The occurrence of the submandibular duct reservoir was reported by Butcher (1972). Its form and functional volume (Schneyer, 1975) and the development of the submandibular complex (Kim, 1975) were studied. The shape of the cells in the epithelial lining of the reservoir had not been determined as yet. So via the techniques of histology and histochemical enzymatic activity, the epithelial lining and the function of the reservoir were investigated. The epithelial lining of the reservoir was not uniform in all regions. The proximal portion of the reservoir was lined by pseudostratified columnar epithelium and the distal portion was lined by stratified columnar or cuboidal epithelium. Acid phosphatase activity in the epithelial lining of the reservoir was observed as well as in the acini, granular convoluted duct and striated duct of the submandibular gland proper.
Acid Phosphatase/metabolism ; Animal ; Epithelial Cells ; Epithelium/enzymology ; Rats/anatomy & histology* ; Submandibular Gland/anatomy & histology* ; Submandibular Gland/cytology

The loss of retinal pigment epithelium (RPE) with aging is related to age-related macular degeneration (AMD). This study was conducted to investigate the mechanism of hydrogen peroxide (H2O2) induced cell death in a human retinal pigment epithelial cell line, ARPE-19. Hydrogen peroxide was added at different concentrations to ARPE-19 cells and cultured. The cytotoxicity was assayed by mitochondrial function using 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) testing. The patterns of cell damage were assessed using an acridine orange-ethidium bromide differential staining method, in situ end labeling (ISEL) assay and transmission electron microscopy (TEM). Catalase, a major antioxidant, was used to prevent cell death. The cleavage of procaspase 3 and poly (ADP-ribose) polymerase (PARP) was determined by western blot analysis. Hydrogen peroxide significantly induced cell death in ARPE-19 cells, whereas pretreatment of the cells with catalase prevented cell death. Application of the ISEL assay and acridine orange/ethidium bromide staining demonstrated that the H2O2-induced cell death occurred by an apoptotic mechanism at lower concentrations of H2O2 (400, 500, 600 microM), whereas higher concentrations of H2O2 induced necrosis rather than apoptosis. Caspase 3 was associated with the apoptotic pathway in human RPE cell death. Western blot analysis confirmed caspase 3 activation and cleavage of substrate proteins in ARPE-19 cells treated with an H2O2 concentration of 600 microM. These results indicate that treatment with H2O2 induces apoptotic and necrotic cell death in ARPE-19, and that caspase 3 is associated with apoptotic cell death. Therefore, H2O2 may induce the destruction of RPE cells in AMD by the combined effects of apoptosis and necrosis.
Apoptosis ; Caspases/metabolism ; Catalase/pharmacology ; Cell Line ; Cell Survival/drug effects ; Enzyme Activation ; Human ; Hydrogen Peroxide/*pharmacology ; Necrosis ; Pigment Epithelium of Eye/*drug effects/enzymology/pathology/*physiology

OBJECTIVE: To construct the recombinant adeno-associated virus(rAAV) vector plasmid pSNAV2.0-TK containing HSV1-TK gene, to produce recombinant adeno-associated virus rAAV2/HSV1-TK, and to detect the integration and expression of HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV1-TK, and to provide foundation for gene therapy of posterior capsular opacification. METHODS: The recombinant vector plasmid constructed by gene recombinant technology was analyzed by PCR and restriction enzyme digestion. The cell strain BHK-21/TK was screened by G418 after the plasmid was transfected into BHK-21 cells,with the helper virus HSV1-rc/UL2 to produce the recombinant virus rAAV2/HSV1-TK. The purity of rAAV2/HSV1-TK was detected by SDS-PAGE and HPLC, and the titre of rAAV2/HSV1-TK was observed by dot blot hybridization. The HSV1-TK gene in lens epithelial cells transfected by rAAV2/HSV-TK was investigated by PCR and RT-PCR. RESULTS: The recombinant plasmid proved successful by PCR and restriction enzyme digestion. The recombinant virus rAAV2/HSV1-TK was produced successfully and its titre was 1 x 10(12) v.g./mL by dot blot hybridization. The HSV1-TK gene was integrated and expressed in lens epithelial cells. CONCLUSION The recombinant adeno-associated virus vector plasmid containing HSV1-TK gene is successfully constructed, and high titre recombinant adeno-associated virus (rAAV2/HSV1-TK) is obtained. The HSV1-TK gene in lens epithelial cells is expressed after being transfected by rAAV2/HSV1-TK.
Animals ; Cloning, Molecular ; Cricetinae ; Dependovirus ; genetics ; metabolism ; Epithelium, Corneal ; cytology ; metabolism ; Genetic Vectors ; Herpesvirus 1, Human ; enzymology ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thymidine Kinase ; biosynthesis ; genetics ; Transfection

