Proteoglycans (PGs) synthesized by chick corneal stromal cells in cell culture and organ culture were metabolically radiolabelled with [ 35 S]sulfate (for glycosaminoglycans) and [ 3 H]leucine (for core proteins). Media, cell extracts and organ extracts from cultures were chromatographed on DEAE-Sephacel columns and separated into three fractions: the pass-through fraction (Fraction 1: nonsulfated PGs, hardly sulfated PGs, or glycoproteins with some oligosaccharides), the fraction eluted with a low salt concentration (Fraction 2: undersulfated PGs), and the fraction eluted with a high salt concentration (Fraction 3: highly sulfated PGs). The PG compositions of each fraction of cell culture and organ culture were then compared. While the proportions of highly sulfated KSPG in Fractions 3 of medium and cell extract of cell culture were both very low compared with those of medium and organ extract of organ culture, respectively, the proportions of highly sulfated CS/DS PG in Fractions 3 of those of cell culture were higher than those of organ culture. On the other hand, the proportions in the 35 S activities of nonsulfated or undersulfated KSPG in Fractions 1 and 2 of medium and cell extract of cell culture were comparable to those of organ culture. Furthermore, the proportions of core proteins of undersulfated KSPG in Fractions 2 were higher in cell culture than in organ culture. These results show that, when the cells are cell-cultured, the degree of sulfation of KS chains decreases markedly, but the syntheses of the glycosaminoglycan backbone and core protein are maintained. S]sulfate and radiolabelled at 37°C for 5 h in a CO 2 -incubator. After radiolabelling, the medium was removed from the culture. The medium fraction, to which solid guanidine chloride was added to make about 4 M, was applied to a PD-10 column to remove unincorporated radioactive precursors as reported previously. 24)The cultured explants were extracted with 4 M guanidine chloride solution containing 0.2 M NaCl, 10 mM EDTA, 6-aminohexanoic acid (AHA), 10 mM N-ethylmaleimide (NEM), and 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) in 0.5 M Tris-HCl, pH 7.5 as reported previously. 24)The cell culture of the stromal cells was done as reported previously.25) The corneal stromas were taken from 114 chicken eyeballs as described above, minced, and digested with 0.25% of collagenase in phosphate-buffered saline, pH 7.2 (8.1 mM Na 2 HPO 4 , 0.14 M NaCl, 1.47 mM KH 2 PO 4 , 2.7 mM KCl, 1ϫ10 5 U penicillin G, and 0.1 g streptomycin sulfate in 1000 ml). The resultant cell suspension was divided into three parts, each of which was placed in 3 ml of Ham's F-12/10% FBS/100 U penicillin G on a culture dish (2.16ϫ 10 6 cells/dish). The cells were cultured in 3 ml of the medium in a CO 2 -incubator (95% air-5% CO 2 ) at 37°C for 8 d. The medium was replaced daily. On the 9th day, the medium was removed, and 1 ml of the medium containing 11.1 MBq of [35 S]sulfate and 3.7 MBq of [ 3 H]leucine was added to each dish. The cultures were ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.