Evidence on the conformation of HeLa-cell 5.8S ribosomal ribonucleic acid from the reaction of specific cytidine residues with sodium bisulphite.
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The reaction of HeLa-cell 5.8S rRNA with NaHSO3 under conditions in which exposed cytidine residues are deaminated to uridine was studied. It was possible to estimate the reactivities of most of the 46 cytidine residues in the nucleotide sequence by comparing 'fingerprints' of the bisulphite-treated RNA with those of untreated RNA. The findings were consistent with the main features of the secondary-structure model for mammalian 5.85S rRNA proposed by Nazar, Sitz, & Busch [J. Biol. Chem (1975) 250, 8591--8597]. Five out of six regions that are depicted in the model as single-stranded loops contain cytidine residues that are reactive towards bisulphite at 25 degrees C (the other loop contains no cytidine). The cytidine residue nearest to the 3'-terminus is also reactive. Several cytidines residues that are internally located within proposed double-helical regions show little or no reactivity towards bisulphite, but the cytidine residues of several C.G pairs at the ends of helical regions show some reactivity, and one of the proposed loops appears to contain six nucleotides, rather than the minimum of four suggested by the primary structure. Two cytidine residues that are thought to be 'looped out' by small helix imperfections also show some reactivity. (+info)
DNA demethylase is a processive enzyme.
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DNA methylation patterns are generated during development by a sequence of methylation and demethylation events. We have recently demonstrated that mammals bear a bona fide demethylase enzyme that removes methyl groups from methylated cytosines. A general genome wide demethylation occurs early in development and in differentiating cell lines. This manuscript tests the hypothesis that the demethylase enzyme is a processive enzyme. Using bisulfite mapping, this report demonstrates that demethylase is a processive enzyme and that the rate-limiting step in demethylation is the initiation of demethylation. Initiation of demethylation is determined by the properties of the sequence. Once initiated, demethylation progresses processively. We suggest that these data provide a molecular explanation for global hypomethylation. (+info)
Sulfitobacter mediterraneus sp. nov., a new sulfite-oxidizing member of the alpha-Proteobacteria.
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Analysis of PCR products of 16S rDNA of 680 isolates from Mediterranean Sea mesocosm experiments with taxon-specific 16S rDNA oligonucleotides revealed that 262 isolates belonged to the alpha subclass of the class Proteobacteria. Partial 16S rDNA sequence analysis of selected isolates and oligonucleotide probing with a Sulfitobacter-specific 16S rDNA probe affiliated 33 strains to the genus Sulfitobacter. Analysis of the HaeIII digest pattern of 16S rDNA revealed the presence of two groups; while 30 strains showed a pattern identical with that obtained for Sulfitobacter pontiacus DSM 10014T, a second group of three strains had a unique pattern that was different from that of the type strain. Five isolates of group 1 and one isolates of group 2, strain CH-B427T, were selected for detailed taxonomic analysis. All six isolates closely resembled the type strain Sulfitobacter pontiacus DSM 10014T in physiological reactions. However, strain CH-B427T differed quantitatively in the composition of fatty acids from Sulfitobacter pontiacus DSM 10014T and showed only 98.2% 16S rDNA sequence similarity with strain DSM 10014T. DNA-DNA reassociation value obtained for strains DSM 10014T and CH-B427T revealed 46% similarity. Based on the results of DNA-DNA reassociation and discrete differences in the nucleotide composition of 16S rDNA, a new species of the genus Sulfitobacter is proposed, designated Sulfitobacter mediterraneus sp. nov., the type strain being strain CH-B427T (= DSM 12244T). (+info)
The relationship between pH and concentrations of antioxidants and vasoconstrictors in local anesthetic solutions.
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pH affects the efficacy of local anesthetics by determining the percentage of the lipid-soluble base form of the anesthetic available for diffusion and penetration of the nerve sheath. The purpose of this study was to determine the relationship between pH and the concentrations of antioxidant and vasoconstrictor in dental local anesthetic solutions over real-time and after accelerated aging. Several batches of lidocaine and mepivacaine with vasoconstrictors were tested. Results showed that, immediately upon receipt from the manufacturers, three batches were below the USP pH limit (pH 3.3), and two batches contained less than the minimum limit of vasoconstrictors (90%). Real-time tests on batches that were within normal limits revealed that solutions were stable past 4 yr. Accelerated aging tests revealed a strong correlation between a decrease in pH and loss of antioxidants and vasoconstrictors. In conclusion, a quality batch of local anesthetic should remain efficacious long past the manufacturer's stated shelf life; a batch that is less than optimal, or one that is exposed to environmental stresses, will degrade rapidly, and efficacy may be affected by decreases in pH and loss of vasoconstrictor. pH may be an inexpensive, readily available screening test for efficacy of local anesthetics. (+info)
CpG island methylator phenotype in colorectal cancer.
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Aberrant methylation of promoter region CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in neoplasia. To understand global patterns of CpG island methylation in colorectal cancer, we have used a recently developed technique called methylated CpG island amplification to examine 30 newly cloned differentially methylated DNA sequences. Of these 30 clones, 19 (63%) were progressively methylated in an age-dependent manner in normal colon, 7 (23%) were methylated in a cancer-specific manner, and 4 (13%) were methylated only in cell lines. Thus, a majority of CpG islands methylated in colon cancer are also methylated in a subset of normal colonic cells during the process of aging. In contrast, methylation of the cancer-specific clones was found exclusively in a subset of colorectal cancers, which appear to display a CpG island methylator phenotype (CIMP). CIMP+ tumors also have a high incidence of p16 and THBS1 methylation, and they include the majority of sporadic colorectal cancers with microsatellite instability related to hMLH1 methylation. We thus define a pathway in colorectal cancer that appears to be responsible for the majority of sporadic tumors with mismatch repair deficiency. (+info)
Confluence-induced alterations in CpG island methylation in cultured normal human fibroblasts.
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Growth constraint of bacterial and human cells has been shown to trigger genetic mutation. We questioned whether growth constraint might also trigger epigenetic mutation in the form of CpG island methylation. Logarithmically growing normal human fibro-blasts (NHF) displayed little (0-15%) CpG methylation in select regions of three CpG islands [estrogen receptor (ER), E-cadherin (ECAD) and O (6)-methylguanine-DNA methyltransferase (MGMT)] examined. NHF grown to and left at confluence for 2-21 days showed little (<10%) CpG methylation in the ER and ECAD CpG islands. These confluent, growth-arrested cells, however, displayed extensive ( approximately 50%) methylation of the MGMT CpG island. CpG methylation in the MGMT CpG island was not associated with cellular senescence. The methylation was, however, heritable, but not permanent, as the level of CpG methylation in the MGMT CpG island of cells 4 population doublings following replating after confluence were no different from those in confluent cultures, but returned to levels noted in logarithmically growing cells by 10 population doublings following replating. These results suggest that growth constraint can trigger transient epigenetic change even in normal non-senescent human cells. (+info)
Mechanism, structure-activity studies, and potential applications of glutathione S-transferase-catalyzed cleavage of sulfonamides.
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The mechanism of sulfonamide cleavage of PNU-109112, a potent HIV-1 protease inhibitor, by glutathione-S-transferase (GST) was investigated in the presence of reduced GSH. GST-catalyzed sulfonamide cleavage takes place via the nucleophilic attack of GSH on the pyridine moiety of the substrate with formation of the GS-para-CN-pyridinyl conjugate, the corresponding amine, and sulfur dioxide. Structure activity studies with a variety of sulfonamides indicate that an electrophilic center alpha to the sulfonyl group is required for cleavage. Substituents that withdraw electron density from the carbon atom alpha- to the sulfonyl group facilitate nucleophilic attack by the GS(-) thiolate bound to GST. The rate of sulfonamide cleavage is markedly affected by the nature of the electrophilic group; replacement of para-CN by para-CF(3) on the pyridine ring of PNU-109112 confers stability against sulfonamide cleavage. On the other hand, stability of sulfonamides is less dependent on the nature of the amine moiety. These principles can be applied to the synthesis of sulfonamides, labile toward cellular GST, that may serve as prodrugs for release of bioactive amines. Tumors are particularly attractive targets for these sulfonamide prodrugs as GST expression is significantly up-regulated in many cancer cells. Another potential application could be in organic synthesis, where protection of amines as the corresponding activated sulfonamides can be reversed by GST/GSH under mild conditions. (+info)
Reduction of adenosine-5'-phosphosulfate instead of 3'-phosphoadenosine-5'-phosphosulfate in cysteine biosynthesis by Rhizobium meliloti and other members of the family Rhizobiaceae.
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We have cloned and sequenced three genes from Rhizobium meliloti (Sinorhizobium meliloti) that are involved in sulfate activation for cysteine biosynthesis. Two of the genes display homology to the Escherichia coli cysDN genes, which code for an ATP sulfurylase (EC 2.7.7.4). The third gene has homology to the E. coli cysH gene, a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase (EC 1.8.99.4), but has greater homology to a set of genes found in Arabidopsis thaliana that encode an adenosine-5'-phosphosulfate (APS) reductase. In order to determine the specificity of the R. meliloti reductase, the R. meliloti cysH homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the A. thaliana reductases, the histidine-tagged R. meliloti cysH gene product appears to favor APS over PAPS as a substrate, with a Km for APS of 3 to 4 microM but a Km for PAPS of >100 microM. In order to determine whether this preference for APS is unique to R. meliloti among members of the family Rhizobiaceae or is more widespread, cell extracts from R. leguminosarum, Rhizobium sp. strain NGR234, Rhizobium fredii (Sinorhizobium fredii), and Agrobacterium tumefaciens were assayed for APS or PAPS reductase activity. Cell extracts from all four species also preferentially reduce APS over PAPS. (+info)