engleski

Sensitivity of transgenic yeast expressing desaturase to oxidative stress

Oxidative stress is defined as a shift in oxidation-reduction balance toward oxidation. One of the consequences of oxidative stress is lipid peroxidation. To provide an in vitro test system for analysis of physiological consequences of lipid peroxidation, yeast, Saccharomyces cerevisiae, was genetically modified with desaturase inserted pYES2 plasmid. Desaturase introduces double bonds to saturated fatty acids, so they become susceptible to lipid peroxidation (caused by oxidative stress). Thus modified yeast can develop endogenous oxidative stress resembling similar stress in mammalian cells. Hydrogen peroxide (H2O2) and 4-hydroxynonenal (HNE) were used as mediators of oxidative stress to see sensitivity of modified strain. Yeast were grown on media with either glucose or with galactose, respectively, to induce desaturase expression linked to GAL1 promotor on pYES2. They were treated with H2O2 (in concentrations 0, 7, 1, 1, 5 and 2 mM) or with HNE (50, 100 and 200 μ M) for 2 hours and afterwards plated on agar in small volume as spots. Further, we wanted to see the antioxidant defense of the cell. Major antioxidant defense in cells against toxic lipid peroxidation product HNE and several ROS is tripeptide glutathione (GSH), so measurements of GSH levels were done. Finally, to see if HNE entered the cells, HNE immunoelectronmicroscopy was performed with genuine monoclonal antibodies against HNE-histidine conjugates. Transgenic strain was more sensitive than the wild type to H2O2 and HNE. This can be explained by cumulative endogenous oxidative stress of desaturase-modified strain combined with endogenous oxidative stress. Modified strain showed increase in GSH level compared to level when grown on glucose and to wild type level grown on galactose. However, electronmicroscopy has shown that neither wild nor transgenic type of yeast had detectable HNE-protein adducts unless they were treated by HNE. These results indicate that endogenous oxidative stress increased glutathione level as a defense mechanism, but due to presence of endogenous oxidative stress further challenge made these cells more susceptible to hydrogen peroxide and HNE, mediators of oxidative stress.

oxidative stress; transgenic yeast; H2O2; HNE

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