DNA methylation changes that could be the cause of the observed genomic instabilities. Of 277 MSAP fragments analyzed, 14% showed methylation patterns that differed between the parental species and the hybrids. We also observed frequent methylation changes in the BC1 progeny. Variation patterns among F1 and BC1 plants suggest that some methylation changes occurred at random. The changes observed may have implications for potato breeding as an additional source of variability.

Animals↗

PubMed2003

Frequent epigenetic inactivation of the RASSF1A tumour suppressor gene in testicular tumours and distinct methylation profiles of seminoma and nonseminoma testicular germ cell tumours.

Testicular germ cell tumours (TGCTs) are histologically heterogeneous neoplasms with variable malignant potential. Previously, we demonstrated frequent 3p allele loss in TGCTs, and recently we and others have shown that the 3p21.3 RASSF1A tumour suppressor gene (TSG) is frequently inactivated by promoter hypermethylation in a wide range of cancers including lung, breast, kidney and neuroblastoma. In order to investigate the role of epigenetic events in the pathogenesis of TGCTs, we analysed the promoter methylation status of RASSF1A and nine other genes that may be epigenetically inactivated in cancer (p16(INK4A), APC, MGMT, GSTP1, DAPK, CDH1, CDH13, RARbeta and FHIT) in 24 primary TGCTs (28 histologically distinct components). RASSF1A methylation was detected in four of 10 (40%) seminomas and 15 of 18 (83%) nonseminoma TGCT (NSTGCT) components (P=0.0346). None of the other nine candidate genes were methylated in seminomas, but MGMT (44%), APC (29%) and FHIT (29%) were frequently methylated in NSTGCTs. Furthermore, in two mixed germ cell tumours, the NSTGCT component for one demonstrated RASSF1A, APC and CDH13 promoter methylation, but the seminoma component was unmethylated for all genes analysed. In the second mixed germ cell tumour, the NSTGCT component was methylated for RASSF1A and MGMT, while the seminoma component was methylated only for RASSF1A. In all, 61% NSTGCT components but no seminoma samples demonstrated promoter methylation at two or more genes (P=0.0016). These findings are consistent with a multistep model for TGCT pathogenesis in which RASSF1A methylation occurs early in tumorigenesis and additional epigenetic events characterize progression from seminoma to NSTGCTs.

Acid Anhydride Hydrolases↗

PubMed1992

A novel GC-rich human macrosatellite VNTR in Xq24 is differentially methylated on active and inactive X chromosomes.

A new X chromosome-specific repetitive sequence, a 3 kilobase HindIII clone with a base composition of 63% C+G, has been isolated. The sequence is organized as a hypervariable tandem repeat cluster ranging in size from 150-350 kilobases, with outlying single copies. This locus, designated DXZ4 and mapped to chromosome band Xq24, may consist of as many as 50 variable-length alleles. It represents a class of variable number of tandem repeat polymorphism which may be termed 'macrosatellite'. The cluster is highly methylated on the active X chromosome and hypomethylated on the inactive X.

Base Composition↗

PubMed1990

[Comparative study of aromatic ring meta-cleavage enzymes in Pseudomonas strains with plasmid and chromosomal genetic control of the catabolism of biphenyl and m-toluate].

It was shown that two different enzymes of aromatic ring oxidative meta-cleavage (2,3-dihydroxybiphenyl-1,2-dioxygenase), DBO and catechol-2,3-dioxygenase, C230) function in Pseudomonas strains with a plasmid and chromosomal genetic control of biphenyl and toluate catabolism. A comparative analysis of DBO's and C230's expressed by the pBS241 biphenyl degradative plasmid in P. putida BS893, pBS311 in P. putida U83, chromosomal genes in P. putida BF and C230 from P. putida PaW160 (pWWO) was carried out. It was found that the DBO's of all strains under study are highly specialized enzymes in respect of 2,3-dihydroxybiphenyl cleavage and are also able to cleave 3-methyl-catechol and catechol (but not 4-methylcatechol) at low rates. In contrast with DBO's, in Pseudomonas strains the substrate specificities of all C230's are variable. The C230's expressed by the D-plasmids pBS241 and pBC311 have a moderate affinity for catechol, 3-methyl- and 4-methylcatechol, but are unable to cleave 2,3-dihydroxybiphenyl. The C230 which is encoded by the chromosomal structure gene from P. putida BF is very similar to C230 which codes for the TOL-plasmid pWWO. These plasmid differ from C230's expressed by biphenyl D-plasmids due to their capability to cleave 2,3-dihydroxybiphenyl in addition to catechol cleavage. All DBO's and C230's under study possess a number of properties that are typical for the enzymes having an oxidative meta-cleaving effect. The different roles of these enzymes in biphenyl and toluate catabolism in Pseudomonas strains are discussed.

