DIFFERENTIAL LOCALIZATION OF CELL SURFACE AND SECRETORY COMPONENTS IN RAT INTESTINAL EPITHELIUM BY USE OF LECTINS

Sections through various levels of small intestine from adult male rats were examined by fluorescence microscopy after treatment with fluorescein isothiocyanate-labeled lectins from Dolichos biflorus, Lotus tetragonolobus, Ricinus communis, and Triticum vulgare (wheat germ). The latter three lectins reacted with the microvillar portion of the epithelial cells lining the crypts and villi in sections of intestine adjacent to the pylorus. This pattern of reactivity was sharply altered along the first 15 cm of intestine so that in sections distal to this point the luminal surfaces of only those epithelial cells in the crypts and at the base of the villi reacted with the L. tetragonolobus and R. communis lectins, whereas the wheat germ lectin reacted with the surfaces of the cells lining the villi. In sections from the distal end of the small intestine, all three lectins reacted with the surfaces of cells only at the base of the villi and in the crypts. These results show a difference in surface components in cells at various portions on the villi and the dependence of these differences on the region of intestine. The D. biflorus lectin reacted with approximately 25% of the goblet cells at each level of intestine studied whereas the reactivities of the goblet cells with the other three lectins were dependent upon the region of intestine.     

Immunohistochemical and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the rice brown planthopper,Nilaparvata lugens (Stal)

Rice brown planthoppers (Nilaparvata lugens) were fed on artificial diet containing snowdrop lectin (Galanthus nivalis agglutinin; GNA), which has been shown to be toxic towards this insect pest. In addition to decreasing survival, the lectin affected development, reducing the growth rate of nymphs by approximately 50% when present at a concentration of 5.3&mgr;M. Immunolocalisation studies showed that lectin binding was concentrated on the luminal surface of the midgut epithelial cells within the planthopper, suggesting that GNA binds to cell surface carbohydrate moieties in the gut. Immunolabelling at a lower level was also observed in the fat bodies, the ovarioles, and throughout the haemolymph. These observations suggest that GNA is able to cross the midgut epithelial barrier, and pass into the insect's circulatory system, resulting in a systemic toxic effect. Electron microscope studies showed morphological changes in the midgut region of planthoppers fed on a toxic dose of GNA, with disruption of the microvilli brush border region. No significant proteolytic degradation of GNA was observed either in the gut or honeydew of planthoppers fed on lectin-containing diet. The presence of glycoproteins which bind GNA in the gut of the brown planthopper was confirmed using digoxigen-labeled lectins to probe blots of extracted gut polypeptides.     

Immunogold localization of ingested kidney bean (Phaseolus vulgaris) lectins in epithelial cells of the rat small intestine

Summary The interactions between dietary kidney bean (Phaseolus vulgaris) lectins and the epithelial cells of the rat small intestine were investigated by immunogold electron microscopy. The results demonstrated that the lectins bind to the glycocalyx of duodenal and jejunal microvilli and that some of these dietary constituents are endocytosed into lysosomal pathways within both absorptive and secretory gut cells. It is concluded that the lysosomal response serves to limit the absorption of nutritionally significant levels of these dietary toxins.     

Immunocytochemical localization of the intrinsic factor-cobalamin receptor in dog-ileum: distribution of intracellular receptor during cell maturation

