Distribution and localization of abnormally expressed OPTN proteins in RGC5 retinal ganglion cells and their effects on subcellular morphology.

The objectives of this study were to investigate the distributions of abnormally expressed optineurin (OPTN) proteins in retinal ganglion cells (RGC5s) of transgenic rats and their effects on subcellular morphological structures. Green fluorescent protein labeled EGFP wild-type (OPTN(WT)), E50K mutant type (OPTN(E50K)), and OPTN siRNA (si-OPTN) eukaryotic expression plasmids were constructed and transfected into RGC5s. Intracellular structures were labeled with organelle specific fluorescent dyes. Construct localization and cell morphologies were visualized by confocal fluorescence microscopy. OPTN(WT) was observed to be distributed as fine punctate fluorescent particles in the cytoplasm around the nucleus, along with exhibiting nuclear expression. OPTNE50K exhibited similar distribution but with non-uniform fluorescence particle size. si-OPTN distribution was similar to that of EGFP: uniform across the cytoplasm and nucleus. Compared with the negative control group, OPTN(WT), and OPTN(E50K) and to a lesser degree pEGFP-transfected cells exhibited fracture and loss of myofilament proteins and mitochondrial swelling and cytoplasmic accumulation, along with abnormal lysosomal distribution and increased volume, and Golgi fragmentation. However, si- OPTN transfected cells exhibited no significant damage. Therefore, we demonstrated that the E50K mutation disrupts the uniformity of OPTN protein distribution upon exogenous overexpression. Furthermore, these results suggested that si-OPTN transfection, and thus potentially OPTN knockdown, did not impact subcellular morphology of RGC5 cells, whereas transfection, especially when combined with wild-type or mutant OPTN expression, led to substantial abnormalities in subcellular morphological structures. These findings lay a foundation for further research into the function of the OPTN protein.

Effects of abnormal optineurin expression on the survival of the rat retinal ganglion cell line RGC-5.

The OPTN gene is thought to be associated with certain types of glaucoma and the function of the protein for which it encodes, optineurin, has been extensively researched, but with contradictory results. We explored the effects of abnormal optineurin expression on the survival of the rat retinal ganglion cell line RGC-5. Plasmids expressing wild-type (WT) or E50K mutant optineurin, or OPTN-specific double-hairpin small interfering RNA (si-RNA), were transfected into RGC-5 cells. The effects on cell survival were monitored by observation of cell morphology and propidium iodide and Hoechst 33342 fluorescent staining, while expression of optineurin was visualized by fluorescence microscopy. Abnormal optineurin expression influenced the survival of RGCs in vitro, as apoptosis was induced by increased WT and E50K mutant optineurin, while a reduction in apoptosis was observed in cells transfected with OPTN-siRNA. Similar results were also observed in transfected cells treated with apoptotic stimuli. Overexpression of WT and mutant E50K protein resulted in greater cell death, while downregulation decreased RGC-5 apoptosis.

Well-balanced commensal microbiota contributes to anti-cancer response in a lung cancer mouse model.

The intestinal microflora affects inflammation and immunity, not only locally at the mucosal level but also systemically, raising the question of whether the microflora affects inflammatory processes that contribute to cancer and its therapy. Prebiotics have also been found to play an antitumor role that is not limited to the gut. We investigated the antitumor roles of the intestinal microbiota using the Lewis lung cancer mouse model. In mice treated with cisplatin combined with ABX (an antibiotic cocktail of vancomycin, ampicillin, and neomycin), which can destroy the host commensal microflora, the tumor size was larger than in mice on a single treatment of cisplatin. Moreover, the survival rate of mice treated with cisplatin combined with ABX was significantly reduced. In contrast, mice treated with cisplatin combined with Lactobacillus bacteria had smaller tumors and an improved survival rate. Further study on gene expression indicated that ABX can partially impair the function of cisplatin by upregulating the expression of VEGFA and downregulating the expression of BAX and CDKN1B. The expression of IFN-γ, GZMB, and PRF1 in the CD8(+) T cells of these mice was reduced by ABX, indicating an immuno-enhancement role of commensal microbiota. Conversely, Lactobacillus co-treatment mice showed an enhanced antitumor response with upregulated IFN-γ, GZMB, and PRF1 expression. We conclude that the commensal microbiota contributes to the anti-lung cancer response and probiotics co-treatment can enhance the antigrowth and proapoptotic effects of cisplatin.

Effect of supplementation of green tea polyphenols on the developmental competence of bovine oocytes in vitro.

The objective of the present study was to examine the effect of green tea polyphenols (GTPs) supplementation during in vitro maturation, in vitro fertilization, and in vitro culture on the developmental competence of bovine oocytes. Cumulus-oocyte complexes aspirated from the ovaries were matured in vitro (38.5 degrees C for 24 h) and fertilized (38.5 degrees C for 15-18 h) and embryos were cultured (38.5 degrees C for 192 h) in a defined conditioned medium with or without GTPs supplementation. The GTPs used in the present study contained 99% catechin derivatives, with the major components being 50% (-)-epigallocatechin gallate, 22% (-)-epicatechin gallate, 18% (-)-epigallocatechin, and 10% (-)-epicatechin. Four replicate trials were done for each type of experiment. GTPs supplementation (15 microM) of the maturation medium led to a significant increase in the rate of blastocyst formation (34.0 vs 21.4%, P < 0.05). However, the rate of blastocyst formation was not improved when higher GTPs concentrations (20 or 25 microM) were added to the in vitro maturation medium. During in vitro fertilization, supplementation with higher GTPs concentrations (20 or 25 microM) significantly reduced the rate of blastocyst formation (P < 0.05). Supplementation of the culture medium with 15 microM GTPs improved the rate of blastocyst formation, while higher GTPs concentrations (25 microM) significantly reduced embryo development (P < 0.05). In conclusion, these results demonstrate that supplementation with GTPs at low concentration (15 microM) during in vitro maturation and in vitro culture improved the developmental competence of bovine oocytes.

Effect of supplementation of green tea polyphenols on the developmental competence of bovine oocytes in vitro

The objective of the present study was to examine the effect of green tea polyphenols (GTPs) supplementation during in vitro maturation, in vitro fertilization, and in vitro culture on the developmental competence of bovine oocytes. Cumulus-oocyte complexes aspirated from the ovaries were matured in vitro (38.5°C for 24 h) and fertilized (38.5°C for 15-18 h) and embryos were cultured (38.5°C for 192 h) in a defined conditioned medium with or without GTPs supplementation. The GTPs used in the present study contained 99 percent catechin derivatives, with the major components being 50 percent (-)-epigallocatechin gallate, 22 percent (-)-epicatechin gallate, 18 percent (-)-epigallocatechin, and 10 percent (-)-epicatechin. Four replicate trials were done for each type of experiment. GTPs supplementation (15 æM) of the maturation medium led to a significant increase in the rate of blastocyst formation (34.0 vs 21.4 percent, P < 0.05). However, the rate of blastocyst formation was not improved when higher GTPs concentrations (20 or 25 æM) were added to the in vitro maturation medium. During in vitro fertilization, supplementation with higher GTPs concentrations (20 or 25 æM) significantly reduced the rate of blastocyst formation (P < 0.05). Supplementation of the culture medium with 15 æM GTPs improved the rate of blastocyst formation, while higher GTPs concentrations (25 æM) significantly reduced embryo development (P < 0.05). In conclusion, these results demonstrate that supplementation with GTPs at low concentration (15 æM) during in vitro maturation and in vitro culture improved the developmental competence of bovine oocytes.