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Di-Calciphor-Dependent Protection Against Cell Death Due to Mitochondrial Failure
Published in John J. Lemasters, Constance Oliver, Cell Biology of Trauma, 2020
Because KCN is a rather non-specific poison, affecting many copper and heme proteins other than cytochrome oxidase, we performed a comparable series of experiments with the specific mitochondrial toxin antimycin A.19 Antimycin A binds stoichiometrically to the cytochrome bc1 complex20 and inhibits electron flow. Addition of antimycin A at concentrations that gave essentially complete inhibition of mitochondrial O2 consumption resulted in rapid loss of ATP and about 50% cell death within 3 h.19 At 1 μM concentration, di-Calciphor gave complete protection against cell death but had no effect on ATP loss.
High-Performance Liquid Chromatography
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
Joel J. Kirschbaum, Adorjan Aszalos
Antimycin A in tissues was chromatographed using a 5-µm octadecylsilane column and precolumn (50 x 4 mm) and a mobile phase of 0.25 M acetate buffer, pH 5-methanol (25:75). Detection of this antifungal agent was either electrochemical (using a glass carbon electrode at +1.00 V versus Ag/AgCl), fluorescence at 365/418 (after derivatization with dimethylaminonaphthalene-5-sulfonyl chloride, or UV at 254 nm. The respective minimum detectable quantities were approximately 0.5, 0.5, and 5 ng [424].
Burkholderia
Published in Dongyou Liu, Handbook of Foodborne Diseases, 2018
Danielle L. Peters, Jaclyn G. McCutcheon, Karlene H. Lynch, Jonathan J. Dennis
Toxoflavin is toxic because it acts as an electron carrier, resulting in the production of hydrogen peroxide. Under normal conditions, NADH and FADH2 produced during glycolysis and the citric acid cycle (in the cytosol and mitochondrial matrix, respectively) transfer their electrons to a series of carriers in the inner mitochondrial membrane, including cytochromes, which are organized into Complex I–IV.94 The electrons are shuttled down this chain based on redox potential, and the energy released is used to pump protons into the intermembrane space of the mitochondria. This transport creates a proton gradient that powers the ATP synthase, which produces the majority of ATP used by the cell in a process called oxidative phosphorylation.94 It was discovered by Latuasan and Berends95 that toxoflavin interferes with this process. When antimycin A, which blocks Complex III of the electron transport chain, is added to yeast, respiration is inhibited.94,95 However, when antimycin A and toxoflavin are added together, no inhibitory effects are observed. Similarly, the addition of potassium cyanide blocks Complex IV and inhibits respiration, but not in the presence of toxoflavin.94,95 These results suggest that toxoflavin facilitates cytochrome-independent electron transfer.95
scRNA-seq reveals ATPIF1 activity in control of T cell antitumor activity
Published in OncoImmunology, 2022
Genshen Zhong, Qi Wang, Ying Wang, Ying Guo, Meiqi Xu, Yaya Guan, Xiaoying Zhang, Minna Wu, Zhishan Xu, Weidong Zhao, Hongkai Lian, Hui Wang, Jianping Ye
CD8+ T cells were isolated from the mouse spleen and activated with CD3/CD28 antibody stimulation in the culture medium. The cells (or the CAR-T cells) were loaded at 1 × 106/well into XF24 plate, which was coated with Cell-Tak (company name, 22.4 μg/mL, in sterile water) for 20 minutes to increase the adhesion of T cells. The plate was centrifuged at 200 × g (zero braking) for 1 minute to let the T cells to adhere to the culture surface. After incubation for 30 minutes at 37°C without CO2 supplementation, Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) were determined with the Seahorse XF Cell Mito Stress Test Kit and Seahorse XF Glycolysis Stress Test kit with the Agilent Technologies equipment. The final concentrations of inhibitors were 1 μM oligomycin, 2 μM FCCP, 0.5 μM rotenone, and antimycin A. In the ECAR assay, the final concentrations of compounds were 10 mM glucose, 1 μM oligomycin, and 50 mM 2-Deoxy-D-glucose. The readings were taken after each sequential injection of corresponding chemicals.
Contributing role of mitochondrial energy metabolism on platelet adhesion, activation and thrombus formation under blood flow conditions
Published in Platelets, 2022
Noriko Tamura, Shinichi Goto, Hideo Yokota, Shinya Goto
The contributing roles of mitochondrial function in platelet adhesion, activation, and thrombus formation were shown by three of mitochondrial function inhibitors with different mechanisms of action. The FCCP is an uncoupler of mitochondrial oxidative phosphorylation [29]. Antimycin A blocks the function of cytochrome-c reductase in mitochondrial complex III [5,30,31]. Oligomycin is a specific inhibitor of F1F0-ATP synthase at mitochondria. All three inhibitors worked at the dose tested because the glucose consumption rates increased in their presence. Thus, our results of inhibited rapid increase in [Ca2+]i upon adhering VWF is likely dependent on the inhibition of mitochondrial function. Mitochondrial function is important for cell signaling and death [32]; one may argue that the reduced rise in [Ca2+]i in platelets adhering to VWF may be based on the vital status of platelets in the presence of mitochondrial functional blockers. To avoid potential cell death induced by the presence of mitochondrial functional blockers, we conducted all experiments within 2 h after drawing blood. Moreover, our experimental results that the addition of mitochondrial functional blockers did not influence platelet adhesion and thrombus formation support the notion that a substantial number of platelets are still alive even in the presence of the three mitochondrial functional blockers we used in our experiments. However, further investigations are necessary for a precise understanding of the role of mitochondria and the rapid increase in [Ca2+]i.
Effects of different standard and special diets on cognition and brain mitochondrial function in mice
Published in Nutritional Neuroscience, 2022
Martina Reutzel, Rekha Grewal, Carsten Esselun, Sebastian Friedrich Petry, Thomas Linn, Annette Brandt, Ina Bergheim, Gunter P. Eckert
Half a brain hemisphere (the frontal part) was used to isolate brain mitochondria. The protocol is described in Hagl et al. [16]. The pellet obtained from the last centrifugation step was dissolved in 250 µl MIRO5. A volume of 80 µl of the resulting cell suspension was injected into the Oxygraph 2k-chamber. The capacity of the oxidative phosphorylation (OXPHOS) was determined using complex-I related substrates pyruvate (5 mM) and malate (2 mM) and ADP (2 mM) followed by the addition of succinate (10 mM). Mitochondrial integrity was measured by the addition of cyctochrom c (10 µM). Oligomycin (2 µg/ml) was added to determine leak respiration (leak (omy)) and afterwards uncoupling was achieved by carbonyl cyanide p-(trifluoromethoxy) phenyl-hydrazone (FCCP, injected stepwise up to 1–1.5 µM). Complex II respiration was measured after the addition of rotenone (0.5 µM). Complex III inhibition was achieved by the addition of antimycin A (2.5 µM) and was subtracted from all respiratory parameters. Cytochrome c oxidase (COX) activity was determined after residual oxygen consumption (ROX) determination by applying 0.5 mM tetramethylphenylenediamine (TMPD) as an artificial substrate of complex IV and 2 mM ascorbate to keep TMPD in the reduced state. Autoxidation rate was determined after the addition of sodium azide (>100 mM), and COX respiration was additionally corrected for autoxidation.