Various kinds of glycosaminoglycans (GAGs) and proteoglycans (PGs) have been known to be involved in structural and space-filling functions, as well as many physiological regulations in skin. To investigate ultraviolet (UV) radiation-mediated regulation of GAGs and PGs in cultured human dermal fibroblasts, transcriptional changes of many types of PGs and GAG chain-synthesizing enzymes at 18 hr after 75 mJ/cm2 of UV irradiation were examined using quantitative real-time polymerase chain reaction methods. Hyaluronic acid synthase (HAS)-1, -2, and -3 and hyaluronidase-2 mRNA expressions were significantly increased by UV irradiation. Expressions of lumican, fibromodulin, osteoglycin, syndecan-2, perlecan, agrin, versican, decorin, and biglycan were significantly decreased by UV irradiation, while syndecan-1 was increased. Expressions of GAG chain-synthesizing glycosyltransferases, xylosyltransferase-1, β1,3-glucuronyltransferase-1, β1,4-galactosyltransferase-2, -4, exostosin-1, chondroitin polymerizing factor, and chondroitin sulfate synthase-3 were significantly reduced, whereas those of β1,3-galactosyltransferase-6, β1,4-galactosyltransferase-3, -7, β-1,3-N-acetylglucosaminyltran sferase-2, and -7 were increased by UV irradiation. Heparanase-1 mRNA expression was increased, but that of heparanase-2 was reduced by UV irradiation. Time-course investigation of representative genes showed consistent results. In conclusion, UV irradiation may increase hyaluronic acid production through HAS induction, and decrease other GAG productions through downregulation of PG core proteins and GAG chain-synthesizing glycosyltransferases in cultured human dermal fibroblasts.
Macrophages can associate with extracellular matrix (ECM) demonstrating nanosequenced cell-adhesive RGD ligand. In this study, we devised barcoded materials composed of RGD-coated gold and RGD-absent iron nanopatches to show various frequencies and position of RGD-coated nanopatches with similar areas of iron and RGD-gold nanopatches that maintain macroscale and nanoscale RGD density invariant. Iron patches were used for substrate coupling. Both large (low frequency) and externally positioned RGD-coated nanopatches stimulated robust attachment in macrophages, compared with small (high frequency) and internally positioned RGD-coated nanopatches, respectively, which mediate their regenerative/anti-inflammatory M2 polarization. The nanobarcodes exhibited stability in vivo. We shed light into designing ligand-engineered nanostructures in an external position to facilitate host cell attachment, thereby eliciting regenerative host responses.
Nanoparticle technology has been a growing field in biomedical research. This is in part due to potential applications in drug delivery, biosensing, diagnostics, and imaging. Our long-term goal is to use protein functionalized AuNPs as a general tool for molecular sensing and drug delivery. The ability to use nanomaterials as biosensors and drug delivery methods in cellular uptake is directly dependent on the amount of protein that is able to bind to the surface of any given nanoparticle. It is hypothesized that electrostatic interactions play a significant role in protein-AuNP interactions, since citrate-stabilized AuNPs carry a net negative charge. Our group has developed an NMR-based approach to rapidly quantify bound protein to AuNP. To understand the above phenomenon, GB3 was chosen as our model protein and it contains seven lysine residues. These positively-charged lysine residues are involved in protein-AuNP binding, and a potential binding site was identified using APBS calculations which contain lysine residues. This hypothesis was tested by mutating the lysine residues to alanine one at a time using site-directed mutagenesis. NMR experiments were carried out to observe how the binding capacities of each of these variants change relative to wild type GB3. Notably K4A, K13A and K50A variants has significantly reduced binding, while the binding capacity of other lysine to alanine variants was on par with wild-type GB3. To obtain better understanding, GB3 variants were competed with wild type GB3 in the same solution with AuNP to observe how the binding capacities vary with wild type GB3. As predicted, the binding capacity ratio was lower for lysine residues in the proposed binding site were changed to alanine. A reduced binding capacity ratio was not observed for other lysine variants. The results reported are significant in establishing our original hypothesis, and suggest that GB3 adopts a specific orientation on the AuNP surface.
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