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Biomedical subjects

J Thomale

Publications and source records attributed to J Thomale.

At least 37 records · Page 2Linked to original sources
PubMed1994

Repair of O6-alkylguanines in the nuclear DNA of human lymphocytes and leukaemic cells: analysis at the single-cell level.

Inter-individual and cell-cell variability of repair of O6-alkylguanines (O6-AlkGua) in nuclear DNA was studied at the single-cell level in peripheral lymphocytes from healthy donors and in leukaemic cells isolated from patients with chronic lymphatic leukaemia (CLL) or acute myeloid leukaemia (AML). Cells were pulse exposed to N-ethyl- or N-(n-)butyl-N-nitrosourea in vitro, and O6-AlkGua residues in DNA were quantified using an anti-(O6-AlkGua) monoclonal antibody and electronically intensified fluorescence. The kinetics of O6-AlkGua elimination revealed considerable inter-individual differences in O6-ethylguanine (O6-EtGua) half-life (t1/2) values in DNA, ranging from 1.5 to 4.5 h (five AML patients), from 0.8 to 2.8 h (five CLL patients) and from 1.2 to 7.3 h (five healthy donors). The elimination from DNA of equimolar amounts of O6-butylguanine was generally 3-5 times slower in comparison with O6-EtGua. The t1/2 values of individual samples varied in parallel for both DNA alkylation products. Upon preincubation with O6-benzylguanine, the activity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AT) in both lymphocytes and leukaemic blasts was reduced to < or = 1%. However, while the rate of O6-EtGua elimination from DNA was decelerated it was not abolished, suggesting the possible involvement of additional repair systems that might be co-regulated with AT. Within individual samples, no major cell subpopulations were observed whose repair kinetics would differ significantly from the remaining cells.

Alkylating Agents↗
PubMed1991

Monoclonal antibody-based, selective isolation of DNA fragments containing an alkylated base to be quantified in defined gene sequences.

We have established a sensitive, monoclonal antibody (Mab)-based procedure permitting the selective enrichment of sequences containing the miscoding alkylation product O6-ethylguanine (O6-EtGua) from mammalian DNA. H5 rat hepatoma cells were reacted with the N-nitroso carcinogen N-ethyl-N-nitrosourea in vitro, to give overall levels of greater than or equal to 25 O6-EtGua residues per diploid genome (corresponding to O6-EtGua/guanine molar ratios of greater than or equal to 10(-8). For analysis, enzymatically restricted DNA from these cells is incubated with an antibody specific for O6-ethyl-2'-deoxyguanosine, the resulting Mab-DNA complexes are separated from (O6-EtGua)-free fragments by filtration through a nitrocellulose (NC) membrane, and the DNA is recovered from the filter-bound complexes quantitatively. The efficiency of Mab binding to DNA fragments containing O6-EtGua is constant over a range of O6-EtGua/guanine molar ratios between 10(-5) and 10(-8). (O6-EtGua)-containing restriction fragments encompassing known gene sequences (e.g., the immunoglobulin E heavy chain gene of H5 rat hepatoma cells used as a model in this study) are subsequently amplified by PCR and quantified by slot-blot hybridisation. The content and distribution of a specific carcinogen-DNA adduct in defined sequences of genomic DNA can thus be analyzed as well as the kinetics of intragenomic (toposelective) repair of any DNA lesion for which a suitable Mab is available.

Animals↗
PubMed1990

Repair of O6-ethylguanine in DNA protects rat 208F cells from tumorigenic conversion by N-ethyl-N-nitrosourea.

O6-Ethylguanine (O6-EtGua) is one of about a dozen different alkylation products formed in the DNA of cells exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea (EtNU). We have evaluated selectively the relative capacity of cells for the specific enzymatic repair of O6-EtGua as a determinant for the probability of malignant conversion. Eleven O6-EtGua-repair-proficient (R+) variant subclones were isolated from the O6-EtGua-repair-deficient (R-) clonal rat fibroblast line 208F by selection for resistance to 1,3-bis-(2-chloroethyl)-1-nitrosourea (frequency, approximately equal to 10(-5). Contrary to the 208F wild-type cells, all variants expressed O6-methylguanine-DNA methyltransferase activity, while both kinds of cells were deficient for repair of the DNA ethylation products O2- and O4-ethylthymine. After exposure to EtNU (less than or equal to 500 micrograms/ml; 20 min), cells were analyzed for the formation of piled-up foci in monolayer culture and of anchorage-independent colonies in semisolid agar medium. Depending on the EtNU concentration, the frequencies of piled-up foci and agar colonies, respectively, in the R+ variants were as low as 1/28th and 1/56th of those in the R- wild type. Contrasting with the cells from R+ variant-derived agar colonies, cells from 208F (R-) agar colonies gave rise to highly malignant tumors when implanted subcutaneously into syngeneic rats. No significant differences in the frequencies of piled-up foci were found between wild-type and variant cells after exposure to the major reactive metabolite of benzo[a]pyrene, (+)-7 beta, 8 alpha-dihydroxy-9,10 alpha-epoxy-7,8,9,10 alpha-tetrahydrobenzo[a] pyrene, for which stable binding to guanine O6 in cellular DNA has not been observed. The relative capacity of cells for repair of O6-alkylguanine is, therefore, a critical determinant for their risk of malignant conversion by N-nitroso carcinogens.

