Purification, identification and characterization of Nag2 N-acetylglucosaminidase from Trichoderma virens strain mango.

BACKGROUND: N-acetylglucosaminidase (NAGase) could liberate N-acetylglucosamine (GlcNAc) from GlcNAc-containing oligosaccharides. Trichoderma spp. is an important source of chitinase, particularly NAGase for industrial use. nag1 and nag2 genes encoding NAGase, are found in the genome in Trichoderma spp. The deduced Nag1 and Nag2 shares ~ 55% homology in Trichoderma virens. Most studies were focus on Nag1 and nag1 previously. RESULTS: The native NAGase (TvmNAG2) was purified to homogeneity with molecular mass of ~ 68 kDa on SDS-PAGE analysis, and identified as Nag2 by MALDI/MS analysis from an isolate T. virens strain mango. RT-PCR analyses revealed that only nag2 gene was expressed in liquid culture of T. virens, while both of nag1 and nag2 were expressed in T. virens cultured on the plates. TvmNAG2 was thermally stable up to 60 °C for 2 h, and the optimal pH and temperature were 5.0 and 60-65 °C, respectively, using p-nitrophenyl-N-acetyl-ß-D-glucosaminide (pNP-NAG) as substrate. The hydrolytic product of colloidal chitin by TvmNAG2 was suggested to be GlcNAc based on TLC analyses. Moreover, TvmNAG2 possesses antifungal activity, inhibiting the mycelium growth of Sclerotium rolfsii. And it was resistant to the proteolysis by papain and trypsin. CONCLUSIONS: The native Nag2, TvmNAG2 was purified and identified from T. virens strain mango, as well as enzymatic properties. To our knowledge, it is the first report with the properties of native Trichoderma Nag2.

Effects of (-)-epigallocatechin gallate on RPE cell migration and adhesion.

PURPOSE: In diseases such as proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR), and age-related macular degeneration (AMD), retinal pigment epithelial (RPE) cells can initiate proliferation and migration and secrete extracellular matrix (ECM) proteins. (-)-Epigallocatechin gallate (EGCG)-a natural anti-oxidant flavonoid that is abundant in green tea-has been shown to suppress the migration and adhesion of many cell types, but its effects on RPE cell migration and adhesion were unknown. Several studies have shown that platelet-derived growth factor (PDGF) enhances proliferation and migration effects on RPE cells in PVR, and that fibronectin is a major ECM component of PVR tissue. Therefore, we investigated the inhibitory effects of EGCG on RPE cell migration induced by PDGF-BB, an isoform of PDGF, and adhesion by fibronectin. METHODS: The migration of RPE cells was detected by an electric cell-substrate impedance sensing (ECIS) migration assay and a Transwell migration assay. Cells were loaded with 2',7'-bis-(carboxyethyl)-5(6')-carboxyfluorescein acetoxymethyl ester (BCECF/AM), and their adhesion to fibronectin was examined. The interactions of EGCG with PDGF-BB were analyzed by a dot binding assay. Cytoskeletal reorganization was examined by immunofluorescence microscopy. The PDGF-BB-induced signaling pathways were detected by western blotting. RESULTS: In the present study, we find that EGCG can inhibit PDGF-BB-induced human RPE cell migration and, in a dose-dependent manner, RPE cell adhesion to fibronectin. Our analysis demonstrates that EGCG does not directly bind to PDGF-BB and the inhibition of EGCG against fibronectin-induced cytoskeletal reorganization is observed. Furthermore, EGCG is shown to suppress PDGF-BB-induced PDGF-beta receptors, downstream PI3K/Akt, and MAPK phosphorylation. CONCLUSIONS: Our results provide the first evidence that EGCG is an effective inhibitor of RPE cell migration and adhesion to fibronectin and, therefore, may prevent epiretinal membrane formation.

Protective effects of myricetin against ultraviolet-B-induced damage in human keratinocytes.

Myricetin is a flavonoid similar to quercetin, which is commonly found in natural foods such as berries, vegetables, teas, wine, and herbs. It is considered to be an antioxidant which is capable of quenching photoaging-causing free radicals within the skin. In this study, we investigated the mechanisms underlying protective effect of myricetin on ultraviolet-B (UVB)-induced damage to keratinocytes. We found that myricetin concentration-dependently attenuated UVB-induced keratinocyte death as determined by a cell viability assay. Pretreatment with myricetin also reduced the UVB-induced malondialdehyde level. Moreover, UVB-induced H(2)O(2) generation in keratinocytes was inhibited by myricetin according to flow cytometry, suggesting that myricetin can act as a free radical scavenger when keratinocytes experience photodamage. Furthermore, UVB-induced activation of c-jun-NH(2) terminal kinase (JNK) in keratinocytes was inhibited by myricetin. UVB-induced pre-G(1) phase arrest leading to apoptotic changes in keratinocytes was blocked by myricetin. Taken together, the protective mechanisms of keratinocyte by myricetin against UVB-induced photodamage occur by the inhibition of UVB-induced intracellular hydrogen peroxide production, lipid peroxidation and JNK activation. Therefore, myricetin is suitable for further development as an anti-aging agent for skin care.

Protective effects of (-)-epigallocatechin gallate on UVA-induced damage in ARPE19 cells.

PURPOSE: Oxidative injury to the retinal pigment epithelium (RPE) has been proposed to play a contributing role in age-related macular degeneration (AMD). Exposure to solar ultraviolet (UV) radiation is believed to cause the production of reactive oxygen species (ROS), which may cause oxidative damage to RPE cells. Studies have shown that (-)-epigallocatechin gallate (EGCG), an abundant and active component in green tea, can protect several cell types from oxidative stress. It may be useful in the prevention of early AMD. METHODS: To determine whether EGCG protects RPE cells from UVA-induced damage, we used a cell viability assay to determine the viability of UVA-treated cells. Intracellular H(2)O(2) levels were measured by flow cytometry. Western blotting was used to detect UVA-induced signaling pathways. RESULTS: The results indicated that EGCG inhibits UVA-induced RPE cell death. In addition, intracellular H(2)O(2) generation in RPE cells irradiated by UVA was inhibited by EGCG in a concentration-dependent manner. EGCG also inhibited UVA-induced extracullar signal-regulated kinase (ERK) and c-jun-NH2 terminal kinase (JNK) activation in RPE cells while a higher concentration of EGCG had an inhibitory effect on UVA-induced p38 activation. Finally, we investigated cyclooxygenase-2 (COX-2) expression in RPE cells exposed to UVA radiation, and EGCG was found to also have inhibited UVA-induced COX-2 expression. CONCLUSIONS: Taken together, our results demonstrate that EGCG inhibits UVA-induced H(2)O(2) production, mitogen-activating protein kinase activation, and expression of COX-2. Moreover, it enhances RPE cell survival after UVA exposure. This suggests EGCG is effective in preventing UVA-induced damage in RPE cells and may be suitable for further developments as a chemoprotective factor for the primary prevention of early AMD.