Complex III was assayed by measuring the reduction of cytochrome c at 550 nm in 50 mM KP (pH 7.5), 2 mM NaN3, 0.1% BSA-EDTA, 50 μM cytochrome c (Roche) and 50 μM decyl-ubiquinol (Sigma), first without antimicyn A (Sigma) and then with 0.01 mg/ml of antimycin A added, so an antimicyn A-sensitive rate of cytochrome c reduction could be calculated.
Complex I was measured by the oxidation of NADH at 340 nm in 20 mM KP (pH 8.0), 200 μM NADH, 1 mM NaN3, 0.1% bovine serum albumin (BSA -EDTA, and 100 μM uBSA -EDTA-1 (Sigmand first without rotenone (Calbiochem, La Jolla, CA, USA), then with 5 μM rotenone added so a rotenone-sensitive rate of NADH oxidation could be calculated.
The activity of CI was measured in a spectrophotometer as the rotenone-sensitive rate of NADH oxidation at 340 nm over 3 min.
Complex III (decylubiquinol cytochrome c oxidoreductase) activity was determined by monitoring the antimycin A-sensitive reduction rate of cytochrome c in the presence of fully reduced CoQ2 as the electron source.
Mitochondrial complex I activity was measured spectrophotometrically (using a Beckman Coulter Spectrophotometer) as the rotenone-sensitive rate of NADH oxidation, and complex IV activity was measured as the cyanide-sensitive pseudo-first-order rate constant for the oxidation of reduced cytochrome c [ 29].
NADH-dependent superoxide generation was assayed at 25°C by superoxide dismutase (SOD -sensitive rate of oxidation of epinephrine to adrenochrome with an increaSOD -sensitivece at 480 nm with 4.0 mM-1 cm-1 as extinction coefficient.
KCN (1 mM) was added to inhibit cytochrome c oxidase activity, which was considered as the cyanide-sensitive rate of cytochrome c oxidation.
Complex I activity (i.e. NADH dehydrogenase) was determined by measuring the rotenone-sensitive rate of oxidation of NADH initiated by coenzyme Q1 [ 23], and citrate synthase activity was measured as described in [ 15].
Recently, evidence for environment-sensitive rates of methylation has been found in humans (Heijmans et al. 2008; Katari et al. 2009; Waterland et al. 2010).
We find that the heritability of a disease can vary greatly depending on rates of transmission of the epigenetic and environmental states and that environment-sensitive rates of epigenetic modification may produce very high heritabilities.
Some environmental thresholds are sensitive to rate of change while others are sensitive to spatial gradients of climate change.
