Subcellular distribution of terminal α-D- and β-D-galactosyl residues in Ehrlich tumour cells studied by lectin-gold techniques

We have studied by high resolution in situ light and electron microscopic lectin-gold techniques the subcellular distribution of α- d -Gal residues using the Griffonia simplicifolia I-B 4 isolectin and compared it with that of β- d -Gal residues as detected with the Datura stramonium lectin in Ehrlich tumour cells grown as ascites or monolayer. The microvillar but not the smooth plasma membrane regions were labelled with the Griffonia simplicifolia I-B 4 isolectin whereas both plasma membrane regions were equally well labelled with the Datura stramonium lectin. Elements of the endocytotic/lysosomal system such as coated membrane invaginations and vesicles, early and late endosomes and secondary lysosomes were positive for both α- d -Gal and β- d -Gal residues. A particular feature of Ehrlich tumour cells is an elaborate tubular membrane system located in the pericentriolar region which is labelled throughout by both lectins and represents part of the endosomal system. In the Golgi apparatus labelling with both lectins was observed to commence in trans cisternae which is indirect evidence for a joint distribution of the sequentially acting β1,4 and α1,3-galactosyl-transferases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45677/1/10719_2004_Article_BF00731358.pd

The Infection of Chicken Tracheal Epithelial Cells with a H6N1 Avian Influenza Virus

Sialic acids (SAs) linked to galactose (Gal) in α2,3- and α2,6-configurations are the receptors for avian and human influenza viruses, respectively. We demonstrate that chicken tracheal ciliated cells express α2,3-linked SA, while goblet cells mainly express α2,6-linked SA. In addition, the plant lectin MAL-II, but not MAA/MAL-I, is bound to the surface of goblet cells, suggesting that SA2,3-linked oligosaccharides with Galβ1–3GalNAc subterminal residues are specifically present on the goblet cells. Moreover, both α2,3- and α2,6-linked SAs are detected on single tracheal basal cells. At a low multiplicity of infection (MOI) avian influenza virus H6N1 is exclusively detected in the ciliated cells, suggesting that the ciliated cell is the major target cell of the H6N1 virus. At a MOI of 1, ciliated, goblet and basal cells are all permissive to the AIV infection. This result clearly elucidates the receptor distribution for the avian influenza virus among chicken tracheal epithelial cells and illustrates a primary cell model for evaluating the cell tropisms of respiratory viruses in poultry

Interaction and uptake of exosomes by ovarian cancer cells

<p>Abstract</p> <p>Background</p> <p>Exosomes consist of membrane vesicles that are secreted by several cell types, including tumors and have been found in biological fluids. Exosomes interact with other cells and may serve as vehicles for the transfer of protein and RNA among cells.</p> <p>Methods</p> <p>SKOV3 exosomes were labelled with carboxyfluoresceine diacetate succinimidyl-ester and collected by ultracentrifugation. Uptake of these vesicles, under different conditions, by the same cells from where they originated was monitored by immunofluorescence microscopy and flow cytometry analysis. Lectin analysis was performed to investigate the glycosylation properties of proteins from exosomes and cellular extracts.</p> <p>Results</p> <p>In this work, the ovarian carcinoma SKOV3 cell line has been shown to internalize exosomes from the same cells via several endocytic pathways that were strongly inhibited at 4°C, indicating their energy dependence. Partial colocalization with the endosome marker EEA1 and inhibition by chlorpromazine suggested the involvement of clathrin-dependent endocytosis. Furthermore, uptake inhibition in the presence of 5-ethyl-N-isopropyl amiloride, cytochalasin D and methyl-beta-cyclodextrin suggested the involvement of additional endocytic pathways. The uptake required proteins from the exosomes and from the cells since it was inhibited after proteinase K treatments. The exosomes were found to be enriched in specific mannose- and sialic acid-containing glycoproteins. Sialic acid removal caused a small but non-significant increase in uptake. Furthermore, the monosaccharides D-galactose, α-L-fucose, α-D-mannose, D-N-acetylglucosamine and the disaccharide β-lactose reduced exosomes uptake to a comparable extent as the control D-glucose.</p> <p>Conclusions</p> <p>In conclusion, exosomes are internalized by ovarian tumor cells via various endocytic pathways and proteins from exosomes and cells are required for uptake. On the other hand, exosomes are enriched in specific glycoproteins that may constitute exosome markers. This work contributes to the knowledge about the properties and dynamics of exosomes in cancer.</p

Avian Influenza Viruses Infect Primary Human Bronchial Epithelial Cells Unconstrained by Sialic Acid α2,3 Residues

Avian influenza viruses (AIV) are an important emerging threat to public health. It is thought that sialic acid (sia) receptors are barriers in cross-species transmission where the binding preferences of AIV and human influenza viruses are sias α2,3 versus α2,6, respectively. In this study, we show that a normal fully differentiated, primary human bronchial epithelial cell model is readily infected by low pathogenic H5N1, H5N2 and H5N3 AIV, which primarily bind to sia α2,3 moieties, and replicate in these cells independent of specific sias on the cell surface. NHBE cells treated with neuraminidase prior to infection are infected by AIV despite removal of sia α2,3 moieties. Following AIV infection, higher levels of IP-10 and RANTES are secreted compared to human influenza virus infection, indicating differential chemokine expression patterns, a feature that may contribute to differences in disease pathogenesis between avian and human influenza virus infections in humans

Correlation Index-Based Responsible-Enzyme Gene Screening (CIRES), a Novel DNA Microarray-Based Method for Enzyme Gene Involved in Glycan Biosynthesis

BACKGROUND: Glycan biosynthesis occurs though a multi-step process that requires a variety of enzymes ranging from glycosyltransferases to those involved in cytosolic sugar metabolism. In many cases, glycan biosynthesis follows a glycan-specific, linear pathway. As glycosyltransferases are generally regulated at the level of transcription, assessing the overall transcriptional profile for glycan biosynthesis genes seems warranted. However, a systematic approach for assessing the correlation between glycan expression and glycan-related gene expression has not been reported previously. METHODOLOGY: To facilitate genetic analysis of glycan biosynthesis, we sought to correlate the expression of genes involved in cell-surface glycan formation with the expression of the glycans, as detected by glycan-recognizing probes. We performed cross-sample comparisons of gene expression profiles using a newly developed, glycan-focused cDNA microarray. Cell-surface glycan expression profiles were obtained using flow cytometry of cells stained with plant lectins. Pearson's correlation coefficients were calculated for these profiles and were used to identify enzyme genes correlated with glycan biosynthesis. CONCLUSIONS: This method, designated correlation index-based responsible-enzyme gene screening (CIRES), successfully identified genes already known to be involved in the biosynthesis of certain glycans. Our evaluation of CIRES indicates that it is useful for identifying genes involved in the biosynthesis of glycan chains that can be probed with lectins using flow cytometry