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  • To the best of our knowledge this is

    2021-11-25

    To the best of our knowledge, this is the first study to examine functional GSTO1‐1 activity in the cornea. Therefore, comparative data for human or animals is not available. Moreover, we observed the nonspecific degradation of the GSTO1‐1 substrate 4NPG via an unknown mechanism, which was corrected for the data presented here. The nonspecific GSTO1‐1 activity due to the degradation of the substrate used constituted a large proportion of the measured activity. Although the nonspecific degradative mechanism appeared to be related to the presence of collagen or extracellular matrix, it is unknown and should therefore be considered when conducting future measurements. Regarding the relative levels of GSTO1‐1 activity in the corneal tissues, it is notable that the excised animal corneas expressed the highest level in their stromal layers. The difference in the GSTO1‐1 activity levels of the corneal cell layers was significant in the porcine cornea (Fig. 5B). In contrast, similar levels of GSTO1‐1 activity were observed in the layers of the Hemicornea construct, which were comparable to the levels of GSTP1‐1 and total GST activity in the epithelial and stromal layers of the Hemicornea construct and the excised animal corneas. Because the levels of GSTO1‐1 activity in the Hemicornea construct and the animal models appeared to be quite different (Figs. 4 B and 5 B), additional measurements of the activity levels in the human cornea are required. Not only was the level of GSTO1‐1 expression found to be comparatively high in the stromal layers of the excised animal corneas but in general, the activity levels of all of the investigated GSTs were found to be unexpectedly high in the stroma of the excised corneas and the stromal equivalent of the Hemicornea construct (Fig. 5). Based on the relatively small number of flavin adenine dinucleotide sale in the stroma, the highest activity levels were presumed to exist in the epithelial cells. In particular, the total GST activity was expected to follow this pattern because the epithelial cell line HCE‐T exhibited a higher level of total GST activity than did the keratocyte cell line HCK (Fig. 5A). This result is unlikely to have resulted from an error in the methodical approach because different methods were used to separate the stromal equivalent of the Hemicornea construct and the stromal layer of the excised corneas (see Section 2.5.1). This result could possibly be explained by the effect of the epithelial cells on the stromal cells in both models. Because a high level of GST activity was also observed in the stromal layer of the two-layered Hemicornea construct, the effect of the endothelium on this activity appears to be unimportant. However, a study of the effect of co-cultivating HCE‐T and HCK cells on the level of GST activity showed no measureable difference compared with those of singly cultivated cells (data not shown). Therefore, it is conceivable that components of the stromal layer may affect its level of GST activity. The effect of culture factors on the GST activity level of cultivated rat hepatocytes, particularly that of the extracellular matrix, has been examined. One of the major relevant findings was that the addition of supplements that are part of the natural environment of hepatocytes, more precisely the addition of collagen type IV, fibronectin and heparin sulfate proteoglycans, did not change the GST activity level compared with that observed using pure collagen type I matrices (Beken et al., 1999). However, compared to that observed in hepatocyte monolayers, the level of GST activity of hepatocytes was increased when they were cultivated in a collagen gel sandwich (Depreter et al., 2000). In addition, a further increase in the level of GST activity was observed when the cultivation period was increased from 7 to 14days (Depreter et al., 2000). No difference in activity was observed when the hepatocytes were cultivated on a single collagen layer or within a collagen sandwich (Richert et al., 2002), the latter of which is comparable to the stromal equivalent of the Hemicornea construct. Furthermore, LeCluyse et al. reported that collagen affected the GST subunit concentration in rat hepatocytes. The levels of Mu and Pi GST expression of hepatocytes cultured in the presence of collagen were increased by two fold and four fold, respectively, compared with the levels in freshly isolated hepatocytes, whereas those of Alpha GST subunits were decreased by approximately two fold (LeCluyse et al., 2000). These findings are consistent with the level of GSTP1‐1 activity of hepatocytes cultured in the presence of collagen being increased by approximately two fold compared with that of freshly isolated hepatocytes (Beken et al., 1999). However, the effect of the extracellular matrix or of only collagen on the GST expression and activity levels depends on the origin of the investigated cells. For instance, studies of SV40-immortalized tracheobronchial epithelial cells revealed an eight-fold increase in total GST activity when they were cultivated on a collagen gel instead of in tissue culture dishes (Reddy et al., 1995). The level of GSTP1‐1 activity of these hepatocytes was increased by fivefold when they were grown on a collagen lattice. In addition, collagen was found to increase their levels of Alpha and Mu GST beyond the limit of detection (Reddy et al., 1995). Thus, collagen may affect the levels of total GST activity and specific GSTP1‐1 activity in the stromal layer of the animal corneas or the stromal equivalent of the Hemicornea construct. Because studies concerning the effect of the extracellular matrix on Omega GST activity are currently lacking, it can only be presumed that collagen might also affect this aspect of GSTO1‐1.