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Biomedical subjects

G Dirheimer

Publications and source records attributed to G Dirheimer.

At least 91 records · Page 5Linked to original sources
PubMed1987

A single base change in the extra-arm of yeast mitochondrial tyrosine tRNA affects its conformational stability and impairs aminoacylation.

The mitochondrial temperature-sensitive mutation tsm-8 maps on a 1.8 kb HpaII fragment of mitochondrial DNA (mt DNA) which contains genes for tRNA(Ala), tRNA(Ile) and tRNA(Tyr). The phenotype of this mutation is, among multiple pleiotropic defects, a temperature-induced reduction of mitochondrial translation. DNA sequencing of the HpaII fragment from the wild type and mutant tsm-8 revealed a single transversion from T to A in position 56 of the mutant tRNA(Tyr) gene. This nucleotide change disrupts a base pairing in the long extra arm of the tRNA cloverleaf. Revertants of the tsm-8 mutant restore correct base pairing in the extra arm by a second-site mutation in the tRNA(Tyr) gene. Analysis of the tRNA(Tyr) transcripts revealed that neither transcription nor processing of the tRNA is affected in the mutant. However, the base alteration destabilizes the conformation of the tRNA and affects its charging parameters. At the non-permissive temperature, the Michaelis-Menten constant of the mitochondrial tyrosyl-tRNA synthetase for the mutant tRNA is increased over 20-fold when compared to the wild-type tRNA. As a consequence, mitochondrial protein synthesis is drastically reduced at the restrictive temperature. Moreover, synthesis of apocytochrome b and of cytochrome oxidase subunit 3 is decreased relative to the other mitochondrially synthesized polypeptides.

Adenine↗
PubMed1987

The Kupffer cell is the first target in ricin-induced hepatitis.

The first target of ricin in the liver appears to be the Kupffer cells which are heavily damaged as early as four hours after the intravenous inoculation of 6 LD100 into mice. At that time, the only endothelial cell damage is constituted by more or less extended interruptions of the fenestrated cytoplasm. Hepatocyte injury affects the endoplasmic reticulum, the glycogen, the mitochondria as well as the plasmic membrane; however, it never results in cytolysis or complete necrosis. The intravascular coagulation observed may be induced either by the endotoxins, which are no longer cleared by the Kupffer cells, or as a result of the loss of the sinusoidal lining with the ensuing platelet activation.

Alanine Transaminase↗
PubMed1986

Changes in urinary and renal tubular enzymes caused by subchronic administration of ochratoxin A in rats.

The activities of 5 enzymes in urine and renal tubules were measured after administration to male Wistar rats of small doses of ochratoxin A (145 micrograms/kg per day for 8-12 weeks, corresponding to 2 ppm in the feed) by intubation. These doses are in the range of natural contaminations found in food and feed. The enzymes examined were gamma-glutamyl transferase (gamma-GT), alkaline phosphatase (ALP), leucine aminopeptidase (LAP), lactate dehydrogenase (LDH), and N-acetyl-beta-D-glucosaminidase (NAG). The doses employed caused increased enzymuria and lower activities of tubular enzymes after 1 week of feeding. This suggests tubular injury. The change of the enzyme activities in the urine and in the tubules appeared in a cyclic way (degeneration and regeneration). Phenylalanine (20 ppm) partially prevented this action of ochratoxin A. The p-[14C]aminohippurate accumulation was inhibited by 60% in the second week but returned to almost normal level 6 weeks after the beginning of the treatment, suggesting an adaptation of the organism or a substitution of damaged cells.

Acetylglucosaminidase↗
PubMed1986

Nucleotide sequence of the gene coding for yeast cytoplasmic aspartyl-tRNA synthetase (APS); mapping of the 5' and 3' termini of AspRS mRNA.

A 3.8 Kb DNA fragment, which contains the structural gene of aspartyl-tRNA synthetase (AspRS) and its flanking regions, has been fully sequenced by the combined M13/dideoxy chain terminator method. From the single open reading frame of correct length (1671 bp) we deduced an amino acid sequence consistent with that of several peptides of AspRS. No significant internal sequence repeats were observed in the primary structure of the protein. The AspRS gene (APS) has a codon usage pattern typical of non abundant proteins. S1 nuclease analysis of APS mRNA showed a major start 17 bases downstream from a "TATA box" and stops near an RNA polymerase terminator sequence.

Amino Acid Sequence↗
PubMed1986

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs.

The sequences of Saccharomyces cerevisiae mitochondrial tRNA Arg1, tRNA Arg2, tRNA Gly, tRNA Lys2, tRNA Leu amd tRNA Pro are reported. Special structural features were found in tRNA Pro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA Lys2, which has a U excluded from base-paring and bulging out from the TpsiC stem. The tRNA Arg1, tRBA Lys2 and tRNA Leu, which belong to two-codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5-carboxymethylaminomethyluridine. tRNA Gly and tRNA Pro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four-codon family. However, tRNA Arg2, which is a minor species and belongs to the CGN four-codon family, has an unmodified A in the wobble position. This unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.

Base Sequence↗
PubMed1986

Morphological alterations induced by patulin on cultured hepatoma cells.

Patulin is a mycotoxin produced by various Penicillium, Aspergillus and Byssochlamys species. To evaluate its inhibitory effect on cells, hepatoma tissue culture cells in suspension were incubated in presence of 30 microM of patulin for 7 h and investigated by transmission and scanning electron microscopy. By transmission electron microscopy, the most significant difference observed between treated and control cells was the disorganization of the cytoplasmic microfilaments in the treated cells. The disappearance of superficial membrane microvilli which contain microfibrillar material was visualized by scanning electron microscopy; the cells also presented protrusions. The effect of this toxin on the cytoskeleton can be compared to that exerted by colchicine or by cytochalasins.

Actin Cytoskeleton↗
PubMed1986

Effect of Ochratoxin A on enzyme activities and macromolecules synthesis in MDCK cells.

Protein, RNA, and DNA synthesis, and enzyme (gamma GT, LDH, LAP, A1-P, NAG) activities were assayed in Madin Darby Canine Kidney cells (MDCK) treated by ochratoxin A, a mycotoxin known to be nephrotoxic in animal and man. Both cellular macromolecules syntheses and enzymatic activities are inhibited by OTA in a dose-dependent manner after 24 h incubation. The protection by 100 microM additional phenylalanine is optimal when MDCK cells were pretreated 4 h before the poisoning by OTA. When OTA 25 microM and phenylalanine 100 microM were added simultaneously the prevention of toxic effects is evident, but not complete in spite of the respective intracellular concentrations of OTA 2.22 microM and phenylalanine 2.4 microM which are normally not in favor of a strong inhibition by OTA, except if one admits that MDCK cells are really very sensitive to OTA.

Animals↗
PubMed1986

Distribution of the [3H]-label from low doses of radioactive ochratoxin A ingested by rats, and evidence for DNA single-strand breaks caused in liver and kidneys.

The distribution of a single low dose of [3H]-ochratoxin A (OTA) in different tissues of male Wistar rats, after administration by intubation, was investigated after 5 h, 24 h and 48 h. This dose corresponds to concentrations encountered in naturally contaminated feed (4 ppm). The distribution of [3H]-label varied with the time elapsed after administration; at 5 h the highest specific label was found in the stomach contents and in decreasing order in: intestinal contents, lung, liver, kidney, heart, fat, intestine, testes, and the lowest in muscles, spleen and brain. With exception of brain, fat, stomach and lung, all tissues showed maximum levels at 24 h, after which time the label decreased steadily, whereas in fat it increased. After a 12-week feeding experiment, with doses of 288.8 micrograms/kg corresponding to an intake of 4 ppm in feed each 48 h, the DNA in liver and kidneys was investigated for damage. By the alkaline elution method combined with micro-spectrofluorimetric determinations of DNA, evidence for DNA single-strand breaks was obtained. These findings support reports on the carcinogenic action of OTA.

Animals↗
PubMed1986

In vitro enzymatic methylation of DNA modified with the mutagenic amine: 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido(1,2-a:3'2'-d )imidazole.

Both the initial velocity and the overall methylation of DNA modified by acetylamino-4,6-dimethyldipyrido(1,2-a:3',2'-d)imidazole (A-Glu-P-3) by rat liver DNA-(cytosine-5-)-methyltransferase are decreased as compared to native DNA. A-Glu-P-3 bound to guanine residues may block the movement of the enzyme along the helix. The modified DNA does not inhibit the enzymatic methylation of native DNA. The enzyme has a lower affinity for the modified DNA than for native DNA. The hypomethylation caused by this carcinogen could have a significance in gene activity, cellular differentiation and cancer induction.

2-Acetylaminofluorene↗
PubMed1986

In vivo covalent binding of aminofluorene derivatives to mitochondrial liver DNA of rats pretreated with phenobarbital.

The covalent binding index (CBI) of aminofluorene derivatives (AF) to both nuclear (n) and mitochondrial (mt) hepatic DNA of rats 16 h after i.p. injection of N-acetyl-2-aminofluorene (AAF) has been determined. It was 69 and 212 respectively, i.e. about 3 times higher in mtDNA than in the nDNA. When rats were pretreated for 5 days with phenobarbital (PB) the two CBI decreased in parallel indicating a detoxification of AAF preventing its binding to both nDNA and mtDNA.

2-Acetylaminofluorene↗
PubMed1986

Action of citrinin on bacterial chromosomal and plasmid DNA in vivo and in vitro.

Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli. In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA. In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium. At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E. coli strain. In an E. coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor. In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml. In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml. Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present. This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.

Bacteriocin Plasmids↗
PubMed1986

[Chemical carcinogens and DNA, covalent binding and chemical modifications].

Chemical carcinogenic agents are usually metabolized into final metabolites susceptible to be DNA bound to form adducts. They may also modify DNA bases. Binding may be studied in vitro in the presence of an enzyme system causing the formation of final metabolites or in vivo. Because of the small amount of adducts formed in vivo, they can only be visualized with radioactive compounds or with a 32P marking technique after DNA hydrolysis. A limit of detection of adduct for 10(8) nucleotides has thus been reached for polycyclic compounds. The covalent binding index enables to quantify this binding. The consequences of DNA alterations vary according to the substitutes. In the case of acetyl-aminofluorene, they lead to mutations by shifting of the reading frame (frameshift). Alkylations lead to inaccurate pairing causing pin point mutations or depurinations.

Carcinogens↗
PubMed1986

Changes in de novo DNA (cytosine-5-)-methyltransferase activity in oncogenically susceptible rat target tissues induced by N-methyl-N-nitrosourea.

The activity of de novo DNA (cytosine-5-)-methyltransferase (DNA methylase) in various rat tissues after administration of a single dose of N-methyl-N-nitrosourea (MNU) has been analyzed. The total and specific activities of the DNA methylase of the brain, where tumor induction is important, are increased. In kidney, the DNA methylase activity first increases up to 16 h and decreases afterwards. Liver DNA methylase activity does not change. This organ is not susceptible to MNU induced cancers. Because organs in which the DNA methylase activity is high or increased after MNU are more prone to carcinogenesis by this compound, we argue that there is a relationship between the effects of MNU and DNA methylase activity.

Animals↗
PubMed1985

Inhibition of aminoacyl-tRNA synthetases by the mycotoxin patulin.

The effect of patulin on tRNA aminoacylation has been determined. This mycotoxin inhibits the aminoacylation process by irreversibly inactivating aminoacyl-tRNA synthetases. At neutral and alkaline pH-values, the inactivation occurs mainly by modification of essential thiol groups of the protein, whereas at acidic pH, where the effect is the most pronounced, the modification of other amino acid residues cannot be excluded.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Yeast mitochondrial tRNAIle and tRNAMetm: nucleotide sequence and codon recognition patterns.

The nucleotide sequence of yeast mitochondrial isoleucine- and methionine-elongator tRNA have been determined. Interestingly, long stretches of almost identical nucleotide sequences are found within these two tRNAs and also within the yeast mt tRNAMetf, suggesting that the 3 tRNAs may have arisen from a common ancestor. Both mt tRNAMetm and tRNAIle contain all the structural characteristics which are present in the standard cloverleaf, except that the mt tRNAMetm contains an extra unpaired nucleotide within the base-paired T psi C stem. This rather unusual feature may have an influence on the decoding properties of the C-A-U anticodon of mt tRNAMetm by conferring the ability to translate not only the codon A-U-G but also A-U-A.

Anticodon↗
PubMed1985

Isolation and characterization of the yeast aspartyl-tRNA synthetase gene.

A yeast genomic library in Escherichia coli, constructed by insertion of Sau3A restriction fragments into the hybrid Saccharomyces cerevisiae-E. coli plasmid pFL1, was screened by a radioimmunoassay (RIA) for colonies expressing yeast aspartyl-tRNA synthetase (AspRS). Four clones were isolated by this technique. Data obtained by Southern and restriction analysis of the inserts showed a common 3.8-kb BamHI restriction fragment which, when inserted into the plasmid pFL1, gave a positive RIA. Several controls showed that this 3.8-kb insert codes for the entire AspRS: (i) S. cerevisiae transformed by the PFL1 plasmid carrying the 3.8-kb fragment overproduces AspRS activity by a factor of ten compared to the wild-type yeast strain; and (ii) a new protein with electrophoretic behaviour similar to AspRS and immuno-reactive toward anti-AspRS appears in crude extracts of transformed yeast and E. coli.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Genotoxicity of ochratoxin A in mice: DNA single-strand break evaluation in spleen, liver and kidney.

Ochratoxin A, a natural contaminant of feed and food, has been shown to induce experimental liver and kidney tumours. In vitro experiments on mice spleen cells showed evidence of DNA single-strand breaks induced by ochratoxin A. We measured single-strand breaks in the DNA of spleen, liver and kidney of ochratoxin A-treated animals. Ochratoxin A induced DNA damage in vivo. This damage reversed with time. The appearance and extent of the damage varied in different tissues. Except for spleen our data correlate with the tumours induced by ochratoxin in mouse liver and kidney. With regard to the spleen, there has been no report to date of experimental leukemia induced by ochratoxin A. Thus our results indicate that this possibility has to be considered.

Animals↗