8 M−1 cm−1). One unit of peroxidase activity is defined as the amount of enzyme required to oxidize 1 μmol of ABTS per 1 min. SOD activity in the cell-free extracts was determined spectrophotometrically at 25 °C using the xanthine oxidase–cytochrome c method (McCord & Fridovich, 1969). The assay mixture in deionized water (1 mL of reaction volume) contained 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA disodium salt, 10 μM cytochrome c (Sigma), 50 μM C59 wnt mw xanthine (Sigma) and 1.7 mU xanthine oxidase (Sigma). The reduction of cytochrome c by the superoxide anion radical, generated from O2 during the oxidation of xanthine in the xanthine oxidase reaction, was recorded by an increase in the absorption
at 550 nm for 5 min. One unit of SOD activity is defined as the amount of enzyme required to inhibit the linear rate of reduction of cytochrome c by 50%. Protein concentrations were determined using the Protein Assay p38 protein kinase Kit (Bio-Rad Laboratories). For total RNA isolation, cell pellets were rinsed three times with 10 mM Tris-HCl (pH 8.0) RNase-free buffer and finally resuspended in 200 μL of 10 mM Tris-HCl, 1 mM EDTA (pH 8.0) RNase-free buffer. Total RNA was isolated using the High Pure RNA Isolation
Kit (Roche Diagnostics) according to the manufacturer’s instructions with an extra DNase I digestion step in order to eliminate contaminating DNA. Extracted RNA (10 μg) was reverse transcribed using a random hexamer primer, dNTPs and Superscript II (Invitrogen) as described previously (Fournier Pomalidomide supplier et al., 2006). cDNA was purified on a microcon YM-30 centrifugal filter unit (Millipore) and stored at −20 °C. qRT-PCR was performed using the LightCycler® FastStart DNA MasterPLUS SYBR Green I Kit (Roche Diagnostics). cDNA was mixed with 0.5 μM of each primer and 2 μL of Master Mix in a 10 μL final volume. The pairs of oligonucleotide primers used to quantify the selected genes expression levels are shown in Supporting Information, Table S1. Real-time PCR runs were carried out on a LightCycler® Real-Time PCR System (Roche Diagnostics), with one cycle at 95 °C for
8 min, followed by up to 45 cycles at 95 °C for 12 s, 60 °C for 10 s and 72 °C for 20 s. For each couple of primers, real-time PCRs were run in triplicate on each cDNA. relative expression software tool (rest) was used to calculate the relative expression of each gene under each condition (Pfaffl et al., 2002). The coefficients of variation of the determined crossing points for each set of replicates were lower than 0.46%. The 16S RNA gene was used as a reference for normalization. The influence of H2O2 on exponentially growing cells in a lactate/sulfate medium is shown in Fig. 1. While the addition of 0.05 mM H2O2 did not significantly perturb D. vulgaris Hildenborough growth, higher concentrations of H2O2 treatment induced both a lower growth rate and a lower final cell density. When 0.