Sites For Wheat Germ Agglutinin Research Articles

Interferon β-1a prevents the effects of lipopolysaccharide on embryonic brain microvessels

By means of light and electron microscopy we have studied the effect of interferon β-1a (IFNβ-1a) in the optic tecta of 20-day-old chick embryos under normal conditions and after exposure to lipopolysaccharide (LPS) which mimics the blood–brain barrier (BBB) disruption in meningoencephalitis. Optic tecta were examined for: (i) ultrastructure by means of transmission electron microscopy; (ii) the immunohistochemical localization of HT7 antigen, a specific marker of differentiation of the brain microvessels; (iii) the brain microvessel permeability, by means of horseradish peroxidase (HRP) tracer; (iv) the expression of microvessel glycoconjugates, by means of lectin histochemistry, using Ricinus communis agglutinin-I (RCA-I), specific for β- d-galactosyl moieties and Wheat Germ agglutinin (WGA) specific for sialyl and N-acetylglucosaminyl moieties. A morphometric evaluation of brain microvessel permeability and of glycoconjugate expression was also performed. In control- and in IFNβ-1a-treated embryos, HRP was confined to the vessel lumina which were sealed by the interendothelial tight junctions. RCA-I binding sites were recognizable both in the basal membranes and in the tight junctions, while WGA sites were present on the luminal side of the endothelial cells. HRP was blocked in the vessels lumina by the interendothelial tight junctions. After LPS treatment, HRP showed an extravascular localization and the labeling of microvessels by anti-HT7 antibodies disappeared. RCA-I binding was only found ultrastructurally and appeared as irregularly clustered gold particles, in the cleft of damaged tight junctions, but were no longer detectable in the endothelial basement membranes. After pretreatment of LPS-treated embryos with IFNβ-1a, the vessel permeability to HRP strongly decreased and the vessels showed the normal pattern of HT7 protein and of the RCA-I binding sites. These results indicate that the changes induced by LPS in the endothelial cells are prevented by IFNβ-1a.

Lectin binding sites during Drosophila embryogenesis.

The spectrum of lectin binding sites as it emerges during embryonic development of Drosophila was analysed by means of fluorescein-labelled lectins. As development and morphogenesis proceed, the reaction pattern becomes more and more complex. Mannose/glucose-, mannose-, N-acetylglucosamine- and poly-N-ace-tylglucosamine-specific lectins bind ubiquitously. Nuclear envelopes only have binding sites for wheat germ agglutinin. N-acetylgalactosamine-binding lectins are specific for ectodermal derivatives. Gaβ-3-N-acetylgalac-tosamine-binding lectins are highly selective markers for neural structures, haemocytes and Garland cells. It is also shown that Drosophila laminin is differentially glycosylated. The possible implications of differential and germ layer-specific glycosylation are discussed.

Role of glycoconjugates in adherence ofSalmonella pullorumto the intestinal epithelium of chicks

1. The distribution of glycoconjugates on the surface of Salmonella pullorum and the ileal epithelium of chicks was demonstrated by lectin cytochemistry. The role of glycoconjugates in adherence of S. pullorum to the ileal epithelium was determined by a sugar inhibition assay using the scanning electron microscope. 2. S. pullorum exhibited binding to Concanavalin A (Con A) and wheat germ agglutinin (WGA) but not to soyabean agglutinin (SBA), Ricinus communis agglutinin (RCA) and Ulex europaeus agglutinin (UEA). 3. The ileal epithelium of chicks bound WGA, Con A and SBA. The binding sites for WGA were on the brush border and cytoplasm of columnar enterocytes as well as on goblet cells. The binding of Con A was confined to the cytoplasm of columnar enterocytes, while SBA bound, in a limited way, to the brush border of columnar enterocytes. 4. After an oral dose of S. pullorum, adherence to the ileal epithelium was inhibited by methyl alpha-D-mannopyranoside.

