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G Dirheimer

Publications and source records attributed to G Dirheimer.

At least 55 records · Page 3Linked to original sources
PubMed1992

Structural specificity of Rn nuclease I as probed on yeast tRNA(Phe) and tRNA(Asp).

A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions.

Base Composition↗
PubMed1992

Use of a dot blot hybridization method for identification of pure tRNA species on different membranes.

The characterization of a tRNA in purification procedures usually involves aminoacylation assays but recently, the hybridization by dot blot with specific oligonucleotides as probes has been used for the tRNA identification. We present here an optimization of a dot blot hybridization method for the tRNA detection by comparing the efficiency of eight different nylon membranes. Neutral 0.22 microns porosity membranes (Nytran, Biodine A) give the best detection efficiency when small quantities of material (less than 40 ng of tRNA) are dotted on filter; by contrast, neutral 0.45 microns porosity membranes (such as Hybond N) are the most efficient when larger quantities of tRNA are dotted on the filter. The described technique allows to detect less than 20 pg of a pure tRNA species. Its use in the identification of Saccharomyces cerevisiae initiator tRNA(Met) in counter-current distribution fractions is shown.

Autoradiography↗
PubMed1992

Inhibition of protein synthesis in liver and kidney of mice by bolesatine: mechanistic approaches to the mode of action at the molecular level.

Protein synthesis was assayed in liver and kidney of mice treated with bolesatine, a toxic glycoprotein from the mushroom Boletus satanas (Lenz) which was previously shown to be an inhibitor of protein synthesis by cell-free systems in vitro and by cultured cell-lines. Protein synthesis in vivo (Swiss mice) is inhibited in a dose-dependent manner in liver and kidney. The mechanism of action does not appear to be due to RNA-N-glycosidase activity of bolesatine or a RNAase activity of this toxin on the ribosomal RNAs. Ribosomes do not appear to be damaged by pretreatment with bolesatine as judged by a poly(U) translation system. Thus bolesatine cannot be included in the group of protein synthesis inhibitors of plant origin known as ribosome-inactivating proteins (RIPs).

Animals↗
PubMed1992

Properties of bolesatine, a translational inhibitor from Boletus satanas Lenz. Amino-terminal sequence determination and inhibition of rat mitochondrial protein synthesis.

Bolesatine is a toxic monomeric glycoprotein of Mr 63,000 isolated from the mushroom Boletus satanas Lenz. Its N-terminal amino acid sequence was established: NH2-Thr-Trp-Arg-Ile-Tyr-Leu-Asn-Asn-Gln-Thr-Val-Lys-Leu-Ala-Leu-Leu-Leu- Pro- Asn-Gly.... It inhibits protein synthesis in isolated rat mitochondria. After 90 min of preincubation with bolesatine, the incorporation of [14C]-leucine into mitochondrial proteins was inhibited with an IC50 of 530 nM.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence of the mitochondrial 5S rRNA gene from lupine (Lupinus luteus).

A lupine mitochondrial clone containing 5S rRNA gene is characterized. The gene is located on the same strand as 18S rRNA and separated from it by 190 nucleotides. The intergenic region in different plants shows high degree of homology. In the case of lupine and soybean 43 nucleotides upstream of 5S rRNA gene exhibits 100% of homology. Comparisons of lupine 5S rRNA gene sequence with other plant mitochondrial 5S rRNA genes displays high degree of homology (from 89.8% to 95.8%).

Animals↗
PubMed1991

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae. Study of its functional organisation by deletion analysis.

Aspartyl-tRNA synthetase (AspRS) from yeast, a homodimer of 125 kDa, was shortened by several residues from the C- and N-termini, via site-directed mutagenesis, to examine the contribution of the removed peptides to the enzyme properties. This study showed that the N-terminal sequence up to amino acid 70 (which confers peculiar ionic properties to the protein) is dispensable for activity. Domains located beyond amino acid 70 appeared to have increasing catalytic importance; the removal of 80 or 90 residues affected the Km values for ATP and deletions of 101 or 140 amino acids profoundly modified the physiochemical properties of AspRS, and by consequence, its structural organisation (extraction of the mutated proteins out of the cells required the presence of SDS). On the C-terminal side, very limited modifications readily affected the enzyme properties. Deletion of as few as three residues increased the Km for ATP and reduced the aminoacylation kcat as well as the thermostability of the adenylate synthesis activity; the kcat of this step was impaired after deletion of two further residues. Finally, shortening the C-terminal decapeptide completely inactivated AspRS, whilst affecting neither its affinity for tRNAAsp nor its dimerisation capacity. These data reveal the role of the C-terminal decapeptide as a determinant in both reactions catalysed by AspRS. This peptide is involved in ATP binding, stabilising the functional conformation of the amino-acid-activating domain and probably maintaining the tRNA-acceptor end in a reactive position with regard to the activated amino acid.

