Coordinated activation of corneal wound response genes in vivo as observed by in situ hybridization

We used subtractive screening of a cDNA library prepared from corneoscleral rims after cauterizing rat corneas. We identified 76 clones whose corresponding mRNA increased during the wound healing process in an in vivo model of injury which damages the corneal epithelium, stroma, and endothelium. Of these clones, 31 sequences encode known proteins. Another 45 clones are novel sequences based on comparison with the GenBank/EMBL databases. Changes in the level of expression of the novel genes, and a selected number of the known genes, were examined by in situ hybridization 22 and 72 hr after corneal injury. The majority produced a ‘wound pattern’ of expression such that the mRNAs were highly induced in all cell types adjacent to the wound site at 22 hr post injury. This signal decreased in intensity with distance from the wound site. In a subset of corneoscleral rims examined by in situ hybridization, the mRNAs for these genes were also highly induced in the limbal epithelium, where the progenitor corneal epithelial stem cells reside. By 72 hr, when acute tissue damage had been repaired, the induced mRNA was only faintly present in the thickened epithelium. Our results provide a useful framework for further studies defining the pathophysiological roles of the known and novel proteins encoded by the isolated cDNA clones

Phytotoxicity and Benzoxazinone Concentration in Field Grown Cereal Rye (Secale cereale L.)

Winter rye (Secale cereale L.) is used as a cover crop because of the weed suppression potential of its mulch. To gain insight into the more effective use of rye as a cover crop we assessed changes in benzoxazinone (BX) levels in rye shoot tissue over the growing season. Four rye varieties were planted in the fall and samples harvested at intervals the following spring. Two different measures of phytotoxic compound content were taken. Seed germination bioassays were used as an estimate of total phytotoxic potential. Dilutions of shoot extracts were tested using two indicator species to compare the relative toxicity of tissue. In addition, BX (DIBOA, DIBOA-glycoside, and BOA) levels were directly determined using gas chromatography. Results showed that rye tissue harvested in March was the most toxic to indicator species, with toxicity decreasing thereafter. Likewise the BX concentration in rye shoot tissue increased early in the season and then decreased over time. Thus, phytotoxicity measured by bioassay and BX levels measured by GC have a similar but not identical temporal profile. The observed decrease in phytotoxic potential and plant BX levels in rye later in the season appears to correlate with the transition from vegetative to reproductive growth