Benzoates↗

PubMed1977

Short circuit current and total conductance measurements on rat ileum.

1. Short circuit current and total conductance measurements were made on preparations of rat terminal ileum, using either the intact tissue or the isolated epithelium.2. The effects of D-glucose, 3-O-methyl-D-glucose, and 3-deoxy-3-fluor-D-glucose, added in suitable concentrations to the external bathing medium, were studied.3. An increase in both short circuit current and total conductance was detected when these sugars were used. It was possible to determine a linear relationship between the two variables and to study the kinetics of the interaction of glucose and 3-O-methyl-D-glucose with the sodium sugar carrier by using increasing concentrations of these substances.4. The increases in short circuit current and total conductance were also verified in isolated epithelium.5. Phlorizin, or the withdrawal of glucose, reversed the increase in short circuit current, but the total conductance remained unchanged.6. Increasing concentrations of 2-deoxy-D-glucose added to the mucosal bathing medium caused a decrease in short circuit current without changing the total conductance, either in preparations of intact ileum or in isolated epithelium.7. The meaning of these variations in the electrical parameters of the preparations is discussed.

Animals↗

PubMed1982

Comparison of the bronchodilatory action and side-effects of ipratropium bromide and thiazinamium methyl sulphate: a long-term, double-blind, crossover trial in patients with chronic bronchitis.

A long-term, double-blind study was carried out in 12 male chronic bronchitics with partially reversible airways obstruction to compare the bronchodilatory effects of ipratropium bromide and thiazinamium methyl sulphate and their influence on micturition and visual accommodation. Patients received either ipratropium bromide (160 micrograms daily) by aerosol plus placebo tablets or thiazinamium methyl sulphate tablets (900 mg daily) plus placebo aerosol for 10 weeks. After a 1-week treatment-free period, patients were crossed over to the alternative medication for a further 10 weeks. Measurements were made before and 1 and 10 weeks after the beginning of each treatment period. The bronchodilatory responses were measured by spirometry (VC and FEV1) and maximal expiratory flow volume (MEFV) curves (FVC, PEF, MEF50 and MEF25). Micturition was estimated from a mictiogram, obtained with a system based on an ultrasonic principle. Subjective clinical findings, heart rate and blood pressure were also obtained. The variables from both the spirometric and MEFV measurements showed the bronchodilatory effect of ipratropium bromide to be superior to that of thiazinamium methyl sulphate. Because the largest differences were found in the variables from the effort-dependent, first part of the forced expiration (PEF and FEV1), the results suggest a difference in effect occurring mainly in central and higher airways. Only the MEF25 value after 10-weeks' treatment was lower in the ipratropium bromide period with respect to the thiazinamium methyl sulphate period, but not significantly so. Although 3 patients on thiazinamium had visual complaints no objective disturbances of micturition or accommodation were established.

Atropine Derivatives↗

PubMed1990

Changes in the allelic methylation patterns of c-H-ras-1, insulin and retinoblastoma genes in human development.

The methylation status of the c-H-ras-1, insulin and retinoblastoma genes was determined in human sperm, hydatidiform mole, fetal tissues, adult lymphocytes and adult kidney. Individual alleles of c-H-ras-1 and insulin were distinguishable due to presence of endogenous variable number of tandem repeat (VNTR) polymorphisms. Both alleles of the latter two genes were extensively methylated in sperm compared to the other tissues. Several sites within these genes were less methylated in fetal tissues and the two alleles were differentially methylated in some cases. The retinoblastoma gene was highly methylated in all tissues examined, with the exception of a single site that was under-methylated in sperm only. The sperm-specific methylation patterns in all three genes could represent imprinting of the parental chromosomes. Since 5-methylcytosine is inherently mutagenic, it is possible that methylation imprinting could alter the susceptibilities of human genes to point mutations.

