safranine-t and thionine

We have developed inhibitors of glutathione reductase that improve on the inhibition of literature lead compounds by up to three orders of magnitude. Thus, analogues of Safranine O and menadione were found to be strong, reversible inhibitors of yeast glutathione reductase. Safranine O exhibited partial, uncompetitive inhibition with Ki and alpha values of 0.5 mM and 0.15, respectively. Thionine O was a partial (hyperbolic) uncompetitive inhibitor with Ki and alpha values of 0.4 microM and 0.15, respectively. LY83583 and 2-anilino-1,4-naphthoquinone also showed (hyperbolic) partial, uncompetitive inhibition with micromolar Ki values. For Nile Blue A a model for two-site binding with (parabolic) uncompetitive inhibition fitted the data with a Ki value of 11 microM and a kinetic cooperativity between the sites of 0.12, increased to 0.46 by preincubation of the enzyme and Nile Blue A in the presence of glutathione disulphide. Analysis of the effects of preincubation on the kinetics and cooperativity indicated the possibility of a slow conformational change in the homodimeric enzyme, the first such indication of kinetic cooperativity in the native enzyme to our knowledge. Further evidence of conformational changes for this enzyme came from studies of the effects of dimethyl sulphoxide which indicated that this co-solvent, which at low concentrations has no apparent effect on initial velocities under normal assay conditions, induced a slow conformational change in the enzyme. Thionine O, Nile Blue A and LY83583 were redox-cycling substrates producing superoxide ion, detectable by means of cytochrome c reduction, but leading to no loss of glutathione reductase activity, under aerobic or anaerobic conditions. The water-soluble Safranine analogues Methylene Blue, Methylene Green, Nile Blue A and Thionine O (5 mg/kg i.p. x 5) were effective antimalarial agents in vivo against P. berghei, but their effect was small and a higher dose (50 mg/kg i.p. x 1) was toxic in mice. Comparison was made with human glutathione reductase and its literature-reported interactions with several tricyclic inhibitors as studied by X-ray diffraction. It is possible that the conformational changes detected in the present study from alterations in detailed kinetic inhibition mechanisms may shed light on information transfer through the glutathione reductase molecule from the dimer interface ligand pocket to the active-site.

Topics: Animals; Antimalarials; Dimethyl Sulfoxide; Glutathione Reductase; Malaria; Mice; Oxazines; Phenazines; Phenothiazines; Saccharomyces cerevisiae; Vitamin K

The usefulness of thionin for staining cartilage sections embedded in glycol methacrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin O stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin O, thionin did not show metachromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.

Topics: Aged; Cartilage, Articular; Chondroitin Lyases; Decalcification Technique; Glycosaminoglycans; Humans; Methacrylates; Paraffin Embedding; Phenazines; Phenothiazines; Plastic Embedding; Sensitivity and Specificity; Staining and Labeling

Strains of Brucella melitensis biovars 1, 2, and 3 isolated in India, Italy, Kuwait, Saudi Arabia, the Federal Republic of Germany, and Zimbabwe were inhibited by thionin (20 micrograms/ml) but not by basic fuchsin (20 micrograms/ml) or safranin O (200 micrograms/ml). In other respects the strains were typical of their species and biovars. The existence of dye-sensitive strains of B. melitensis suggests that the present system of classification requires modification.

Topics: Bacterial Typing Techniques; Brucella; Coloring Agents; Drug Resistance, Microbial; Humans; Phenazines; Phenothiazines; Rosaniline Dyes

A microtitre-plate method for evaluating the inhibitory effect of dyes on the growth of mycoplasmas in fluid medium is described. Different species were shown to differ in their sensitivity to dyes. Statistical analysis (a) compared the general sensitivity and resistance of different mycoplasma species to the dyes and (b) showed that the dyes fell into two main groups in their effects on the mycoplasma species.

Topics: Acholeplasma; Acriflavine; Coloring Agents; Gentian Violet; Methylene Blue; Microbial Sensitivity Tests; Mycoplasma; Oxazines; Phenazines; Phenothiazines; Rosaniline Dyes; Xanthenes