Absorption of cobalamin is facilitated by the binding of the intrinsic factor-cobalamin complex (IF-cbl) to specific receptors in the ileum. The physical and biochemical characteristics of this ligand-receptor binding reaction have been extensively studied, but little is known about the cellular mechanisms or receptor synthesis, intracellular transport, and expression on the microvillus surface membrane. We attempted to delineate these mechanisms by using ultrastructural immunocytochemistry to localize the IF-cbl receptor in the crypt, mid-villus, and villus tip regions of mucosal biopsies obtained from the ileum of anesthetized dogs. Prior to initiating the ileal localization studies, the antisera to purified canine IF-cbl receptor that was employed in our studies was shown to have specificity for site (e.g., ileal enterocytes vs. other cells within the gastrointestinal tract) and immunohistochemical specificity. Receptor synthesis in endoplasmic reticulum begins in crypt enterocytes, but continues in cells throughout the villus. In the mid-villus region synthesized receptor translocates vectorially to the microvillus surface associated with membranous vesicles and then inserts into the microvillus pit. Receptor remains fixed to the microvillus pit and does not distribute uniformly over the brush border membrane. All villus tip enterocytes contained IF-cbl receptor in microvillus pits, vesicles, and endoplasmic reticulum, but in addition extensive perinuclear membrane staining was evident as well as re-internalized receptor associated with multivesicular bodies. Basolateral membranes contained no receptor at any level of the villus. These observations suggest that the IF-cbl receptor (a) translocates to the apical cell surface at the mid-villus region by transport in vesicles, (b) directly inserts into and then remains fixed in microvillus pits, (c) is elaborated on the luminal surface most extensively in villus tip cells, and (d) although reinternalized, does not move IF and/or cbl to the basolateral cell surface.     

Water-soluble luminal contents of the gut of the earthworm Lumbricus terrestris L. and their physiological significance.

In the post-gizzard gut of the earthworm Lumbricus terrestris, distinguishing the functions of the luminal epithelium from those of the chloragogenous tissue has been hindered by the close apposition of these two tissues. Moreover, both tissues may have different functions from the anterior to the posterior of the animal. We analyzed the gut luminal contents of L. terrestris so as to gain a better understanding of the function of the luminal epithelium. The intestine was divided into four regions from anterior to posterior, and the water-soluble portion of the luminal contents of these four regions was analyzed for protease and amylase activity, calcium and ammonium ions, and protein. The same four regions of the gut wall were analyzed for glutamate dehydrogenase (GDH) and serine dehydratase (SDH) to determine their location with reference to the site of ammonia production. We observed high levels of proteases, amylase, protein and calcium ions in the gut luminal contents of the first two regions, and a significant decline of all four variables in region III. Conversely, ammonia was low in the gut contents of regions I and II but rose sharply in region III, which was also the region to which the tissue enzymes GDH and SDH were localized. The ammonia content of earthworm casts was observed to be much higher than that of the surrounding soil. These data are presented as partial evidence for the proposal that the excretory ammonia produced by feeding earthworms is a product of the luminal epithelium of region III of the gut. It is also proposed that ammonia and calcium may function as ion-exchangers in the absorptive function of the earthworm gut.     

Lectin binding reveals divergent carbohydrate expression in human and mouse Peyer's patches

The nature of cell-associated carbohydrates in the human intestine that may mediate transepithelial transport of bacterial and dietary lectins and their processing by the lymphoid cells of Peyer's patches is not known. Because the cell surface carbohydrate receptors for lectins may vary in different species, the glycoconjugates of human and mouse follicle-associated epithelium and gut-associated lymphoid tissue were compared. A panel of 27, mainly recently isolated, lectins were used to identify glycoconjugate expression in M-cells, enterocytes, goblet cells, lymphocytes and macrophages in mouse and human intestine. Mouse M-cells were exclusively labelled by fucose-specific lectins but in human follicle-associated epithelium no distinct M-cell staining pattern was observed. In the human Peyer's patches,Bryonia dioica lectin bound selectively to paracortical T-lymphocytes andChelidonium majus lectin to germinal centre B-cells. Certain mannose-specific lectins (Galanthus nivalis, Hippeastrum hybrid) stained the tingible body macrophages in the germinal centre of human Peyer's patches but labelled the macrophages in the paracortical T-cell region of the mouse. The results indicate distinct differences in glycosylation between mouse and human Peyer's patches and their associated lymphoid cells. When considering cell surface glycoconjugates as target molecules for the gut immune system, care has to be taken to choose the appropriate lectin for each species.     

