The mechanism of irreversible inactivation of lysozyme at neutral pH at 100 degrees C, and effects of additives on the inactivation were investigated. The thermoinactivation of lysozyme at neutral pH was caused by intra- and intermolecular disulfide exchange and the production of irreversibly denatured lysozyme, which was destabilized by multiple chemical reactions other than disulfide exchange. In addition, independently, deamidation slightly affected the inactivation by causing a decrease of electrostatic interaction between positive charges of lysozyme and negative charges of the bacterial cell wall. As for the effects of additives on the inactivation, a small amount of copper ion suppressed intra- and intermolecular disulfide exchange by catalyzing air oxidation of heat-induced trace amounts of free thiols, and organic reagents (acetamide, ethanol, and glycerol) changed the mechanism of the inactivation to that under acidic conditions by shifting the pKa values of dissociable residues and also suppressed intermolecular disulfide exchange by decreasing hydrophobic interactions.
The glucocorticoid receptor regulates gene expression mainly by two mechanisms; transactivation and transrepression. A ligand with strong transrepression and weak transactivation activity is predicted to be a beneficial agent with potent anti-inflammatory activity and minor adverse effects. Recently, the profile of a synthetic steroid, RU24858, has been reported to fulfill this condition in vitro, but others have reported no dissociation between the anti-inflammatory activity and side effects in vivo. To gain further information on the profile of this compound, we evaluated its transactivation ability using a reporter gene analysis both in vitro and in vivo. In the in vitro analysis, RU24858 demonstrated only a weak transactivation activity in HeLa cells, when compared with prednisolone. In CV-1 cells, however, these two glucocorticoids exhibited equivalent transactivation activities. This behavior is independent of whether the reporter gene is regulated by mouse mammary tumor virus promoter or multiple copies of glucocorticoid response element. When the reporter plasmid was inoculated into mouse abdominal skin using a gene gun, followed by orally administration of glucocorticoids, RU24858 induced significantly higher reporter enzyme activity than prednisolone. These results suggest that the profile of RU24858 is divergent and its transactivation ability is comparable to prednisolone depending on the cell-type both in vitro and in vivo.
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