Biomedical subjects

S Som

Publications and source records attributed to S Som.

At least 19 recordsLinked to original sources

PubMed2002

Antibiotic resistance of subgingival species during and after antibiotic therapy.

AIM: The purpose of the present investigation was to determine the percentage and identity of antibiotic-resistant species in subgingival plaque and saliva samples from chronic periodontitis patients treated by scaling and root planing followed by orally administered amoxicillin or metronidazole. METHOD: In all, 20 chronic periodontitis patients were selected for study. After clinical and microbiological monitoring, subjects were randomly assigned to receive either orally administered amoxicillin at the dosage of 500 mg, 3 times daily for 14 days or orally administered metronidazole at the dosage of 250 mg, 3 times daily for 14 days. For the antibiotic resistance determinations, subgingival plaque samples were taken from six posterior teeth at baseline, and 90 days; and from two randomly selected teeth at 3, 7 and 14 days during and after antibiotic administration. Samples were plated on enriched blood agar plates with or without either 2 micro g/mL metronidazole or 2 micro g/mL amoxicillin. Colonies were counted at 7 days. Significant differences in percentage of resistant organisms over time were determined by the Quade test. Microbial growth was washed from antibiotic-containing media and the identity of species determined using checkerboard DNA-DNA hybridization. Data were compared with those obtained in a previous study from subjects receiving SRP only or SRP followed by 14 days of orally administered doxycycline. The level of doxycycline used to determine antibiotic resistance in that study was 4 micro g/mL. RESULTS: The mean percentage of resistant isolates increased during antibiotic administration and returned to baseline levels by 90 days post therapy. The mean percentages (+/- SEM) of isolates resistant to 2 micro g/mL metronidazole were 53 +/- 9, 65 +/- 9, 79 +/- 4 and 69 +/- 7 at baseline, 3, 7 and 14 days during antibiotic administration, and 57 +/- 4, 64 +/- 5, 62 +/- 7 and 47 +/- 6 at 3, 7, 14 and 90 days after antibiotic administration. At the same time points, the percentage of resistant isolates to amoxicillin was 0.5 +/- 0.2, 22 +/- 12, 14 +/- 5 and 37 +/- 11 during, and 31 +/- 11, 8 +/- 3, 3 +/- 2 and 3 +/- 0.6 after, administration. Antibiotic-resistant isolates of resistant species detected during or after therapy were also detected prior to therapy. The most prevalent resistant species in the metronidazole-treated group were: A. naeslundii 1, S. constellatus, A. naeslundii 2, S. mitis, S. oralis, A. odontolyticus, S. sanguis, and in the amoxicillin-treated group: S. constellatus, P. nigrescens, E. saburreum, A. naeslundii 1, S. oralis, P. melaninogenica and P. intermedia. CONCLUSIONS: Systemic antibiotic administration transiently increased the percentage of resistant subgingival species, but a major component of subgingival plaque remained sensitive to the agents during their administration. Antibiotic-resistant isolates of resistant species could be detected in samples both prior to and after therapy. However, % antibiotic-resistant isolates returned to baseline levels 90 days after antibiotic administration.

Adult↗

PubMed2001

Change in subgingival microbial profiles in adult periodontitis subjects receiving either systemically-administered amoxicillin or metronidazole.