Animals↗
PubMed1989

1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-deoxycytidine detected by monoclonal antibodies in lung and liver DNA of rats exposed to vinyl chloride.

1,N6-Etheno-2'-deoxyadenosine (epsilon dAdo) and 3,N4-etheno-2'-deoxycytidine (epsilon dCyd) are formed in vitro by reaction of DNA with the electrophilic metabolites of vinyl chloride (VC), chloroethylene oxide and chloroacetaldehyde. To detect and quantitate these DNA adducts in vivo, we have raised a series of specific monoclonal antibodies (Mab). Among those, Mab EM-A-1 and Mab EM-C-1, respectively, were used for detection of epsilon dAdo and epsilon dCyd by competitive radioimmunoassay (RIA), following pre-separation of the etheno adducts from DNA hydrolysates by high performance liquid chromatography. At 50% inhibition of tracer-antibody binding, both Mab had a detection limit of 187 fmol and antibody affinity constants (K) of 2 x 10(9) l/mol. The levels of epsilon dAdo and epsilon dCyd were quantitated in the DNA of lung and liver tissue of young Sprague-Dawley rats exposed to 2000 p.p.m. of VC for 10 days. The epsilon dAdo/2'-deoxyadenosine and epsilon dCyd/2'-deoxycytidine molar ratios were 1.3 x 10(-7) and 3.3 x 10(-7), respectively, in lung DNA, and 5.0 x 10(-8) and 1.6 x 10(-7) in liver DNA. When hydrolysates of 3 mg of DNA were analyzed by RIA at 25% inhibition of tracer-antibody binding, epsilon dAdo and epsilon dCyd were not detected in liver DNA from untreated rats above the limiting epsilon dAdo/2'-deoxyadenosine and epsilon dCyd/2'-deoxycytidine molar ratios of 2.2 x 10(-8) and 3.1 x 10(-8), respectively.

Animals↗
PubMed1988

Alternative pathways for the in vivo repair of O6-alkylguanine and O4-alkylthymine in Escherichia coli: the adaptive response and nucleotide excision repair.

The in vivo removal of three different O-alkylated bases from DNA was measured in Escherichia coli. Using monoclonal antibodies specific for O6-methylguanine, O6-ethylguanine and O4-ethylthymine we have monitored the removal of these lesions from six different strains to assess the relative contributions of the adaptive response and of nucleotide excision repair. During the first hour after DNA alkylation, O6-methylguanine, O6-ethylguanine and O4-ethylthymine lesions were repaired almost exclusively by nucleotide excision, except when the adaptive response was being constitutively expressed. In wild-type E. coli the adaptive response began to contribute to O6-methylguanine repair about one hour after alkylation, the time required for the full induction of the ada DNA methyltransferase. In contrast, the adaptive response did not play such a large role in the repair of O6-ethylguanine and O4-ethylthymine in wild-type E. coli, presumably because DNA ethylation damage is a poor inducer of the adaptive response; possible reasons for this poor induction are discussed. The repair of all three O-alkylated lesions was virtually absent in ada- uvr- bacteria suggesting that no alternative pathway is available for their repair, at least during the first two hours after alkylation. When the repair of O-alkylated bases was compromised by an ada- or by a uvr- mutation, the bacteria became more sensitive to alkylation induced killing and mutation.

Alkylation↗
PubMed1987

Acquisition of resistance to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride in V79 cells through increased removal of O6-alkylguanine.