The effects of bisphosphonates on the resorption cycle of isolated osteoclasts

Binding sites for wheat germ agglutinin (WGA)-lectin have been shown to become revealed in the demineralized resorption lacunae that osteoclasts excavate on bone substrate. Peroxidase-conjugated WGA-lectin, which binds to bone matrix glycoconjugates and proteoglycans, was used in pit formation assays to assess the activity of isolated osteoclasts cultured on either 3-amino-1,1-hydroxypropylidene-bisphosphonate (APD)-or dichloromethylene bisphosphonate (Cl2MBP)-covered bone slices. Immunofluorescence and histochemical techniques were also used to study the effects of bone-bound bisphosphonates on isolated rat osteoclasts. Neither APD nor Cl2MBP interfered with the special organization of actin or vinculin in osteoclasts when the cells were initializing their resorption cycle. After 24 hours of culture, the number of resorbing osteoclasts increased strongly on control slices, but remained low on either APD- or Cl2MBP-treated slices. At this time, the actin and vinculin rings in osteoclasts also started to exhibit abnormal, more diffuse staining. Both bisphosphonates studied resulted in signs of cytotoxicity: the number of osteoclasts decreased on APD- or Cl2MBP-covered bone during the course of the study and those remaining attached exhibited severe cytoplasmic retractions. The total areas of resorption remained at significantly lower levels in both experimental groups studied, and this was due to decreases in both the number and sizes of individual resorption pits. The size of the most extensive lacunae detected on the Cl2MBP slices did not exceed 5 x 10(3) microns 2, whereas on the control slices, resorption pits bigger than 15 x 10(3) microns 2 were frequently discovered.

The role of lymphocyte surface binding sites for wheat germ agglutinin in the negative regulation of cancer patients.

The role of lymphocyte surface binding sites for wheat germ agglutinin (WGA) in the negative regulation of cancer patients was investigated. The number of WGA binding sites on the surface of each lymphocyte ranged from 10(7) to 10(8). Fluorescein isothiocyanate (FITC)-conjugated WGA, bound to the majority of peripheral blood lymphocytes (PBL) with two peaks of fluorescent intensity was expressed either dimly or brightly. The increase in lymphocytes brightly expressing WGA fluorescent intensity (WGA bright lymphocytes) significantly correlated with the number of WGA binding sites. The suppression of lymphocyte proliferation mediated by the purified soluble suppressor factor (SSF) significantly correlated with an increase in the WGA bright lymphocyte population (P < 0.05). A significantly greater number of WGA bright lymphocytes in PBL was found in patients with esophageal, gastric, breast, or colon cancer, than in those with benign diseases or in healthy controls. Furthermore, an increase in WGA bright lymphocytes was found in subsets expressing the antigens CD8 dimly or CD16. Thus, it is suggested that the number of WGA binding sites may increase mainly on the surface of effector cells such as NK cells and CD8-positive killer T cells in cancer patients, triggering the negative regulation mediated by SSF.

Histochemical characterisation of carbohydrate residues during the morphogenesis of the urogenital system in Caretta caretta

The distribution of carbohydrates in the urogenital system of Caretta caretta during embryogenesis and post-natal development was investigated by classical carbohydrate histochemical techniques and by the use of fluorescein-labelled lectins. PAS positive glycoconjugates were observed in nephrons of the mesonephros at an earlier stage of development than those from the metanephros. The lectin binding pattern of both kidney precursors revealed that the appearance of terminal carbohydrates in various parts of the nephron are dependent on the developmental stage of the animal. Binding sites for Wheat germ agglutinin and Maclura pomifera agglutinin were the most abundant throughout the developing kidney, whereas reactivity for Soy bean agglutinin and phythaemagglutinin was limited to fewer sites. The results indicate that the compartmentalised expression of cell glycoconjugates in the kidneys of juvenile Caretta caretta reflects the morphological and functional maturation of the nephron during development. Less pronounced lectin binding pattern of the gonad and genital ducts and the absence of changes during organogenesis indicate that terminal carbohydrates in the genital system of Caretta caretta do not play a major role in sexual differentiation.

Ultrastructural localization of WGA, RCA I, LFA and SBA binding sites in the seven-day-old mouse embryo

In the present investigation we localized binding sites for the lectins WGA (wheat germ agglutinin), RCA I (Ricinus communis agglutinin), LFA (Limax flavus agglutinin) and SBA (soya bean agglutinin) in the 7-day-old mouse embryo at the ultrastructural level. Lectin binding sites were localized on formaldehyde fixed embryos, embedded in LR-Gold, using gold-labelled lectins. Binding sites for WGA and RCA I were observed at the surface of the embryonic ectoderm oriented towards the proamnion cavity and the outer surface of the extraembryonic and the embryonic endoderm. Staining for SBA and LFA binding sites was seen in the basement membrane of the ectoderm. Moreover, binding sites for LFA were observed in the nucleoli of cells of the ectodermal, the mesodermal and the endodermal layer and in free ribosomes located in the cytoplasm of these cells.

