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G Dirheimer

Publications and source records attributed to G Dirheimer.

At least 37 records · Page 2Linked to original sources
PubMed1995

Postlabeling: a sensitive method for studying DNA adducts and their role in carcinogenesis.

The covalent binding of xenobiotics to DNA is an important trigger of the multistage process that leads to carcinogenesis. 32P-postlabeling represents a highly sensitive method for biomonitoring exposure to genotoxic agents and for cancer risk assessment; it is capable of detecting less than one DNA adduct per human genome. Recent improvements to the technique have shown that the resistance of adducted DNA to enzyme digestion may lead to an overestimation of the number of different adducts present in a sample.

Animals↗
PubMed1995

Structural specificity of nuclease from wheat chloroplasts stroma.

A single-strand-specific nuclease from wheat chloroplasts (ChS nuclease) was tested as a tool for RNA secondary and tertiary structure investigations, using yeast tRNA(Phe) and yeast tRNA(Asp) as models. In tRNA(Phe) the nuclease introduced main primary cleavages at positions U33, A35 and A36 in the anticodon-loop and G18 and G19 in the D-loop. In tRNA(Asp) the main primary cleavages occurred at positions U33, G34 and U35 in the anticodon-loop and the lower one at position C20:1 in the D-loop. No primary cleavages were observed within the double-stranded stems. Because ChS nuclease has (i) a low molecular weight, (ii) a wide pH range of action (5.0 to 7.5) (iii) no divalent cation requirement in the reaction mixture and (iv) can be obtained as a pure protein in rather large quantities it appeared to be a very good tool for secondary and tertiary structural studies of RNAs.

Base Sequence↗
PubMed1994

Deficiency of queuine, a highly modified purine base, in transfer RNAs from primary and metastatic ovarian malignant tumors in women.

The tRNAs from rapidly growing tissues, particularly from neoplasia, often exhibit queuine deficiency. In order to check whether different kinds of ovarian tumors display queuine deficiencies we have analyzed tRNA samples from 16 ovarian malignancies. The tRNAs from histologically normal myometrium (4 samples) and myoma (6 samples) were taken as healthy tissue and benign tumor references. Queuine deficiency was determined by an exchange assay using [8-3H]guanine and tRNA:guanine transglycosylase from Escherichia coli. The mean values of queuine deficiencies in tRNAs were: 10.95 +/- 2.21 (SD) pmol/A260 in gonadal and germ cell tumors (5 cases); 23.75 +/- 7.89 pmol/A260 in primary epithelial tumors (9 cases); and 34.58 +/- 7.18 pmol/A260 in metastatic tumors (2 cases). These values displayed statistically significant differences (P = 0.0003, Kruskal-Wallis test). The queuine deficiencies in tRNAs significantly increased when moving from well-differentiated through moderately differentiated to poorly differentiated tumors, with the highest values found in poorly differentiated metastatic tumors (P = 0.0002, Kruskal-Wallis test). Queuine deficiency determination in tRNAs is proposed as a factor for clinical outcome prognosis of ovarian malignancies.

Adolescent↗
PubMed1994

Transfer RNA binding protein in the nucleus of Saccharomyces cerevisiae.

A yeast nuclear protein that binds to tRNA was identified using a RNA mobility shift assay. Northwestern blotting and N-terminal sequencing experiments indicate that this tRNA-binding protein is identical to zuotin which has previously been shown to bind to Z-DNA [(1992) EMBO J. 11, 3787-3796]. Labeled tRNA and poly(dG-m5dC) stabilized in the Z-DNA form identify the same protein on a Northwestern blot. In a gel retardation assay poly(dG-m5dC) in the Z-form strongly diminishes the binding of tRNA to zuotin. These studies establish that zuotin is able to bind to both tRNA and Z-DNA. Zuotin may be transiently associated with tRNA in the nucleus of yeast cells and play a role in its processing or transport to the cytoplasm.

Amino Acid Sequence↗
PubMed1994

Yeast serine isoacceptor tRNAs: variations of their content as a function of growth conditions and primary structure of the minor tRNA(Ser)GCU.