Chromosome Mapping↗

PubMed2006

Histone modification pattern of the T-cellular Herpesvirus saimiri genome in latency.

Herpesvirus saimiri (HVS) subgroup C strains are able to growth transform human T lymphocytes in vitro. The stably persisting and nonintegrating HVS episome represents an optimal prerequisite for the investigation of the epigenetic state of latent herpesvirus genomes in vitro. Quantitative chromatin immunoprecipitation experiments using seven different histone acetylation- or methylation-specific antibodies revealed repressive marks at four lytic gene promoters and a variable pattern at the weakly transcribed LANA/orf73 promoter. The constitutive stpC/tip promoter regulating the viral oncoproteins and, more interestingly, the noncoding repetitive H-DNA elements flanking the coding region, showed a permissive chromatin structure. This study provides an appropriate model for the analysis of epigenetic herpesvirus genome modifications and their dynamics in T cells.

Acetylation↗

PubMed2007

Variability in human skin permeability in vitro: comparing penetrants with different physicochemical properties.

Appreciating and compensating for the inherent variability associated with percutaneous absorption is essential in optimizing (trans)dermal therapy. In this study, the variability in human skin permeability associated with model penetrants of differing lipophilicity (caffeine (CF), methyl paraben (MP), and butyl paraben (BP)) was examined in a standardized intra-laboratory study (Franz cell experiments) using epidermal tissue from various donors. Experimentally derived permeability coefficients (K(P)) were also compared to that derived from two skin permeation models namely, Potts & Guy and Robinson (revised) models in order to further validate the Franz diffusion method employed and also elucidate the potential permeation pathway(s) employed by the model penetrants. Intra-subject variability associated with skin permeation of the model penetrants was generally found to be lower than inter-subject variability. Experimental K(P) values were found to be the same order of magnitude as predicted by the mathematical models. Calculated residual variance suggested the Potts and Guy's model to be relatively accurate in predicting skin permeability of the two parabens whilst the Robinson (revised) model was more effective for CF. The high variability in CF permeation compared to the parabens may suggest the in vitro skin permeation of solutes becomes more sensitive to intra- and/or inter-subject variation in skin lipid content, appendageal density, and imperfections (pores, cracks) as the hydrophilic nature of the solute increases. Such variability in skin permeability suggests a difference in CF permeation kinetics relative to the parabens. As such when performing in vitro drug permeation studies, it is essential that the variability in the absorption of the model permeants, according to their physicochemical properties, is considered when they are used to normalize or standardize any resulting data.

Caffeine↗

PubMed1998

Induction of cytochrome P450 1A1 in rat liver slices by 7-ethoxycoumarin and 4-methyl-7-ethoxycoumarin.

7-Ethoxycoumarin (EC) is widely used as a model substrate for monooxygenase function, its O-deethylation representing cytochrome P450 (P450) activity mainly of 1A but also of 2B isoforms. Reports on investigations of its own capacity to induce or suppress P450 activities, however, have not been found in biomedical literature. To avoid the influence of in vivo pharmacokinetics, studies can well be undertaken with liver slice incubation. Therefore in the present investigation precision-cut rat liver slices from male 43-63-day-old male HAN:Wistar outbred rats were incubated at 30 degrees C in carbogen saturated William's Medium E for 24 h. EC was added previously to final concentrations of 10, 25, 50, 75 or 100 microM. After incubation, homogenate was prepared from slices and used for model reactions (7-ethoxyresorufin O-deethylation [EROD] and 7-pentoxyresorufin O-depentylation [PROD]). EROD, indicating activities of 1A isoforms, was enhanced by incubation with EC at 25 and 50 microM to about doublefold but showed control or lower values at 75 and 100 microM. Incubation with beta-naphthoflavone in comparison led to variable increases (3-5-fold of controls). For PROD as an indicator of the phenobarbital inducible P450 isoforms 2B1 and 2B2 no enhancement was found, but a decrease by incubation with 75 and 100 microM EC. To further investigate the correlation between enzyme activity and gene expression after slice incubation, P450 1A1 mRNA content was measured by RT-PCR. Induced gene expression for 1A1 was seen with different EC concentrations to a variable extent, though not as strong as with BNF. Similar incubation with 4-methyl-7-ethoxycoumarin revealed an even stronger induction of EROD activity with maxima at about 10-32 microM, reaching BNF values. In contrast incubation with 7-benzyloxycoumarin had no evident inducing or suppressing effect, neither on EROD nor on PROD activity.

