Phenazine Methosulfate Decreases HIF-1α Accumulation during the Exposure of Cells to Hypoxia

2012 ◽  
Vol 76 (9) ◽  
pp. 1682-1687 ◽  
Author(s):  
Akiko YAMAKI ◽  
Haruhiro MURATSUBAKI
Keyword(s):  
Phenazine Methosulfate

Optimization of a Colorimetric Assay to Determine Lactate Dehydrogenase B Activity Using Design of Experiments

2020 ◽  
pp. 247255522095658
Author(s):  
Christos Papaneophytou ◽  
Maria-Elli Zervou ◽  
Anastasis Theofanous
Keyword(s):  
Lactate Dehydrogenase ◽  
High Throughput Screening ◽  
Color Change ◽  
Colorimetric Assay ◽  
Phenazine Methosulfate ◽  
Ph Values ◽  
Absorbance Change ◽  
Final Volume ◽  
Protein Pellet ◽  
Purple Formazan

Lactate dehydrogenase B (LDH-B) is overexpressed in lung and breast cancer, and it has been considered as a potential target to treat these types of cancer. Herein, we propose a straightforward incomplete factorial (IF) design composed of 12 combinations of two reaction buffers, three pH values, three salt (NaCl) concentrations, and three incubation times, which we called IF-BPST (Buffer/pH/Salt/Time), for the optimization of a colorimetric LDH-B assay in a final volume of 100 µL using 96-well plates. The assay is based on the absorbance change at ~570 nm and the color change of the reaction mixture due to the release of NADH that reacts with nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS), resulting in the formation of a blue-purple formazan. The results obtained using the IF-BPST were comparable with those obtained by response surface methodology. Our work revealed that the NBT/PMS assay with some modifications can be used to measure the activity of LDH-B and other dehydrogenases in a high-throughput screening format at the early stages of drug discovery. LDH-B containing lysates cannot be assayed directly, however, due to the sensitivity of the method toward detergents. Thus, we suggest precipitating the proteins in the lysates to remove the interfering detergents, and then to dissolve the protein pellet in a suitable buffer and carry out the assay.

Metabolically induced permeability changes across mesothelium and endothelium

1964 ◽  
Vol 206 (2) ◽  
pp. 373-382 ◽  
Author(s):  
Joseph Cascarano ◽  
Arnold D. Rubin ◽  
William L. Chick ◽  
Benjamin W. Zweifach
Keyword(s):  
Carotid Artery ◽  
Tetrazolium Salt ◽  
Phenazine Methosulfate ◽  
Depth Of Penetration ◽  
Tissue Slices ◽  
Tetrazolium Chloride ◽  
Quantitative Studies ◽  
Permeability Changes ◽  
Inner Surface

The ability of mesothelial and endothelial membranes to influence solute passage into tissue preparations was investigated under in vitro conditions. It is possible by using the Warburg formula for the diffusion of solutes into tissue slices to calculate the concentration of solute bathing the subepithelial tissue when the depth of penetration and tissue activity are known. These conditions were met by selecting an electron acceptor with histochemical properties—the tetrazolium salt, 2- p-iodophenyl-3- p-nitrophenyl-5-phenyl tetrazolium chloride (INT). Quantitative studies with hemidiaphragm demonstrate that the concentration of INT which reaches the inner surface of the mesothelial layer is only 9% of that in the medium. Addition of a redox dye, phenazine methosulfate (PM), increases this value by 6.7 times. (Qualitatively similar results were obtained across mesothelial surfaces of liver and heart, as well as at endothelial surfaces of heart and carotid artery.) The ability of PM to increase permeability is counteracted by malate or succinate, and in turn restored when the latter is blocked with malonate. These results suggest that oxidative metabolism and adenosine triphosphate formation are intimately linked with this phenomenon.

scholarly journals Vesiculation of sickle erythrocytes during thermal stress

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 1060-1063 ◽  
Author(s):  
BH Rank ◽  
NL Moyer ◽  
RP Hebbel
Keyword(s):  
Thermal Stress ◽  
Membrane Protein ◽  
Strong Correlation ◽  
Phenazine Methosulfate ◽  
Thiol Oxidation ◽  
Protein Thiol ◽  
Functional Abnormality ◽  
Sickle Erythrocytes ◽  
Protein Thiol Oxidation

Abstract Since it is not known why sickle RBCs tend to undergo microvesiculation, we have investigated their susceptibility to thermal stress. While normal RBCs start to vesiculate at 49.0 +/- 0 degrees C (n = 14), sickle RBCs begin to vesiculate at 47.9 +/- 0.5 degrees C, with a range of 46.5 to 48.5 degrees C (n = 14). This abnormality is reproduced by treating normal RBCs with phenazine methosulfate (PMS), which stimulates the excess intracellular generation of superoxide characteristic of sickle RBCs. For PMS-treated RBCs, there is a strong correlation between membrane protein thiol oxidation and vesiculation temperature (r = .977, P less than .001). The abnormal vesiculation temperature of both unmanipulated sickle RBCs and PMS-treated RBCs is significantly improved by treatment of the RBCs with dithiothreitol. The most dense sickle RBCs are most prone to vesiculation during thermal stress, and they are the subpopulation having the greatest amount of thiol oxidation. We conclude that the tendency of sickle RBCs to vesiculate during thermal stress is further evidence for functional abnormality of the RBC cytoskeleton due to thiol oxidation.

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