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Biomedical subjects

R Wirth

Publications and source records attributed to R Wirth.

At least 55 records · Page 3Linked to original sources
PubMed1986

Effective chelation therapy after incorporation of neptunium-239 in rats.

It was demonstrated in rats that it is possible to reduce the retention of 239Np in all body tissues by an early combined treatment with small doses of DTPA and DFOA. The content of 239Np can be decreased in soft tissues even if treatment is delayed. Promptly administered LICAM(C) proved more effective than the above chelate combination in reducing 239Np retention in the bones but increased that in the muscles and especially in the kidneys. This side effect of LICAM(C) could be partly prevented by simultaneous treatment with DTPA.

Animals↗
PubMed1986

Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector.

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.

Cloning, Molecular↗
PubMed1985

Identification of transferrin as the principal neptunium-binding protein in the blood serum of rats.

The binding of 239Np(V) to blood serum components of rats was examined in vivo and in vitro. After gel filtration of the serum using a Sephacryl S-300 column, 98% of the applied activity appeared with protein fractions representing coeluted albumins and transferrin. A separation of the albumin- and transferrin-proteins by ion-exchange chromatography using DEAE-cellulose showed the 239Np being entirely bound to the iron-carrier protein transferrin. The high elution yields from the ion-exchange columns, greater than 90%, suggest that the binding may be quite strong. The binding capacity of transferrin for neptunium in vivo was found to decline when the iron level in blood serum was increased. Precipitation experiments showed that 84 +/- 2% of the 239Np was precipitated with 10% (w/v) trichloracetic acid, 77 +/- 3% with 90% ethanol but only 6 +/- 1% with saturated ammonium sulphate at pH 7.4. The available data indicate that as for plutonium, thorium, americium and curium, the iron transport protein, transferrin, may be the main carrier protein for neptunium in mammalian blood serum.

Animals↗
PubMed1985

Gastrointestinal absorption of Np in rats.

The effect of Np mass and the acidity of the administered Np solutions as well as the age, sex and nutritional status of the animals injected or gavaged with 239Np or 237Np were determined. The latter factor proved to be dominant for absorption of Np from the gut. Thus in fasting weanling and young adult male rats, the absorption of 239Np was sixfold higher (0.18% and 0.12%, respectively) than in fed ones (0.03% and 0.02%, respectively). Absorption by fasted adult females was 0.05% of the administered 239Np, about half of that of adult males. Raising the Np-mass gavaged to fasted female rats to 1 and 10 mg 237Np/kg resulted in an absorption of 0.23% and 0.26%, respectively. Thus, an increased absorption of Np in adult rats seems to be expected only if a large mass is ingested. No dependence of the absorption of Np on nitric acid concentration was found. The data obtained after oral administration of 238Pu and 239Np to adult rats suggest that the f1 factor recommended by the ICRP for fractional absorption of soluble Np compounds from the gut should be decreased, whereas the f1 factor for soluble Pu compounds should be raised.

Animals↗
PubMed1984

Regulation of the synthesis of hydrogenase (formate hydrogen-lyase linked) of E. coli.

The regulation of synthesis of the hydrogenase which is a component of the formate hydrogen-lyase complex was studied by means of a strain of Escherichia coli possessing a transcriptional fusion of the hydrogenase gene (hyd) with the lacZ gene (hyd::lac fusion). Formation of active hydrogenase in the wild strain requires the presence of nickel in the medium; transcription of the hyd gene, however, is independent from the presence of Ni2+. Ni2+ addition to Ni2+-prestarved cells did not lead to any activation of presumptive hydrogenase apoprotein. Regulatory mutants were isolated in which nitrate repression of hyd::lac expression was relieved. Two main classes of regulatory mutants were identified: (i) Mutants with a defect in nitrate reductase; (ii) mutants with a cis-dominant regulatory mutation closely linked to the hyd::lac fusion. In the presence of formate which acts as an inducer, the hyd::lac fusion was also expressed under aerobic conditions. The results infer that nitrate repression of transcription of the hydrogenase structural gene is not effected by nitrate itself but requires the function of the electron transport chain leading to nitrate and that mutations in the promoter/operator region of the hyd cistron may confer insensitivity to redox control both by oxygen and nitrate.

Aldehyde Oxidoreductases↗
PubMed1984

Long term retention of 237Np in rats.

This interim report summarizes the results of observations during the first year after a single injection of 237Np nitrate (0.2 or 1.0 mg/kg body weight) into adult female rats and further preliminary data obtained with young animals. The retention of 237Np was followed by whole body counting and serial sacrifice of groups of animals. The retention data could be fitted to three-component exponential equations which show no major differences between the two 237Np dose levels. The half-times and extrapolated initial fractions calculated from the first two exponential terms indicate that one fraction, representing about 40 per cent of the injected 237Np was excreted within the first 5 days and an additional 15 per cent within the first 5 months, while the rest was excreted with a half-time of about 3.5 years. This final long term component is assumed to indicate the rate of loss of 237Np from the skeletal compartment. In young animals both whole-body and skeletal retention of 237Np during the first 5 months of observation was about 50 per cent higher than in the adults. Several soft tissue tumours, mostly mammary tumours, have appeared to approximately the same extent in both control and 237Np treated adult rats but no osteosarcomas were detected up to 15 months after injection of 237Np.

Animals↗
PubMed1983

On the redox control of synthesis of anaerobically induced enzymes in enterobacteriaceae.