The molecular mechanism of acquisition of resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride (ACNU) was investigated using ACNU-resistant clones (ACNUr-1-4) isolated from the V79 cell line. The binding level of alkyl cyanate, a decomposition product of ACNU, to protein in ACNUr-1 cells was not less than that in the parental V79 cells, indicating that the acquired resistance was not due to a reduced intracellular concentration of ACNU. Because O6-chloroethylguanine, an intermediate in cytotoxic interstrand cross-link formation by ACNU, is known to be repaired by the same mechanism as O6-ethyldeoxyguanosine (O6-EtdGuo), we quantitated O6-EtdGuo by radioimmunoassay at various times after exposure of cells to 100 micrograms/ml N-ethyl-N-nitrosourea for 20 min. In V79 cells, elimination of O6-EtdGuo was negligible, but in all four resistant clones, 30 to 59% of the O6-EtdGuo was removed within 24 hr after exposure. This increased removal of O6-EtdGuo among the resistant clones was associated with the activity of O6-alkylguanine DNA alkyltransferase (O6-AGT) determined using cell extracts. The present results indicate that increased removal of O6-chloroethylguanine in ACNU-resistant clones by O6-AGT is mechanistically linked to the acquisition of resistance to ACNU.

Animals↗
PubMed1985

O(6)-methyldeoxyguanosine in oesophageal DNA among individuals at high risk of oesophageal cancer.

A radioimmunoassay using monoclonal antibodies (MAb) against 0(6)-medGuo and 0(6)-etdGuo enabled the presence of these DNA adducts to be detected in human tissues. With this methodology and with 1 mg of hydrolysed DNA it is possible to detect approximately 25 fmol 0(6)-medGuo or 12.5 fmol 0(6)-etdGuo per mg DNA. The analysis comprised a total of 37 human tissue specimens derived from patients who underwent surgery for cancer of the oesophagus in Linxian County (People's Republic of China); for comparison 12 tissue samples obtained from hospitals in Europe were similarly analysed. Seventeen samples from Linxian County showed a level of 0(6)-medGuo ranging from 15 to 50 fmol/mg DNA, 10 showed higher levels up to 160 fmol/mg DNA, and the remaining 10 samples were below the limit of detection. All the tissue samples from Europe showed levels below 45 fmol 0(6)-medGuo/mg DNA, 7 being below the limit of detection. No 0(6)-etdGuo was detected in any of the samples. 0(6)-alkylguanine DNA alkyltransferase activity observed in tissue extracts from Linxian County ranged from 190 to 326 fmol (mean values) of 0(6)-medGuo removed per mg protein and lower values were obtained for the removal of 0(6)-etdGuo. The findings obtained using this approach appear promising in assessing the role of alkylating agents, e.g. nitrosamines, in the aetiology of human cancer.

China↗
PubMed1985

Induction of malignant transformation by various chemicals in Balb/3T3 clone A31-1-1 cells and biological characterization of some transformants.

Balb/c A31-1-1 cells were used for the study of transformation induction by chemicals with different mutagenic specificities. We show that survival of these cells and therefore the calculated transformation frequency per cells at risk is dependent upon the cell density at the time of treatment. It is suggested that equal cell densities should be used for measuring survival values and transformation induction. The quantitative results obtained are discussed in the light of the known mutagenic mechanisms of the chemicals tested. We also characterized morphologically transformed foci induced by different chemicals with respect to some biological properties. Anchorage independence was determined by testing growth in soft agar, loss of contact inhibition was quantitated by measuring maximum cell densities and malignancy was tested by tumor induction in nude mice. Although no very close correlation between these parameters and morphology was observed, the most malignant clones are also the ones with the highest values in the other tests. Our data make one or few genetical targets for transformation induction likely. We therefore speculate that the diverse phenotypes obtained might be due to differential activation of one or very few transforming genes in these cells.

Animals↗
PubMed1984

Excretion of modified nucleosides during development of malignant lymphomas in mice after whole body irradiation.

During x-ray-induced development of malignant lymphomas in mice their urinary excretion of eight modified nucleosides was monitored and the values were compared to the results of the histological examination of the animals at time of their sacrifice. It was found that the pathologically augmented excretion of modified nucleosides begins as much as several weeks before the malignant lymphomas can be diagnosed clinically. Thus some mice had increased levels of modified nucleosides even 10 weeks before sacrifice, though at the time of sacrifice the histological investigation revealed only some small foci of reticulum cell neoplasm in their spleen. It is therefore stressed that the usefulness of the determination of urinary modified nucleosides as an early noninvasive screening test for cancer in man and as an in vivo carcinogenicity test should be evaluated.

Animals↗
PubMed1983

Increasing urinary levels of modified nucleosides and bases during tumor development in mice.

Based on the fact that human cancer patients excrete increased amounts of various modified nucleosides and bases in their urine, we investigated whether the same phenomenon takes place in mice bearing experimentally induced tumors. We did indeed find that mice with MCA-induced skin tumors and mice exhibiting leukemia after X-ray irradiation excrete severalfold higher levels of modified nucleosides and bases than do untreated control mice. Comparison of the time course of altered urinary excretion of these RNA catabolites with the appearance of a recognizable tumor after MCA application revealed that the onset of the altered excretion rate of these compounds precedes tumor diagnosis. At present, the time-course studies in our mice exposed to a single X-ray dose to induce lymphoblastic leukemia seem to indicate a similar situation. Mice exhibiting preleukemic histological features already excrete increased amounts of various modified nucleosides and bases. Confirmation of our results by analysis of further irradiation-exposed mice in our present detailed time-course studies and of tumors experimentally induced in other organs of mice and other species will be taken as a basis for developing an in vivo test for carcinogenicity. Furthermore, the results could provide a foundation for the setting up of a noninvasive, early screening method for cancer in human beings.

Animals↗
PubMed1983

Increased urinary levels of RNA catabolites in mice as early indicators for malignant growth.

It is known that human cancer patients exhibit an altered urinary excretion pattern of modified nucleosides and bases, that mice bearing skin tumors excrete increased amounts of various modified nucleosides and bases, and that the onset of altered excretion of modified RNA constituents precedes tumor diagnosis. We have now obtained similar results in mice for lymphoblastic leukemia induced by a single exposure to X-ray irradiation. Mice showing severe leukemic symptoms excrete severalfold amounts of modified nucleosides in their 24-hr urine, when compared with untreated controls. X-ray-treated mice, showing no visible symptoms except increased excretion rates of these RNA constituents, were sacrificed and histologically examined. (Pre)leukemic features in spleen and lymph nodes were observed. Our results provide further evidence in support of the use of RNA catabolites as a basis for the development of an early noninvasive screening method for cancer in human beings.

Animals↗
PubMed1982

Elevated urinary excretion of RNA catabolites as an early signal of tumor development in mice.

Determination of the urinary excretion rates of 12 modified nucleosides and bases in mice after tumor induction by application of a single dose of 3-methylcholanthrene revealed that mice bearing tumors in advanced stages excrete many-fold amounts of these nucleic acid catabolites compared with the control values. The excretion rate of several of these nucleic acid constituents like ac4C, m1A, PsU, and m2Gua increased before the tumor was diagnosable. Untreated control mice as well as mice having received the carcinogen, but not developing a tumor, did not show an alteration in the excretion values of any of the modified nucleosides and bases determined.

Animals↗
PubMed1980

A new method for the simultaneous analysis of unmodified and modified urinary nucleosides and nucleobases by high performance liquid chromatography.

Details are given of a new, rapid and simple pre-fractionation method and an isocratic high performance liquid chromatography system suitable for parallel analysis of nucleosides and nucleobases from urine and other biological fluids. The quantitative recovery and excellent reproducibility of the method is demonstrated by analysis of representative standard RNA catabolites. The advantage of this new method for application to biological samples is discussed.

Adult↗
PubMed1980

Non-coordinate regulation of enzymes involved in transfer RNA metabolism in Escherichia coli.

During different steady state growth conditions in Escherichia coli the level of the three tRNA-modifying enzymes, the tRNA(m5Urd)-, tRNA(m1Guo)- and tRNA(mam5s2Urd)methyltransferase and of five aminoacyl-tRNA synthetases, the leucyl-, valyl-, isoleucyl-, arginyl- and threonyl-tRNA-synthetase, has been determined. It is shown that those two classes of tRNA affecting enzymes are not coordinately regulated and that even within these two groups of enzymes the constituents are regulated independently of each other. Furthermore it is demonstrated that none of the aminoacyl-tRNA synthetases and only one of the three tRNA-methyltransferases, the tRNA(m5Urd)methyltransferase, is under control of the relA+-gene.

Amino Acyl-tRNA Synthetases↗
PubMed1980

Genetic analysis of mutations causing borrelidin resistance by overproduction of threonyl-transfer ribonucleic acid synthetase.

