Mouse/Rat Galanin ELISA Kit

Wnt/beta-catenin signaling regulates critical, context-dependent transcription in numerous physiological events. Among the well-documented mechanisms affecting Wnt/beta-catenin activity, modification of N-glycans by L-fucose is the newest and the least understood. Using a combination of Chinese hamster ovary cell mutants with different fucosylation levels and cell-surface fucose editing (in situ fucosylation [IU]), we report that alpha(1-3)-fucosylation of N-acetylglucosamine (GlcNAc) in the Gal beta(1-4)-GlcNAc sequences of complex N-glycans modulates Wnt/beta-catenin activity by regulating the endocytosis of low-density lipoprotein receptor-related protein 6 (LRP6). Pulse-chase experiments reveal that ISF elevates endocytosis of lipid-raft-localized LRP6, leading to the suppression of Wnt/beta-catenin signaling. Remarkably, Wnt activity decreased by ISF is fully reversed by the exogenously added fucose. The combined data show that in situ cell-surface fucosylation can be exploited to regulate a specific signaling pathway via endocytosis promoted by a fucose-binding protein, thereby linking glycosylation of a receptor with its intracellular signaling.

Purpose We assessed whether mitomycin-C (MMC) has different antifibrotic mechanisms in trabeculectomy wound healing. Methods We identified 2 concentrations of MMC as "low-dose" by using WST-1 assay, Lactic dehydrogenase assay, and fluorescence-activated cell sorting flow cytometry. Senescence-associated beta-galactosidase (SA-beta-gal) and fibrotic gene expression was examined through immunocytochemistry, flow cytometry, real-time quantitative reverse transcription polymerase chain reaction, Western blotting, zymography, and modified scratch assay in vitro. In vivo, 0.1 mL of MMC or normal saline was injected to Tenon's capsule before trabeculectomy in a rabbit model. SA-beta-gal expression, apoptotic cell death, and collagen deposition in sites treated and not treated with MMC were evaluated using terminal dUTP nick end labeling assay and histochemical staining. Bleb function and intraocular pressure (IOP) levels were examined 3, 7, 14, 21, 28, and 35 days after trabeculectomy. Results In vitro, human Tenon's fibroblast (HTF) senescence was confirmed by observing cell morphologic change, SA-beta-gal accumulation, formation of senescence-associated heterochromatin, increased p16(INK4a)and p21(CIP1/WAF1)expression, lower percentage of Ki-67-positive cells, and decreased COL1A1 release. Increased expression of alpha-SMA,COL1A1, and Smad2 signaling in TGF-beta 1-induced stress fibers were passivated in senescent HTFs. In addition, cellular migration enhanced by TGF-beta 1was inactivated. In vivo, histological examination indicated increased SA-beta-gal accumulation, lower apoptosis ratios, and looser collagen deposition in sites treated with 0.2 mu M MMC. Low-dose MMC-induced cellular senescence prolonged trabeculectomy bleb survival and reduced IOP levels in a rabbit model. Conclusion Low-dose MMC-induced cellular senescence is involved in the antifibrotic mechanism of trabeculectomy wound healing.