PubMed · 13111259
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E BUKOVICS, F X WOHLZOGEN. 1953-12-05. Sequential sampling in biological assay.. https://doi.org/10.1038/1721058a0
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The current studies describe a new, robust cell-based functional assay useful to characterize L-type voltage-dependent calcium channels and their antagonists. The basis of this assay is measurement in plate format of Ca2+ influx through the L-type Ca2+ channel complex (alpha1C, alpha2delta, and beta2a subunits) in response to potassium-mediated depolarization; EC(50)=11 mM [K+](o). The Ca2+ influx was inhibited by the L-type Ca2+ channel antagonist, nimodipine; IC(50)=59 nM. These cells were also transfected with the Kir2.3 inward rectifier K(+) channel, which allows for changing the cell membrane potential by modulation of extracellular [K](o); -65 mV in physiological [K](o) and -28 mV in 30 mM [K](o) containing buffer. The conformational state of the voltage-sensitive Ca2+ channel is altered under these different conditions. Under the depolarized condition, nimodipine was a more potent antagonist, inhibiting Ca2+ influx with an IC(50) value of 3 nM. The results demonstrate that the interaction of nimodipine and other antagonists with the channel is modulated by changes in membrane potential and thus the state of the channel. Overall, this novel assay can be used to identify state-dependent calcium channel antagonists and should be useful for evaluating state-dependent inhibitory potency of a large number of samples.
To probe the specificity of a camalexin detoxifying enzyme(s) produced by Rhizoctonia solani, the putative 5-camalexin hydroxylase (5-CAHY), the naturally occurring phytoalexin 1-methylcamalexin and designer phytoalexins in which the H-5 of camalexin was replaced with either a methyl group or a fluorine atom were synthesised. This investigation showed that biotransformation of 5-fluorocamalexin by R. solani was substantially slower than that of camalexin (12 days vs. six to eight hours), 5-methylcamalexin (5-6 days) or 1-methylcamalexin (5-6 days). Antifungal bioassays showed that 5-fluorocamalexin, 5-methylcamalexin and 1-methylcamalexin were more inhibitory to R. solani than camalexin, whereas their metabolic products displayed substantially lower inhibitory activity. It was concluded that detoxification via oxidation of the indole moiety of camalexins is predominant in the biotransformation of both camalexin and 5-methylcamalexin and likely catalysed by a specific 5-CAHY. By contrast, the pathways for detoxification of 1-methylcamalexin and 5-fluorocamalexin are likely catalysed by non-specific "house-keeping" enzymes. Most importantly, because 1- methylcamalexin showed stronger antifungal activity and was metabolised at substantially slower rate than camalexin this work suggested that, from a plant's perspective 1-methylcamalexin could be a more effective antifungal defence than camalexin.
Colorectal cancer is often lethal when invasion and/or metastasis occur. Tumor progression to the metastatic phenotype is mainly dependent on tumor cell invasiveness. Secondary bile acids, particularly deoxycholic acid (DCA), are implicated in promoting colon cancer growth and progression. Whether DCA modulates beta-catenin and promotes colon cancer cell growth and invasiveness remains unknown. Because beta-catenin and its target genes urokinase-type plasminogen activator receptor (uPAR) and cyclin D1 are overexpressed in colon cancers, and are linked to cancer growth, invasion, and metastasis, we investigated whether DCA activates beta-catenin signaling and promotes colon cancer cell growth and invasiveness. Our results show that low concentrations of DCA (5 and 50 microM) significantly increase tyrosine phosphorylation of beta-catenin, induce urokinase-type plasminogen activator, uPAR, and cyclin D1 expression and enhance colon cancer cell proliferation and invasiveness. These events are associated with a substantial loss of E-cadherin binding to beta-catenin. Inhibition of beta-catenin with small interfering RNA significantly reduced DCA-induced uPAR and cyclin D1 expression. Blocking uPAR with a neutralizing antibody significantly suppressed DCA-induced colon cancer cell proliferation and invasiveness. These findings provide evidence for a novel mechanism underlying the oncogenic effects of secondary bile acids.