Capping of saccharides on the plasma membrane of lymphocytes as studied by fluorescein-labelled lectins

The capping of saccharides on the plasma membrane of rat splenic lymphocytes was studied by means of fluorescein-labelled lectins. Treatment of unfixed splenic lymphocytes with any one of the three lectins, concanavalin A (Con A), Ricinus communis agglutinin (RCA) and wheat germ agglutinin (WGA) led to the formation of caps of each saccharide receptor on the plasma membrane. Treatment of unfixed lymphocytes with Con A was found to result in the formation of caps of saccharide receptors for RCA, whereas cap formations were never noted in such double treatment of the cells with all other combined uses of two lectins. These results are taken to indicate that the saccharide receptors for Con A are associated with those for RCA in the plasma membrane of rat splenic lymphocytes.     

An ultrastructural search for lectin-binding sites on surfaces of spinach leaf organelles

Organelles isolated from leaves of spinach (Spinacia oleracea L.) were prefixed in glutaraldehyde and then incubated with ferritin conjugates of four lectins — Concanavalin A (Con A), Ricinus communis L. agglutinin, MW 120,000 (RCA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA) — in order to probe their cytoplasmic surfaces for saccharide residues. In each case the major leaf organelles, including microbodies, mitochondria and chloroplast derivatives, failed to exhibit labeling when examined with the electron microscope. Tobacco (Nicotiana tabacum L.) leaf protoplasts, incubated simultaneously with and under identical conditions to the spinach organelles, showed specific labeling of their plasma membranes with all four lectin conjugates, thus establishing the efficacy of the procedure for demonstrating the presence of binding sites when they exist. Further attempts to show binding of one of the lectins, Con A, by labeling with fluorescein-Con A and by organelle agglutination, yielded results consistent with the absence of ultrastructural labeling. It is concluded that no saccharide residues recognized by the four lectins are present on the cytoplasmic surfaces of organelles and that those residues reported to be constituents of intracellular membranes, therefore, are most likely exposed on the luminal (extracytoplasmic) surfaces.Abbreviations Con A Concanavalin A - RCA Ricinus communis agglutinin, MW 120,000 - SBA soybean agglutinin - WGA wheat germ agglutinin     

Cell-to-cell binding induced by different lectins

The cell-to-cell binding induced by concanavalin A (Con A) and the lectins from wheatgerm, soybean, and waxbean has been analyzed by measuring the ability of single cells to bind to lectin-coated cells immobilized on nylon fibers. The cells used were lymphoma, myeloid leukemia, and normal fibroblast cells. With all lectins, cell-to-cell binding was inhibited if both cells were prefixed with glutaraldehyde. However, in most cases cell-to-cell binding was enhanced when only the lectin-coated cell was prefixed. With normal fibroblasts, treatment of either one or both cells with trypsin enhanced the cell-to-cell binding induced by Con A and the wheatgerm lectin. Neuraminidase, which increases the number of receptors for soybean agglutinin, increased cell-to-cell binding only if both cells were treated. Although cell-to- cell binding induced by the lectins from soybean and wheatgerm could be partially reversed by the appropriate competitive saccharide inhibitor, binding induced by Con A could not be reversed. The experiments indicate that cell-to-cell binding induced by a lectin can be prevented by an insufficient density of receptors for the lectin, insufficient receptor mobility, or induced clustering of receptors. These effects can explain the differences in cell-to-cell binding and agglutination observed with different cell types and lectins. They also suggest that cell-to-cell binding induced by different lectins with a variety of cell types is initiated by a mechanism involving the alignment of complementary receptors on the colliding cells for the formation of multiple cell-to-lectin-to-cell bridges.     

Trypanosoma rhodesiense Bloodstream Trypomastigote and Culture Procyclic Cell Surface Carbohydrates1, 2, 3

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRATat 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diaminobenzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A < PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WGA, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M. The expression of lectin binding cell surface saccharides of T. rhodesiense WRATat 1 is related to the parasite stage. Sugars resembling α-D-mannose are on the surface of bloodstream trypomastigotes and culture procyclics; n-acetyl-D-galactosamine and D-galactose residues are on bloodstream forms; and n-acetyl-D-glucosamine-like sugars are on procyclic stages.     