The primary structure of Saccharomyces cerevisiae tRNA(Ser)GCU is presented (EMBL database accession No. X74268 S. cerevisiae tRNA-Ser). In addition, quantitation of the relative amounts of serine isoaccepting tRNAs in yeast grown on different media showed that the minor tRNA(Ser)GCU decreased while the major tRNA(Ser)AGA increased as the growth rate and the cellular protein content increased. The minor species, tRNA(Ser)CGA and tRNA(Ser)UGA, were not separated by our gel system, however, taken together they appeared to vary in the same way as tRNA(Ser)GCU. These data suggest a growth rate dependence of yeast tRNAs similar to that previously described for E. coli tRNAs.

Anticodon↗
PubMed1994

Effect of superoxide dismutase and catalase on the nephrotoxicity induced by subchronical administration of ochratoxin A in rats.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other molds. It is a natural contaminant of mouldy food and feed. OTA has a number of toxic effects, the most prominent being nephrotoxicity. Furthermore, OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. OTA inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction. Recently, lipid peroxidation induced by OTA has been reported, indicating that the lesions induced by this mycotoxin could be also related to oxidative pathways. It was then interesting to study effects of the superoxide dismutase (SOD) and catalase on the nephrotoxicity induced by OTA in rats. The two enzymes (20 mg/kg body weight each) were given to rats by subcutaneous injection, every 48 h, 1 h before gavage by OTA (289 micrograms/kg b.w. every 48 h), for 3 weeks. SOD and catalase prevented most of the nephrotoxic effects induced by ochratoxin A, observed as enzymuria, proteinuria, creatinemia and increased urinary excretion of OTA. Altogether these results indicate (i) that superoxide radicals and hydrogen peroxide are likely to be involved in the damaging processes of OTA in vivo, (ii) that SOD and catalase might be used for prevention of renal lesions in cases of ochratoxicosis.

Administration, Oral↗
PubMed1993

Overproduction and purification of native and queuine-lacking Escherichia coli tRNA(Asp). Role of the wobble base in tRNA(Asp) acylation.

Escherichia coli tRNA(Asp) was overproduced in E. coli up to 15-fold from a synthetic tRNA(Asp) gene placed in a plasmid under the dependence of an isopropyl-beta,D-thiogalactopyranoside-inducible promoter. Purification to nearly homogeneity (95%) was achieved after two HPLC DEAE-cellulose columns. E. coli tRNA(Asp)[G34] (having guanine instead of queuine at position 34) was obtained by the same procedure except that it was overproduced in a strain lacking the enzyme responsible for queuine modification. Nucleoside analysis showed that, except for the replacement of Q34 by G34 in mutant-derived tRNA(Asp), the base modification levels of both tRNAs are the same as those in wild-type E. coli tRNA(Asp). Kinetic properties of tRNA(Asp)[Q34] and [G34] with yeast AspRS compared to those in the homologous reactions in yeast and E. coli clearly indicate that the major identity elements are the same in both organisms: the conserved discriminant base and the anticodon triplet. In connection with this, we explored by site-directed mutagenesis the functional role of the interactions which, as revealed by the crystallographic structure, occur between the wobble base of yeast tRNA(Asp) and two residues of yeast AspRS. Their absence strongly affected aspartylation and the kd of tRNA(Asp). Each contact individually restores almost completely the wild-type acylation properties of the enzyme; thus, wobble base recognition in yeast appears to be more protected against mutational events than in E. coli, where only one contact is thought to occur at position 34.

Anticodon↗
PubMed1993

Role of dimerization in yeast aspartyl-tRNA synthetase and importance of the class II invariant proline.

Cytoplasmic aspartyl-tRNA synthetase (AspRS; EC 6.1.1.12) from yeast is, as are most class II synthetases, an alpha 2 dimer. The only invariant amino acid in signature motif 1 of this class is Pro-273; this residue is located at the dimer interface. To understand the role of Pro-273 in the conserved dimeric configuration, we tested the effect of a Pro-273-->Gly (P273G) substitution on the catalytic properties of homo- and heterodimeric AspRS. Heterodimers of AspRS were produced in vivo by overexpression of their respective subunit variants from plasmid-encoded genes and purified to homogeneity in one HPLC step. The homodimer containing the P273G shows an 80% inactivation of the enzyme and an affinity decrease for its cognate tRNA(Asp) of one order of magnitude. The P273G-mutated subunit recovered wild-type enzymatic properties when associated with a native subunit or a monomer otherwise inactivated having an intact dimeric interface domain. These results, which can be explained by the crystal structure of the native enzyme complexed with its substrates, confirm the structural importance of Pro-273 for dimerization and clearly establish the functional interdependence of the AspRS subunits. More generally, the dimeric conformation may be a structural prerequisite for the activity of mononucleotide binding sites constructed from antiparallel beta strands.