AIM: The current investigation evaluated changes in levels and proportions of 40 bacterial species in subgingival plaque samples during, immediately after and up to 1 year after metronidazole or amoxicillin therapy combined with SRP. METHOD: After baseline clinical and microbiological monitoring, 17 adult periodontitis subjects received full mouth SRP and 14 days systemic administration of either metronidazole (250 mg, TID, n=8) or amoxicillin (500 mg, TID, n=9). Clinical measurements including % of sites with plaque, gingival redness, bleeding on probing and suppuration, pocket depth (PD) and attachment level (AL) were made at baseline, 90, 180 and 360 days. Subgingival plaque samples were taken from the mesial surface of all teeth in each subject at baseline, 90, 180 and 360 days and from 2 randomly selected posterior teeth at 3, 7, and 14 days during and after antibiotic administration. Counts of 40 subgingival species were determined using checkerboard DNA-DNA hybridization. Significance of differences over time was determined using the Quade test and between groups using ANCOVA. RESULTS: Mean PD was reduced from 3.22+/-0.12 at baseline to 2.81+/-0.16 (p<0.01) at 360 days and from 3.38+/-0.23 mm to 2.80+/-0.14 mm (p<0.01) in the amoxicillin and metronidazole treated subjects respectively. Corresponding values for mean AL were 3.21+/-0.30 to 2.76+/-0.32 (p<0.05) and 3.23+/-0.28 mm to 2.94+/-0.23 mm (p<0.01). Levels and proportions of Bacteroides forsythus, Porphyromonas gingivalis and Treponema denticola were markedly reduced during antibiotic administration and were lower than baseline levels at 360 days. Counts (x10(5), +/-SEM) of B. forsythus fell from baseline levels of 0.66+/-0.16 to 0.04+/-0.02, 0.13+/-0.04, 0.10+/-0.03 and 0.42+/-0.19 in the amoxicillin group at 14, 90, 180 and 360 days respectively (p<0.001). Corresponding values for metronidazole treated subjects were: 1.69+/-0.28 to 0.02+/-0.01, 0.20+/-0.08, 0.22+/-0.06 and 0.22+/-0.08 (p<0.001). Counts of Campylobacter species, Eubacterium nodatum, Fusobacterium nucleatum subspecies, F. periodonticum and Prevotella nigrescens were also detected at lower mean levels during and immediately after therapy, but gradually increased after withdrawal of the antibiotics. Members of the genera Actinomyces, Streptococcus and Capnocytophaga were minimally affected by metronidazole. However, amoxicillin decreased the counts and proportions of Actinomyces species during and after therapy. CONCLUSIONS: The data suggest that metronidazole and amoxicillin are useful in rapidly lowering counts of putative periodontal pathogens, but must be accompanied by other procedures to bring about periodontal stability.

Actinomyces↗

PubMed2001

Reduced dental plaque accumulation on composite gold alloy margins.

Restoration of tooth surfaces with materials that inhibit formation of heavy bacterial plaque accumulations could be important in the treatment of patients with existing oral disease or in reducing the likelihood for periodontal disease. Captek is a dental gold composite material used to produce copings for ceramometal restorations that has been reported to inhibit plaque accumulation. In this study, the oral bacteria of nine periodontally healthy subjects with a total of 42 gold composite copings were sampled. Contralateral teeth with normal tooth surfaces were also sampled as controls. The quantitative presence of forty bacteria was determined in each sample by DNA:DNA hybridization. The results indicated that the porcelain/gold composite alloy coping surfaces had significantly fewer bacteria than the control normal tooth surfaces (71% reduction). The percentage composition, however, did not differ significantly between surfaces.

Analysis of Variance↗

PubMed2000

The effect of repeated professional supragingival plaque removal on the composition of the supra- and subgingival microbiota.

BACKGROUND, AIMS: The purpose of the present investigation was to determine the effect of weekly professionally administered supragingival plaque removal on the composition of the supra and subgingival microbiota. METHODS: 18 adult subjects with periodontitis who had been treated and were in a maintenance phase of therapy were clinically and microbiologically monitored at baseline, 3, 6 and 12 months. After the baseline visit, the subjects received scaling and root planing followed by professional supragingival plaque removal every week for 3 months. Clinical measures of plaque accumulation, bleeding on probing (BOP), gingival redness, suppuration, pocket depth and attachment level were made at 6 sites per tooth at each visit. Separate supra (N = 1804) and subgingival (N = 1804) plaque samples were taken from the mesial aspect of all teeth excluding third molars in each subject at each time point and evaluated for their content of 40 bacterial taxa using checkerboard DNA-DNA hybridization. Significance of changes in mean counts, prevalence and proportions of bacterial species over time in both supra and subgingival samples were determined using the Quade test and adjusted for multiple comparisons. RESULTS: Mean % of sites exhibiting plaque, gingival redness and BOP were significantly reduced during the course of the study. Significant decreases in mean counts were observed in both supra and subgingival samples. Mean total DNA probe counts (x10(5), +/-SEM) at baseline, 3, 6 and 12 months were: 133+/-19, 95+/-25, 66+/-6, 41+/-6 (p<0.001) for supragingival samples and 105+/-22, 40+/-10, 19+/-4, 13+/-3 (p<0.001) for subgingival samples. Mean counts of 22 of 40 and 34 of 40 species tested were significantly reduced in the supra and subgingival samples respectively over the monitoring period. For example, mean counts of Porphyromonas gingivalis x10(5) at baseline, 3, 6 and 12 months in the subgingival plaque samples were 2.0+/-0.4, 0.5+/-0.2, 0.6+/-0.3, 0.3+/-0.1 (p<0.001); Bacteroides forsythus 2.0+/-0.6, 0.4+/-0.1, 0.4+/-0.2, 0.1+/-0.2 (p<0.001); Treponema denticola 3.4+/-1.1, 0.8+/-0.3, 0.4+/-0.2, 0.3+/-0.3 (p<0.01). Similar reductions were seen in supragingival plaque samples. While counts were markedly reduced by professional plaque removal, the proportion and prevalence of the 40 test species were marginally affected. CONCLUSIONS: Weekly professional supragingival plaque removal profoundly diminished counts of both supra- and subgingival species creating a microbial profile comparable to that observed in periodontal health. This profile was maintained at the final monitoring visit, 9 months after completion of therapy.

