Biomedical subjects
Publications and source records attributed to A Ejchart.
A combination of two-dimensional total correlation spectroscopy (TOCSY) and rotating-frame nuclear Overhauser enhancement spectroscopy (ROESY) is suggested as the optimum strategy for determining the primary structure of peracetylated oligosaccharides.
Transferred nuclear Overhauser enhancement was used to examine the conformation of NAD+ and NADP+ bound to glucose-6-phosphate dehydrogenase and glutamate dehydrogenase and of NAD+ bound to lactate dehydrogenase. The results demonstrate that the conformation of the nicotinamide-ribose bond is anti for dehydrogenases with A stereospecificity and syn for dehydrogenases with B stereospecificity. In those dehydrogenases that bind both NAD+ and NADP+, significant differences occur in the conformations of the bound nicotinamide coenzymes.
Phosphorus-31 and especially Carbon-13 NMR measurements have recently become primary input to the understanding of DNA solution dynamics. While the 31P measurements are inherently easier, the quality of 31P dynamics information is suspect and therefore 13C measurements are preferred. In fact, it is necessary to obtain several kinds of 13C data (T1s, NOE's, linewidths, integrated peak intensities) over a wide range of magnetic fields (13C NMR frequencies) in order to identify major features of DNA internal motions. Further information comes from variation of temperature and DNA fragment length and/or concentration. Most of our 13C measurements have been performed at 37.7-90.6 MHz on fully double stranded monomer size (147 base pair) DNA at concentrations in phosphate buffer of approximately less than 10 to approximately greater than 200 mg ml-1; temperatures studied range from 6 to 55 degrees C. Other measurements have been performed on monomer-size single-stranded DNA at 85 and 92 degrees. The large data set we have acquired appears to answer some important questions about the nature and extent of DNA overall and internal motional dynamics. However, the picture remains incomplete and a number of questions arise from these results: 1. Overall motion of the double stranded DNA fragments follows expected hydrodynamic behavior; 2. Restricted but rapid internal motion along the DNA structure is well represented by a spaghetti-like wobbling-in-a-cone model; 3. DNA-DNA Interactions and solvent ordering, present at relatively low DNA concentrations, partially quench the internal motion, consistent with hinge-model structural changes (and the spaghetti model above) but not as compatible with in-plane torsional motion models; 4. The deoxyribose C-2' sites undergo additional motion which is partially uncoupled from the internal wobbling motions: 5. At high DNA concentrations, a phase transition occurs, resulting in ordered structures which drastically affect DNA internal dynamics; 6. DNA interacting with ethidium does not greatly change its conformational mobility; 7. DNA interacting with Hg2+ ions shows less than anticipated change in internal DNA dynamics. The remaining challenge is to interpret our current results in terms of specific conformational processes and to understand why the conformational mobility of double stranded DNA is relatively unhindered by major structural perturbants such as intercalating ethidium and mercury ion.
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UV efficiently induces mutations in mitDNA , conferring resistance to erythromycin. Mitochondrial chloramphenicol-resistant mutants are probably also induced by UV, but almost 90% of mutants with such phenotype are non-mitochondrial; therefore it is possible to estimate accurately the frequences of the induced presumptive mitochondrial capr mutations.
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A medium was found in which manganese efficiently induces erythromycin-resistant mitochondrial mutations, and which is suitable for measuring Mn2+ uptake and the labelling of DNA (fig. 1). Mn2+ uptake is stimulated by glucose and slowed down by cycloheximide (Fig 2). Mg2+ competes with Mn2+ uptake much stronger than does Zn2+ (Fig. 3). All of the conditions which favour Mn2+ uptake also favour induction of erythromycin-resistant mutations (Tables 3, 4). Mn2+ strongly inhibits protein synthesis (Table 1). Nuclear DNA replication is also strongly inhibited by this cation, while mitochondrial DNA replication is only weakly inhibited during the first 3 h of labelling, but there is small if any increase of the label incorporation between the 3rd 6th h of labelling (Table 2). The relation between label incorporation into mitDNA and mutation induction by manganese is not straightforward (Table 5). From among 11 divalent cations tested, only Mn2+ was capable of inducing mitochondrial erythromycin-resistant mutations (Table 6).
