Subcompartment sugar residues of gastric surface mucous cells studied with labeled lectins

Summary We examined the intracellular localization of sugar residues of the rat gastric surface mucous cells in relation to the functional polarity of the cell organellae using preembedding method with several lectins.In the surface mucous cells, the nuclear envelope and rough endoplasmic reticulum (rER) and cis cisternae of the Golgi stacks were intensely stained with Maclura pomifera (MPA), which is specific to -Gal and GalNAc residues. In the Golgi apparatus, one or two cis side cisternae were stained with MPA and Dolichos biflorus (DBA) which is specific to terminal -N-acetylgalactosamine residues, while the intermediate lamellae were intensely labeled with Arachis hypogaea (PNA) which is specific to Gal 1,3 GalNAc. Cisternae of the trans Golgi region were also stained with MPA, Ricinus communis I (RCA I) which is specific to -Gal and Limax flavus (LFA) which is specific to -NeuAc. Immature mucous granules which are contiguous with the trans Golgi lamellae were weakly stained with RCA I, while LFA stained both immature and mature granules.The differences between each lectin's reactivity in the rough endoplasmic reticulum, in each compartment of the Golgi lamellae and in the secretory granules suggest that there are compositional and structural differences between the glycoconjugates in the respective cell organellae, reflecting the various processes of glycosylation in the gastric surface mucous cells.     

Post-embedding staining of rat gastric mucous cells with lectins

Summary The mucous cells of the rat stomach were stained with lectins by two post-embedding staining methods for electron microscopy. The mucous granules of surface mucous cells and foveolar mucous cells were stained weakly by Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of mucous neck cells were stained by concanavalin A-ferritin, Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of pyloric gland cells showed an affinity for wheat germ agglutinin-ferritin and concanavalin A-ferritin, while Ricinuscommunis agglutinin-ferritin only slightly stained the granules. The granules of mucous neck cells and pyloric gland cells were also stained by the concanavalin A-horseradish peroxidase-colloidal gold method, but the granules of surface and foveolar mucous cells were not stained by this method. Periodic acid oxidation of the sections before the standard concanavalin A-ferritin procedure enhanced the staining of the granules of mucous neck cells and pyloric gland cells slightly. Reduction of the sections after the periodic acid oxidation weakened the staining. Similar results were obtained using the concanavalin A-horseradish peroxidase-colloidal gold method. Though the staining with Ricinus communis agglutinin-ferritin was inhibited by periodic acid oxidation of the sections before staining, the staining with wheat germ agglutinin-ferritin was not inhibited by the oxidation. It is suggested that the paradoxical staining is closely related to the position of the concanavalin A-binding sugar residues in the carbohydrate chains.This work was supported in part by a grant-in-aid (No. 457008) from the Ministry of Education, Science and Culture, Japan and a grant-in-aid for cancer research (55-21) from the Ministry of Health and Welfare, Japan     

Ultrastructural localization of cell surface sugar residues in ericoid mycorrhizal fungi by gold-labeled lectins

Summary Surface sugar residues were ultrastructurally localized in two strains ofHymenoscyphus ericae, one having a strong tendency to form ericoid mycorrhiza, the other, very little. The strains were studied both in the presence and absence of the host plant. Wheat germ agglutinin (WGA) and Concanavalin A (Con A)-colloidal gold complexes were used as cytochemical markers.N-acetylglucosamine residues were localized exclusively on septa and on the inner electron-transparent layer of longitudinal walls, confirming the presence of chitin in well defined regions of the fungal cell wall, both in the infective and in the noninfective strain.Con A-binding sites were detected on extracellular material commonly radiating from the wall of the infective strain. They were particularly abundant when the infective strain was in contact with the host, but were uncommon on the surface of the noninfective strain, whether this was in contact with the host or not.The extracellular material presumed to contain glucose and mannose residues appears to be important in establishing contact between fungus and host.     