The role of nitric oxide (NO) in the ocular surface remains unknown. We investigated the conditions leading to an increase of NO generation in tear and the main sources of NO in ocular surface tissue. We evaluated the dual action (cell survival or cell death) of NO depending on its amount. We measured the concentration of nitrite plus nitrate in the tears of ocular surface diseases and examined the main source of nitric oxide synthase (NOS). When cultured human corneal fibroblast were treated with NO producing donor with or without serum, the viabilities of cells was studied. We found that the main sources of NO in ocular surface tissue were corneal epithelium, fibroblast, endothelium, and inflammatory cells. Three forms of NOS (eNOS, bNOS, and iNOS) were expressed in experimentally induced inflammation. In the fibroblast culture system, the NO donor (SNAP, S-nitroso-N-acetyl-D, L-penicillamine) prevented the death of corneal fibroblast cells caused by serum deprivation in a dose dependent manner up to 500 micrometer SNAP, but a higher dose decreased cell viability. This study suggested that NO might act as a doubleedged sword in ocular surface diseases depending on the degree of inflammation related with NO concentration.
Animals ; Apoptosis/drug effects/physiology ; Aqueous Humor/metabolism ; Blood Proteins/pharmacology ; Cell Survival/drug effects/physiology ; Cells, Cultured ; Epithelium, Corneal/*cytology/*enzymology ; Fibroblasts/cytology/enzymology ; Humans ; Nitric Oxide/biosynthesis/*physiology ; Nitric Oxide Donors/pharmacology ; Nitric Oxide Synthase/metabolism ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; Penicillamine/*analogs & derivatives/pharmacology ; Peroxynitrous Acid/biosynthesis ; Rabbits ; Tears/metabolism ; Uveitis/metabolism

This study evaluated the effects of glucose in human retinal pigment epithelial (RPE) cells to investigate the cause of diabetic retinal complications. Human RPE cells were cultured in media containing 5.5 mM, 11.0 mM, and 16.5 mM D-glucose. The present study performed proliferation and migration assays, and conducted western blotting for the protein expression, as well as RT-PCR for the mRNA expression, of MMP-2 and -9, and TIMP-1 and -2. The results of the western blotting analysis showed that increasing glucose concentration significantly increased the expression of MMP-2 and -9, but significantly decreased the expression of TIMP-1 and -2. Moreover, the RT-PCR results indicated significant increases in the mRNA expression of MMP-2 and -9, as well as of TIMP-1 and -2, by raising glucose concentration. This study provides fundamental data for future research on the mechanism of retinal complication in diabetic patients.
Blotting, Western ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Comparative Study ; Dose-Response Relationship, Drug ; Glucose/*pharmacology ; Humans ; In Vitro ; Matrix Metalloproteinases/genetics/*metabolism ; Pigment Epithelium of Eye/*drug effects/enzymology ; RNA, Messenger/metabolism ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Tissue Inhibitor of Metalloproteinases/genetics/*metabolism

OBJECTIVEOxidative stress plays an important role in retinal pigmental epithelium (RPE) death during aging and the development of age-related macular degeneration. Although early reports indicate that reactive oxygen species (ROS) including H₂O₂ can trigger apoptosis at lower concentrations and necrosis at higher concentrations, the exact molecular mechanism of RPE death is still unclear. The purpose of this study was to investigate the molecular pathways involved in RPE death induced by exogenous ROS, especially at higher concentrations.