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The induction of antibiotic-resistant mutations in yeast mitochondrial DNA by manganese is decreased when the manganese-containing medium is additionally supplemented with magnesium. At equimolar concentrations of manganese and magnesium the former is no longer mutagenic. Amino acid starvation, cycloheximide, chloramphenicol and erythromycin have very little, if any, effect on the mutagenicity of manganese. Hydroxyurea itself seems to be slightly mutagenicity of manganese. Our results show that manganese acts as an error-producing factor in DNA replication probably through a direct interaction with mitDNA polymerase(s).
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When yeast cells were incubated for 4 to 8 h in yeast extract-peptone-glucose medium, pH 6, containing 8 mM-manganese, and then plated on selective media, there was a strong induction of antibiotic-resistant mutations. Indirect evidence suggests that practically all resistant mutants selected were of independent origin. The analysis of manganese-induced resistant mutants showed that most were extranuclear, while those tested showed recombination with known mitochondrial markers. Our results suggest that manganese can be considered as a mutagen which specifically induces mitochondrial mutations in Saccharomyces cerevisiae.
The influence of catalase on the genotoxic effect of bleomycin (BLM) has been evaluated in three cell lines which differ in catalase activity. CRL1307, cells from Xeroderma pigmentosum patient and CLV102, normal embryonic cells have catalase activity 3.5 and 5 times lower then CRL2088, normal skin fibroblasts. Genotoxicity of BLM (0.5-50 micrograms/ml, 2 h treatment) measured with in vitro micronucleus test did not differ in three tested lines. BLM at concentration range from 1 to 25 micrograms/ml (2 h treatment), tested in comet assay, caused similar degree of DNA damage in CLV102 and CRL2088 cells. Exogenous catalase (300 and 900 u/ml) added to the assay medium with BLM did not influence the micronuclei induction. The absence of endo- and exogenous catalase influence on BLM genotoxicity suggests that not hydrogen peroxide but other reactive oxygen species are formed in reaction of activated BLM with molecular oxygen.
The mutagenic properties of tofisopam, the member of the 2,3-benzodiazepine family, were evaluated on the basis of Ames test with Salmonella typhimurium TA1537, TA97, TA98, TA100 and TA102 strains. The genotoxic properties of tofisopam were estimated on L929 cell line with the cytokinesis-block technique. Under the experimental conditions, no mutagenic activity of tofisopam in tester bacteria strains was found, and no genotoxic activity was observed.
Riboflavin was shown to inhibit mutagenicity of benzo(a)pyrene and 2-acetylaminofluorene in the presence of S9 liver fractions deriving from B10.A mice as well as from DBA/2 mice and had no influence on mutagenicity of methyl methanesulfonate. The above findings confirm the supposition that antimutagenicity of riboflavin results from its interaction with enzymes responsible for metabolic activation of promutagens. The antimutagenic effects of riboflavin were more drastic in the presence of liver fractions from B10.A mice than in the presence of those from DBA/2 mice.
The mutagenic and genotoxic activities of binazine and hydralazine were studied. In the Ames test, both with and without S-9 fraction, hydralazine was mutagenic in strains Salmonella typhimurium TA100 and TA1537, whereas binazine was not mutagenic in these strains. Both drugs were negative in mice micronucleus test.
The mutagenicity (Ames test) and genotoxicity (SOS Chromotest) of hydralazine were studied. Hydralazine was found to be genotoxic to E.coli PQ37. In experiments with E.coli MD332 it was genotoxic in responsive temperature (30 degrees C) but not genotoxic in non-responsive temperature (42 degrees C). Hydralazine was mutagenic to S.typhimurium TA100 and TA104 but not mutagenic to TA102. Different active oxygen species scavengers did not influence the genotoxicity and mutagenicity of hydralazine.
The role of reactive oxygen species in adriamycin and bleomycin-induced mutagenicity was investigated in Salmonella typhimurium TA98 and TA102 respectively. Activity of superoxide dismutase (SOD) was inhibited by preincubation of bacteria with diethyldithiocarbamate (DEDTC). Results of Ames test may suggest the involvement of active oxygen species in bleomycin induced mutagenesis and an absence of their participation in adriamycin induced mutagenesis.
The influence of catalase activity in adriamycin and bleomycin mutagenesis was investigated in Salmonella typhimurium TA98 and TA102, respectively. The activity of catalase in bacterial cells was inhibited by sodium azide. Mutagenicity of both drugs was not changed in bacterial cells with depressed catalase activity.