Ammonia-induced apoptosis is accelerated at higher pH in gastric surface mucous cells

Gastric luminal ammonia produced by Helicobacter pylori has been shown to cause gastric mucosal injury. This study was conducted to examine the mechanisms by which gastric luminal ammonia causes apoptosis of gastric epithelial cells. Monolayers of GSM06 cells, developed from murine gastric surface mucous cells, were cultured in the absence or presence of 10-30 mM NH(4)Cl at ambient pH of 5.0, 6.0, and 7.0. In the presence of luminal NH(4)Cl, GSM06 cells showed 1) cell shrinkage and nuclear chromatin condensation, 2) DNA fragmentation into oligonucleosomes, 3) leakage of cytochrome c into cytosolic fraction without affecting bax expression, and 4) increases in activity of caspases-3 and -9. These changes were accentuated when the cells were cultured at pH 7.0. In the absence of NH(4)Cl, none of these changes was detected at any pH examined. These results suggest that gastric luminal ammonia, at concentrations detected in H. pylori-infected subjects, induces apoptosis of gastric epithelial cells by release of cytochrome c from mitochondria, followed by activation of caspases-9 and -3, especially at higher ambient pH.     

Human sperm surface mapping with lectins.

Ten fluorescein isothiocyanate (FITC)-linked lectins [Bauhimia purpurea, Concanavalin A, Dolichos biflorus (DBA), Griffonia simplicifolia I, Griffonia simplicifolia II, Maclura pomifera, Arachis hypogea (PNA), Glycine max, Ulex europaeus (UEA) and Triticum vulgaris agglutinin] have been used to study their binding features on the human ejaculate spermatozoa. Qualitative changes in the labeling pattern have been observed in unfixed and acetone-treated spermatozoa. Furthermore, ultrastructural localization of some of the colloidal gold-linked lectins, namely PNA, UEA and DBA, has been attempted to delineate the binding domains of the specific sugars on the sperm surface. It needs to be emphasized that flow-cytometric methods employed in our study, which provide quantitative slant to qualitative data, should be utilized to evaluate the functional status of the spermatozoa.     

Leguminous lectins as tools for studying the role of sugar residues in leukocyte recruitment

The natural physiological ligands for selectins are oligosaccharides found in glycoprotein or glycolipid molecules in cell membranes. In order to study the role of sugar residues in the in vivo lectin anti-inflammatory effect, we tested three leguminous lectins with different carbohydrate binding affinities in the peritonitis and paw oedema models induced by carrageenin in rats. L. sericeus lectin was more anti-inflammatory than D. virgata lectin, the effects being reversed by their specific binding sugars (N-acetylglucosamine and alpha-methylmannoside, respectively). However, V. macrocarpa, a galactose-specific lectin, was not anti-inflammatory. The proposed anti-inflammatory activity of lectins could be due to a blockage of neutrophil-selectin carbohydrate ligands. Thus, according to the present data, we suggest an important role for N-acetylglucosamine residue as the major ligand for selectins on rat neutrophil membranes.     

Co-localization of TFF2 with gland mucous cell mucin in gastric mucous cells and in extracellular mucous gel adherent to normal and damaged gastric mucosa

Trefoil factor 2 (TFF2) is mucin associated peptide that has a mucosal barrier function in addition to participating in repair and healing. We examined the localization of TFF2 and gastric mucins in gastric mucous cells, the surface mucous gel layer (SMGL) adherent to normal gastric mucosa, and in the mucoid cap covering gastric erosions. Carnoy’s solution, or formalin/picric acid-fixed paraffin embedded materials from resected stomachs and formalin-fixed paraffin embedded gastric biopsy materials were used. Sections were immunostained for the TFF2 and histochemically stained for gastric mucins. In addition, thick sectioned gastric mucosa fixed in Carnoy’s solution were stained with FITC-labeled GSA-II lectin specific for gland mucous cell mucin and examined for three-dimensional images of the SMGL using a confocal laser scanning microscope. The TFF2 and gland mucous cell mucin were found intermixed together in the gastric gland mucous cells, in the SMGL in laminated layers, and in the mucoid cap. A laminated arrangement of continuous sheets of gland mucous cell mucin in the SMGL was demonstrated in the three-dimensional images. Co-localization of the TFF2 with gland mucous cell mucin suggests a physical interaction between the TFF2 and gland mucous cell mucin. The TFF2 trapped in the adherent mucins may be responsible for mucosal defense, healing, and repair.     

Coexistence of gland mucous cell-type mucin and lysozyme in gastric gland mucous cells

Class III mucin, identified by paradoxical concanavalin A staining, is confined to gastric gland mucous cells and is an essential component of the gastric surface mucous gel layer. The pretreatment required has hampered the application of this method to electron microscopic studies. Antibody HIK1083 reacts selectively with class III mucins. The present study was undertaken to explore, electron microscopically, the immunoreactivity of the human stomach to HIK1083. We examined normal mucosa from resected human stomachs (five cases; formalin-fixed, paraffin-embedded) and gastric biopsy specimens from patients with early gastric cancer [nine cases; glutaraldehyde- and osmium-fixed, epoxy-embedded (seven cases) and half-strength Karnovsky’s solution-fixed, Lowicryl K4M-embedded (two cases)]. Immunostaining with HIK1083 and anti-lysozyme antibody was examined under light and electron microscopes. Gland mucous cells were labeled with HIK1083, and lysozyme was detected in some gland mucous cells and surface mucous cells. Electron microscopically, the secretory granules of gland mucous cells contained a single electron-dense core. HIK1083-positive mucins and lysozyme coexisted in the secretory granules of gastric gland mucous cells. HIK1083-reactive mucins and lysozyme were distributed in the matrix and in the dense core of these secretory granules, respectively. HIK1083 can be used for electron immunohistochemistry. Accepted: 1 December 1999     

Chromatographic measurement of mucin production in cultures of gastric mucous cells.

The use of a miniature column chromatographic assay (using Sepharose CL-4B columns) for measuring mucin production in guinea pig gastric mucous cell cultures is described. The assay was based upon the ability of radiolabelled precursors ([14C]serine and [3H]galactose) to incorporate with high specificity into mucins which thereby appeared in the excluded material. Rates of excluded material radiolabelling by both precursors were constant for incubations up to 24 hours, and substantially reduced by cycloheximide co-incubation (25 microM). Labelled excluded material was completely degraded by mild alkaline borohydride treatment, only partially degraded by HNO2 (pH 1.5), and not degraded by chondroitinase ABC. Thus the major radiolabelled product measured in this system was mucin, although we found that it was less glycosylated than gastric mucins obtained from other sources. In addition, the technique employed to separate and measure mucin production proved rapid and consistent.     

Cell surface charge and sugar residues of Crithidia fasciculata and Crithidia luciliae.

The surface charge of Crithidia fasciculata and Crithidia luciliae was analysed by measurement of the zeta-potential and labelling of the protozoan surface with cationized ferritin particles. Both trypanosomatids have a net negative surface charge, with a zeta-potential of -10.39 mV and -11.12 mV for C. luciliae and C. fasciculata, respectively. Enzyme treatment showed that phosphate groups, but not sialic acid, significantly contributed to the negative surface charge. Lectin-induced agglutination was used to analyse the presence of surface-exposed carbohydrates in C. fasciculata and C. luciliae. The cells did not agglutinate when incubated in the presence of lectins which recognized L-fucose, N-acetyl-D-glucosamine and sialic acid. However, lectins which bind to N-acetyl-D-galactosamine, D-galactose and D-mannose agglutinated both protozoa.     

Characterization of surface sugars on algal cells with fluorescein isothiocyanate-conjugated lectins

Summary. We used qualitative and quantitative fluorescence microscopy of the fluorescein isothiocyanate-conjugated lectins Concanavalin A, phytohaemagglutinin-erythroagglutinin, pokeweed mitogen, and peanut agglutinin to examine sugar composition on the cell surface and cell-associated mucilage (where present) in a number of cultured and environmental algae. Lectin-binding activity was markedly different between laboratory-cultured and environmental samples of the same species. Sugar composition of the cyanobacterium Anabaena cylindrica varied with growth cycle, although no clear pattern of change was observed. Akinetes typically showed lectin-binding activity higher than that of the vegetative cells or heterocysts throughout the growth cycle. Algae with mucilage showed greater lectin binding, indicating that mucilage contained more surface sugars accessible to the lectin probe compared with the cell wall surface. A low level of galactose and N-acetyl galactosamine (detected by peanut agglutinin) was associated with the surface mucilage of most algal species. Relatively high amounts of mannose, glucose, and N-acetyl glucosamine (detected by Concanavalin A, phytohaemagglutinin, and pokeweed mitogen) were also present. Lectin binding was shown to be a highly specific and sensitive approach to the examination of cell surface chemistry of both cultured and environmental algae and to the study of biodiversity in phytoplankton. Correspondence: School of Biological Sciences, University of Manchester, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, U.K.     