A differential response elicited in macrophages on interaction with lectins

The interaction of several lectins, both native and chemically modified, with mouse peritoneal macrophages was studied. Surface distribution and interiorization of the lectins was assessed quantitatively using their radioactively-labeled derivatives, and qualitatively by employing fluorescein-labeled lectins. On the basis of their effect on the macrophages, the lectins tested fall into two classes: lectins that induce vacuole formation in the cells (concanavalin A (ConA), wax bean agglutinin (WBA) and wheat germ agglutinin (WGA)) and lectins that in their native form do not induce vacuolation (soybean agglutinin (SBA), peanut agglutinin (PNA) and the lectin from Lotus tetragonolobus (LT)). Neuraminidase treatment of the cells did not change their response to the lectins, though in the case of SBA and PNA binding was observed only with neuraminidase-treated macrophages. Incubation of the latter cells with SBA and subsequently with ConA resulted in significantly higher vacuolation than that observed with ConA alone. Glutaraldehyde-crosslinked polymers of SBA and of PNA, which are multivalent with respect to sugar binding, induced vacuolation in neuraminidase-treated macrophages. On the other hand, succinylation of ConA, which reduces the number of sugar binding sites per mole from four to two, abolished its ability to induce vacuole formation. These data suggest that multivalency of lectins and probably also their size are important factors in inducing vacuolation, by causing extensive crosslinkage of membrane receptors which is prerequisite for triggering of vacuole formation. Quantitative binding and internalization data indicate that vacuole formation is not directly related to the number of lectin receptors on the macrophages nor to the extent of their internalization.     

Distribution of glycoconjugates in normal human skin using biotinyl lectins and avidin-horseradish peroxidase

Lectin binding patterns in normal human skin were studied using five different biotinyl lectins and avidin-horseradish peroxidase. The staining pattern was specific for each lectin. In the epidermis, peanut agglutinin (PNA) and soybean agglutinin (SBA) preferentially stained the cell membranes of keratinocytes in the spinous and granular cell layers, indicating changes in the saccharide residues during keratinocyte differentiation. In the secretory segment of an eccrine sweat gland, the superficial cells gave a strong granular staining with Ricinus communis agglutinin (RCA). Dolichos biflorus agglutinin (DBA) and SBA, on the other hand, strongly stained the basal cells. With these lectins, two types of cells in the secretory segment were clearly distinguished. These results show that (1) PNA and SBA binding sites increase during the course of keratinocyte differentiation, and (2) RCA, DBA, and SBA are good markers to distinguish two types of cells in the secretory segment of an eccrine sweat gland.     

Distribution of cell surface saccharides on pancreatic cells. II. Lectin-labeling patterns on mature guinea pig and rat pancreatic cells

The surface saccharide composition of collagenase-dispersed pancreatic cells from adult guinea pig and rat glands was examined by using eight lectins and their ferritin conjugates: Concanavalin A (ConA); Lens culinaris (LCL); Lotus tetragonolobus (LTL); Ricinus communis agglutinins I and II (RCA I, RCA II); Soybean agglutinin (SBA); Ulex europeus lectin (UEL); and wheat germ agglutinin (WGA). Binding studies of iodinated lectins and lectin-ferritin conjugates both revealed one population of saturable, high-affinity receptor sites on the total cell population (approximately 95% acinar cells). Electron microscopy, however, revealed differences in lectin-ferritin binding to the plasmalemma of acinar, centroacinar, and endocrine cells. Whereas acinar cells bound heavily all lectin conjugates, endocrine and centroacinar cells were densely labeled only by ConA, LCL, WGA, and RCA I, and possessed few receptors for LTL, UEL, and SBA. Endocrine and centroacinar cells could be differentiated from each other by using RCA II, which binds to centroacinar cells but not to endocrine cells. Some RCA II receptors appeared to be glycolipids because they were extracted by ethanol and chloroform-methanol in contrast to WGA receptors which resisted solvent treatment but were partly removed by papain digestion. RCA I receptors were affected by neither treatment. The apparent absence of receptors for SBA on endocrine and centroacinar cells, and for RCA II on endocrine cells, was reversed by neuraminidase digestion, which suggested masking of lectin receptors by sialic acid. The absence of LTL and UEL receptors on endocrine and centroacinar cells was not reversed by neuraminidase. We suggest that the differential lectin-binding patterns observed on acinar, centroacinar, and endocrine cells from the adult pancreas surface-carbohydrate-developmental programs expressed during morphogenesis and cytodifferentiation of the gland.     