Effects of the sterol demethylase inhibitor, cyproconazole, on hyphal tip cells of Sclerotium rolfsii: III. Cell wall cytochemistry

Hyphae of Sclerotium rolfsii were grown on cyproconazole-amended agar at fungistatic concentrations of 0.1, 0.75, and 1.0 μg/ml, which respectively produced approximately 50, 80, and 95% inhibition of hyphal extension. Hyphal tip cells were prepared for transmission electron microscopy by cryofixation and freeze substitution methods. Cell walls of control and fungicide-treated hyphal tip cells were compared cytochemically using periodic acid-thiocarbohydrazide-silver protein, gold-conjugated lectins, and immunocytochemical staining. Cell walls of both control and fungicide-treated cells reacted positively to periodic acid-thiocarbohydrazide-silver protein staining although the patterns of staining were different. Apical walls of control cells were generally thinner than those in fungicide-treated hyphae. Two distinct cell wall layers were identified in the subapical walls of control hyphae while distinct wall layers were typically not observed in fungicide-treated hyphae. Staining characteristics of abnormal cell wall deposits were variable after periodic acid-thiocarbohydrazide-silver protein stain. In regions of mature cell wall of control hyphal tip cells, wheat germ agglutinin-binding sites were localized along the inner layer of the cell wall. In fungicide-treated hyphae, binding sites for wheat germ agglutinin occurred throughout the cell wall except in electron opaque wall inclusions. Concanavalin A-binding sites were localized along the plasma membranes of both control and cyproconazole-treated hyphae in subapical cell regions. Electron opaque inclusions in abnormal cell wall deposits had affinities for concanavalin A. Immunocytochemical localization of β-1,3 glucans occurred in mature cell walls of control and fungicide-treated cells. No β-1,3 glucans were detected in electron opaque wall inclusions of hyphae exposed to cyproconazole. Alterations in the cell wall are discussed in light of the mode of action of the fungicide and aspects of cell wall deposition.

Ultrastructural demonstration of lectin binding sites in the Golgi apparatus of rat epiphyseal chondrocytes.

Binding sites for wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Ricinus communis I agglutinin (RCA I) and Limax flavus agglutinin (LFA) have been ultrastructurally detected in rat epiphyseal chondrocytes by a post-embedding cytochemical technique using colloidal gold as marker. The four lectins labelled exclusively the Golgi apparatus of chondrocytes embedded in Lowicryl K4M resin by two different methods. WGA binding sites were localized in medial and trans cisternae as well as in immature secretory vesicles, whereas those for DBA were seen concentrated in cis and medial cisternae. Labelling with both RCA I and LFA lectins was distributed throughout all the cisternae of the Golgi stack, and the latter also in vesicles and tubules at the trans face. Neuraminidase pretreatment of the sections abolished LFA staining, decreased reaction with WGA and increased that with RCA I, while it did not affect DBA staining. After chondroitinase ABC treatment only the RCA I reaction was modified, revealing new binding sites in the trans Golgi face, secretory granules and extracellular matrix. These results indicate that the distribution of subcompartments in the Golgi apparatus of chondrocytes is different from that in cells secreting glycoproteins as major products.

Effect of in vivo γ-irradiation on the binding of wheat germ agglutinin on lymphocyte plasma membranes

Using quantitative fluorimetry with fluoresceinated wheat germ agglutinin, we have been able to investigate in vivo gamma radiation-induced damage at the outer membrane level of rat splenic lymphocytes, namely damage to the glucosidic moieties of membrane glycoproteins and glycolipids. This paper demonstrates that below an irradiation level of 1 gray (Gy), removal of sialic acid is the major feature leading to new exposed specific binding sites for wheat germ agglutinin, since this lectin is specific for sialic acid and N- acetyl- D-glucosamine . Our studies also suggest that above 1 Gy of irradiation more internal damage occurs, since we observed a striking decrease in wheat germ agglutinin binding sites.

Alterations in glomerular lectin binding sites of human kidney as detected by fluorescence microscopy

Different lectins were used to study frozen sections of kidney samples showing alterations in routine immunofluorescence studies. Arachis hypogaea agglutinin (peanut lectin, PNA), lacking binding sites in normal glomeruli, bound to the glomeruli in two of the five samples studied, giving a granular fluorescence pattern. Concomitantly with the appearance of PNA-binding, binding sites for wheat germ agglutinin (WGA) appeared to be lost at glomeruli. Furthermore, changes in the expression of glomerular binding sites for Wistaria floribunda (WFA), Helix pomatia (HPA) and Dolichos biflorus (DBA) agglutinins could be seen in the kidneys studied, whereas the binding sites for Ulex europaeus agglutin (UEA I) in vascular endothelia seemed to be unaltered. The results show that kidney specimens presenting changes in routine immunofluorescence studies may also present altered binding for certain lectins. On this basis we propose that certain lectins may aid in characterizing these changes and are thus of potential use in studying diseased kidneys.

Cytochemical analysis of oligosaccharide processing in frog photoreceptors.

Lectin cytochemistry, together with exoglycosidase enzyme digestion, has been used to characterize partially glycoconjugates of several intracellular compartments in frog photoreceptors. In order to obtain uniform access of reagents to all intracellular compartments, the experiments were performed directly on semi-thin sections of Xenopus laevis retinal tissue embedded in a hydrophilic plastic resin. In the rod, the major photoreceptor intracellular binding sites for wheat germ agglutinin (WGA) are the outer segment, the Golgi complex, and other inner segment organelles which are probably involved in the transport of glycoconjugates from the Golgi complex to the outer segment. In addition, shed outer segment tips (phagosomes) are uniformly labelled with WGA. The WGA-binding sites of the outer segment and of the presumed transport organelles are resistant to neuraminidase digestion. This is consistent with the possibility that glycoconjugates (primarily opsin) are transported from the Golgi complex to the outer segment without further oligosaccharide processing. Specific staining of rod outer segments and of phagosomes is also obtained with the N-acetylglucosamine-specific lectin, succinyl-WGA (S-WGA). Outer segments and phagosomes stain the same with WGA, S-WGA and a variety of other lectins tested suggesting that no major post-Golgi oligosaccharide processing accompanies the shedding-phagocytosis event. Concanavalin A (Con A) staining of intracellular sites in rod inner segments reveals a striking difference compared to WGA staining in that the Con A binding sites are concentrated in the photoreceptor axon and presynaptic terminal. These results, and results from previous studies, indicate that the photoreceptor may utilize different mechanisms of oligosaccharide processing from the level of a single Golgi complex to the opposite ends of this cell. Furthermore, those glycoconjugates destined for the presynaptic terminal may undergo post-Golgi processing at or near their sites of insertion into the presynaptic plasma membrane.

Lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in the membrane of differentiating HL-60 and U-937 cells assessed with fluorescence recovery after photobleaching (FRAP).

The promyelocytic leukemia cell line HL-60 and the histiocytic cell line U-937 were grown in suspension culture. They were induced to differentiate during 5-d cultivation in the presence of dimethylsulfoxide (DMSO; 1.3% w/v) or phorbol-12-myristate-acetate (PMA; 10(-7) M), which yields granulocyte- and macrophage-like cells, respectively. Differentiation was evidenced by increased capacity to recognize and phagocytize IgG- or complement-coated yeast particles. Aliquots taken from the cultures with and without DMSO (or PMA) were spun down directly on glass microscope slides, washed, labeled with fluoresceinated wheat germ agglutinin (WGA), and directly examined at room temperature for the rate of fluorescence recovery after photobleaching (FRAP). It was found that cultivation of the HL-60 and the U-937 cells in the presence of DMSO, which yields granulocyte-like cells, reduced the average value of lateral diffusion coefficient D (X 10(10] from 1.72 +/- 0.13 cm2s-1 to 0.97 +/- 0.13 cm2s-1 and from 1.77 +/- 0.11 cm2s-1 to 0.82 +/- 0.13 cm2s-1, respectively. U-937 cells grown with PMA also showed a reduction of D(X 10(10] to 0.88 +/- 0.10 cm2s-1. There was a larger immobile fraction of fluorescence in the HL-60 cells than in the U-937 cells, viz., 70-80% compared to 10-50%. The total number of binding sites for WGA was not altered, but the surface density changed, since the HL-60 and the U-937 cells became smaller and larger, respectively, when grown in the presence of DMSO. It is concluded that differentiation reduces the average lateral mobility of the WGA-binding membrane component by a factor around 2.

