ABSTRACT
BACKGROUND/AIMS: To investigate if galectin-3: (1) enhances adhesion of rat corneal epithelial cells onto a collagen IV substrate and (2) promotes wound healing in rat corneal explants. METHODS: Primary cultures of rat corneal epithelial cells were fixed and immunostained with galectin-3 antibody. To test cellular adherence onto plates coated with collagen type IV, isolated corneal epithelial cells from rats were cultured for 24 h with or without recombinant galectin-3. The attached cells were counted after fixing and staining with 0.1% crystal violet. Direct binding of galectin-3 to collagen IV was tested using a biotin label transfer method. To evaluate wound healing, explants with a 3.5-mm diameter wound in the central corneal epithelium from rats were incubated for 16 h with or without recombinant galectin-3. Changes in the size of the wound were measured with a digital microscope after staining with 5% fluorescein sodium. RESULTS: In rat corneal epithelial cells, galectin-3 was stained throughout the cytoplasm, with increasing density adjacent to the plasma membrane. Exogenous galectin-3, but not epidermal growth factor (EGF), significantly promoted adhesion of corneal epithelial cells onto the collagen IV substrate. Galectin-3 directly bound to collagen IV in vitro. Exogenous galectin-3 significantly enhances wound healing in the corneal explants, which was partially inhibited by ß-lactose. CONCLUSION: Galectin-3 promotes adhesion of corneal epithelial cells onto collagen IV and enhances wound healing in corneal explants. Since galectin-3 functions in promoting wound healing by a different mechanism than that used by EGF, exogenous galectin-3 may be a candidate drug for enhancing epithelial cell wound healing in disorders of the cornea.
Subject(s)
Collagen Type IV/metabolism , Epithelium, Corneal/physiology , Galectin 3/pharmacology , Wound Healing/drug effects , Animals , Cell Adhesion/drug effects , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique, Indirect , Immunoblotting , Male , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Organ Culture Techniques , Rats , Rats, Sprague-Dawley , Recombinant Proteins/pharmacologyABSTRACT
The electrophoretic pattern of G6PD changes in sea urchin egg supernatants, gels, and pellets (homogenized in 0.01 M MgCl2, 0.01 M Tris, pH8, and centrifuged at 4 X 10(4)g) within the first minute following sperm penetration. Therefore, the changes in pattern are an early event of egg activation and are not changes in gene activity. Calcium ionophore activation causes similar changes in G6PD in unfertilized eggs, so calcium is probably involved directly or indirectly. Treatment with 1 mg/ml of G6P and/or NADP, DTT, or papain also causes changes in the isozyme patterns, but the resulting patterns are different from patterns obtained from the fertilized fractions, and each treatment produces a unique pattern. The results lead to the conclusion that the different patterns of unfertilized and fertilized eggs are due to enzyme posttranslational modification. Different patterns may result from intrinsic differences in 1) -SH reduction or levels of glutathione, 2) substrate and cofactor levels, or 3) proteolytic enzymes released naturally or as a result of homogenization.
