5 Star
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02 June 2018
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In-tissue mitochondrial function analysis

Freshly harvested tissues (~10 mg) were immersed in ice-cold BIOPS buffer (10 mM CaK2-EGTA, 7.2 mM K2-EGTA, 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM dithiothreitol, 6.5 mM MgCl2, 5.8 mM ATP, and 15 mM creatine phosphate; pH 7.1). Myofiber bundles were transferred to 2 ml of MIR05 buffer (0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, and 1 mg/ml fatty acid free bovine serum albumin, pH 7.1) containing 50-?g/ml saponin, and incubated at 4°C for 30 min to achieve chemical permeabilization of the sarcolemma membrane. Permeabilized myofiber bundles (~2 mg) were washed with MIR05 buffer, and used for measuring mitochondrial respiration by using an Oxygraph-2k (O2K) respirometer (Oroboros Instruments, Innsbruck Austria). Oxygen concentration was determined at 2 sec intervals and used to compute oxygen flux per mg of tissue by Oroboros DatLab software. Briefly, after recording the baseline respiration with myofiber bundles alone, 5 mM pyruvate, 2 mM malate and 10 mM glutamate (P+G+M) were added, and complex I driven state 4 respiration was recorded. Electron transfer was coupled to phosphorylation by the addition of 5 mM ADP, and state 3 respiration was recorded. Maximal state 3 respiration with parallel electron input from complex I and complex II was recorded with addition of 10 mM succinate, and complex II supported respiration was measured in presence of 6.25 ?M rotenone (inhibits complex I) [22].

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There may be no bigger question than whether we are alone in our solar system. As our spacecraft find new clues about the presence of liquid water now or in the past on Mars, the possibility of some kind of life there looks ...

Hospital cleaning protocol ineffective against A. baumannii

November 30, 2012 .
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