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.     

An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture

The gastric surface epithelium is situated at an air-liquid interface because the luminal surface of the alimentary tract is in continuity with the air phase. However, the effects of this microenvironment on the gastric epithelium remain unclear. The aim of this study was to clarify the effects of an air-liquid interface on gastric epithelial cell biology. Gastric surface mucous cells (GSM06) were cultured at an air-liquid interface. Cultured cells were examined by histology, histochemistry, and transmission electron microscopy. When the cells were cultured at an air-liquid interface, the surface cells on the collagen gel became tall columnar and secreted periodic acid-Shiff-positive substances at the apical surface. These cells indicated many mucous granules in the apical cytoplasm and organized the basal lamina at the contact side with the gel. In contrast, under immersed condition, the surface cells showed immature features. This is the first report of an air-liquid interface promoting the differentiation of gastric surface mucous cells in a reconstruction culture of the gastric surface epithelial layer, suggesting that an air-liquid interface may function as a crucial luminal factor to maintain the homeostasis of gastric mucosa.     

Identification of an apical Cl-/HCO3- exchanger in gastric surface mucous and duodenal villus cells

The molecular identity of the apical HCO3(-)-secreting transporter in gastric mucous cells remains unknown despite its essential role in preventing injury and ulcer by gastric acid. Here we report the identification of a Cl-/HCO3- exchanger that is located on apical membranes of gastric surface epithelial cells. RT-PCR studies of mouse gastrointestinal tract mRNAs demonstrated that this transporter, known as anion exchanger isoform 4 (AE4), is expressed in both stomach and duodenum. Northern blot analysis of RNA from purified stomach epithelial cells indicated that AE4 is expressed at higher levels in mucous cells than in parietal cells. Immunoblotting experiments identified AE4 as a approximately 110- to 120-kDa protein in membranes from stomach epithelium and apical membranes from duodenum. Immunocytochemical staining demonstrated that AE4 is expressed in apical membranes of surface cells in both mouse and rabbit stomach and duodenum. Functional studies in oocytes indicated that AE4 functions as a Cl-/HCO3- exchanger. These data show that AE4 is an apical Cl-/HCO3- exchanger in gastric mucous cells and duodenal villus cells. On the basis of its function and location, we propose that AE4 may play an important role in mucosal protection.     

Bioconversion of sugar cane crop residues with white-rot fungiPleurotus sp.

Four mushroom strains ofPleurotus spp. were cultivated on sugar cane crop residues for 30 days at 26°C. Biochemical changes affected the substrate as a result of fungal growth, in terms of nitrogen, lignin, cellulose and hemicellulose contents. All strains showed a strong ligninolytic activity together with variable cellulolytic and xylanolytic action.Pleurotus sajor-caju attacked lignin and cellulose at the same rate, showing a degradation of 47% and 55%, respectively. A better balance was shown by theP. ostreatus-P. pulmonarius hybrid, which exhibited the poorest cellulolytic action (39%) and the highest ligninolytic activity (67%). The average composition of mushroom fruit bodies, in terms of nitrogen, carbohydrates, fats and amino acid profiles, was determined. Crude protein and total carbohydrate varied from 23% to 33% and 36% to 68% of dry matter, respectively. Fat ranged from 3.3% to 4.7% and amino acid content from 12.2% to 22.2%. Slight evidence for a nitrogen fixing capability was encountered in the substrate to fruit body balance.     

In situ localization of galactosyltransferase in surface mucous cells of the rat stomach.

Cryostate sections of the rat stomach fundus were incubated in the presence of uridine 5'-diphosphate-[3H]-galactose. The radioactivity in the surface mucous epithelial cells was shown by autoradiography to be specifically incorporated into a supranuclear area, the area where in these cells the Glogi system is situated. The incorporation lasted only 5-6 min and was Mn++ dependent. Galactose was probably incorporated into a beta-glycosidic bond.