Lectin Analysis of Trypanosoma congolense Bloodstream Trypomastigote and Culture Procyclic Surface Saccharides by Agglutination and Electron Microscopic Technics*

SYNOPSIS. Living, intact bloodstream trypomastigotes and culture procyclic forms of Trypanosoma congolense were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose binding protein (FBP). Similar experiments were conducted with living bloodstream and culture forms treated with trypsin or dextranase. Parasites were incubated for 30 min at 25 C in various concentrations of each lectin, then examined for agglutination by dark-field microscopy. Control preparations consisted of parasites incubated alone or with 0.5 M of the specific competing sugar, with or without the corresponding lectin. Electron-microscopic localization of lectin binding sites on the surface of intact and dextranase-treated bloodstream and intact culture forms was accomplished with Con A, reacted with horseradish peroxidase (HRP) and then diaminobenzidine (DAB). In addition, FBP and SBA were coupled to HRP, then utilized for the localization of binding saccharides on the surface of blood-stream forms by the DAB technic. Similar studies were conducted with culture procyclics incubated with WGA-, SBA, PP- or FBP-HRP conjugates and then reacted with DAB. Controls were utilized to confirm the sugar specificity of all positive reactions. Intact living bloodstream forms were agglutinated in a concentration-dependent manner with all the lectins tested. Agglutination levels were scored as Con A > FBP > WGA = PP = SBA. Sugars resembling α-D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and α-L-fucose are evidently present on the surface of the parasites. No agglutination was noted in any control preparations. Identical lectin-induced agglutinations were obtained with trypsin- or dextranase-treated bloodstream forms. Trypsin disrupted but did not entirely remove the surface coat of bloodstream forms, while dextranase did not alter the ultrastructure of the parasites. Con A-, SBA- and FBP-binding saccharides were distributed uniformly on the surface coat of intact bloodstream forms; a similar distribution of Con A receptors was noted also on the surface of dextranase-treated cells. No lectin-binding saccharides were visualized by electron microscopy on any control preparations. Intact, trypsin- or dextranasetreated, procyclics were agglutinated in a concentration-dependent fashion by Con A and WGA, but not by the other lectins tested. Control preparations did not agglutinate and the enzymes did not affect the ultrastructure of the parasites. Con A- and WGA-specifically binding saccharides were uniformly distributed on intact procyclics and control preparations were lectin-negative. Thus, T. congolense procyclics retained surface saccharides resembling α-D-mannose and N-acetyl-D-glucosamine but lost sugars resembling N-acetyl-D-galactosamine (or D-galactose) and α-L-fucose. The failure of dextranase to remove the lectin-binding saccharides from the surface of bloodstream and procyclic forms suggests that α-1,6-glucan bonds do not link these carbohydrates. The results are contrasted with lectin research on other trypanosome species and discussed with relation to the biology of T. congolense.     

