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Roberto Docampo - One of the best experts on this subject based on the ideXlab platform.

  • Inhibition of protein synthesis and amino acid transport by Crystal Violet in Trypanosoma cruzi.
    The Journal of eukaryotic microbiology, 1995
    Co-Authors: Maria E. Hoffmann, Silvia N. J. Moreno, Junhyuk Jang, Roberto Docampo
    Abstract:

    ABSTRACT. [35S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of Crystal Violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM Crystal Violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although Crystal Violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, Crystal Violet was able to inhibit total [35S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against Crystal Violet inhibition. Crystal Violet also inhibited total [3H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by Crystal Violet is probably due to inhibition of amino acid uptake.

  • Inhibition of Trypanosoma cruzi trypanothione reductase by Crystal Violet
    Molecular and Biochemical Parasitology, 1994
    Co-Authors: Silvia N. J. Moreno, Eva Gunilla Skare Carnieri, Roberto Docampo
    Abstract:

    A trypanothione reductase activity is present in all the main differentiation stages of Trypanosoma cruzi, amastigotes having the highest activity, and trypomastigotes the lowest. Trypanothione reductase could not be induced in epimastigotes exposed to H2O2. The trypanocidal drug Crystal Violet was a potent inhibitor of T. cruzi trypanothione reductase in vitro. The inhibition was competitive with respect to trypanothione with a Ki of 5.3 ± 0.5 μM, uncompetitive with NADPH, and increased below pH 7.0 and above pH 8.0. Crystal Violet, however, was not able to decrease the level of total reduced thiols in intact cells. Dihydrotrypanothione but not reduced glutathione, protected the enzyme from inhibition by Crystal Violet.

Silvia N. J. Moreno - One of the best experts on this subject based on the ideXlab platform.

  • Inhibition of protein synthesis and amino acid transport by Crystal Violet in Trypanosoma cruzi.
    The Journal of eukaryotic microbiology, 1995
    Co-Authors: Maria E. Hoffmann, Silvia N. J. Moreno, Junhyuk Jang, Roberto Docampo
    Abstract:

    ABSTRACT. [35S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of Crystal Violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM Crystal Violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although Crystal Violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, Crystal Violet was able to inhibit total [35S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against Crystal Violet inhibition. Crystal Violet also inhibited total [3H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by Crystal Violet is probably due to inhibition of amino acid uptake.

  • Inhibition of Trypanosoma cruzi trypanothione reductase by Crystal Violet
    Molecular and Biochemical Parasitology, 1994
    Co-Authors: Silvia N. J. Moreno, Eva Gunilla Skare Carnieri, Roberto Docampo
    Abstract:

    A trypanothione reductase activity is present in all the main differentiation stages of Trypanosoma cruzi, amastigotes having the highest activity, and trypomastigotes the lowest. Trypanothione reductase could not be induced in epimastigotes exposed to H2O2. The trypanocidal drug Crystal Violet was a potent inhibitor of T. cruzi trypanothione reductase in vitro. The inhibition was competitive with respect to trypanothione with a Ki of 5.3 ± 0.5 μM, uncompetitive with NADPH, and increased below pH 7.0 and above pH 8.0. Crystal Violet, however, was not able to decrease the level of total reduced thiols in intact cells. Dihydrotrypanothione but not reduced glutathione, protected the enzyme from inhibition by Crystal Violet.

  • Disruption of Ca2+ Homeostasis In Trypanosoma Cruzi By Crystal Violet
    The Journal of eukaryotic microbiology, 1993
    Co-Authors: Roberto Docampoa, Silvia N. J. Moreno, Fernanda R. Gadelha, Gustavo Benaim, Maria E. Hoffmann, Anibal E. Vercesi
    Abstract:

    We have demonstrated previously that Crystal Violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that Crystal Violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca2+ from the mitochondria. In addition, Crystal Violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca2+ uptake by digitonin-permeabilized epimastigotes. Crystal Violet also induced Ca2+ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, Crystal Violet inhibited Ca2+ uptake and the (Ca(2+)-Mg2+)-ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca2+ homeostasis by Crystal Violet may be a key process leading to trypanosome cell injury by this drug.

