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James T Stewart - One of the best experts on this subject based on the ideXlab platform.

  • high performance thin layer chromatographic determination of digoxin and related compounds digoxigenin bisdigitoxoside and Gitoxin in digoxin drug substance and tablets
    Journal of Chromatography A, 1994
    Co-Authors: Garratt W Ponder, James T Stewart
    Abstract:

    Abstract A high-performance thin-layer chromatographic (HPTLC) method for the determination of digoxin and its related compounds digoxigenin bisdigitoxoside (DBD) and Gitoxin in digoxin drug substance and tablets was developed. Separation of the three compounds was accomplished on a C18 wettable reversed-phase plate using water-methanol-ethyl acetate (50:48:2 v/v/v) as the mobile phase. The analytes were determined by densitometry using absorbance for digoxin and fluorescence for the two related compounds. All peaks were quantified by peak-height analysis. Linear regression analysis of the data was performed for all three compounds. The calibration range for digoxin was set at 320–480 ng per 5-mm band, equivalent to 80–120% (w/w) of a 400-ng band load, that for DBD was set at 4–12 ng per 5-mm band, equivalent to 1–3% (w/w) of the digoxin load, and that for Gitoxin was set at 0.4–1.6 ng per 5-mm band, equivalent to 0.1–0.4% (w/w) of the digoxin load. The limit of quantification (LOQ) for digoxin was 64 ng per 5-mm band with a limit of detection (LOD) of 8 ng per 5-mm band. The LOQs for both DBD and Gitoxin were 0.12 ng per 5-mm band with LODs of 0.4 ng per 5-mm band. The linearity range for the digoxin peak height in the absorbance mode was 0–5000 ng per 5-mm band. The linearity range for DBD and Gitoxin peak heights in the fluorescence mode was 0–2000 ng per 5-mm band.

Walter Berger - One of the best experts on this subject based on the ideXlab platform.

  • Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na^+/K^+-ATPase
    Molecular Cancer, 2013
    Co-Authors: Laetitia Moreno Y Banuls, Adriana Katz, Walter Miklos, Alessio Cimmino, Elena Ainbinder, Martin Zehl, Ernst Urban, Antonio Evidente, Brigitte Kopp, Walter Berger
    Abstract:

    Background Surface-expressed Na^+/K^+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC_50 in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK. Methods The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, diGitoxin, Gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. Results Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. Conclusions Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth.

Roderick Mackinnon - One of the best experts on this subject based on the ideXlab platform.

  • AGitoxin footprinting the Shaker potassium channel pore
    Neuron, 1996
    Co-Authors: Adrian Gross, Roderick Mackinnon
    Abstract:

    In voltage-dependent K+ channels, each of the four identical subunits contributes one pore loop to the central ion selectivity unit at the interface between the subunits. The pore loop is also the target for scorpion venom peptide inhibitors. These inhibitors bind at the pore entryway between the four subunits and can assume any one of four orientations. The orientations become distinguishable only if the binding site symmetry is disrupted. We have used mutagenesis and site-directed chemical modification to alter pore loop amino acids in either one or four subunits. The effects of these alterations on inhibitor affinity define the eccentricity of amino acids in the pore entryway and imply a different secondary structure for the amino and carboxyl ends of the pore loop.

  • purification and characterization of three inhibitors of voltage dependent k channels from leiurus quinquestriatus var hebraeus venom
    Biochemistry, 1994
    Co-Authors: Maria L Garcia, Margarita Garciacalvo, Patricia Hidalgo, Roderick Mackinnon
    Abstract:

    : Three new toxins from the venom of the scorpion Leiurus quinquestriatus var. hebraeus have been identified on the basis of their ability to block the Shaker K+ channel. These toxins have been purified using HPLC techniques and characterized as 38 amino acid peptides by mass spectroscopy, amino acid analysis, and sequence determination. Their chemical identity was confirmed by producing fully functional synthetic toxins using recombinant methods. These peptides are potent inhibitors of the Shaker K+ channel (Kd < 1 nM) as well as the mammalian homologues of Shaker. They are related to other previously described K+ channel toxins, but form a new subclass within the larger family of K+ channel inhibitors derived from scorpion venom. We have named these toxins aGitoxin 1, 2, and 3, respectively.

Garratt W Ponder - One of the best experts on this subject based on the ideXlab platform.

  • high performance thin layer chromatographic determination of digoxin and related compounds digoxigenin bisdigitoxoside and Gitoxin in digoxin drug substance and tablets
    Journal of Chromatography A, 1994
    Co-Authors: Garratt W Ponder, James T Stewart
    Abstract:

    Abstract A high-performance thin-layer chromatographic (HPTLC) method for the determination of digoxin and its related compounds digoxigenin bisdigitoxoside (DBD) and Gitoxin in digoxin drug substance and tablets was developed. Separation of the three compounds was accomplished on a C18 wettable reversed-phase plate using water-methanol-ethyl acetate (50:48:2 v/v/v) as the mobile phase. The analytes were determined by densitometry using absorbance for digoxin and fluorescence for the two related compounds. All peaks were quantified by peak-height analysis. Linear regression analysis of the data was performed for all three compounds. The calibration range for digoxin was set at 320–480 ng per 5-mm band, equivalent to 80–120% (w/w) of a 400-ng band load, that for DBD was set at 4–12 ng per 5-mm band, equivalent to 1–3% (w/w) of the digoxin load, and that for Gitoxin was set at 0.4–1.6 ng per 5-mm band, equivalent to 0.1–0.4% (w/w) of the digoxin load. The limit of quantification (LOQ) for digoxin was 64 ng per 5-mm band with a limit of detection (LOD) of 8 ng per 5-mm band. The LOQs for both DBD and Gitoxin were 0.12 ng per 5-mm band with LODs of 0.4 ng per 5-mm band. The linearity range for the digoxin peak height in the absorbance mode was 0–5000 ng per 5-mm band. The linearity range for DBD and Gitoxin peak heights in the fluorescence mode was 0–2000 ng per 5-mm band.

Brigitte Kopp - One of the best experts on this subject based on the ideXlab platform.

  • Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na^+/K^+-ATPase
    Molecular Cancer, 2013
    Co-Authors: Laetitia Moreno Y Banuls, Adriana Katz, Walter Miklos, Alessio Cimmino, Elena Ainbinder, Martin Zehl, Ernst Urban, Antonio Evidente, Brigitte Kopp, Walter Berger
    Abstract:

    Background Surface-expressed Na^+/K^+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC_50 in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK. Methods The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, diGitoxin, Gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. Results Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. Conclusions Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth.

  • Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na+/K+-ATPase.
    'Springer Science and Business Media LLC', 2013
    Co-Authors: Moreno L. Y Banuls, Adriana Katz, Walter Miklos, Alessio Cimmino, Elena Ainbinder, Martin Zehl, Ernst Urban, Antonio Evidente, D.m. Tal, Brigitte Kopp
    Abstract:

    BACKGROUND: Surface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50 in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human alpha1beta1 NaK. METHODS: The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, diGitoxin, Gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human alpha1beta1, alpha2beta1 and alpha3beta1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. RESULTS: Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the alpha2beta1 and alpha3beta1 than for the alpha1beta1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for alpha1beta1 than for the alpha2beta1 and alpha3beta1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. CONCLUSIONS: Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the alpha2beta1 and alpha3beta1 than for the alpha1beta1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth