Glycoconjugate expression in follicle-associated epithelium (FAE) covering the nasal-associated lymphoid tissue (NALT) in specific pathogen-free and conventional rats.
We examined lectin-histochemically the glycoconjugate expression in the follicle-associated epithelium (FAE) covering the nasal-associated lymphoid tissue (NALT) in the rat under specific pathogen-free (SPF) and conventional (CV) conditions and compared the results for SPF and CV rats as well as for membranous (M) cells and adjacent ciliated respiratory epithelial (CRE) cells in FAE. N-acetylgalactosamine-specific lectins, Dolichos biflorus (DBA), Helix pomatia (HPA), Glycine max (SBA) and Vicia villosa (VVA), and alpha-L-fucose-specific lectin, Ulex europaeus (UEA-I), preferentially bound to M cells mainly in the luminal surface compared with CRE cells in SPF rats, whereas DBA and UEA-I showed signs of preferential binding to the apical and basolateral cytoplasm as well as to the luminal surface of M cells in CV rats. In addition, HPA, SBA and VVA more frequently and extensively labeled M cells than CRE cells in CV rats with the same subcellular staining pattern as DBA and UEA-I. On the whole, the changes in lectin binding frequency and strength were more prominent in M cells than in CRE cells in both SPF and CV rats. The present results indicate that DBA and UEA-I are useful as markers of M cells in NALT. Furthermore, the pattern of expression of carbohydrate residues recognized by such lectins in SPF and CV rats suggests that M cells are highly sensitive to environmental changes. (+info)
Characterization of mannooligosaccharide caps in mycobacterial lipoarabinomannan by capillary electrophoresis/electrospray mass spectrometry.
A new analytical approach based on capillary electrophoresis-electrospray mass spectrometry (CE/ESI-MS) has provided new insight into the characterization of mannooligosaccharide caps from lipoarabinomannans (LAMs), which are key molecules in the immunopathogenesis of tuberculosis. This analytical approach requires oligosaccharide labeling with the fluorophore 1-aminopyrene-3,6,8-trisulfonate (APTS) by reductive amination at the reducing termini. Optimization of the separation and ionization conditions, such as the choice of capillary electrophoresis (CE) electrolyte buffers, is presented and discussed. Anionic separation of the mono and oligosaccharide APTS derivatives was finally achieved with aqueous triethylammonium formate buffer. It was found that in contrast to the triethylammonium phosphate buffer, the triethylammonium formate buffer was appropriate for CE/ESI-MS coupling analysis of APTS-carbohydrate derivatives. In this case, negative ESI-mass spectra of APTS-carbohydrate adducts showed mainly (M-2H)2-pseudomolecular ions and some sequence fragment ions allowing their non-ambiguous structural characterization at the picomolar level. This analytical approach was successfully applied to more complex mixtures of carbohydrates released by mild acid hydrolysis of the lipoarabinomannans from Mycobacterium bovis BCG. The APTS-mannooligosaccharide cap adducts were separated by CE and their structural characterization achieved by CE/ESI-MS analyses. Mannooligosaccharide caps were routinely analyzed by capillary electrophoresis-laser induced fluorescence (CE-LIF) from 50 fmol of lipoarabinomannans with mannosyl capping (ManLAMs) but sensitivity was about 50 times lower using ESI-MS detection. (+info)
Altered expression profile of the surface glycopeptidolipids in drug-resistant clinical isolates of Mycobacterium avium complex.
Members of the Mycobacterium avium complex are the most frequently encountered opportunistic bacterial pathogens among patients in the advanced stage of AIDS. Two clinical isolates of the same strain, numbers 397 and 417, were obtained from an AIDS patient with disseminated M. avium complex infection before and after treatment with a regimen of clarithromycin and ethambutol. To identify the biochemical consequence of drug treatment, the expression and chemical composition of their major cell wall constituents, the arabinogalactan, lipoarabinomannan, and the surface glycopeptidolipids (GPL), were critically examined. Through thin layer chromatography, mass spectrometry, and chemical analysis, it was found that the GPL expression profiles differ significantly in that several apolar GPLs were overexpressed in the clinically resistant 417 isolate at the expense of the serotype 1 polar GPL, which was the single predominant band in the ethambutol-susceptible 397 isolate. Thus, instead of additional rhamnosylation on the 6-deoxytalose (6-dTal) appendage to give the serotype 1-specific disaccharide hapten, the accumulation of this nonextended apolar GPL probably provided more precursor substrate available for further nonsaccharide substitutions including a higher degree of O-methylation to give 3-O-Me-6-dTal and the unusual 4-O-sulfation on 6-dTal. Further data showed that this alteration effectively neutralized ethambutol, which is known to inhibit arabinan synthesis. Thus, in contrast with derived Emb-resistant mutants of Mycobacterium smegmatis or Mycobacterium tuberculosis, which are devoid of a surface GPL layer, the lipoarabinomannan from resistant 417 isolate grown in the presence of this drug was not apparently truncated. (+info)
Identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza C virus and bovine coronavirus.