Multiple forms of (Na+ + K+)-ATPase in the chicken. Selective detection of the major nerve, skeletal muscle, and kidney form by a monoclonal antibody.

Evidence is presented that monoclonal gamma-immunoglobulin secreted by hybridoma-24 recognizes the sodium and potassium ion-stimulated ATPase of neurons, muscle fibers, and kidney tubule cells in the chicken. The antigen consists of alpha (Mr = 105,000) and beta (Mr = 47,000) subunits. Only the beta subunit bears major oligosaccharide additions (including binding sites for wheat germ agglutinin) to its polypeptide core (Mr = 32,000). The detergent-solubilized antigen sediments as a 7 S complex of alpha beta or alpha beta 2. The antigen has a basolateral distribution in the plasma membrane of renal tubule cells, and in myogenic cell cultures there is up-regulation of the antigen in low potassium medium. Monoclonal antibody-24 specifically recognizes purified gull salt gland (Na+ + K+)-ATPase as well as the major candidate molecule for the (Na+ + K+)-ATPase in enriched preparations from chicken kidney. On cultured myotubes, the number of ouabain-binding sites (4.8 X 10(5)/nucleus) and antigenic sites (4.4 X 10(5)) are approximately equal. However, the antigenic sites on fibroblasts (1.1 X 10(4)) account for only about 4% of the ouabain-binding sites, and there is little or no antibody binding to capillary endothelial cells, Schwann cells, or erythrocytes. Evidence is presented that the antigenic determinant is proteinaceous. It is concluded that at least two antigenically distinct (Na+ + K+)-ATPases exist in the chicken.

Interaction of sulfated glycosaminoglycans with lectins.

The sulfated glycosaminoglycans, such as keratan sulfate and chitin sulfate having 3-hydroxy free N-acetyl-beta-D-glucosaminyl residues as constituents, reacted with wheat germ agglutinin and Solanum tuberosum agglutinin by sugar-specific interaction. The glycosaminoglycans showed different inhibitory activities to the hemagglutination reaction of these lectins and keratan sulfate and its modified products formed insoluble complexes with both of the lectins at pH 7.0 in physiological saline solutions (0.15 M NaCl). S. tuberosum agglutinin was precipitated within a particularly narrow concentration range of keratan sulfate, and the formation of a soluble complex was observed by gel chromatography. These interactions were specifically inhibited by N,N'-diacetylchitobiose but not by 2 M NaCl. The specific interactions of the glycosaminoglycans with S. tuberosum agglutinin were confirmed by their ultraviolet difference spectra with two peaks at 285 and 298 nm attributable to the tryptophan residues in the binding site of the agglutinin. It was also found that S. tuberosum agglutinin and wheat germ agglutinin have different binding specificities. The presence of sulfate groups in either keratan sulfate or chitin sulfate did not interfere with their specific interactions with S. tuberosum agglutinin as strongly as with wheat germ agglutinin. The N-acetylneuraminic acid residues in keratan sulfate were found to be receptor sites for wheat germ agglutinin but not for S. tuberosum agglutinin.

Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes.