Trichostrongylus colubriformis: effects on villi and crypts along the whole small intestine in infected rabbits

In control and Trichostrongylus colubriformis-infected rabbits histological samples were taken from the small intestine at 25-cm intervals from the pylorus. Length of villi, surface of glands of Lieberkühn, and number of epithelial cells per unit length were measured with a digitized table. Two zones were so delimited along the parasitized mucosa: in the proximal part of the gut, both shortened villi and dilated crypts were found; in the distal region of the intestine, which was also the area with the lowest density of parasites, dilatation of crypts was associated with an increase in the size of villi. In both regions, the size of enterocytes was unchanged. Correlation coefficients calculated between worm burdens and relative variation in length of villi were significant and negative. Conversely, the same coefficients were significant and positive between worm burdens and surface of crypts. The possibility of local and general actions of Trichostrongylus colubriformis on both villi and crypts is evoked to explain the changes in the two zones. The functional importance of the hypertrophy in the distal region is still to be investigated during a T. colubriformis infection.     

Lectins as markers for cells infiltrating human renal glomeruli

This study describes a simple method for detecting mononuclear cells in human renal glomeruli using labeled lectins as probes. The lectins used in this study showed prominent binding to different cell types among the nonresident glomerular cells but not to normal glomerular elements. Monoclonal antibodies against monocytes/macrophages (OKM 1), T-cells (OKT 11), suppressior/cytotoxic T-cells (OKT 8), and anti-lysozyme antibodies were used in double-fluorescence studies with the lectins in an attempt to identify the lectin-positive cells. The results indicate that Bandeiraea simplicifolia I isolectin and Lotus tetragonolobus agglutinin in particular are useful for screening of kidney biopsies for cells of their monocyte/macrophage series and T-cells invading human renal glomeruli.     

Lectin binding of surface epithelia and concomitant glands of gallbladder and biliary ducts in the guinea pig

Summary Complex carbohydrate components of secretory granules and the glycocalix were analysed in surface epithelia, endoepithelial glands and exoepithelial tubuloalveolar glands of the biliary-ductular system (guinea pig). Brunner glands and pyloric glands were studied for comparison. The columnar epithelial cells of the gallbladder and biliary ducts displayed a well-developed PAS-positive apical glycocalix. These materials strongly bound Ricinus communis AI, Ulex europaeus I, Lotus tetragonolobus A and wheat-germ-A lectins. With the exception of Lotus A lectin which did not bind at all, the same lectins stained the basolateral cell surface. The secretory granules in the supranuclear regions of surface epithelia and in the exoepithelial glands strongly bound Ricinus A I, Ulex europaeus I, wheat-germ-A and Helix pomatia lectins. Concanavalin A was less intensively bound by the secretions of tubuloalveolar glands than by the secretory granules in surface epithelia. The luminal and basolateral cell surfaces of glandular cells in the exoepithelial glands were stained by the same spectrum of lectins as were the less distinct. In the guinea pig, the lectin-binding patterns of tubuloalveolar glands in the biliary ducts closely resembled those of Brunner glands and pyloric glands. The secretions of the tubuloalveolar glands were different from the secretion of surface epithelia, as they bound Concanavalin A less intensively.     

Changes in rates of tissue protein synthesis in rats induced in vivo by consumption of kidney bean lectins

1. Plantaris and gastrocnemius muscles from rats fed a diet containing raw kidney beans lost proportionately more weight and protein than muscles from rats fed a protein free diet, resulting in a progressive reduction in the ratio muscle mass: body weight over a period of 6 days. Rates of muscle protein synthesis were also lower in rats fed raw beans.2. Destruction of the bean lectins by boiling abolished both these effects.3. Two hours after administration of a single dose of purified kidney bean lectin, rates of protein synthesis were unchanged in the muscle and liver but were increased in the gut mucosa.     

Kidney bean lectin-induced Escherichia coli overgrowth in the small intestine is blocked by GNA, a mannose-specific lectin

The reversible and dose-dependent hyperplastic growth of the small intestine and accelerated epithelial cell turnover caused by feeding rats with diets containing kidney bean lectin (PHA) increased the proportion of immature cells on the villi whose membrane and/or cytoplasm contained mainly simple, polymannosylated glycans. These new α-linked mannosyl terminals, particularly of the damaged epithelium, facilitated the preferential adherence of opportunistic Escherichia coli with mannose-sensitive Type 1 fimbriae, and other coliforms, to the glycocalyx. Accordingly, the growth of the gut was accompanied by a reversible and PHA dose-dependent overgrowth with E.coli. As expected from their common carbohydrate specificity, the inclusion in the diet of the mannose-specific agglutinin from snowdrop ( Galanthus nivalis ) bulbs (GNA) significantly reduced the extent of E.coli overgrowth, but abolished neither the growth nor the damage caused by PHA to the small intestine. Thus, GNA and perhaps other mannose-specific lectins, especially when used in a preventive mode, can be used to specifically block the proliferation of Type 1 E.coli in the small intestine.     

