After wounding, the capacity for epithelial cell proliferative and migration appears to be as active in the central cornea as in the periphery/limbus. Central and peripheral epithelial recovery remains equal even after ablation of the limbus. Central human corneal epithelial cells are therefore capable of corneal epithelial regeneration, at least in the first 12 hours after wounding.

Both limbal and central epithelial cells are capable of forming spheres in cultures that have stem cell properties. Central and limbal epithelial cells cannot be differentiated using FACS, but younger donor tissues give rise to greater numbers of large cells with high dye efflux. Therefore, results indicate that human central corneal epithelium contains cells with stem/progenitor properties, and these stem properties decline with age.

Human beta-glucuronidase, due to low intrinsic immunogenicity in humans, is an attractive enzyme for tumor-specific prodrug activation, but its utility is hindered by low activity at physiological pH. Here we describe the development of a high-throughput screening procedure for enzymatic activity based on the stable retention of fluorescent reaction product in mammalian cells expressing properly folded glycoproteins on their surface. We utilized this procedure on error-prone PCR and saturation mutagenesis libraries to isolate beta-glucuronidase tetramers that were up to 60-fold more active (k(cat)/K(m)) at pH 7.0 and were up to an order of magnitude more effective at catalyzing the conversion of two structurally disparate glucuronide prodrugs to anticancer agents. The screening procedure described here can facilitate investigation of eukaryotic enzymes requiring posttranslational modifications for biological activity.

The roles of the gap junction protein connexin31.1 (Cx31.1) are poorly understood, especially as the protein appears to form non-functional channels. Cx31.1 specific antisense oligodeoxynucleotides (ODNs) were designed to evaluate its roles in a corneal epithelium model. Expression of Cx31.1 in corneal epithelium extends from the suprabasal layers of polyhedral wing cells through to the flat squamous cells of superficial layers which are shed into the tear film. Deoxyribozymes (Dzs) were tested for cleavage efficacy using in vitro transcribed Cx31.1 mRNA. Cleavage results showed a putative tertiary structure for Cx31.1 mRNA with one region appearing to have a higher potential for antisense targeting. Application of antisense ODNs designed to this region caused Cx31.1 knockdown in rat and human corneal organotypic culture models, leading to a reduction in apoptosis and a thickening of the corneal epithelium (p=0.0045). Cx31.1 appears to play a role in triggering cell death; knocking it down may provide a novel approach for tissue repair and engineering.

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