ABSTRACT
PURPOSE: To identify novel proteins which bind to myocilin, using a yeast two-hybrid screening system. METHODS: A bait plasmid of myocilin was constructed, and then transformed into yeast AH109. The transformed yeast cells were mated with yeast Y187 containing human skeletal muscle cDNA library plasmids in 2 X YPD medium. Mated diploid yeasts were plated on synthetic dropout medium (SD/-Trp-Leu-His and SD/-Trp-Leu-His-Ade). The positive clones grown on the selective medium were amplified, sequenced, and analyzed using the database of GenBank. The expression of selected genes in trabecular meshwork (TM) cells was detected by RT-PCR. RESULTS: The expression of bait protein in yeast was confirmed by Western blot analysis. Extensive screening of a cDNA library led to the selection of twenty-four positive clones which represent eight genes, including six of cytoskeleton associated proteins such as alpha-actin, myosin regulatory light chain 2A, dynactin, syntrophin alpha1, microtubule associated protein 1B, and myosin binding protein C. CONCLUSIONS: Myocilin may interact with cytoskeleton associated proteins in TM cells. Further studies on their interactions will provide functional clues of myocilin.
Subject(s)
Humans , Actins , Blotting, Western , Carrier Proteins , Clone Cells , Cytoskeleton , Databases, Nucleic Acid , Diploidy , Gene Library , Mass Screening , Microtubules , Muscle, Skeletal , Myosin Light Chains , Myosins , Patient Dropouts , Plasmids , Trabecular Meshwork , YeastsABSTRACT
To investigate whether the abnormal expression of matrix metalloproteinases(MMPs) in the aqueous humor may case the development of glaucoma, the activity of MMP in the aqueous humor of galucoma and cataract patients were measured and compared. Six primary open angle glaucoma(POAG), 2 chronic angle closureglaucoma(CACG), 2 normal tension glaucoma(NTG), and 14 cataract patients were enrolled. Aqueous humor of each patients were collected during surgery, and total protein concentration and activity of gelatinase A in the aqueous humor were measured by protein assay kit and zymography, respectively. Total protein concentration and gelatinase A activity of POAG patients were increased by 2.1 times and 3.9 times, respectively compared to those of cataract patients. However, there were no statistically significant changes in total protein concentration and gelatinase A activity of CACG and NTG patients compared to those of cataract patients. In conclusion, the development of POAG may be associated with the abnormal expression of MMP in the aqueous humor.