Scanning electron microscopic study of the gut mucosa of the rainbow trout Salmo gairdneri Richardson.

The topological characteristics of the entire gut surface of the rainbow trout were investigated utilizing the scanning electron microscope. The mucosa exhibited longitudinal ridges in the oesophagus and stomach, villi in the intestine, fine longitudinal ridges in the caeca and annular folds in the rectum, arranged as a stack of caudally-directed funnels starting from the intestino-rectal valve to the vent. Detailed study revealed taste pores in the anterior oesophagus and the sculpting of the luminal plasmalemma of the surface cells into micro-ridges with complicated patterns. The surfaces of the posterior oesophagus and stomach were demarcated into polygons by rows of stubby microvilli—each polygon representing the luminal surface of an epithelial cell. Each rectal fold consisted of a smooth, caudally-directed apex and a base which was supported by perpendicular buttress-like secondary folds. The functional significance of these features which emphasize the vast difference in the physical length of the gut and the effective surface area is discussed.     

Surface carbohydrate changes on Onchocerca lienalis larvae as they develop from microfilariae to the infective third-stage in Simulium ornatum

Use was made of seven FITC labelled lectins as tools to investigate the surface of Onchocerca lienalis larvae as they develop through to the infective third-stage in a natural vector, Simulium ornatum. The lectins were derived from Canavalia ensiformis (Con A), Lens culinaris (lentil), Triticum vulgaris (wheat germ), Arachis hypogaea (peanut), Helix pomatia, Phaseolus vulgaris (kidney bean) and Tetragonolobus purpureus (asparagus pea). Between 70 and 100 living parasites were examined for each developmental stage; i.e. skin microfilariae, late first-stages, second-stages, preinfective third-stages and infective third-stages isolated from the mouth parts of the flies. None of the lectins used bound to the surface of the microfilariae. However, progressive binding to the cuticle of the first- and second-stages was observed using Con. A, lentil lectin and wheat germ agglutinin (WGA). Following moulting to the third-stage, binding of these three lectins declined. Furthermore, as these lectins decreased, peanut and Helix pomatia lectins progressively increased in their binding, despite the fact that they showed little or no binding to the first- and second-stages; stages at which Con A, lentil and WGA were at their maximum. Asparagus pea and kidney bean lectins failed completely to bind to any of the larvae examined. Carbohydrate inhibition tests showed that the lectin was indeed binding specifically to glycoconjugates on the parasite surface. WGA binding was not inhibited by prior incubation with N-acetyl-D-glucosamine, even at high concentrations, but neuraminic acid did completely inhibit its binding. Judging from the patterns of binding on the nematodes themselves, the carbohydrates may not be vector in origin, but derive from the worms. The lectin specificities indicate that initially mannose/glucose type derivatives are present on the surface. Following moulting to the third-stage these are progressively replaced, or overlaid with galactosamine type derivatives, also present on the infective third-stage as it enters the bovine host. The availability of these surface glycoconjugates to attack mediated by natural insect lectins may be of importance in the parasite regulatory mechanisms of the blackfly. Variability in these surface carbohydrates, and in the response to them could well be a contributing factor in the cytospecific variation in S. damnosum susceptibility to geographical variants of O. volvulus.     