Adenosine Triphosphate↗
PubMed1991

Effect of cobalamin derivatives on in vitro enzymatic DNA methylation: methylcobalamin can act as a methyl donor.

5-Methylcytosine synthesis in DNA involves the transfer of methyl groups from S-adenosyl-methionine to the 5'-position of cytosine through the action of DNA (cytosine-5)-methyltransferase. The rate of this reaction has been found to be enhanced by cobalt ions. We therefore analyzed the influence of vitamin B12 and related compounds containing cobalt on DNA methylation. Vitamin B12, methylcobalamin, and coenzyme B12 were found to enhance significantly the de novo DNA methylation in the presence of S-adenosylmethionine for concentrations up to 1 microM, but at higher concentrations these compounds were found to inhibit DNA methylation. Methylcobalamin behaves as a competitive inhibitor of the enzymatic methylation reaction (Ki = 15 microM), the Km for S-adenosylmethionine being 8 microM. In addition, the use of radioactive methylcobalamin shows that it can be used as a methyl donor in the de novo and maintenance DNA methylation reactions. Thus, two DNA methylation pathways could exist: one involving methylation from S-adenosylmethionine and a second one involving methylation from methylcobalamin.

Animals↗
PubMed1991

Site-specific hypomethylation of c-myc protooncogene in liver nodules and inhibition of DNA methylation by N-nitrosomorpholine.

The protooncogene c-myc was investigated in N-nitrosomorpholine-induced rat liver nodules to elucidate the role of altered DNA methylation in chemical carcinogenesis. Furthermore, Micrococcus luteus DNA and chicken erythrocyte DNA were modified in vitro by reactive metabolites of N-nitrosomorpholine, generated by P450-dependent monooxygenases. The modified DNAs were less methylated in vitro than control DNAs by DNA-(cytosine-5)-methyltransferase (DNA methylase). The DNA methylase assay and 32P-postlabeling analysis revealed lowered levels of DNA methylation in nodular DNA. In nodular tissue, c-myc messenger RNA levels were found to be increased compared to normal liver. DNA methylation analysis using the restriction endonucleases HpaII/MspI indicated hypomethylation in the first intron of c-myc DNA in liver nodules. The results suggest that genotoxic lesions may cause stably inherited, aberrant DNA methylation patterns which may be responsible for site-specific hypomethylation of the c-myc protooncogene in liver nodules.

Animals↗
PubMed1991

Orellanine inhibits protein synthesis in Madin-Darby canine kidney cells, in rat liver mitochondria, and in vitro: indication for its activation prior to in vitro inhibition.

Pure orellanine, a nephrotoxic compound extracted from the mushroom Cortinarius orellanus, which is known to induce severe kidney damage several days or weeks after ingestion, is found to inhibit strongly the synthesis of macromolecules (proteins, RNA and DNA) in Madin-Darby canine kidney (MDCK) cells and in rat liver mitochondria, although the uptake of labelled precursors of the above macromolecules is not significantly altered. Direct addition of orellanine to a cell-free system of rabbit reticulocyte lysate does not produce any inhibition of protein synthesis. However, when orellanine is pre-incubated with activating rat liver microsomal systems, this inhibition occurs. Thus, the in vivo inhibition of protein synthesis is most likely due to a metabolite of orellanine.

2,2'-Dipyridyl↗
PubMed1991

Cysteinyl-tRNA synthetase: determination of the last E. coli aminoacyl-tRNA synthetase primary structure.

