PURPOSE: To determine whether the expression of mutant myocilin can lead to death of human trabecular meshwork (HTM) cells and to determine whether the mechanism by which this occurs is apoptosis. METHODS: HTM cells were transduced with a recombinant adenovirus expressing human mutant (Q368X) myocilin. The apoptotic death of HTM cells caused by expression of mutant myocilin was examined using a cell proliferation assay, flow cytometry, Western blot analysis, and immunocytochemistry. RESULTS: It appeared that the expression of mutant myocilin itself was not sufficient to cause HTM cell death. Furthermore, the expression of mutant myocilin did not lead to apoptosis of HTM cells although it did elicit a protein unfolding response. CONCLUSIONS: Our data suggest that the mechanism of myocilin glaucoma is not apoptotic death of HTM cells caused by mutant myocilin expression, and that the actual mechanism remains unknown.
Adenoviridae ; Apoptosis ; Blotting, Western ; Cell Death ; Cell Proliferation ; Cytoskeletal Proteins ; Eye Proteins ; Flow Cytometry ; Glaucoma ; Glycoproteins ; Humans ; Protein Unfolding ; Trabecular Meshwork

PURPOSE: To assess whether the expression of heat shock protein 72 (Hsp72) protects rat retinal ganglion cells (RGC-5) from apoptotic cell death. METHODS: Hsp72 expression in RGC-5 cells transduced with replication-deficient recombinant adenovirus was analyzed by Western blot analysis and immunofluorescence. The effect of Hsp72 expression on etoposide-induced apoptotic cell death was examined by microscopic analysis and confirmed by cell proliferation assay. RESULTS: Western blot analysis and immunofluorescence clearly showed adenovirus-mediated Hsp72 expression in RGC-5 cells. Treatment with etoposide resulted in the death of a proportion of the cells by apoptosis. However, this apoptotic cell death was significantly reduced in cells expressing Hsp72, with the reduction in cell death correlating to the level of Hsp72 expression. CONCLUSIONS: Over-expression of Hsp72 alone is sufficient to rescue neuronal cells from apoptotic cell death, suggesting that fine-tuning its expression may be an effective neuroprotective approach in retinal degenerative disease.
Animals ; Blotting, Western ; Cell Death/*genetics ; Cell Survival ; Cells, Cultured ; DNA/*genetics ; Disease Models, Animal ; Etoposide/toxicity ; *Gene Expression Regulation ; HSP72 Heat-Shock Proteins/biosynthesis/*genetics ; Immunohistochemistry ; Rats ; Retinal Degeneration/*genetics/metabolism/pathology ; Retinal Ganglion Cells/drug effects/*metabolism/pathology

No abstract available.
Hematopoietic Stem Cell Transplantation* ; Humans

BACKGROUND: YMDD motif variants of the hepatitis B virus (HBV) emerge in some chronic hepatitis B patients after prolonged lamivudine treatment. HBV DNA breakthrough may be accompanied by the emergence of YMDD variants. The detection of YMDD motif variants will be necessary since Adefovir dipivoxil was recently approved to be an effective treatment for lamivudine-resistant patients. METHODS: Samples were chosen from twenty-one patients who experienced the DNA breakthrough after an undetectable HBV DNA period by HBV DNA hybrid-capture assay. We tested the samples of each stage for detection of YMDD motif variants by a sequencing method using Accu-Typer(TM) HBV YMDD typing Kit (DNA Link, Seoul, Korea) and ABI PRISM 3700 DNA Analyzer. RESULTS: All 17 samples that were collected before treatment had the wild-type YMDD motif. Of 20 samples amplified, which were from the undetectable HBV DNA period, three (15%) samples showed YMDD mutation. After DNA breakthrough, YMDD mutants were detected in 13 (63%) of 21 samples (YIDD 8 cases, YVDD 5 cases). CONCLUSION: We could reconfirmed that YMDD motif variants were remarkably related to the lamivudin resistance. YMDD motif variants; however, were not detected in one-third of the lamivudine resistance. The sequencing method of our study would be useful in providing the neighboring nucleotide information other than the YMDD motif in patients experiencing DNA breakthrough.
DNA ; Hepatitis B virus ; Hepatitis B, Chronic ; Humans ; Lamivudine ; Seoul