Aspartate-tRNA Ligase↗
PubMed1993

Some effects of ochratoxin A, a mycotoxin contaminating feeds and food, on rat testis.

In previous toxicokinetic studies, ochratoxin A (OTA) was found in testicles of laboratory animals. Therefore its possible adverse effects on the rat testis have been investigated. Male Wistar rats 300 g +/- 20 g, randomised in groups of 5 animals, were given OTA: 289 micrograms/kg in 0.1 M NaHCO3 every 48 h for 2, 4, 6 and 8 weeks, by gastric intubation. This corresponds to a contamination of 2 ppm/day in the feeds. Controls were given only the vehicle. Some testicular enzymic activities and the stages of association of germinal cells (according to Leblond and Clermont) were determined. Some of these enzymic activities vary with the ochratoxin A poisoning in the homogenate of testicles: alpha-amylase, from 1905 +/- 145 units/g to 3190 +/- 128 units/g, alkaline phosphatase from 259 +/- 20 units/g to 323 +/- 15 units/g, gamma-glutamyltransferase (gamma GT), from 170 +/- 59 units/g to 900 +/- 65 units/g after 8 weeks. In adult rats, the increase of these enzymes, especially gamma GT, may be associated with an impairment of spermatogenesis and an accumulation of premeiotic germinal cells induced by OTA. Concerning the number of association stages of germinal cells, a decrease is observed for stages I and VII whereas stages XII and XIII increase after OTA treatment, indicating also a possible impairment of spermatogenesis. All these changes seem to be due to an earlier modification of the androgen status, the testosterone level in testes being 10.4 +/- 3.6 micrograms/g of protein after 3 weeks OTA treatment instead of 5.3 +/- 1 micrograms/g in control animals.

Alkaline Phosphatase↗
PubMed1993

Kinetic properties of pure overproduced Bacillus subtilis phenylalanyl-tRNA synthetase do not favour its in vivo inhibition by ochratoxin A.

Ochratoxine A (OTA) inhibits growth of Bacillus subtilis at pHs below 7. Since OTA is a phenylalanine analogue, this effect could be due to inhibition of phenylalanine-tRNA synthetase (PheRS) by competition of this mycotoxin with the amino acid. Homogeneous PheRS was purified from Bacillus subtilis and from E. coli transformed with the PheRS gene. The latter produced about 40 times more PheRS than B. subtilis. The Km and Ki values of PheRS, respectively, for phenylalanine and OTA were measured and their concentrations within the cell determined. It appears that the concentration of OTA in the cell, in spite of a 25-fold accumulation, remained too low to significantly compete with phenylalanine. This does not suggest PheRS to be the target of OTA in cell growth and protein synthesis inhibition in Bacillus subtilis. It was also shown that the 2-3-fold increase of PheRS in OTA-treated cells is not due to phenylalanine-controlled attenuation regulation.

Bacillus subtilis↗
PubMed1993

Rye nuclease I as a tool for structural studies of tRNAs with large variable arms.

A single-strand-specific nuclease from rye germ (Rn nuclease I) was used for secondary and tertiary structure investigations of tRNAs with large variable arms (class II tRNAs). We have studied the structure in solution of two recently sequenced tRNA(Leu): yeast tRNA(Leu)(ncm5UmAA) and bovine tRNA(Leu)(XmAA) as well as yeast tRNA(Leu)(UAG), tRNA(Leu)(m5CAA) and tRNA(Ser)(IGA). The latter is the only tRNA with a long variable arm for which the secondary and tertiary structure has already been studied by use of chemical probes and computer modelling. The data obtained in this work showed that the general model of class II tRNAs proposed by others for tRNA(Ser) can be extended to tRNAs(Leu) as well. However interesting differences in the structure of tRNAs(Leu) versus tRNA(Ser)(IGA) were also noticed. The main difference was observed in the accessibility of the variable loops to nucleolytic attack of Rn nuclease I: variable loops of all studied tRNA(Leu) species were cut by Rn nuclease I, while that of yeast tRNA(Ser)(IGA) was not. This could be due to differences in stability of the variable arms and the lengths of their loops which are 3 and 4 nucleotides in tRNA(Ser)(IGA) and tRNAs(Leu) respectively.

