PubMed · 9634833
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M Gordon. 1996. Taxol supply problem? What problem?. https://doi.org/10.1038/nbt1296-1635b
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The cytotoxicity and the electrophysiological effects against MRC-5 fibroblasts of 2-alkylpyrrolidine, (-)-R-bgugaine [1], a natural alkaloid, extracted from Arisarum vulgare Targ., (Araceae) were studied. This compound showed a cytotoxic potential against MRC-5 fibroblasts at 10 microg/ml. The electrophysiological study of this molecule on MRC-5 cells was carried out using the technique of patch-clamp and showed that (-)-R-bgugaine [1] has a sub-maximum effect on the outward potassium (current) at the concentration of 1 microM (0.281 microg/ml). In this study we show that R-bgugaine [1] activates the outwards potassium (current) activated by intracellular calcium.
To examine the role of early carbohydrate recognition/trimming reactions in targeting endoplasmic reticulum (ER)-retained, misfolded glycoproteins for ER-associated degradation (ERAD), we have stably expressed the cog thyroglobulin (Tg) mutant cDNA in Chinese hamster ovary cells. We found that inhibitors of ER mannosidase I (but not other glycosidases) acutely suppressed Cog Tg degradation and also perturbed the ERAD process for Tg reduced with dithiothreitol as well as for gamma-carboxylation-deficient protein C expressed in warfarin-treated baby hamster kidney cells. Kifunensine inhibition of ER mannosidase I also suppressed ERAD in castanospermine-treated cells; thus, suppression of ERAD does not require lectin-like binding of ER chaperones calnexin and calreticulin to monoglucosylated oligosaccharides. Notably, the undegraded protein fraction remained completely microsome-associated. In pulse-chase studies, kifunensine-sensitive degradation was still inhibitable even 1 h after Tg synthesis. Intriguingly, chronic treatment with kifunensine caused a 3-fold accumulation of Cog Tg in Chinese hamster ovary cells and did not lead to significant induction of the ER unfolded protein response. We hypothesize that, in a manner not requiring lectin-like activity of calnexin/calreticulin, the recognition or processing of a specific branched N-linked mannose structure enhances the efficiency of glycoprotein retrotranslocation from the ER lumen.
Antileukemic interactions between the nucleoside analog 1-beta-D-arabinofuranosylcytosine (ara-C) and the kinase inhibitor 7-hydroxystaurosporine (UCN-01) have been examined in relation to Bcl-2 expression/phosphorylation, mitochondrial damage, caspase activation, and loss of clonogenic potential. Subsequent exposure of ara-C-pretreated U937 cells (1 microM; 6 hr) to UCN-01 (300 nM; 24 hr) resulted in marked potentiation of pro-caspase-3 and -9 cleavage/activation, poly(ADP-ribose)polymerase degradation, diminished mitochondrial membrane potential (Deltapsi(m)), enhanced cytochrome c release, reduction in the S-phase fraction, and induction of classic apoptotic morphologic features. Enforced expression of full-length Bcl-2 significantly protected cells (at 24 hr) from ara-C/UCN-01-induced caspase activation and apoptosis, but was ineffective in preventing loss of Deltapsi(m) and cytochrome c release. Ectopic expression of a Bcl-2 N-terminal phosphorylation loop-deleted protein (Bcl-2Delta(32-80)) was more potent than its full-length counterpart in blocking drug-induced loss of Deltapsi(m, ) caspase activation, and apoptotic morphology, but not cytochrome c release. Examination of cells at later intervals revealed that ectopic expression of Bcl-2 or Bcl-2Delta(32-80) could only delay, but not prevent, mitochondrial damage, caspase activation, and cell death induced by ara-C/UCN-01 treatment. Despite their initial ability to inhibit apoptosis, neither full-length nor truncated Bcl-2 protein restored clonogenic potential to drug-treated cells. These findings indicate that subsequent exposure of ara-C-pretreated human leukemia cells to UCN-01 potently triggers mitochondrial damage and apoptosis, and that these events are postponed but not prevented by ectopic expression of Bcl-2 or its phosphorylation loop-deleted counterpart.