Adult↗

PubMed1999

Systemic doxycycline administration in the treatment of periodontal infections (I). Effect on the subgingival microbiota.

Systemic doxycycline is one of the more common antimicrobial agents used in the treatment of periodontal infections and yet little is known of its effect on subgingival plaque composition during and after its administration. The purpose of the present investigation was to evaluate changes in subgingival plaque composition during and after 14 days of doxycycline administration. 20 subjects with adult periodontitis were randomly assigned to test (n = 10) and control (n = 10) groups. The subjects received full mouth clinical assessment of pocket depth, attachment level, BOP, gingival redness, suppuration and plaque accumulation at baseline and 90 days. All subjects received full mouth SRP at baseline and, additionally, the test group received 100 mg doxycycline daily for 14 days. Subgingival plaque samples were taken from the mesial surface of up to 28 teeth in each subject at baseline and 90 days. In addition, plaque samples were taken from 2 randomly selected teeth at 3, 7 and 14 days during and after antibiotic administration. Control subjects were sampled at the same time points. Counts of 40 subgingival species were determined using checkerboard DNA-DNA hybridization and fluorescent detection. Significance of differences between test and control groups was determined at each time point using the Mann Whitney test. Significance of changes over time within test and control groups was determined using the Quade test. A modest but significant reduction in mean pocket depth from baseline to 90 days occurred in both test and control groups. A significant decrease in the % of sites with gingival redness occurred in the test group. There were no significant differences in proportions between test and control groups for 33 of the test species at any time point. Test subjects exhibited lower proportions of 4 Actinomyces species and an increase in 3 Streptococcus species during antibiotic administration. After cessation of doxycycline, Actinomyces sp. increased while Streptococcus sp. returned to baseline proportions. The relationship between these 2 genera appeared to be reciprocal; an increase in one was accompanied by a decrease in the other. Periodontal pathogens including B. forsythus, P. gingivalis, T. denticola and A. actinomycetemcomitans were not significantly altered by oral administration of doxycycline using conventional therapeutic dosage.

Adult↗

PubMed1999

Systemic doxycycline administration in the treatment of periodontal infections (II). Effect on antibiotic resistance of subgingival species.

The purpose of this investigation was to determine the proportion and prevalence of doxycycline resistant species in subgingival plaque samples taken during and after doxycycline administration. 20 subjects with adult periodontitis were randomly assigned to test (n = 10) or control groups (n = 10). Saliva samples as well as subgingival plaque samples taken from the distal surface of 6 posterior teeth were collected at baseline. All subjects received full mouth SRP and the test group systemic doxycycline at the dosage of 100 mg/day for 14 days. Saliva samples and plaque samples from the distal surface of 2 randomly selected teeth were taken at 3, 7 and 14 days during and after antibiotic administration. Control subjects were sampled at the same time points. Samples were anaerobically dispersed and serially diluted in PRAS Ringer's solution and plated on enriched Trypticase soy blood agar plates with or without 4 microg/ml doxycycline. After 7 days of anaerobic incubation, colonies were counted on both sets of plates. Microbial growth was washed from the doxycycline-containing media and the species identified using 40 DNA probes and checkerboard DNA-DNA hybridization. Differences in proportions of resistant species between test and control groups were tested for significance at each time point using the Mann Whitney test and over time within each group using the Quade test. The mean % (+/-SEM) of isolates resistant to 4 microg/ml doxycycline in the plaque samples of the test subjects increased from 6+/-2 to 48+/-9% during doxycycline administration, decreasing to 25+/-6% 2 weeks later and 9+/-2% at 90 days. In saliva, the % of resistant isolates rose from 13+/-1% to 81+/-10% during doxycycline administration falling to 46+/-8% 2 weeks later and 22+/-5% at 90 days. The % of resistant isolates did not change significantly in plaque or saliva samples of the control subjects at the same time points. For all subject visits combined, the most prevalent resistant species were: Streptococcus anginosus, Streptococcus oralis, Streptococcus intermedius, Streptococcus sanguis, Streptococcus mitis, Veillonella parvula, Actinomyces gerencseriae, Streptococcus constellatus, Actinomyces naeslundii genospecies 2, Streptococcus gordonii, Eikenella corrodens and Actinomyces naeslundii genospecies 1. Doxycycline resistant strains of these species were detected in both plaque and saliva samples prior to therapy and in the control group. Despite the finding of increased resistance, approximately 50% of the organisms present at periodontal sites at the end of 14 days of doxycycline administration tested sensitive to the agent.

