The use of HAI-1 and AI-2 in ratios characteristic of longer cultivation times resulted in a linear increase in inhibition although the slope was lower than for AI-2 alone

tes. To obtain state 2 respiration mitochondria were added to the chamber containing the KCl buffer. For respiration linked to NADH oxidation 5 mM pyruvate and 2.5 mM L-malate were added and for respiration linked to fatty acid b-oxidation 10 mM palmitoylcarnitine was added instead of pyruvate and the L-malate concentration was reduced to 1 mM. When a steady state of O2 consumption was reached a measurement of state 2 was taken. From the stable rate the O2 consumption was determined in either state 3.5 or state 3. For state 3.5 respiration 5 mM creatine, 40 mg creatine kinase and 400 mM ATP were added to the chamber. To obtain state 3 respiration 1.5 mM ADP was added. When the O2 consumption again reached a stable rate 10 mM cytochrome c was added to determine the amount of outer mitochondrial membrane damage. Hydrogen peroxide get PF-8380 production by isolated cardiac mitochondria. Mitochondria prepared using the protease method were used to measure the rate of hydrogen peroxide 12695532 production in state 3.5 from respiration linked to NADH oxidation and fatty acid b-oxidation and supplemented with 30 mM Amplex Red and 0.1 mgmL21 peroxidase, as previously described. The samples were excited at 540 nm and emission measured at 585 nm with a multi-plate fluorescent plate reader at 37uC. Hexokinase activity assay. Hexokinase assay was performed on isolated mitochondria prepared using the Polytron method, as described previously. The lysis buffer contained 33 mM KH2PO4, 50 mM dithiothreitol, protease inhibitor and pH 7.2. The assay was performed at 37uC and the mitochondria were diluted to 2 mgmL21. The hexokinase buffer consisted of 100 mM Tris-HCl containing 0.4 mM NADP+, 10 mM MgCl2, 5 mM ATP and 0.3% Triton X100. Mitochondria were added into a cuvette containing 1 mL final volume of hexokinase buffer supplemented with 0.5 unitsmL21 glucose-6-phosphate dehydrogenase. The Non-Obesogenic High-Fat Diet and Cardiac Remodeling reaction was started after 2 min by addition 22408714 of 1 mM glucose. For one mole of glucose used by hexokinase there was one mole of NADPH produced and therefore absorbance was recorded at 340 nm for 2 min with a spectrophotometer. Citrate synthase activity assay. Citrate synthase activity assay was performed on isolated mitochondria prepared using the Polytron method, as described previously. The assay was performed at 37uC and the mitochondria were diluted to 0.1 mgmL-1 in lysis buffer, as above. The citrate synthase buffer consisted of 50 mM Tris-HCl, 150 mM DTNB, 0.3% Triton X-100 and pH 7.4. Mitochondria were added to a cuvette containing 1 mL final volume of citrate synthase buffer supplemented with 0.3 mM acetyl CoA. The samples were incubated at 37uC for 2 min, a blank measurement taken and then 0.5 mM oxaloacetate was added. Absorbance was recorded at 412 nm for 2 min with a spectrophotometer. The effect of high-fat diet on insulin resistance, atherosclerosis, cardiac pump function, cardiac hypertrophy and cardiac apoptosis There was no evidence for a diabetic phenotype in the high-fat diet group as shown by similar non-fasting blood glucose and confirmed using an intra-peritoneal insulin tolerance test. Histological studies demonstrated that despite elevated blood lipids the aortic sinus, brachiocephalic artery and coronary arteries had no lesions even after longer periods of high-fat feeding. There were no signs of cardiac hypertrophy in the high-fat diet group compared to the normal diet group as shown by wet and dry heart weight t