Membrane carbohydrate characterization of Acanthamoeba astronyxis, A. castellanii and Naegleria fowleri by fluorescein-conjugated lectins

A comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living Acanthamoeba castellanii, Naegleria fowleri and A. astronyxis, respectively from sewage sludge in India was carried out by means of fluorescein-conjugated lectin binding using eight lectins. Two lectins, viz. Concanavalin A and Phytohaemagglutinin P, could bind all free-living amoebae at different concentrations. The most notable feature of the study is that peanut agglutinin (PNA) and wheatgerm agglutinin (WGA) can differentiate between the pathogenic A. castellanii and non-pathogenic A. astronyxis strain, respectively. However, Ulex agglutinin I (UEA I) was the only lectin positive to both pathogenic A. castellanii and N. fowleri. During in vitro conversion from trophozoites to cysts, A. castellanii and N. fowleri cysts gained WGA-specific saccharide whereas A. castellanii; A. astronyxis and N. fowleri lost or reduced Dolichos biflorus agglutinin, PNA; WGA and ConA, and UEA I-specific saccharides, respectively. Neuraminidase could not alter the fluorescein-lectin binding to WGA and PNA. These demonstrated that only two lectins can recognize the factors giving Acanthamoeba their pathogenic (PNA-specific) and non-pathogenic (WGA-specific) status. More interestingly, UEA I can only differentiate between pathogenic and non-pathogenic amoebae. It is also suggested that during stage conversion the surface of the organism exhibited replacement of saccharides.     

Lectin binding and uptake and glycoprotein characterization of isolated porcine aortic endothelial and smooth muscle cells

Endothelial and smooth muscle cells were isolated from porcine aorta and kept in short-term culture. To determine the terminal carbohydate composition of the plasma membranes from both cell populations, the cells were incubated with a panel of fluorescein-labelled lectins. Both cell populations shared a number of terminal carbohydrates, but the N-galactosamine specific lectin Wistaria floribunda agglutinin labelled only the endothelial cells. A lectin which selectively labelled smooth muscle cells was not found. Western blot analysis of isolated endothelial cell membrane glycoproteins indicated that most membrane glycoproteins are labelled by Wistaria floribunda agglutinin.     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae

Summary The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone wich was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.     

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas

Summary Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): l-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis-to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.     

Location of sialoglycoconjugates containing the Sia(alpha)2-3Gal and Sia(alpha)2-6Gal groups in the rat hippocampus and the effect of aging on their expression.

The histochemical distribution of sialoglycoconjugates in the CA1 region in the hippocampus formation of 9-week-old rats and 30-month-old rats was examined using electron microscopy in combination with two lectins, Maackia amurensis lectin, specific for Sia(alpha)2-3Gal, and Sambucus sieboldiana agglutinin, specific for Sia(alpha)2-6Gal. Each lectin stained the plasma membranes of pyramidal cells, indicating that the Sia(alpha)2-3Gal and Sia(alpha)2-6Gal groups were expressed on their plasma membranes. These lectins also bound to synapses in the stratum lacunosum molecular. The staining intensity of the lectins in the synapses in these layers was downregulated in the 30-month-old rats. These results indicated that both the Sia(alpha)2-3Gal and Sia(alpha)2-6Gal groups are expressed on these synapses and that the expression of these sialyl linkages decreases in the aged brain     

Changes in the cell surface coat during the development ofXenopus laevis embryos,detected by lectins

Summary The composition of the surface coat in embryonic cells ofXenopus laevis was examined by agglutination and fluorescent staining with lectins.Cells of early and mid gastrula stages were agglutinated by lectins specific for D-mannose, D-galactose, L-fucose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. No differences in agglutinability among ectoderm, mesoderm and endoderm cells were observed with lectins specific for D-mannose, D-galactose and N-acetyl-D-galactosamine, though agglutination of gastrula cells with fluorescent lectins revealed considerable differences in the intensity of lectin binding among cells within an aggregate. These differences in amount of lectin bound were not related to cell size or morphology. Patches of fluorescent material formed on the cells, suggesting that lectin receptors are mobile in the plane of the plasma membrane.In the early cleavage stages intensive lectin binding occurs only at the boundary between preexisting and nascent plasma membranes. The external surface of the embryo has few lectin receptors up to the late gastrula stage. The unpigmented nascent plasma membranes, when exposed to fluorescent lectins, do not assume any fluorescence distinguishable from the background autofluorescence of yolk, in stages up to the mid-blastula. From this stage onwards lectin binding was observed on the membranes of the reverse side of surface layer cells and on the membranes of deep layer cells. During gastrulation there is an accumulation of lectin-binding material on surfaces involved in intercellular contacts.The significance of lectin binding material for morphogenesis is discussed.     

