A system was devised which allows the efficient substitution of cysteine residues in a protein by selenocysteine. It involves overexpression of the respective gene with the aid of the T7 ...promotor/polymerase system in a cysteine auxotrophic strain. The induction of the T7 polymerase formation was performed in cysteine-supplemented medium followed by wash-out of the cysteine and production of the desired gene product in the presence of selenocysteine. The system was applied to substitute the two cysteine residues in Escherichia coli thioredoxin. Analysis of the purified gene product by electrospray mass spectrometry and HPLC revealed that both cysteine residues were replaced in approximately 75-80% of the protein, only one cysteine residue was substituted in about 5-10%, and no substitution had taken place in 12-17% of the protein. The occurrence of diselenide, seleno-sulfur, and disulfide bridges in the purified gene product was revealed by ES/MS and chemical modification studies. The diselenide bridge represents an entity in protein structures which has hitherto not been described. The redox property of the selenocysteine variant of thioredoxin (Se) sub(2)-thioredoxin was found to be substantially different from that of thioredoxin. Only the latter could be reduced under native conditions in the presence of an excess of beta -mercaptoethanol. The oxidized (Se) sub(2)-thioredoxin was then separated from the selectively reduced and carboxymethylated protein by anion-exchange chromatography. The purity of the isolated (Se) sub(2)-thioredoxin was at least 92%.
Nährstoffaufnahme bei gesunden Betagten Schlettwein-Gsell, D.; Decarli, B.; Amorim Cruz, J. A. ...
Zeitschrift für Gerontologie und Geriatrie,
6/1999, Letnik:
32, Številka:
S1
Journal Article
Fifty years ago the first author of this paper described the entity known as idiopathic medial necrosis of the aorta. The present report describes two cases of ruptured aneurysm of the main pulmonary ...artery. The histologic alterations of the arterial wall were the same as in the much more common aneurysms of the aorta, viz. severe medial necrosis and mucoid degeneration. The findings are discussed and compared with 20 cases documented in the literature. Physical (hypertension) as well as biochemical factors appear to play an essential role in the pathogenesis of medial necrosis.
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