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Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K.
Elajaili HB, Hernandez-Lagunas L, Ranguelova K, Dikalov S, Nozik-Grayck E
(2019) J Vis Exp :
MeSH Terms: Animals, Antimycin A, Bleomycin, Bronchoalveolar Lavage Fluid, Cattle, Cell Compartmentation, Electron Spin Resonance Spectroscopy, Lung, Mice, Mitochondria, Oxidation-Reduction, RAW 264.7 Cells, Superoxides, Temperature
Show Abstract · Added March 26, 2019
The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments. In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD, allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide). Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 °C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 °C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.
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14 MeSH Terms
Survivin mediates renal proximal tubule recovery from AKI.
Chen J, Chen JK, Conway EM, Harris RC
(2013) J Am Soc Nephrol 24: 2023-33
MeSH Terms: Acute Kidney Injury, Animals, Anti-Bacterial Agents, Antimycin A, Cell Line, Inhibitor of Apoptosis Proteins, Kidney Tubules, Proximal, Mice, Mice, Inbred BALB C, Mice, Knockout, Receptor, Notch2, Recovery of Function, Reperfusion Injury, Repressor Proteins, STAT3 Transcription Factor, Survivin, Up-Regulation
Show Abstract · Added January 28, 2014
AKI induces the renoprotective upregulation of survivin expression in kidney epithelial cells, but the underlying mechanisms have not been identified. To determine the role of survivin in renal recovery from AKI, we generated mice with renal proximal tubule-specific deletion of survivin (survivin(ptKO)). Renal survivin expression increased substantially in response to ischemia-reperfusion (I/R) injury in control littermates but remained minimal in survivin(ptKO) mice. Functional and histologic data indicated similar degrees of renal injury in survivin(ptKO) and control mice 24 hours after reperfusion, but recovery was markedly delayed in survivin(ptKO) mice. In MCT cells, a mouse renal proximal tubule cell line, ATP depletion by antimycin A treatment upregulated survivin expression through a phospho-STAT3-dependent pathway. In wild-type mice, inhibition of STAT3 kinase diminished I/R-induced upregulation of STAT3 phosphorylation and survivin expression and delayed recovery. Furthermore, I/R injury activated Notch-2 signaling, and a γ-secretase inhibitor suppressed I/R-induced Notch-2 signaling, STAT3 phosphorylation, and survivin expression and delayed recovery. In MCT cells, inhibition of γ-secretase similarly attenuated antimycin A-induced Notch-2 activation, upregulation of survivin, and phosphorylation of STAT3, but STAT3 kinase inhibition did not prevent Notch-2 activation. Therefore, these data suggest that STAT3 phosphorylation and subsequent upregulation of survivin expression mediated by Notch-2 signaling in renal proximal tubule epithelial cells aid in the functional and structural recovery of the kidney from AKI.
1 Communities
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17 MeSH Terms
Platelet-collagen adhesion-membrane fluidity and the development of high affinity adhesion through multiple interacting sites.
Santoro SA, Cunningham LW
(1981) Coll Relat Res 1: 517-26
MeSH Terms: Antimycin A, Binding Sites, Blood Platelets, Collagen, Deoxyglucose, Humans, Membrane Fluidity, Platelet Aggregation, Surface Properties, Tissue Adhesions
Show Abstract · Added March 5, 2014
Multiple, linked interactions between the platelet surface and collagen fibers have been implicated in the initiation of platelet secretion and subsequent aggregation. The formation of such multiple simultaneous interactions could give rise to high affinity adhesion of platelets to collagen even though the affinity of the individual interactions may be much weaker. This concept has been tested by measuring the adhesion of platelets to collagen under conditions which could effect the formation of multiple interactions. Adhesion is markedly diminished at 4 degrees C but not at 23 or 37 degrees C. Metabolic inhibitors such as 2-deoxyglucose and Antimycin A do not inhibit adhesion although they virtually abolish subsequent aggregation. Brief formaldehyde fixation of platelets greatly reduces adhesion. These results are consistent with the concept that the formation of multiple linked interactions between the platelet surface and collagen are important in platelet-collagen adhesion and that mobility of platelet membrane components is required for the clustering of these interactions in focussed regions on the platelet surface.
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10 MeSH Terms