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Vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in MDCK cells.
We studied the cell-surface delivery pathways of newly synthesized membrane glycoproteins in MDCK cells and for this purpose we characterized an endogenous apical integral membrane glycoprotein. By combining a pulse-chase protocol with domain-selective cell-surface biotinylation, immune precipitation, and streptavidin-agarose precipitation (Le Bivic et al. 1989. Proc. Natl. Acad. Sci USA. 86:9313-9317), we followed the appearance at the cell surface of a major apical sialoglycoprotein, gp114, a basolateral protein, uvomorulin, and a transcytosing protein, the polyimmunoglobulin receptor (pIg-R). We determined that both gp114 and uvomorulin appeared to be delivered directly to their respective surface, with mistargeting levels of 8 and 2%, respectively. Using the same technique, the pIg-R was first detected on the basolateral domain and then on the apical domain, to be finally released into the apical medium, as described (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). To directly determine whether the gp114 pool present on the basolateral surface was a precursor of the apical gp114, we compared it with the equivalent pIg-R pool, by labeling with sulfo-NHS-SS-biotin, a cleavable, tight junction-impermeable probe, and by following the fraction of this probe that became resistant to basal glutathione and accessible to apical glutathione during incubation at 37 degrees C. We found that, contrary to pIg-R, basolateral gp114 was poorly endocytosed and was not transcytosed to the apical side. These results demonstrate that an endogenous apical integral membrane glycoprotein of Madin-Darby canine kidney cells is sorted intracellularly and is vectorially targeted to the apical surface
Protective Activity of Broccoli Sprout Juice in a Human Intestinal Cell Model of Gut Inflammation
Benefits to health from a high consumption of fruits and vegetables are well established
and have been attributed to bioactive secondary metabolites present in edible plants. However,
the effects of specific health-related phytochemicals within a complex food matrix are difficult to
assess. In an attempt to address this problem, we have used elicitation to improve the nutraceutical
content of seedlings of Brassica oleracea grown under controlled conditions. Analysis, by LC-MS, of
the glucosinolate, isothiocyanate and phenolic compound content of juices obtained from sprouts
indicated that elicitation induces an enrichment of several phenolics, particularly of the anthocyanin
fraction. To test the biological activity of basal and enriched juices we took advantage of a recently
developed in vitro model of inflamed human intestinal epithelium. Both sprouts’ juices protected
intestinal barrier integrity in Caco-2 cells exposed to tumor necrosis factor under marginal zinc
deprivation, with the enriched juice showing higher protection. Multivariate regression analysis
indicated that the extent of rescue from stress-induced epithelial dysfunction correlated with the
composition in bioactive molecules of the juices and, in particular, with a group of phenolic
compounds, including several anthocyanins, quercetin-3-Glc, cryptochlorogenic, neochlorogenic and
cinnamic acids
Virtual Summer School: Alternative Methods and Models in Science: A Multidisciplinary In Vitro Approach
Virtual Summer School: Alternative methods and models in science : a multidisciplinary in vitro approach
Vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in MDCK cells.
Abstract. We studied the cell-surface delivery pathways of newly synthesized membrane glycoproteins in MDCK cells and for this purpose we characterized an endogenous apical integral membrane glycoprotein. By combining a pulse-chase protocol with domainselective cell-surface biotinylation, immune precipitation, and streptavidin-agarose precipitation (Le Bivi
Soybean-and lupin-derived peptides inhibit DPP-IV activity on in situ human intestinal Caco-2 cells and ex vivo human serum
Recent investigations have focused on food-derived peptides as novel natural inhibitors of dipeptidyl peptidase IV (DPP-IV), a new target for diabetes. This study aimed to optimize fast, sensitive, and cost-effective DPP-IV assays in situ on human intestinal Caco-2 cells and ex vivo on human serum. Both assays were applied to investigate the inhibitory activity of soy and lupin peptides. The best conditions for in situ DPP-IV activity in Caco-2 cells were obtained using 2-day cells and 50 \ub5M Gly-Pro-AMC. Sitagliptin, used as reference inhibitor, showed a dose-dependent response with a 50% inhibition concentration (IC50) of 0.6 \ub5M. A lower IC50(0.2 \ub5M) was obtained for sitagliptin on human serum incubated with the substrate for 24 h. Both assays were applied to assess the activity of Lup1 (LTFPGSAED) and Soy1 (IAVPTGVA) on DPP-IV. Lup1 and Soy1 inhibited DPP-IV in situ, with IC50values of of 207.5 and 223.2 \ub5M, respectively, and maintained their inhibitory activity ex vivo on circulating DPP-IV with a slightly lower potency. These assays can be used to characterize the DPP-IV inhibitory activity of food-derived molecules more accurately than in vitro biochemical tests. This combined approach also considers their effects on the circulating form of DPP-IV, correlated to metabolic diseases
Extra virgin olive oil phenolic extract on human hepatic HEPG2 and intestinal CACO-2 cells: Assessment of the antioxidant activity and intestinal trans-epithelial transport
Hypocholesterolaemic Activity of Lupin Peptides : Investigation on the Crosstalk between Human Enterocytes and Hepatocytes Using a Co-Culture System Including Caco-2 and HepG2 Cells
Literature indicates that peptic and tryptic peptides derived from the enzymatic hydrolysis of lupin protein are able to modulate cholesterol metabolism in human hepatic HepG2 cells and that part of these peptides are absorbed in a small intestine model based on differentiated human Caco-2 cells. In this paper, a co-culture system, including Caco-2 and HepG2 cells, was investigated with two objectives: (a) to verify whether cholesterol metabolism in HepG2 cells was modified by the peptides absorption through Caco-2 cells; (b) to investigate how lupin peptides influence cholesterol metabolism in Caco-2 cells. The experiments showed that the absorbed peptides, not only maintained their bioactivity on HepG2 cells, but that this activity was improved by the crosstalk of the two cells systems in co-culture. In addition, lupin peptides showed a positive influence on cholesterol metabolism in Caco-2 cells, decreasing the proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion
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