The gene coding for E. coli cysteinyl-tRNA synthetase (cysS) was isolated by complementation of a strain deficient in cysteinyl-tRNA synthetase activity at high temperature (43 degrees C). Sequencing of a 2.1 kbp DNA fragment revealed an open reading frame of 1383 bp coding for a protein of 461 amino acid residues with a Mr of 52,280, a value in close agreement with that observed for the purified protein, which behaves as a monomer. The sequence of CysRS bears the canonical His-Ile- Gly -His (HIGH) and Lys-Met-Ser-Lys-Ser (KMSKS) motifs characteristic of the group of enzymes containing a Rossmann fold; furthermore, it shows striking homologies with MetRS (an homodimer of 677 residues) and to a lesser extent with Ile-, Leu-, and ValRS (monomers of 939, 860, and 951 residues respectively). With its monomeric state and smaller size, CysRS is probably more closely related to the primordial aminoacyl-tRNA synthetase from which all have diverged.

Amino Acid Sequence↗
PubMed1991

Characterization of bolesatine, a toxic protein from the mushroom Boletus satanas Lenz and it's effects on kidney cells.

Protein, DNA, and RNA syntheses were assayed in Madin Darby canine kidney cells (MDCK) treated by bolestaine, a toxic glycoprotein from the mushroom Boletus satanas (single chain, Mr 63,000 +/- 3000, pI 8.3 +/- 0.1, disulphide intrachain bridge) previously shown to be an inhibitor of in vitro protein synthesis. Cellular protein and DNA syntheses are inhibited in a dose-dependent manner after 24 h of incubation, whereas the uptake of the labelled precursors of proteins, DNA and RNA biosyntheses into the cells is not affected. The IC50 of bolesatine for protein synthesis is 0.14 microM in the cell culture medium. RNA synthesis is not inhibited at this concentration. The IC50 for DNA synthesis is 0.32 microM. When galactose is added to the culture medium, it decreases or even abolishes the toxic effects, indicating that it prevents the toxin from binding on the membrane and penetrating inside the cells. The profiles of polysomes in MDCK cells treated with bolesatine, compared to the untreated ones, show an increasing pool of polysomes indicating that the toxin acts on the peptidyl elongation step in the protein synthesis.

Animals↗
PubMed1991

Sequence of a new tRNA(Leu)(U*AA) from brewer's yeast.

The nucleotide sequence of a new tRNA(Leu)(anticodon U*AA) from Saccharomyces cerevisiae which could recognize exclusively the UUA codon has been determined. Its primary structure is: pGGAGGGUUGm2GCac4CGAGDGmGDCDAAGGCm2(2)GGCAGACmUU*AAm1GA++ + psi CUGUUGGACGGUUGUCCGm5CGCGAGT psi CGm1A(orA)ACCUCGCAUCCUUCACCA. This tRNA has a large extraloop and contains 15 modified nucleotides. So far it is the third isoacceptor tRNA for leucine in yeast. It has 61% homology with tRNA(Leu)(anticodon m5CAA) and 63% homology with tRNA(Leu)(anticodon UAG), the two other known yeast tRNAs(Leu).

Base Sequence↗
PubMed1991

Disposition of the toxic protein, bolesatine, in rats: its resistance to proteolytic enzymes.

1. Bolesatine is a toxic protein (LD50 oral 3.3 mg/kg in mice) isolated from the mushroom Boletus satanas Lenz, which inhibits protein synthesis in vitro. It induces gastroenteritis in human. 2. 14C-Bolesatine, given orally to rats (30 micrograms/kg), is distributed in the gastrointestinal, tract, kidney, liver and, to a lesser extent, in the thymus, spleen and lung. Bolesatine is eliminated in faeces and urine (80% in 24h). 3. The material excreted in urine is not proteolysed, and no protease (trypsin, chymotrypsin, pronase, proteinase K, Staphylococcus aureus (strain V8) protease and pepsin) is found to hydrolyse bolesatine in either its native or denatured form. However, thermolysin hydrolysed denatured bolesatine to a protein having a Mr of about 55 kD. 4. Bolesatine is found in all the following rat liver and kidney subcellular fractions: cytoplasm, mitochondria, ribosomes, microsomes and nuclei.

Animals↗
PubMed1991

Human ochratoxicosis in France.

The prevalence of human ochratoxicosis in France is being determined using serum and plasma collected from apparently healthy people. The analytical method is based on the partition coefficient of ochratoxin A in aqueous and organic solvents, according to pH. High-performance liquid chromatography and spectrofluorimetry are used for detection and quantification (limit of detection, greater than 0.2 ng/ml). The presence of ochrotoxin A is confirmed by the action of carboxypeptidase to yield ochratoxin alpha or by derivatization of ochratoxin A with boron trifluoride. The significance of the interim values obtained and the number of positive samples is discussed. A comparison with the distribution of known values in Germany and Scandinavia could be helpful in risk assessment with a view to prevention.