Adult↗

PubMed1997

Characterization of the intergenic region which regulates the MspI restriction-modification system.

The 110-bp intergenic region between mspIM and mspIR, the genes encoding the MspI modification (M.MspI) and restriction (R.MspI) enzymes, respectively, was fused, in both orientations, with lacZ. Expression of a single-copy mspIM-lacZ fusion is more than 400-fold stronger than expression of an mspIR-lacZ fusion. M.MspI in trans represses expression of the mspIM-lacZ fusion by binding to the DNA but does not affect expression of the mspIR-lacZ fusion. Transcription start sites of the genes were identified, and a set of nonoverlapping promoters was assigned. DNase I footprinting showed that M.MspI binds to a site within the intergenic region that includes only the mspIM regulatory elements.

DNA Footprinting↗

PubMed1994

Regulation of EcoRII methyltransferase: effect of mutations on gene expression and in vitro binding to the promoter region.

EcoRII Methyltransferase (M.EcoRII) which methylates the second C in the sequence CCWGG (W = A/T) is autogenously regulated by binding to the 5' regulatory region of its gene. DNase I footprinting experiments demonstrated that purified M.EcoRII protected a 47-49 bp region of DNA immediately upstream of the ecoRIIM coding region. We have studied this interaction with mutants of the enzyme, in vitro by DNA binding and in vivo by investigating the repression in trans of expression of beta-galactosidase from an ecoRIIM-lacZ operon fusion. Two catalytically active mutants failed to repress expression of the fusion whereas catalytically inactive mutants had repressor activity. However, with one of the catalytically inactive mutants, C186S, in which the catalytic Cys was replaced with Ser, and which bound unmethylated CCWGG sequences, repression could only be demonstrated when those sequences in cellular DNA were methylated by supplying a cloned dcm gene in trans. In vitro binding of the DNA fragment containing the ecoRIIM regulatory region was detected only with the mutants that showed repressor activity, including C186S. Results indicate that down-regulation of the gene in vivo and binding to the promoter in vitro are not dependent on the catalytic properties of M.EcoRII. Mobility shift experiments with C186S also revealed that it could bind either the promoter or unmethylated CCWGG sites, but not both. We conclude that the concentration of unmethylated CCWGG sites controls expression from the ecoRIIM promoter.

Amino Acid Sequence↗

PubMed1994

Inhibition of transcription in vitro by binding of DNA (cytosine-5)-methylases to DNA templates containing cytosine analogs.

DNA(cytosine-5)-methylases form tight complexes at their methylation sites when the target cytosine residue is substituted by analogs such as 5-azacytosine or 5-fluorocytosine. To test whether such complexes can block RNA transcription in vitro, template DNA-containing methylation sites were prepared, in which cytosine residues in either the (+)- or (-)-strand were substituted by the analogs. Such templates, irrespective of the strand in which substitution was made, could effectively block the elongation of RNA at specific sites when complexed with EcoRII methylase. The protein-DNA complex probably prevents the unwinding of the template strands or might directly present itself as a steric block to the advancing RNA polymerase. RNA synthesis was also inhibited at specific sites due to complex formation between azacytosine-containing DNA and two other methylases, HhaI and HpaII. The 3' ends of the interrupted transcripts were mapped and were found to lie within 13-14, 13, and 23 nucleotides of the binding sites on the (-)-strand for HhaI, HpaII, and EcoRII methylases, respectively. Exonuclease III footprinting revealed that the boundaries of the complexed methylases, HhaI, HpaII, and EcoRII, on the (-)-strand were within 2-3, 1-2, and 9-10 nucleotides, respectively, of the last nucleotide copied by the RNA polymerase.