Immunolocalization of transferrin and transferrin receptor in mouse small intestinal absorptive cells

The mechanisms by which the duodenal mucosa absorbs iron are unknown. Insorption into absorptive cells of luminal iron bound to transferrin via receptor-mediated endocytosis has been hypothesized, but transferrin and transferrin receptor are absent in apical microvillous brush borders of small bowel biopsies taken from fasted patients and normal volunteers. We hypothesized that a normal iron-containing diet might induce the transient appearance of transferrin and transferrin receptor in apical brush borders of small intestinal absorptive cells in a normal mouse that was provided iron-containing chow until the moment of sacrifice. Light and electron microscopic immunolocalization of transferrin and transferrin receptor in proximal small intestinal absorptive cells was limited to basolateral membranes and coated pits of cells predominantly in the crypts and basal regions of the villi. Transferrin and transferrin receptor were not detected in apical microvillous brush border membranes of these enterocytes. In parallel immunolocalization protocols designed to show the ability to immunodetect other antigens at these locations, maltase and proteoglycan were demonstrated in apical microvillous brush border membranes and in basolateral membranes, respectively, in absorptive cells of small intestinal villous tip, base, and crypt regions. Furthermore, transferrin and transferrin receptor were immunolocalized in hepatocyte sinusoidal microvillus membranes. We conclude that food does not induce the appearance of immunodetectable transferrin and transferrin receptor in the apical microvilli of small intestinal absorptive cells and, therefore, that these iron transport proteins are not involved in the apical microvillous membrane transport of luminal dietary iron.     

The nature of lectins fromDolichos lablab

The lectins from the seedsof Dolichos lablab var.lignosus (field bean) andDolichos lablab var.typicus (lablab bean) have been isolated in a homogeneous form by affinity chromatography on D-mannose linked Sepharose. Both the lectins are glycoproteins and have a molecular weight of 60,000 andS _20,w value of 5.2 and seem to be made up of 4 similar subunits (apparent molecular weight 15,000). The carbohydrate content ofthe lectins is mostly fucose (2–5 mol per mol of protein), mannose (5–8 mol per mol of protein) and N-acetyl glucosamine (1–2 mol per mol of protein). The amino acid composition of both the lectins was similar and methionine and half cystine could not be detected, Both the lectins have similar tryptic peptide map. Alanine and serine were the only N and C-terminal amino acids for both lectins. The lectins were found to contain low amounts of bound metals such as manganese, magnesium and calcium. The near ultra-violet circular dichroism spectra of the lectins are similar to that of Sainfoin. Circular dichroism data indicate that tyrosine and tryptophan residues are involved in sugar binding. The lectins are nonspecific for human blood groups and they agglutinate a variety of other erythrocytes. Among a number of sugars, D-glucose and D-mannose inhibited the haemag-glutinating activity ofthe lectins. The lectins were antigenically similar     

On the hemagglutination reaction in methanotrophic bacteria

The reaction of hemagglutination with trypsin-treated rabbit erythrocytes was used to reveal lectins on the cell surface of methanotrophic bacteria and in their culture liquids. By this method, no lectins were detected on the cell surface ofMethylococcus capsulatus IMV B-3001 andMethylomonas rubra IMV B-3075 or in the culture liquid of any of the species studied. With intact cells ofMethylocystis parvus IMV B-3491, the positive hemagglutination reaction observed was nonspecific and most probably occurred due to the high cell surface hydrophobicity characteristic of this species.     

Metagenomic assessment of the Cebus apella gut microbiota

Cebus Apella (C. apella) is a species of Nonhuman Primate (NHP) used for biomedical research because it is phylogenetically similar and shares anatomical commonalities with humans. Here, the gut microbiota of three C. apella were examined in the different regions of the intestinal tract. Using metagenomics, the gut microbiota associated with the luminal content and mucus layer for each intestinal region was identified, and functionality was investigated by quantifying the levels of short chain fatty acids (SCFAs) produced. The results of this study show a high degree of similarity in the intestinal communities among C. apella subjects, with multiple shared characteristics. First, the communities in the lumen were more phylogenetically diverse and rich compared to the mucus layer communities throughout the entire intestinal tract. The small intestine communities in the lumen displayed a higher Shannon diversity index compared to the colon communities. Second, all the communities were dominated by aero‐tolerant taxa such as Streptococcus, Enterococcus, Abiotrophia, and Lactobacillus, although there was preferential colonization of specific taxa observed. Finally, the primary SCFA produced throughout the intestinal tract was acetic acid, with some propionic acid and butyric acid detected in the colon regions. The small intestine microbiota produced significantly less SCFAs compared to the communities in the colon. Collectively, these data provide an in‐depth report on the composition, distribution, and SCFA production of the gut microbiota along the intestinal tract of the C. apella NHP animal model.     