Cell-surface changes during mitogenic stimulation of lymphocytes assessed by the binding of wheat-germ agglutinin and other plant lectins

Lymphocytes from murine lymph node, cultured in the presence of an optimally mitogenic dose of phytohaemagglutinin, were stained with fluoresceinated lectins and analysed by flow cytometry. A marked increase in the ability of lymphocytes to bind wheat-germ agglutinin was observed that is particularly pronounced for the blast cells, reaching a maximum at about 40 h, when they are 5.5-times brighter than cells at zero time. The corresponding intensification of the small cells is 2-fold. Much smaller increases in binding accompanying blast transformation were observed when fluoresceinated concanavalin A or Lens culinaris haemagglutinin were used. Polyacrylamide gel electrophoresis of plasma membranes followed by treatment of the gels with radioactively labelled lectins and autoradiography also showed a very distinct increase in the binding of wheat-germ agglutinin to membranes from mitogen-stimulated porcine lymphocytes. Less marked changes in the binding of concanavalin A Lens culinaris heamagglutin and Ricinus communis agglutinin 120 were also noted. The apparent multiplicity of glycoproteins that bind each lectin, suggests that in each case the sites are heterogeneous. We conclude that lymphocytes stimulated by the T-cell mitogen phytohaemagglutinin expose new glycoprotein receptors for wheat-germ agglutinin that are most abundant on blast cells at 40 h. Attempts to characterize the receptor biochemically suggest that the carbohydrate moiety recognised by wheat-germ agglutinin is present on a glycoprotein of approx. 120 kDa molecular mass and also possibly on glycoproteins of 170–190 kDa.     

Localization of the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes

Freeze-etch electron microscopy has been utilized to localize the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes and to determine the relation of these different glycoprotein receptors to the intramembranous particles. A. bisporus lectin, which could be visualized directly on the surface of erythrocyte membranes, and ferritin conjugates of wheat germ agglutinin showed a distribution that correlates exactly with the intramembranous particles at all lectin concentrations tested. The binding sites for both of these lectins are located on the major sialoglycoprotein of the membrane. The R. communis agglutinin-ferritin conjugate which binds to receptors on membrane glycoproteins that are distinct from the major sialoglycoprotein showed a close correlation with the intramembranous particles at low lectin concentrations and a poor correlation at high lectin concentrations. High concentrations resulted in virtually complete coating of the surface of trypsinized ghosts which displayed marked aggregation of the intramembranous particles. We conclude that the intramembranous particles of erythrocyte membranes contain at least two glycoproteins and that some membrane lectin receptors are not associated with the intramembranous particles.     

Lectin binding to parietal cells of human gastric mucosa

A light microscopic and ultrastructural analysis of lectin receptors on parietal cells from human gastric mucosa was performed utilizing 12 biotinylated lectins in conjunction with an avidin-biotin-peroxidase complex. Peanut agglutinin conjugated directly to peroxidase was also used. Several fixatives and fixation regimens were evaluated for optimal preservation of parietal cell saccharide moieties. Formalin proved to be the most practical fixative for light microscopic studies. A periodate-lysine-paraformaldehyde (PLP) combination provided good preservation of lectin binding capacity but yielded relatively poor ultrastructure. Conversely, glutaraldehyde provided excellent preservation of ultrastructure but a somewhat diminished lectin binding activity, which was overcome by using long incubation times and high concentrations of reagents. Parietal cells reacted strongly with Bandieraea simplicifolia, Dolichos biflorus, peanut agglutinin, and soybean agglutinin (all specific for galactosyl/galactosaminyl groups) and weakly with Ulex europaeus (specific for fucose). At the light microscopic level a beaded, perinuclear staining pattern was observed which, ultrastructurally, corresponded to an intense staining of intracytoplasmic canaliculi. The membranes of the intracytoplasmic canaliculi were characterized by an abundance of galactosyl residues, a paucity of fucosyl groups, and a lack of mannosyl and glucosyl residues. The biochemical and physiological significance of these findings is discussed.     

