engleski

Sensitivity to oxidative stress of transgenic yeast expressing w6-desaturase - a model of cellular adjustment to lipid peroxidation

Oxidative stress is defined as a shift in oxidation-reduction balance toward oxidation, causing lipid peroxidation of biomembranes. To provide an in vitro test system for analysis of physiological consequences of lipid peroxidation, yeast, Saccharomyces cerevisiae, was genetically modified with w6-desaturase. Desaturase introduces double bonds to saturated fatty acids, so they become susceptible to lipid peroxidation. Thus modified yeast can develop endogenous oxidative stress resembling the analogous stress in mammalian cells. Yeast strains were grown on media with either glucose (controls) or galactose (inducer of desaturase expression). Before testing sensitivity to oxidants, real time PCR was performed to see if desaturase is expressed, and gas chromatography to see if it is active. Influence of short-term and long-term desaturase induction to oxidative stress sensitivity was examined by treatment with H2O2 or 4-hydroxynonenal (HNE) and plating them on agar as spots. Experiments showed that the desaturase was expressed and active only in modified strain grown on galactose, as expected. Short-term desaturase expression made transgenic strain more sensitive to oxidant challenge than wild type. On the other hand, long-term expression made desaturase strain less sensitive than wild type. These observations could be due to cumulative effect of of endogenous and exogenous oxidative stress in short-time expression, while long-term expression of desaturase induces adaptation mechanisms. Hence, the cellular adjustment to low level of endogenous oxidative stress with consequential lipid peroxidation may lead to increased resistance to harmful effects of oxidative stress.

4-hydroxy-2-nonenal; transgenic yeast; omega-6-desaturase

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