Diallyl disulfide (DADS), a component of garlic, has been shown to induce growth inhibition and apoptosis in human cancer cell types. The present studies were designed to investigate the effects of DADS on mouse-rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, cell cycle analysis, reactive oxygen species (ROS), Ca2+ production, mitochondria membrane potential, apoptosis induction, associated gene expression and caspases-3 activity were examined by flow cytometric assay and/or Western blot. After 24-h treatment with DADS, a dose- and time-dependent decrease in the viability of N18 cells was observed and the approximate IC50 was 27.6 microM. The decreased percentage of viable cells are associated with the production of ROS then followed by the production of Ca2+ which is induced by DADS. DADS induced apoptosis in N18 cells via the activation of caspase-3. DADS increased the protein levels of p53, cytochrome c and phosphated JNK within 24 h of treatment and it decreased the levels of Bcl-2 and those factors may have led to the mitochondria depolarization of N18 cells. DADS induced apoptosis were accompanied by increased levels of Ca2+ and decreased mitochondrial membrane potential which then led to release the cytochrome c, cleavage of pro-caspase-3. Deleted levels of Ca2+ by BAPTA-AM 10 microM (intracellular calcium chelator) then led to decrease DADS-induced apoptosis. Inhibition of caspase-3 activation by inhibitor (z-VAD-fmk) completely blocked DADS-induced apoptosis on N18 cells. The results indicated that oxidative stress modulates cell proliferation and Ca2+ modulates the cell death induced by DADS.

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Genetic factors and the influence of superoxide are known to play roles in the etiology of glaucoma. We evaluated the association between primary open angle glaucoma (POAG) and two polymorphisms in the epithelial nitric oxide synthase (eNOS) gene, and one polymorphism in the myeloperoxidase (MPO) gene. We enrolled 66 patients with POAG and 100 healthy volunteers in this study. The polymorphisms in the eNOS and the polymorphism MPO -463 G-to-A in the MPO gene were resolved by polymorphism polymerase chain reaction (PCR). There were no significant differences in the distribution of the eNOS intron -4 (P=0.481), eNOS promotor -786 (P=0.555), and MPO -463 (P=0.292) gene polymorphisms between the POAG patients and the volunteers (P-values=0.481, 0.555, and 0.292, respectively). None of the alleles from either gene differed between the groups (P-values=0.483, 0.554, and 0.183, respectively). Superoxide is closely related to glaucoma, and eNOS and MPO are two important enzymes in the free radical pathway. However, polymorphisms of the eNOS intron-4, eNOS promotor -786, and MPO -463 gene polymorphisms did not reveal significant differences between POAG patients and controls in our study. The use of these agents and other superoxide-related genes for clinical applications requires further investigation.

The benzodiazepine receptor (BZDR) of the embryonic chick brain contained three subunit proteins with molecular weights of 48-kilodalton (KD), 50-KD and 51-KD at a pI of 5.6, as demonstrated by two-dimensional gel electrophoresis and fluorography of the 3H-flunitrazepam (FNZ)-photolabeled receptor. Monoclonal antibodies (mAB) against the receptor were produced by using the spleen cells of one mouse immunized with the three subunit proteins extracted from SDS-PAGE gels. When the radioligand-labeled membranes were subjected to two-dimensional gel electrophoresis followed by immunoblotting using the mAB 2C3, both 50-KD and 51-KD bands with a pI of 5.6 were immunoreactive and radioactive. Thus, the mAB 2C3 recognized a common epitope on the 50-KD and 51-KD subunits of the BZDR. In addition, the mAB 2C3 was used with immunocytochemistry to determine the distribution of the receptor in the chick embryo brain. The BZDR immunoreactivity was observed among various brain areas, including hippocampus, optic tectum and cerebellum. The reaction product was localized in the neuronal membranes and cytoplasm. Certain neurons in the culture derived from embryonic chick brains were also immunoreactive as detected by immunocytochemical staining.

Purpose: E-cadherin (E-CDH) is one of the most important cell surface glycoproteins involved in cell morphogenesis. In primary open angle glaucoma (POAG), the extracellular matrixes of trabecular meshwork and lamina cribrosa in the optic nerve head are out of balance. We suspected that E-CDH by way of metalloproteinases is closely related to POAG. We therefore investigated the relationship between CDH-1 gene 3′ untranslated region (3′-UTR) polymorphism and POAG patients in order to support this hypothesis. Patients and Methods: We enrolled 60 POAG patients and 103 healthy volunteers from the Department of Ophthalmology at the China Medical University Hospital, Taichung, Taiwain, ROC. None of the control subjects had a history of eye disease and all underwent the same examination as the POAG patients. PCR-based analysis of the restriction fragment length polymorphism was used to test the CDH-1 gene 3′-UTR polymorphism. All statistical analyses were performed by the χ² test. Result: There was a significant difference in the distribution of the CDH-1 gene 3′-UTR C/T polymorphism between POAG patients and the normal controls (p <0.000). The odds ratio of the ‘C’ allele was al so significantly different between both groups (odds ratio = 5.510, 95% confidence interval = 3.171–9.574). Conclusion: CDH-1 is closely related to metalloproteinase and plays an important but not well-understood role in the onset and progression of POAG. The exact role of CDH-1 in POAG could be resolved by the posttranslated products of the gene and the protein-protein interaction of the gene products in the future.