To assess the subcellular distribution of oestrogen-binding components in their native state, plasma membrane and other cell fractions were prepared from hepatocytes in the absence of [(3)H]oestradiol-17beta. Cells from livers of ovariectomized rats were disrupted, with submaximal homogenization in buffered isotonic sucrose with CaCl(2) and proteinase inhibitor, and fractionated by using isotonic media. Fractions were characterized by determinations of enzyme activities, biochemical constituents and ligand binding. Specific binding of 2nm-[(3)H]oestradiol-17beta to intact cells and their fractions was detemined after equilibration for 1.5h at 4 degrees C. More than 92% of the radioactivity from representative preparations was verified as authentic oestradiol by thin-layer chromatography. Activities of plasma-membrane marker enzymes as well as binding sites for oestrogen and for wheat germ agglutinin were present principally in particulate fractions, rather than in 105000g-supernatant fractions. However, by using alternative homogenization procedures (i.e. hypotonic media), known to fragment and strip structural components, oestradiol-binding sites and activities of plasma-membrane marker enzymes were distributed predominantly into cytosol. By using the more conservative procedures, plasma membranes of low (rho=1.13-1.16) and high (rho=1.16-1.18) density were purified from crude nuclear fractions. A second low-density subfraction of plasma membrane was prepared from microsome-rich fractions. Activities of plasma-membrane marker enzymes were enriched to about 28 and four times that of the homogenate in plasma membranes of low and high density respectively. Binding sites for wheat germ agglutinin and oestradiol were concentrated in low-density plasma membranes to 46-63 times that of the homogenate. Specific binding of oestrogen in low-density plasma membranes purified from crude nuclei was saturable, with an apparent association constant of 3.5nm. At saturation, such oestradiol receptors corresponded to 526fmol/mg of membrane protein. A Hill plot showed a moderate degree of positive co-operativity in the interaction of hormone with plasma membranes. Specific binding of [(3)H]oestradiol-17beta was reduced by a 200-fold molar excess of unlabelled oestradiol-17beta, oestriol or diethylstilbestrol, but not by oestradiol-17alpha, cortisol, testosterone or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 60 degrees C, but remained essentially undiminished by extraction of membranes with either hypotonic or high-salt buffers. Extraction with 0.1% (v/v) Triton X-100 partially solubilized the oestrogen-binding component(s) of plasma membranes. Particle-free extracts were resolved on 5-20% (w/v) sucrose density gradients with either 0.01m- or 0.4m-KCl, and the fractions were analysed by adsorption to hydroxyapatite. In low-salt gradients macromolecule-bound oestrogen sedimented at predominantly 7.4S and binding was 1560 times that of the homogenate. Under high-salt conditions oestradiol-binding activity occurred at both 3.6S and 4.9S.

Lectin receptors as markers for Trypanosoma cruzi. Developmental stages and a study of the interaction of wheat germ agglutinin with sialic acid residues on epimastigote cells.

Trypanosoma cruzi at various stages of maturation and differentiation have been isolated by conventional cellular fractionation procedures and characterized by cell surface markers using 30 highly purified lectins encompassing all known sugar specificities. Cell surface carbohydrates of the various T. cruzi stages were analyzed by agglutination and lectin-binding assays. Specific receptors for wheat germ agglutinin (WGA), Helix pomatia, Sophora japonica, and Bandeiraea simplicifolia lectin II were found only in culture epimastigotes, whereas peanut agglutinin (PNA) sites were present exclusively in amastigotes, those for Phaseolus vulgaris in bloodstream trypomastigotes and amastigotes, and for Wistaria floribunda hemagglutinin predominantly in culture forms of T. cruzi. The N-acetylgalactosamine (DGalNAc)-binding lectin from Bauhinia purpurea agglutinated and inhibited the movement of epimastigotes and bloodstream trypomastigotes, but it only inhibited--without agglutinating--culture trypomastigotes. Because both the agglutination and inhibition of movement were reversed by specific sugar haptens, Bauhinia purpurea sites were present in all the flagellated parasites. On the other hand, PNA sites were detectable on epimastigotes after the cells were treated with sialidase, whereas, at the same time, WGA receptors were completely removed and those for the other sialic acid-binding proteins, Aaptos papillata lectin II and Limulus polyphemus, were partially eliminated; moreover, the activity of Wistaria floribunda hemagglutinin, a DGalNAc-binding lectin, increased 4,000 times. Trypsinization and lyzozyme treatment of epimastigote cells did not significantly affect lectin agglutination or lectin binding. WGA reacted solely with sialic acid residues on epimastigote cell surface with an apparent association constant of 2 x 10(6) M-1, each epimastigote having an estimated average of 3 x 10(6) WGA sites, as determined by binding experiments and a minimum of 7.7 x 10(6) sialic acid residues, as calculated by colorimetric method after sialidase digestion. Evidences are presented that the sialyl residues are rapidly regenerated (in approximately 4 h) and that they, at least for the most part, are not adsorbed from the culture medium. The receptor for the D-mannose-binding lectins (concanavalin A [Con A] and Lens culinaris) must either be on the same carbohydrate moiety having the WGA site, or, if in a distinct molecule, both carrier molecules of Con A and WGA sites must be located close to each other in the plasma membrane of the parasite.