Mutations leading to borrelidin resistance in Escherichia coli by overproduction of threonyl-transfer ribonucleic acid synthetase were anaylzed genetically. The regulatory mutations were closely linked to the treonyl-transfer ribonucleic acid synthetase structural gene (thrS), located clockwise to it. The mutation that causes the threefold-increased enzyme level was more distant from thrS than the mutation responsible for the ninefold overproduction. Both mutations were cis dominant in merodiploid strains, indicating that they affected promoter-operator-like control elements. Overproduction was restricted to threonyl-transfer ribonucleic acid synthetase and was not observed for the products of genes neighboring thrS (e.g., infC, pheS, pheT, and argS), providing evidence that thrS is transcribed singly and that gene amplificationis not a likely basis for increased thrS experession.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Escherichia coli mutants overproducing phenylalanyl- and threonyl-tRNA synthetase.

The structural genes for threonyl-tRNA synthetase (ThrRS) and phenylalanyl-tRNA synthetase (PheRS) are closely linked on the Escherichia coli chromosome. To study whether these enzymes share a common regulatory element, we have investigated their synthesis in mutants which were selected for overproduction of either ThrRS or PheRS. It was found that mutants isolated previously for overproduction of ThrRS as strains resistant to the antibiotic borrelidin (strains Bor Res 3 and Bor Res 15) did not show an elevated level of PheRS. PheRS-overproducing strains were then isolated as revertants of strains with structurally altered enzymes. Strain S1 is a temperature-resistant derivative of a temperature-sensitive PheRS mutant, and strain G118 is a prototrophic derivative of a PheRS mutant which shows phenylalanine auxotrophy as a consequence of an altered K(m) of this enzyme for the amino acid. In both kinds of revertants, S1 and G118, the concentration of PheRS and ThrRS was increased by factors of about 2.5 and 1.8, respectively, whereas the level of other aminoacyl-tRNA synthetases was not affected by the mutations. Genetic studies showed that the simultaneous overproduction of PheRS and ThrRS in revertants G118 and S1 is based upon gene amplification, since this property was easily lost after growing the cells in the absence of the selective stimulus, and since this loss could be prevented by the presence of the recA allele. By similar criteria, the four- and eightfold overproduction of ThrRS in strains Bor Res 3 and Bor Res 15, respectively, was very stable genetically, indicating that it is caused by a mutational event other than gene amplification. From these results, we conclude that the concomitant increase of PheRS and ThrRS in strains G118 and S1 is an expression of gene duplication and not of a joint regulation of these two aminoacyl-tRNA synthetases. This conclusion is further supported by the result that, in mutant G118 as well as in its parental strain G1, growth in minimal medium lacking phenylalanine led to an additional twofold increase of their PheRS concentration. This increase was restricted to the PheRS, since the level of other aminoacyl-tRNA synthetases, including the ThrRS, stayed unchanged.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Alteration of the intracellular concentration of aminoacyl-tRNA synthetases and isoaccepting tRNAs during amino-acid limited growth in Escherichia coli.

Growth of Escherichia coli AB 2271 under threonine or isoleucine deficiency leads to a depression of the threonyl-tRNA synthetase and isoleucyl-tRNA synthetase respectively. During this amino-acid-limited growth the concentrations of isoaccepting fractions of the cognate tRNA species were changed, as demonstrated by their altered reversed-phase-5 chromatograms. But, in addition, the profiles of the isoacceptors of all other tRNA species investigated, i.e. of tRNAsLeu, tRNAsSer and tRNAsArg were also altered. This means that, if there is a correlation between regulation of the level of an aminoacyl-tRNA synthetase and its cognate isoaccepting tRNAs, it is superimposed by the effect of amino acid limitation upon the concentration of all isoaccepting tRNAs. So far drastic changes in profiles of isoaccepting tRNAs have only been observed under unbalanced growth in relaxed cells or during treatment with antibiotics. Here we demonstrate that similar heavy alterations in patterns of isoaccepting tRNAs occur in a proven stringent E. coli strain growing exponentially under amino acid limitation. Thus the observed changes in the profiles of isoaccepting tRNAs during amino acid limitation signal a meaningful biological function of those newly or increasingly occurring isoaccepting tRNAs. During the growth under amino acid limitation the total acceptor activity of eight investigated tRNA species, however, stayed unchanged, except that under threonine-limited growth the total amount of tRNAIle was reduced to about half and that of tRNAGlu increased; both tRNA species of these isoacceptors are known [30,31] as spacers between ribosomal RNAs.

Amino Acids↗