Human MDDCs were FBS-starved for 24 hr, to set the basal state of the GTPase activation, and then treated under various conditions. Total RNA was extracted using the RNeasy Mini Kit and the RNase-Free DNase Set to eliminate genomic DNA, all from Qiagen (Manchester, UK). The supernatants were removed and used for total sialic acid quantification. In this study we focus on the expression and function of sialic acids in pulmonary endothelium. The relative mRNA levels were normalized against the arithmetic mean of the β-actin and GAPDH expression and calculated by the adapted formula 2−ΔCt × 1000, which infers the number of mRNA molecules of the gene of interest per 1000 molecules of the endogenous controls.31 ΔCt represents the difference between the cycle threshold of the target gene and that of the endogenous control genes. The term “operably linked” refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence. Expression of cytokine genes was analysed by real-time PCR. After the phagocytosis assay, MDDCs were co-cultured with T lymphocytes at a DC : T-lymphocyte ratio of 1 : 4. T-lymphocyte stimulation was assessed by determining interferon-γ (IFN-γ) gene expression after 48 hr of co-culture.
Human MDDCs were adhered to cover-slip glasses, fixed and then permeabilized, blocked with 3% BSA for 15 min and then stained with rabbit anti-nuclear factor-κB (NF-κB) p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1 : 100, for 1 hr at room temperature. In some experiments, phagocytosis was conducted with human MDDCs incubated with 50 μg/ml of either SNA or MAA lectins, or, alternatively, in the presence of 10 μm cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-5-NeuAc) (Sigma). “heterologous gene”, as used herein, is one that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form. It will be understood that heterologous genes that can be introduced may originates from different sources, such as E. coli, Neisseria, Campylobacter species, as well as mammals or yeasts. Methods of transforming prokaryotes other than E. coli are well known. In the context of the two glycoproteins recognized in the array, AGP and apo-transferrin, the terminal α2,3/α2,6 trisaccharide motif is linked to several other sugars before they are N-linked to the protein, and thus the length of the chain is not likely to be limiting for binding.
A: schematic of the endothelial glycocalyx composed of glycoproteins (dark blue), glycolipids (green-gray), proteoglycans (dark gray), and glycosaminoglycans (light gray) covering the endothelial plasma membrane. B: schematic of an N-linked, triantennary oligosaccharide found on mature glycoproteins. The surface of vascular endothelium bears a glycocalyx comprised, in part, of a complex mixture of oligosaccharide chains attached to cell-surface proteins and membrane lipids. The endothelial glycocalyx consists of glycoproteins, glycolipids, proteoglycans and glycosaminoglycans, which coat the cell surface (18, 23) (Figure 1A). The carbohydrate network that contributes to the glycocalyx is very complex, and, to date, the structure of these complex carbohydrates and their role in endothelial barrier function are poorly understood. Importantly, understanding of the structure and function of the endothelial glycocalyx is poorly understood. Molecular structure of sialic acids. Following treatment of PAECs with neuraminidase from Vibrio cholerae-positive lectin, staining was still observed, indicating that not all α(2,3)-linked sialic acids were hydrolyzed (Fig. 5E). We observed the same pattern of lectin staining when the cells were treated with neuraminidase from Clostridium perfringens.