Base Sequence↗

PubMed1993

Autogenous regulation of the EcoRII methylase gene at the transcriptional level: effect of 5-azacytidine.

mRNA of the EcoRII methylase (M.EcoRII), a type II modification enzyme, was induced when Escherichia coli carrying a cloned M.EcoRII gene was exposed to the bacteriocidal drug 5-azacytidine. Induction occurred only when transcription was initiated from its own promoter. When the 5' promoter sequences were deleted or replaced with the lac promoter sequences, no induction occurred. The induction was independent of the template DNA level, but the presence of an intact M.EcoRII protein was a requirement. The drug is incorporated into DNA which then inhibits M.EcoRII by binding tightly to the enzyme. A deletion within the M.EcoRII coding region caused a marked increase in the basal level of mRNA transcribed from the M.EcoRII promoter, but no induction occurred upon 5-azacytidine treatment. The level could be reduced to normal by M.EcoRII in trans. In vitro, the enzyme bound to the sequences upstream of the transcription start sites and inhibited the initiation of transcription. These experiments indicate that expression of the M.EcoRII gene was autogenously regulated at the transcriptional level. Similar regulation is also noted in another DNA (cytosine-5) methylase, M.MspI.

Azacitidine↗

PubMed1993

Induction of EcoRII methyltransferase: evidence for autogenous control.

The cytosine analog 5-azacytidine kills Escherichia coli cells that carry plasmids expressing EcoRII DNA (cytosine 5)methyltransferase under control of its own promoter. We previously showed that this enzyme binds tightly to azacytidine-containing DNA in vitro and proposed that such binding is lethal in vivo. In support of this proposal, we now show that the enzyme sediments with the nucleoid of azacytidine-treated cells. Azacytidine treatment led to an increase in the amount of enzyme, and this increase required sequences in the ecoRIIM promoter region. Enzyme inducibility correlated with drug sensitivity: plasmids carrying the methyltransferase gene but lacking the wild-type promoter did not confer sensitivity. These results suggested that the ecoRIIM gene was under autogenous control. Transcriptional ecoRIIM'-lacZ fusions in E. coli were, therefore, constructed. They showed that expression from the ecoRIIM promoter was inhibited when EcoRII DNA (cytosine-5)methyltransferase was introduced into the cell in trans and inhibition was reversed by treating the cells with azacytidine. These results provide evidence that the expression of the ecoRIIM gene is under autogenous regulation and that cell death induced by azacytidine is due, in part, to the disruption of autoregulation.

Azacitidine↗

PubMed1991

The core element of the EcoRII methylase as defined by protease digestion and deletion analysis.

Binding of the EcoRII DNA methyltransferase to azacytosine-containing DNA protects the enzyme from digestion by proteases. The limit digest yields a product having a Mr on SDS-PAGE 20% less than the intact protein. The N terminus of the tryptic digestion product was sequenced and found to be missing the N terminal 82 amino acids. Under the conditions used unbound enzyme was digested to small peptides. Protection of the enzyme from protease digestion implies that the enzyme undergoes major conformational changes when bound to DNA. The trypsin sensitive region of the EcoRII methyltransferase occurs prior to the first constant region shared with other procaryotic DNA(cytosine-5)methyltransferases. To determine if this region played a role in substrate binding or specificity, N-terminal deletion mutants were studied. Deletion of 97 amino acids resulted in a decrease of enzyme activity. Further deletions caused a complete loss of activity. Enzyme deleted through amino acid 85 was purified and found to have the same specificity as wild type however there was an increase in Km for both S-adenosylmethionine (AdoMet) and DNA of 27 and 18 fold respectively. The N-terminus of the EcoRII methylase, although a variable region present in many procaryotic DNA(cytosine-5)methylases, plays no role in determining enzyme specificity, although it does contribute to the interaction with both AdoMet and DNA.

Amino Acid Sequence↗

PubMed1991

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186.

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.

Amino Acid Sequence↗

PubMed1990

Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine.

Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either [methyl-3H]AdoMet or [35S]AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of [3H]AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported (Friedman, S. (1986) Nucleic Acids Res. 14, 4543-4556). The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.

Adenosine↗

PubMed1988

Survival and mutagenic effects of 5-azacytidine in Escherichia coli.

