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AJ Quantock, V Siegler, C Hughes, B Caterson; Keratan Sulphate Levels During Development of the Embryonic Avian Cornea . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3222.
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Purpose: During the latter stages of development, the secondary chick corneal stroma undergoes significant structural, transparency and compositional changes. Previously, it was shown that the sulphated form of lumican, the cornea's main keratan sulphate (KS) containing proteoglycan, begins to accumulate in the embryonic chick cornea between day 12 and 15 of development (Cornuet et al. (1994) IOVS;35:870-77). The current study was designed to provide information about the amount of keratan sulphate in the secondary chick cornea during development. Methods: Corneas, freshly excised from chick embryos at developmental days 13-18 were extracted in 4M GdnHCl containing proteolytic inhibitors for 48 h at 4ºC. The supernatants were dialysed, dried, reconstituted in water and assayed for KS by ELISA using Mab 5-D-4(Caterson et al. (1983) J. Biol. Chem. 258: 8848-8854). The corneas were further digested with papain, overnight at 60ºC, followed by boiling for 15 minutes and subsequent addition of iodoacetamide to inactivate the enzyme. Papain digested supernatants were assayed for KS using Mab 5-D-4. For KS-assay by ELISA, absorbance readings were taken at 405nm. Standard curves were plotted and concentration of cornea KS were calculated using these curves. Results: The amount of extractable sulphated GAG from developing chick corneas by 4M guanidine was relatively low as measured using the DMMB assay (Farndale et al. (1986) Biochim. Biophys. Acta. 833:173-177.). However, a considerable proportion of the total matrix KS was in this extract. During developmental days 13-16 immunodetectable keratan sulphate levels were unchanged. By day 17 an approximate 8-fold increase in the amount of extractable keratan sulphate was measured using the 5-D-4 ELISA, this result paralleling changes in the regularity of collagen fibril arrangement. Conclusions: The increase in extractable keratan sulphate between day 16 and 17 of development reported here is consistent with both increased histochemical staining of sulphated proteoglycan, as well as the establishment of a more closely packed array of regularly arranged collagen fibrils. A causal relationship has not yet been established, but a functional role for sulphated keratan sulphate in tissue restructuring at this time is a clear possibility.
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