Stimulation of cell surface phospholipase A2 and prostaglandin synthesis in 3T3 mouse fibroblasts by phallolysin, a toxin from Amanita phalloides

Phallolysin, a cytotoxic glycoprotein from the poisonous mushroom Amanita phalloides, stimulates high levels of cellular phospholipase A 2 in 3T3 Swiss mouse fibroblasts biosynthetically labeled by incorporation of [5,6,8,9, 11,12,14,15- 3H]arachidonic acid. Up to 39.9% of labeled cell lipids were hydrolyzed in 30 min with concomitant synthesis of prostaglandins (up to 5.3 and 1.7% of incorporated label released in 30 min as chromatographically identifiable prostaglandins E 2 and F 2 α , respectively). A similar extent of hydrolysis of labeled phosphatidylcholine was observed in cells biosynthetically labeled with [1,2- 14C]choline, with production of an equivalent amount of lysophosphatidylcholine. No detectable hydrolysis of sphingomyelin was observed, indicating (1) that phospholipases B, C and D and sphingomyelinases were not significantly activated, and (2) that the phospholipase A 2 activated did not exhibit a high degree of specificity for arachidonic acid moieties in its substrates. An analysis of the Ca 2+ requirement for activation suggests that phallolysin activates primarily cell surface, Ca 2+-requiring phospholipase A 2. Several lines of evidence suggest that stimulation of phospholipase A 2 by phallolysin involves binding to receptor sites which are also sites for wheat germ agglutinin. The extent of cell death, as assessed by failure to exclude trypan blue dye, correlated with the percent of maximal activation of phospholipase A 2 by a range of concentrations of phallolysin, suggesting that the toxin kills cells by inducing hydrolysis of membrane phospholipids.

Influence of Concanavalin A, wheat germ agglutinin, and soybean agglutinin on the fusion of myoblasts in vitro.

Although muscle cell fusion was shown to be an energy-requiring process, release of myoblasts from an EGTA fusion block could be accomplished with Earle's balanced salt solution (containing 1.8 mM Ca++) free of glucose or any other energy-produced metabolite. The effect of concanavalin A, abrin, and the lectins from wheat germ, soybean, and Lens culinaris on myoblast fusion was examined with synchronized myoblast cultures upon release from fusion block. At a concentration of 15 mug/ml, these lectins were found to inhibit the fusion process to the extent of 62%, 41%, 32%, 8%, and 19%, respectively. Concanavalin A inhibition could be prevented by alpha-methyl-D-mannoside. The inhibitory effect of all the lectins except abrin could be reversed by changing to the normal, serum-containing medium. The number of binding sites was 3.4 X 10(7), 6.1 X 10(7), and 1.7 X 10(6), respectively. Although myoblasts were found to have about twice as many binding sites for wheat germ agglutinin as for concanavalin A, concanavalin A was determined to be twice as effective as wheat germ agglutinin as an inhibitor of myoblast fusion. These findngs raise the possibility that specific cell surface glycoproteins may be an important factor in this process.

Isolation and partial chemical characterization of cell-surface glycopeptides from AS-30D rat hepatoma which possess binding sites for wheat germ agglutinin and concanavalin A.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIsolation and partial chemical characterization of cell-surface glycopeptides from AS-30D rat hepatoma which possess binding sites for wheat germ agglutinin and concanavalin ADavid F. Smith, Giovanni Neri, and Earl F. Walborg, Jr.Cite this: Biochemistry 1973, 12, 11, 2111–2118Publication Date (Print):May 1, 1973Publication History Published online1 May 2002Published inissue 1 May 1973https://doi.org/10.1021/bi00735a015RIGHTS & PERMISSIONSArticle Views26Altmetric-Citations38LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (901 KB) Get e-Alerts Get e-Alerts