Mutants of Escherichia coli were isolated in which transcription of the structural genes for hydrogenase (hyd) and for one of the components of formate dehydrogenase (fdh) (of the formate hydrogen-lyase complex) is coupled with that of the lacZ gene. They were--together with lac fusions of the nifH and nifL genes from Klebsiella--used to study regulation by redox control, of the expression of the respective structural genes. The following results were obtained: (i) beta-galactosidase synthesis was fully repressed in the presence of O2 or nitrate (anaerobically), and induced in the absence of an external electron acceptor. Fumarate as terminal electron acceptor only marginally affected nif expression and partially repressed hyd and fdh expression. Redox control of the synthesis of hydrogenase and formate dehydrogenase, therefore, (as well as that of nif) acts at the level of transcription; the size of the redox potential seems to be correlated with the amount of repression; (ii) beta-galactosidase synthesis in the hyd:: lac and fdh::lac fusion strains is induced by formate. At high concentrations formate reverses the repression by nitrate and fumarate but not that by oxygen.

Anaerobiosis↗
PubMed1982

Ribosomal protein S20 purified under mild conditions almost completely inhibits its own translation.

The efficiency of ribosomal protein S20 to act as repressor of its own synthesis in an in vitro system was found to depend greatly on the procedures employed to purify this protein. Whilst conventionally purified r-protein S20 inhibited its own synthesis by some 30%, up to 90% inhibition was observed if "milder" purification conditions were used. Evidence is presented that the latter preparation shows also a higher binding affinity to 16S rRNA.

Gene Expression Regulation↗
PubMed1982

Regulation of formation of threonyl-tRNA synthetase, phenylalanyl-tRNA synthetase and protein synthesis initiation factor 3 from Escherichia coli in vivo and in vitro.

The expression of the structural genes for the protein synthesis initiation factor 3 (IF-3), threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase carried by the transducing phage lambda p2 was studied in a DNA-dependent transcription-translation system in vitro and the results were compared to the regulatory pattern in vivo. In vitro, the DNA of the phage lambda p2 gives rise to the formation of the two forms of IF-3 (IF-31 and IF-3S) which are known to be present in vivo. The kinetics of synthesis indicate an interconversion of IF-31 into IF-3S. Addition of excess purified IF-31 does not significantly repress IF-3 synthesis but does stimulate the rate of conversion of IF-31 into IF-3S. This apparent lack of autoregulation in vitro is in accordance with gene-dosage-dependent synthesis in vivo. The fact that strains with more than one copy of the IF-3 structural gene contain a higher relative amount of IF-3S than do haploid ones suggests that the proteolytic conversion of IF-31 into IF-3S may occur predominantly in the free (non-ribosome-bound) state. In vivo, the amount of IF-3 varies with the growth rate much like elongation factor Tu or aminoacyl-tRNA synthetases. As with the aminoacyl-tRNA synthetases, IF-3 synthesis is not significantly subject to a stringent control system. This coordinated regulatory response in vivo, however, is not paralleled by the susceptibility of synthesis in vitro to guanosine 3'-diphosphate 5'-diphosphate (ppGpp), since IF-3 formation is inhibited by ppGpp whereas that of threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase is stimulated.

Amino Acyl-tRNA Synthetases↗
PubMed1981

Factors modulating transcription and translation in vitro of ribosomal protein S20 and isoleucyl-tRNA synthetase from Escherichia coli.

The DNA-dependent protein-synthesizing system developed by Zubay [Zubay, G. (1973) Annu. Rev. Genet. 7, 267--287] was optimized for the transcription and translation of genes from the 0.5-min region of the Escherichia coli chromosome carried by transducing lambda phages. The E. coli gene products synthesized were isoleucyl tRNA synthetase, ribosomal protein S20, dihydrodipicolinic acid reductase and (possibly) the two subunits carbamoyl-phosphate synthetase. Formation of ribosomal protein S20 is specifically stimulated by the addition of 16-S rRNA and not by 5-S or 23-S rRNA. 16-S rRNA increases the rate of S20 synthesis, the final yield of product depends on the duration of persistence of the RNA added. Addition of 16-S rRNA to the separate transcription and translation systems showed that it is the translation of the S20 mRNA which is enhanced. Furthermore, S20 synthesis is stimulated more than fourfold when concomitant synthesis of rRNA occurs from a plasmid carrying an rrn transcriptional unit. The results described are explained in terms of a model which suggests that ribosomal protein S20 feedback inhibits its synthesis at the translational level and that removal of S20 into ribosomal assembly (i.e. binding to 16-S rRNA) releases inhibition. The model postulates a direct link between synthesis of ribosomal RNA and ribosomal protein and between the rates of ribosomal assembly and ribosomal protein synthesis. The stimulatory effect of guanosine 3'-diphosphate 5'-diphosphate on isoleucyl-tRNA synthetase formation and its inhibition of the synthesis of ribosomal protein S20 in vitro occurs at the level of transcription. Its relevance in vivo, however, remains to be demonstrated. Formation of isoleucyl-tRNA synthetase in vitro is not influenced either by the addition of a surplus of purified enzyme nor by the limitation of protein synthesis by the addition of anti-(isoleucyl-tRNA synthetase) serum. There is no evidence, therefore, that isoleucyl-tRNA synthetase is autogenously regulated.

Amino Acyl-tRNA Synthetases↗
PubMed1980

Regulation of synthesis of ribosomal protein S20 in vitro.

Addition of 16S rRNA to a coupled transcription-translation in vitro system stimulated the synthesis of ribosomal protein S20 from transducing phage lambda DNA. The S20 made under this condition was found to be bound to 16S rRNA. These results may indicate a connection between ribosomal assembly and the rate of ribosomal protein synthesis.

Bacteriophage lambda↗