Trichostrongylus colubriformis: epithelial cell migration in the proximal and distal small intestine of infected rabbits

In Trichostrongylus colubriformis-infected rabbits, epithelial cell migration rates and cell transit times along the villi were compared by radioautography on histological slides to normal values from noninfected small intestine. Regions of gut with high (upper jejunum) and low (ileum) burdens of worms were both examined. In the control rabbits, the estimated values for the cell migration rates in the proximal and distal parts of gut were respectively 5.8 and 2.8 microns/hr. Seventy-two hours after the thymidine injection, the labeled epithelial cells were near the tip of the villi in the jejunum whereas only 60% of the villous length was labeled in the ileum. In the infected rabbits, the presence of T. colubriformis was associated with a two-fold increase of the cell velocity, in the main site of infection. Although less prominent than in the proximal region, a significant acceleration in the cell migration was also noticed in the ileum. The cell transit time was markedly reduced in the parasitized jejunum, but no variation of this parameter was found in the distal part of gut. These changes in the dynamics of epithelial cells in both regions of the gut appeared to underlie the morphological and enzymological changes of the parasitized mucosa. They particularly contribute to create an adaptive region in the small intestine beyond the main site of infection.     

Protein blotting: detection of proteins with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes

Methods to detect "native" proteins immobilized on nitrocellulose membranes in spot tests or on blots prepared from polyacrylamide slab gels after electrophoretic separation are described. Gold sols were found to be useful as general stains for proteins: They are polychromatic, yield an indelible record, and are complementary to india ink as protein stains because these two stains have different sensitivities for a number of proteins tested. For detection of wheat germ lectin (WGL)-binding glycoproteins, avidin-peroxidase was an effective enzyme probe, because the glycoportion of the avidin moiety possesses binding affinity to WGL. Glycocomponents in human parotid saliva were detected with this probe and with the following biotin-conjugated lectins as intermediary probes: soybean lectin, Bandeiraea simplicifolia lectin, Lotus tetragonolobus lectin, and kidney bean lectin. Autoclaving blots prior to probing eliminated endogenous peroxidase activity. Concanavalin A and WGL were separated by isoelectric focusing and detected on blots with horseradish peroxidase and avidin-peroxidase, respectively. The versatility of the biotin/avidin system was used to detect other lectins on similar blots using biotin-conjugated glycoproteins as intermediary probes: Helix pomatia lectin and B. simplicifolia lectin were detected with biotinyl neoglycoproteins, and kidney bean lectin with biotin-conjugated components of parotid saliva.     

Acceptance and suitability of four plant substrates for rearing Orius sauteri (Hemiptera: Anthocoridae)

Orius sauteri (Poppius) is an important hemipterous predator that has been mass-reared for biological control of numerous pests in protected crop-production systems. To find a good oviposition substrate for mass-rearing this predator under insectary conditions [25°C, 65 ± 5% relative humidity, and a photoperiod of 16:8 (L:D) h], we compared kidney bean, soybean, broad bean sprouts and fresh leaves of kidney bean. We found that O. sauteri made more punctures and laid more eggs in kidney bean sprouts than in the other substrates examined. However, there were no significant differences among substrates in the proportion of punctures receiving eggs. Female O. sauteri laid the most eggs (as many as 68 eggs) in kidney bean sprouts and also had the shortest pre-oviposition period on this plant material. In addition, there were no significant differences in total oviposition durations or female longevity among the four plant substrates. The hatch rates of nymphs in the sprouts and leaves of kidney bean (>90%) were higher than those in soybean and broad bean sprouts. Thus, we found that the kidney bean sprout was the most suitable substrate for mass-rearing of O. sauteri.