Changes in number, mobility, and topographical distribution of lectin receptors during maturation of chick erythroid cells

Plant lectins have been used to probe changes in cell surface characteristics that accompny differentiation in a complete series of chick erythroid cells. Dramatic differences in lectin receptor mobility were observed between the most immature cells of the series, the proerythroblasts, and cells at the next stage of maturation, the erythroblasts. Both concanavalin A and Ricinus communis agglutinin form caps on proerythroblasts, whereas they develop a patchy distribution on erythroblasts. Erythroid cells at later developmental stages show a homogeneous distribution of surface-bound R. communis agglutinin. Concanavalin A also shows a uniform distribution on the cell periphery, but appears to be concentrated in a ring above the perinuclear region of the cell. In addition to changes in mobility of lectin receptors, a large reduction (50-70%) in the number of lectin receptors per cell accompanies maturation of proerythroblasts to erythroblasts. Pretreatment of the cells with neuraminidase results in enhanced binding of R. communis agglutinin to proerythroblasts. The number of additional R. communis agglutinin receptors exposed by enzyme treatment remains relatively constant during subsequent cell maturation.     

Lectin histochemical studies of mouse granulated metrial gland cells

A lectin histochemical study has been carried out on mouse granulated metrial gland cells, the major leucocyte population that differentiates in the uterine wall in pregnancy. The binding characteristics of 26 lectins were examined using light microscopical methods. Fourteen of the lectins, with affinities ranging through N-acetylgalactosamine, galactose, N-acetylglucosamine, mannose and sialic acid residues, bound to the cytoplasmic granules of granulated metrial gland cells, and each appeared to bind to the limiting membrane of the granules. The binding characteristics of three of these lectins (Wheat germ agglutinin, Concanavalin A and Helix pomatia agglutinin) were examined using electron microscopical methods. These showed a different binding pattern to the cytoplasmic granules of granulated metrial gland cells compared with that found using light microscopical methods, as they appeared to bind evenly across the granule's matrix. This binding pattern corresponds to the reactivity of the granule matrix in the periodic acid--Schiff technique. Six lectins bound to the cell membranes of granulated metrial gland cells. These included the E and L isoforms of Phaseolus vulgaris agglutinin, with affinities for complex carbohydrates, whose binding differences were related to the stage of differentiation of the granulated metrial gland cells. The lectin binding described presents additional markers of granulated metrial gland cells and tools for investigating carbohydrate moieties in the functional activities of granulated metrial gland cells This revised version was published online in November 2006 with corrections to the Cover Date.     

Ca2+ binding sites in plasma membranes of rat liver and hepatoma cells, and effect of concanavalin A on the Ca2+ binding sites and cellular uptake of Ca2+

1. Plasma membranes isolated from rat livers and ascites hepatoma cells (AH-130, AH-7974) were assayed for specific Ca2+ binding sites using 45Ca2+ and a Millipore filtration technique. The presence of higher (Kd = 1.4--1.5 . 10(-5) M) and lower (Kd = 0.9--1.0 . 10(-4) M) affinity sites in both liver and hepatoma membranes was observed. The hepatoma plasma membranes however, showed 1.4--2.1-fold as many Ca2+ binding sites (higher and lower affinity sites) as the liver plasma membranes on the basis of protein. 2. Concanavalin A stimulated the specific Ca2+ binding to liver and hepatoma plasma membranes, showing a maximal stimulation (3--5-fold) at 100 microgram/ml. Succinyl concanavalin A was less effective, whereas wheat germ agglutinin and ricinus lectin were ineffective. 3. Concanavalin A stimulated the Ca2+ uptake by AH-7974 cells. The concanavalin A-mediated stimulation of Ca2+ uptake showed lectin-concentrations and Ca2+-concentration dependencies similar to those in the concanavalin A-mediated stimulation of Ca2+ binding.