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Role of Mitochondrial Injury During Oxidative Injury to Hepatocytes: Evidence of a Mitochondrial Permeability Transition by Laser Scanning Confocal Microscopy
Published in John J. Lemasters, Constance Oliver, Cell Biology of Trauma, 2020
Anna-Liisa Nieminen, Roberto Imberti, Alice K. Saylor, Samuel A. Tesfai, Brian Herman, John J. Lemasters
In freshly isolated rat hepatocytes, cyanide caused progressive cell killing over 2½ hours (Figure 1). Fructose protected completely against this cell killing. To test the hypothesis that protection was mediated by glycolytic ATP generation, we examined the effect of the mitochondrial uncoupler, CCCP, on fructose protection. CCCP collapses the mitochondrial proton electrochemical gradient and stimulates the mitochondrial F1F0-ATPase. Thus, if fructose protection is mediated through intracellular ATP generation, accelerated ATP hydrolysis induced by CCCP should block fructose protection. This is what was found experimentally (Figure 1). To further test the hypothesis that cell killing is ATP-linked, we evaluated the effect of oligomycin. Oligomycin is an inhibitor of the mitochondrial F1F0- ATPase. By itself, oligomycin is cytotoxic,3 but in the presence of fructose and CCCP, oligomycin should block accelerated ATP hydrolysis and prevent cell killing. Again, the experiments confirmed this expectation (Figure 1). In parallel experiments, we measured cellular ATP levels. Cyanide caused ATP to fall below measurable levels (Figure 2). Fructose partially restored ATP, but CCCP blocked the fructose-induced restoration of ATP, an effect reversed by oligomycin. In every case a positive correlation was observed between preservation of cell viability and ATP.
Ultraviolet and Light Absorption Spectrometry
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
Zoltan M. Dinya, Ferenc J. Sztaricskai
The antibiotic oligomycin A (26-methylrutamycin) contains a 26-membered lactone ring. Its UV spectrum shows two characteristic absorptions [236] at 225 nm (ε = 2020 m2/mol; conjugated diene, π ⃗ π*) and at 232 nm (ε= 1820 m2/mol; α,βunsaturated lactone,π ⃗ π*). In oligomycins A, B, and C there are differences in the intensity of the two bands. Primicin contains a 36-membered macrocycle substituted with a side chain bearing a guanidine group. Its UV spectrum displays a high-intensity absorbance (ε= 3777 m2/mol) at 209 nm.
Cold stored platelets in the management of bleeding: is it about bioenergetics?
Published in Platelets, 2023
Chloe E. George, Christine V. Saunders, Alex Morrison, Tom Scorer, Sarah Jones, Nina C. Dempsey
The use of modulators of the ETC in the XF analyzer has the potential to provide a more precise understanding of platelet concentrate metabolism during storage through the isolation of individual parts of the ETC. The use of the inhibitor oligomycin inhibits ATP synthase, resulting in a reduction in the OCR that reflects the fraction of oxygen consumption as a result of ATP synthesis via OXPHOS. The subsequent addition of rotenone (a complex I inhibitor) and antimycin A (a complex III inhibitor) shuts down mitochondrial respiration and, with the simultaneous measurement of the acidification rate, enables the calculation of glycolysis-driven ATP production [87]. The respiratory reserve of mitochondria can be measured with the addition of a proton uncoupler, which allows protons to bypass the ATP synthase and consume oxygen at the maximum potential rate. By subtracting the basal OCR from this maximal rate, it is possible to quantify the ability of the cells to respond to a stressor with increased ATP production [88].
Chronic exposure to dim artificial light disrupts the daily rhythm in mitochondrial respiration in mouse suprachiasmatic nucleus
Published in Chronobiology International, 2023
Prabha Rajput, Dhanananajay Kumar, Sairam Krishnamurthy
Mitochondrial respiration was assessed as previously described by (Rajput and Krishnamurthy 2022; Samaiya and Krishnamurthy 2015), with a Clark-type electrode in a sealed, thermostatically controlled chamber at 37°C. Briefly, the mitochondria were added to the respiratory chamber states were evaluated with suitable substrates and inhibitors. Purified mitochondrial protein was suspended in a respiration buffer in a final volume of 250 μL. State II respiration was initiated by adding pyruvate/malate; (P/M) shows a basal respiration rate. The addition of adenosine diphosphate-initiated state III respiration; (ADP); the high level of oxygen utilization indicates that ADP is converted into ATP. The addition of oligomycin measured state IV. The addition of FCCP measured state V. This causes uncoupling of the ETC to ATP synthesis and represents the maximum respiration rate. Rotenone was then added to shut down complex I-mediated respiration. The addition of succinate determined state V. This is the maximum respiration rate via complex II since FCCP is present. The RCR was calculated by dividing state III respiration (presence of ADP) by state IV respiration (absence of ADP).
Thiopental sodium loaded solid lipid nano-particles attenuates obesity-induced cardiac dysfunction and cardiac hypertrophy via inactivation of inflammatory pathway
Published in Drug Delivery, 2020
Canzhan Zhu, Wanjing Li, Xinhong Wang, Jiahong Xue, Ling Zhao, Yafan Song, Tian Zhou, Mingjuan Zhang
Briefly, for the estimation of cardiac mitochondria respiratory rate by using the Oxygraph-2k at 37 °C and for the estimation of respiratory rates Datlab software was used. Mitochondrion (1 mg) was mixed into each chamber after respiration stabilization and after that, the 5 mM glutamate and substrates (2-mM malate) were added for the estimation of leak respiration of complex I (CI LEAK). 5 mM ADP was added for the estimation of oxidative phosphorylation of CI (CI OXPHOS) and 10 μM cytochrome C was mixed for the determination of outer membrane integrity of mitochondria. For the estimation, the oxidative phosphorylation capacity of CI + II (CI + II OXPHOS, state P) 2.5 mM succinate was added. Further, 1 μg/ml oligomycin added after the CI + II OXPHOS estimation. After that, the ability of electron transport chain was titrated after the addition of FCCP and finally, the CII respiration was scrutinized after mixed the 0.5 μM rotenone (CI inhibitor), by suppressing the respiration complex I. for the estimation the consumption of residual oxygen via CIII inhibition with the 2.5 μM antimycin A.