Maria E. Hoffmann - One of the best experts on this subject based on the ideXlab platform.

  • Inhibition of protein synthesis and amino acid transport by Crystal Violet in Trypanosoma cruzi.
    The Journal of eukaryotic microbiology, 1995
    Co-Authors: Maria E. Hoffmann, Silvia N. J. Moreno, Junhyuk Jang, Roberto Docampo
    Abstract:

    ABSTRACT. [35S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of Crystal Violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM Crystal Violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although Crystal Violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, Crystal Violet was able to inhibit total [35S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against Crystal Violet inhibition. Crystal Violet also inhibited total [3H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by Crystal Violet is probably due to inhibition of amino acid uptake.

  • Disruption of Ca2+ Homeostasis In Trypanosoma Cruzi By Crystal Violet
    The Journal of eukaryotic microbiology, 1993
    Co-Authors: Roberto Docampoa, Silvia N. J. Moreno, Fernanda R. Gadelha, Gustavo Benaim, Maria E. Hoffmann, Anibal E. Vercesi
    Abstract:

    We have demonstrated previously that Crystal Violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that Crystal Violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca2+ from the mitochondria. In addition, Crystal Violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca2+ uptake by digitonin-permeabilized epimastigotes. Crystal Violet also induced Ca2+ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, Crystal Violet inhibited Ca2+ uptake and the (Ca(2+)-Mg2+)-ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca2+ homeostasis by Crystal Violet may be a key process leading to trypanosome cell injury by this drug.

Anibal E. Vercesi - One of the best experts on this subject based on the ideXlab platform.

  • Disruption of Ca2+ Homeostasis In Trypanosoma Cruzi By Crystal Violet
    The Journal of eukaryotic microbiology, 1993
    Co-Authors: Roberto Docampoa, Silvia N. J. Moreno, Fernanda R. Gadelha, Gustavo Benaim, Maria E. Hoffmann, Anibal E. Vercesi
    Abstract:

    We have demonstrated previously that Crystal Violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that Crystal Violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca2+ from the mitochondria. In addition, Crystal Violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca2+ uptake by digitonin-permeabilized epimastigotes. Crystal Violet also induced Ca2+ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, Crystal Violet inhibited Ca2+ uptake and the (Ca(2+)-Mg2+)-ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca2+ homeostasis by Crystal Violet may be a key process leading to trypanosome cell injury by this drug.

J.p. Dean Goldring - One of the best experts on this subject based on the ideXlab platform.

  • Crystal Violet stains proteins in SDS-PAGE gels and zymograms.
    Analytical biochemistry, 2018
    Co-Authors: Robert G.e. Krause, J.p. Dean Goldring
    Abstract:

    Abstract Coomassie brilliant blue R250, an anionic dye is the most popular stain to detect proteins resolved in SDS-PAGE gels. Crystal Violet, a cationic dye was found to be versatile and stained proteins in SDS-PAGE gels and in zymograms. Stained proteins can be transferred to nitrocellulose and the stained proteins on the western detected with enzyme coupled antibodies. Staining can be reversed. Staining takes 3 h at RT or 30 min at 60 °C. Crystal Violet stained some E. coli high and low molecular weight proteins not stained by Coomassie blue R250. Crystal Violet stained down to 16 ng of protein, some five-fold lower than Coomassie blue, though the two stains had a similar linear dynamic range. The staining sensitivity could be increased to 2 ng when Crystal Violet and Coomassie blue were combined in a double staining/counterion dye formulation. The low concentrations of the dye without a destaining step reduces the costs of the technique and results in a more environmentally friendly stain compared to traditional staining methods.