Survival and mutagenesis caused by 5-azacytidine was studied in Escherichia coli. Survival was partially lexA- and recA-dependent and was decreased by the presence of a DNA (cytosine-5)methyltransferase. The dcm, MspI, and EcoRII methyltransferase genes all decreased survival. There was no direct relationship between amount of methylase enzyme present and cell survival, but only plasmids containing a methylase gene sensitized cells to 5-azacytidine. Survival was not affected by uvrA, uvrB or umuCD mutations. Induction of sulA::lacZ fusions by 5-azacytidine was inhibited in strains containing elevated levels of DNA methylase. Cells resistant to 5-azacytidine when they contained a plasmid specifying the EcoRII methylase were sensitive if the plasmid specified the complete EcoRII restriction-modification system. The mechanism of cell death in these situations is therefore different. Mutation of the rpoB gene by 5-azacytidine was studied. The mutation rate was decreased by the presence of recA and lexA mutations. Mutation in umuCD had little effect on the mutation rate. The recA430 mutation, which does not support SOS-dependent mutagenesis induced by UV light, does support 5-azacytidine induced mutagenesis. The presence of DNA (cytosine-5)methyltransferase had no effect on the mutation rate caused by 5-azacytidine treatment. The mutagenic and lethal lesions caused by 5-azacytidine in the absence of methylase therefore differ from the lethal lesions that occur in the presence of methylase. The former could be due to the opening of the 5-azacytosine ring in DNA. Cell death in the presence of methylase could be due to tight binding of methylase to azacytosine containing DNA as well as inhibition of induction of the SOS response.

Azacitidine↗

PubMed1987

Nucleotide sequence and expression of the gene encoding the EcoRII modification enzyme.

The gene coding for the EcoRII modification enzyme has been cloned and the nucleotide sequence of 1933 base pairs containing the gene has been determined. The gene codes for a protein of 477 amino acids. Two transcriptional start sites have been mapped by S1 mapping. One deletion that removes 34 N-terminal amino acids was found to have partial enzyme activity. Comparison of the EcoRII methylase sequence with other cytosine methylases revealed several domains of partial homology among all cytosine methylases. Cloning the gene in multicopy pUC vectors increased the expression by 6-18 fold. A 40 fold overproduction of the EcoRII methylase was obtained by cloning the gene in the expression vector carrying the lambda PL promoter.

Amino Acid Sequence↗

PubMed1987

Metabolism of 7,12-dimethylbenz[a]anthracene by mouse mammary cells in serum-free organ culture medium.

The murine mammary gland is a prime target organ for 7,12-dimethylbenz[a]anthracene, (DMBA)-induced carcinogenesis. We analyzed the metabolism of 3H-DMBA in a cell-free microsomal activation system derived from mouse mammary microsomes and in a whole mammary organs in culture. The in vitro microsomal activation system failed to show the more polar diol derivative of DMBA after HPLC. The metabolites obtained directly from the 3H-DMBA-treated whole mammary organs, however, revealed the presence of both the diol as well as the phenolic derivatives of DMBA. Analysis of the glands and the culture medium further showed that nearly 95% of the radioactivity added to the culture medium was associated with the adipose tissue and complete solubilization of the fat pad released substantial amounts of DMBA and its metabolites. It appears that a large portion of DMBA and its metabolites remain entrapped in the adipose tissue surrounding the parenchyma. Formic acid digestion of the gland releases the DMBA and the metabolites allowing their ethylacetate extraction, and HPLC characterization.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1984

Beta-carotene inhibition of 7,12-dimethylbenz[a]anthracene-induced transformation of murine mammary cells in vitro.

The chemopreventive action of beta-carotene during chemically-induced transformation of the epithelial cells in organ culture of the whole mammary glands from BALB/c female mice was studied. The mammary epithelial cells in the whole mammary organ in a hormone supplemented, serum-free medium were transformed after 24 h exposure to 7.8 microM 7,12-dimethylbenz[a]anthracene (DMBA) between 3rd and 4th day of a total 10 day culture period. The transformation process was associated with appearance of nodule-like alveolar lesions (NLAL) in glands in vitro. The epithelial cells transformed by DMBA are potentially neoplastic, and NLAL serves as a morphological marker of preneoplasia in the glands in vitro. Treatment with beta-carotene (10(-6) M) during DMBA exposure (3rd-4th day) caused 68% inhibition in the number of glands with incidence of NLAL. A 49% inhibition of NLAL was evident when the glands were incubated with beta-carotene (days 4-10) after exposure to DMBA. Results indicate that beta-carotene inhibits DMBA-induced transformation of the mammary glands in vitro acting both at the initiation and the promotional stages. This inhibitory effect is likely due to the action of beta-carotene itself since no accumulation of retinol, the metabolic derivative of the vitamin A precursor, was detectable in the mammary glands during the 10 day culture period.

9,10-Dimethyl-1,2-benzanthracene↗