Animals↗

PubMed1995

The diazo group as an example of an isolated 15N-14N nuclear spin pair: a static and variable spinning frequency investigation.

The 15N-14N heteronuclear spin pair present in the polycrystalline compound 5-methyl-2-diazobenzenesulphonic acid hydrochloride singly labelled with 15N at either the alpha- or the beta-position is investigated by solid-state nuclear magnetic resonance. The 15N spectrum is obtained both under static and spinning conditions. Unusually well resolved fine structure is observed in both cases and, in particular, the static spectra allow a direct and straightforward measurement of the 15N-14N internuclear dipolar coupling in addition to values of the principal components of the shielding tensors for 15N labelling (at either the alpha- or the beta-positions of the diazo moiety) and their orientation relative to a molecular frame. We also present a preliminary theoretical discussion concerning the origin of the observed spinning frequency dependence of the sideband fine structure.

Anisotropy↗

PubMedDate not supplied

[Genomic variability of sugar beet morphogenic and non-morphogenic callus].

Cytogenetic characters and the level of DNA methylation were studied in sugarbeet callus cultures with different morphogenic potential which were obtained from the same leaf explant. It was shown that non-morphogenic callus in comparison with the morphogenic one was characterized by the higher level of cell ploidy and of the frequency of chromosome structural rearrangements even at the early stages of cultivation. Overspiralization of chromosomes and formation of condensed chromatin were observed in polyploid cells of non-morphogenic callus. At the same time cytosine hypermethylation occurs in site-specific CpCpGpG-sequences of DNA indicating the possible role of this DNA modification in the processes of genome rearrangements.

Beta vulgaris↗

PubMed1994

Sequence-specific methylation inhibits the activity of the Epstein-Barr virus LMP 1 and BCR2 enhancer-promoter regions.

We reported earlier that variable expression of the Epstein-Barr virus (EBV) encoded membrane protein LMP 1 in nasopharyngeal carcinoma and the host-cell-phenotype-dependent activity of the BCR2 promoter (one of the possible initiator sites for transcripts of Epstein-Barr nuclear antigens) in Burkitt's lymphoma (BL) lines can be related to the methylation status of the 5'-flanking regulatory regions of the BNLF 1 and BCR2 promoter, respectively. Here we report that clones of the BL line Mutu that differ in expression of LMP 1 also show a differential methylation pattern of the LMP 1 regulatory sequences: this region is hypomethylated in an LMP 1 expressing (group III) clone but methylated in a group I clone that does not express LMP 1. We introduced in vitro methylated reporter plasmids carrying BNLF 1 and BCR2 enhancer-promoter sequences into the BL line Raji and found that overall methylation of 5'-CG-3' sequences by the Spiroplasma methylase Sssl significantly reduced their activity compared to unmethylated or mock-methylated controls. Methylation of 5-CCGG-3' sequences by Hpall methyltransferase gave similar results. On the contrary, methylation of 5'GCGC-3' sequences by Hhall methyltransferase gave similar results. On the contrary, methylation of 5'-GCGC-3' sequences by Hpal methyltransferase resulted only in a moderate reduction of BNLF 1 enhancer-promoter activity. These data support the notion that methylation at discrete sites within control regions of latent, growth-transformation associated EBV genes may contribute to silencing their expression.

Burkitt Lymphoma↗