METHODSCultured ARPE-19 cells were treated with H₂O₂ at different concentrations and cell viability was measured with the MTT assay. Cell death was morphologically studied by microscopy using APOPercentage assay and PI staining. Furthermore, the impact of oxidative stress on ARPE-19 cells was assessed by HO-1 and PARP-1 Western blotting and by the protection of antioxidant EGCG. Calcium influx was determined using the fura-2 calcium indicator and the role of intracellular calcium overload in ARPE-19 cell death was evaluated following cobalt treatment to block calcium effects.

RESULTSH₂O₂ reduced the viability of ARPE-19 cells in a concentration-dependent manner, which was presented as a typical s-shaped curve. Cell death caused by high concentrations of H₂O₂ was confirmed to be programmed necrosis. Morphologically, dying ARPE-19 cells were extremely swollen and lost the integrity of their plasma membrane, positively detected with APOPercentage assay and PI staining. 24-hour treatment with 500 μmol/L H₂O₂ induced remarkable up-regulation of HO-1 and PARP-1 in ARPE-19 cells. Moreover, antioxidant treatment using EGCG effectively protected cells from H₂O₂-induced injury, increasing cell viability from 14.17%±2.31% to 85.77%±4.58%. After H₂O₂ treatment, intracellular calcium levels were highly elevated with a maximum concentration of 1200 nM. Significantly, the calcium channel inhibitor cobalt was able to blunt this calcium influx and blocked the necrotic pathway, rescuing the ARPE-19 cell from H₂O₂-induced death.

CONCLUSIONSAt high concentrations, H₂O₂ induces ARPE-19 cell death through a regulated necrotic pathway with calcium overload as a critical step in the cell death program.

Antioxidants ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Cell Culture Techniques ; Cell Line ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Heme Oxygenase-1 ; metabolism ; Humans ; Hydrogen Peroxide ; toxicity ; Necrosis ; drug therapy ; Oxidative Stress ; drug effects ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Retinal Pigment Epithelium ; drug effects ; enzymology ; metabolism ; pathology

Interleukin 1 beta (IL-1 beta), a proinflammatory cytokine, is related with inflammatory diseases and it up-regulates MUC2 gene expression and mucin secretion. This study was designed to investigate the signal transduction pathway of the IL-1 beta-mediated MUC2 gene expression and mucin secretion in human airway epithelial cells. In cultured human airway NCI-H292 epithelial cells, the steady state of the mRNA level of MUC2 gene expression and mucin secretion induced by IL-1 were determined by reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunoblot analysis. To observe the signal pathway of the IL-1 beta-mediated MUC2 gene expression and mucin secretion, we used several specific inhibitors. PD98059 (MEK/ERK inhibitor) suppressed IL-1 beta-mediated MUC2 gene expression and mucin secretion, while SB203580 (p38 inhibitor) did not. Ro31-8220 (PKC inhibitor) inhibited IL-1 beta-mediated MUC2 gene expression and mucin secretion. It inhibited ERK phosphorylation, but did not inhibit p38 phosphorylation. LY294002 (PI3K inhibitor) also suppressed MUC2 expression, but did not inhibit any MAPKs phosphorylation. These results suggest that the IL-1 -mediated MUC2 gene expression and mucin secretion in NCI-H292 cells are regulated through activation of the PKC-MEK/ERK pathway, and that PI3K is also involved in the IL-1 beta-mediated MUC2 gene expression and mucin secretion.
1-Phosphatidylinositol 3-Kinase/*metabolism ; Cell Line ; Chromones/pharmacology ; Dose-Response Relationship, Drug ; Enzyme Activation ; Enzyme Inhibitors/pharmacology ; Epithelium/*enzymology ; Flavonoids/pharmacology ; Humans ; Imidazoles/pharmacology ; Immunoassay ; Immunoblotting ; Indoles/pharmacology ; Interleukin-1/metabolism/*physiology ; Lung/cytology/*metabolism ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase Kinases/*metabolism ; Morpholines/pharmacology ; Mucin-2 ; Mucins/*biosynthesis/metabolism ; Phosphorylation ; Protein Kinase C/*metabolism ; Protein Structure, Tertiary ; Pyridines/pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Time Factors