Chromatography, High Pressure Liquid↗
PubMed1991

Mechanism of action of ochratoxin A.

Ochratoxin A has a number of toxic effects in mammals, the most notable of which is nephrotoxicity. It is also immunosuppressive, teratogenic and carcinogenic. The biochemical and molecular aspects of its action were first studied in bacteria. The appearance of 'magic spots' (ppGpp and pppGpp) pointed to inhibition of the charging of transfer ribonucleic acids (tRNA) with amino acids. This suggestion was confirmed by the demonstration that ochratoxin A inhibits bacterial, yeast and liver phenylalanyl-tRNA synthetases. The inhibition is competitive to phenylalanine and is reversed by an excess of this amino acid. As a consequence, protein synthesis is inhibited, as shown with hepatoma cells in culture, with Madin Darby canine kidney cells (which are much more sensitive) and in vivo in mouse liver, kidney and spleen, the inhibition being more effective in the latter two organs. An excess of phenylalanine also prevents inhibition of protein synthesis in cell cultures and in vivo. Analogues of ochratoxin A in which phenylalanine has been replaced by other amino acids have similar inhibitory effects on the respective amino acid-specific aminoacyl tRNA synthetases. 4R-Hydroxyochratoxin A, a metabolite of ochratoxin A, has a similar action, whereas ochratoxin alpha (the dihydroisocoumarin moiety) and ochratoxin B (ochratoxin A without chlorine) have no effect. Ochratoxin A might act on other enzymes that use phenylalanine as a substrate. We showed recently that it inhibits phenylalanine hydroxylase. In addition, the phenylalanine moiety of ochratoxin A is partially hydroxylated to tyrosine by incubation with hepatocytes and in vivo. This competitive action with phenylalanine might explain why this amino acid prevents the immuno-suppressive effect of ochratoxin A and partially prevents its teratogenic and nephrotoxic actions. The effect of ochratoxin A on protein synthesis is followed by an inhibition of RNA synthesis, which might affect proteins with a high turnover. Ochratoxin A also lowers the level of phosphoenolpyruvate carboxykinase, a key enzyme in gluconeogenesis; this inhibition is reported to be due to a specific degradation of mRNA that codes for this enzyme. Recently, ochratoxin A was also found to enhance lipid peroxidation both in vitro and in vivo. This inhibition might have an important effect on cell or mitochondrial membranes and be responsible for the effects on mitochondria that have been shown by several authors. Finally, the recent results of Pfohl-Leszkowicz et al. (this volume), who showed the formation of DNA adducts mainly in kidney but also in liver and spleen, explain the DNA single-strand breaks observed previously in mice and rats after acute and chronic treatment.

Animals↗
PubMed1991

DNA adduct formation in mice treated with ochratoxin A.

Several authors have reported the occurrence of renal and hepatic tumours in mice and rats exposed to ochratoxin A in long-term studies. The compound was not mutagenic, however, in various microbial and mammalian gene mutation assays, either with or without metabolic activation. Contradictory results were obtained for induction of unscheduled DNA synthesis and sister chromatid exchange. We showed previously that ochratoxin A causes DNA damage, manifested as single-strand breaks in mouse spleen cells and in vivo. These findings, which suggest that ochratoxin A is weakly genotoxic to mammalian cells, prompted us to search for DNA adducts using a modified 32P-postlabelling method, the sensitivity of which was improved by treatment with nuclease P1. DNA was isolated from liver, kidney and spleen excised from mice 24, 48 and 72 h after oral treatment with ochratoxin A at 0.6, 1.2 and 2.5 mg/kg body weight. Several adducts were found in the DNA of the three organs, the levels varying greatly. After administration of 2.5 mg/kg body weight, 40 adducts per 10(9) nucleotides were found in kidney DNA and 7 adducts per 10(9) nucleotides in liver after 72 h. The levels of most of the adducts increased from 24 to 72 h, but those of others diminished after 24 or 48 h. Adducts were found in spleen only at 24 and 48 h. These results confirm the genotoxicity of ochratoxin A.

Administration, Oral↗