Recently we found that a single administration of T-2 toxin (T-2), a trichothecene mycotoxin, into mice induced DNA fragmentation, a biochemical hallmark of apoptosis, in the thymus. In this study, we investigated the effective chemical structure(s) of T-2-derived metabolites capable of inducing thymic apoptosis in vivo in mice. Metabolic conversion of T-2 to 3'-hydroxy-T-2 toxin (3'-OH-T-2) did not diminish the apoptosis-inducing activity, since essentially the same level of fragmented DNA was detected in the thymus taken from mice injected with either T-2 or 3'-OH-T-2. In contrast, hydrolysis of T-2 and 3'-OH-T-2 at the carbon-4 (C-4) position to HT-2 toxin (HT-2) and 3'-hydroxy-HT-2 toxin (3'-OH-HT-2), respectively, greatly decreased the level of DNA fragmentation. Similarly, hydrolysis of T-2 at the carbon-8 (C-8) position to neosolaniol strongly diminished its ability to induce DNA fragmentation. T-2 tetraol, having no ester groups, was unable to induce apoptosis. Based on the data presented in this study, we concluded that both the acetyl group at the C-4 position and the isovaleryl or 3'-hydroxyisovaleryl group at the C-8 position of the T-2 molecule are important for inducing cell death through apoptosis in the thymus.
We established an expression system for large amount of recombinant sheep angiotensinogen (sAngn) in Chinese hamster ovary cells. The recombinant sAngn (21.6 mg) was purified by a single column chromatography to a homogeneous level on sodium dodecyl sulphate-polyacrylamide gel electrophoresis from 2.71 of conditioned medium. Human renin cleaved only a Leum-Leu" bond in sAngn, although Leu"-Val" was another candidate for scissile peptide bond. The Km and km, values for the reaction of human renin with sAngn were 0.21 ,uM and 150 min", respectively. Recombinant rat renin could also react with the sAngn. The Km and kg, for the reaction were 0.84 tag and 78 min", respectively. Purified sAngn was stable at temperature up to 50°C, but rapidly inactivated at that higher than 55°C.
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