We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substrates p-nitrophenyl acetate, alpha-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse alpha1 macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested. (+info)
Helicobacter pylori attaches to NeuAc alpha 2,3Gal beta 1,4 glycoconjugates produced in the stomach of transgenic mice lacking parietal cells.
Helicobacter pylori infection of the human stomach is associated with altered acid secretion, loss of acid-producing parietal cells, and, in some hosts, adenocarcinoma. We have used a transgenic mouse model to study the effects of parietal cell ablation on H. pylori pathogenesis. Ablation results in amplification of the presumptive gastric epithelial stem cell and its immediate committed daughters. The amplified cells produce sialylated oncofetal carbohydrate antigens that function as receptors for H. pylori adhesins. Attachment results in enhanced cellular and humoral immune responses. NeuAc alpha 2,3Gal beta 1,4 glycoconjugates may not only facilitate persistent H. pylori infection in a changing gastric ecosystem, but by promoting interactions with lineage progenitors and/or initiated cells contribute to tumorigenesis in patients with chronic atrophic gastritis. (+info)
IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo.
Goblet cell metaplasia and mucus hypersecretion are important features in the pathogenesis of asthma. The cytokine IL-4 has been shown to play a role in animal models of asthma, where it induces Th2 lymphocyte differentiation and B lymphocyte IgE class switch. IL-4 has also been implicated in the differentiation of goblet cells via effects on lymphocytes and eosinophils. In this study we hypothesized that IL-4 induces airway epithelial cell mucin gene expression and mucous glycoconjugate production by direct action on these cells. In vitro, cultured airway epithelial cells (NCI-H292) expressed IL-4R constitutively, and IL-4 (10 ng/ml) induced MUC2 gene expression and mucous glycoconjugate production. In vivo, mouse airway epithelial cells expressed IL-4R constitutively, and IL-4 (250 ng) increased MUC5 gene expression and Alcian blue/periodic acid-Schiff-positive staining at 24 h; IL-4 did not increase inflammatory cell numbers in airway tissue or in bronchoalveolar lavage. TNF-alpha and IL-1beta levels in bronchoalveolar lavage were not increased in response to IL-4 instillation. These results indicate that airway epithelial cells express IL-4R constitutively and that IL-4 directly induces the differentiation of epithelium into mucous glycoconjugate-containing goblet cells. (+info)
Up-regulation of the alpha2,6-sialyltransferase messenger ribonucleic acid increases glycoconjugates containing alpha2, 6-linked sialic acid residues in granulosa cells during follicular atresia of porcine ovaries.
The sugar chains in cellular glycoconjugates have many biological functions. Extensive morphological development and remodeling occur in the ovary of female animals. This caused us to study glycobiological characteristics of ovarian cells, particularly granulosa cells that undergo apoptosis during follicular atresia. The lectin Sambucus sieboldiana agglutinin (SSA) specific for Siaalpha2,6Gal/GalNAc showed positive staining for granulosa cells only in atretic follicles of porcine ovaries by lectin histochemistry. Lectin blot analysis for SSA demonstrated specific glycoproteins only in atretic follicles. Furthermore, we performed analysis of backbone structures of SSA-positive glycans carried by granulosa cell glycoproteins increased during atresia by glycosidase treatment. Most of these structures were Siaalpha2,6Galbeta1,4GlcNAc on complex-type N-glycans, suggesting that only ST6Gal I of four distinct alpha2,6-sialyltransferases catalyzes alpha2,6-sialic acid transfer in most of the increased glycoproteins of granulosa cells during follicular atresia. Reverse transcription-polymerase chain reaction analysis demonstrated that the expression of ST6Gal I mRNA was up-regulated in granulosa cells during atresia. These results suggested that the alteration of glycoconjugates by ST6Gal I in granulosa cells during atresia is involved in some processes of ovarian follicular atresia and granulosa cell apoptosis. (+info)
UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation.
Modification of cell surface molecules with sialic acid is crucial for their function in many biological processes, including cell adhesion and signal transduction. Uridine diphosphate-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) is an enzyme that catalyzes an early, rate-limiting step in the sialic acid biosynthetic pathway. UDP-GlcNAc 2-epimerase was found to be a major determinant of cell surface sialylation in human hematopoietic cell lines and a critical regulator of the function of specific cell surface adhesion molecules. (+info)