Animals↗
PubMed1993

Differential DNA adduct formation and disappearance in three mouse tissues after treatment with the mycotoxin ochratoxin A.

Ochratoxin A (OTA) is a mycotoxin which has been implicated in Balkan endemic nephropathy, a disease characterized by a high incidence of urinary tract tumors. It induces DNA single-strand breaks and has been shown to be carcinogenic in two rodent species. For a better understanding of the OTA genotoxic effect, OTA-DNA adduct formation and disappearance has been measured using the 32P-post-labelling method after oral administration of 2.5 mg/kg of OTA to mice. In kidney, liver and spleen, several modified nucleotides were clearly detected in DNA, 24 h after administration of OTA, but their level varied significantly in a tissue and time dependent manner over a 16-day period. Total DNA adducts reached a maximum at 48 h when 103, 42 and 2.2 adducts per 10(9) nucleotides were found respectively in kidney, liver and spleen, indicating that kidney is the main target of the genotoxicity and likely carcinogenicity of OTA. The major adduct differed between kidney and liver. All adducts disappeared in liver and spleen 5 days after compound administration, whereas some adducts persisted for at least 16 days in the kidney. Some adducts were organ specific. The finding that the adducts are not quantitatively and qualitatively the same in the three organs examined is likely due to differences of metabolism in these organs, leading to different ultimate carcinogens and may also result from differences in the efficiency of repair processes.

Animals↗
PubMed1993

Ochratoxin A-related DNA adducts in urinary tract tumours of Bulgarian subjects.

Ochratoxin A (OTA), a natural contaminant of mouldy food and feed, is suspected of being one of the etiological agents responsible for Balkan endemic nephropathy (BEN) and the associated urinary tract tumours. We have previously shown that ochratoxin A is genotoxic as expressed by DNA single-strand breaks. DNA-OTA adducts have been detected in various mouse organs after ochratoxin A treatment. Tumorous tissues from three kidneys and five bladders of Bulgarian patients undergoing surgery for cancer and from three non-malignant kidneys collected from French subjects were analysed for DNA adducts. Several adducts with the same RF values as those obtained from mouse kidney after treatment with OTA (one major and some minor adducts) were detected, mainly in kidney but also in bladder tissues from Bulgaria. No adducts were detected in French kidney tissues. These results provide new evidence of the possible role of OTA in the development of tumours of the urinary tract in Bulgaria.

Animals↗
PubMed1993

DNA adduct detection: some applications in monitoring exposure to environmental genotoxic chemicals.

In the assessment of genotoxic risk factors in the environment, the measurement of DNA adducts in aquatic organisms and in plants may have considerable implications. Using 32P-postlabelling, we have detected DNA adducts in the liver of carp (Chondrostoma nasus) from the River Rhône (France), both downstream and upstream from a polychlorinated biphenyl incineration plant. Some of the DNA adducts were specific to downstream fish, suggesting a differential pattern of exposure. We have also detected DNA damage in needles in a declining spruce forest. We found that, in the declining forest, the amounts of DNA adducts increase in relation to the degree of damage to the needles whereas, in a healthy forest, the levels of DNA adducts were low. We have also found DNA adducts in the leaves of hops grown in fields where heptachlor residues persisted.

Animals↗
PubMed1992

Isolation and structure determination of natural analogues of the mycotoxin ochratoxin A produced by Aspergillus ochraceus.

Three new natural mycotoxins, analogues of ochratoxin A, in which the phenylalanine moiety is replaced by serine, hydroxyproline or lysine, were isolated from cultures of Aspergillus ochraceus by TLC followed by HPLC column chromatography. Their structures were determined after acidic hydrolysis by the characterisation of both their amino acid moieties and of ochratoxin alpha, the chlorinated dihydroisocoumarin moiety of ochratoxin A. These structures were confirmed by mass spectrum analysis.

Aspergillus ochraceus↗