Studies of lectin binding to the human cervix uteri: II. Cervical intraepithelial neoplasia and invasive squamous carcinoma

Summary The cell surface carbohydrate profile of formalin-fixed paraffin-embedded tissue sections of neoplastic cervical squamous epithelium was evaluated using lectins ofBauhinia purpurea (BPA),Canavalin ensiformis (Con A),Griffonia simplicifolia I (GS I),Griffonia simplicifolia II (GS II),Maclura pomifera (MPA),Archis hypogaea (PNA),Glycine max (SBA),Ulex europaeus I (UEA I) andTriticum vulgaris (WGA). Three lectins (BPA, Con A and PNA) showed a similar pattern of staining in both normal squamous epithelium and in cervical intraepithelial neoplasia (CIN). Variable alterations were seen in lectin-binding patterns in CIN with seven lectins (GS I, GS II, MPA, PNA, SBA, UEA I and WGA). A significant difference was seen between the intensity of staining of normal squamous epithelium and CIN with all lectins except WGA. The alteration in GS II-binding pattern and intensity was significantly related to grade of CIN. No correlation was found between lectin binding and the presence of koilocytes in squamous epithelium. Cases of invasive squamous carcinoma showed a heterogeneous lectin-binding pattern and no siginificant association was found between lectin binding and tumour differentiation or patient survival. These results indicate that neoplasia in cervical squamous epithelium is associated with alterations in terminal -Man residues, - and -GalNAc residues, - and -GlcNAc residues, - and -Gal residues, and -Fuc-containing residues, present in the outer parts of bothN-linked andO-linked glycoconjugates. The implications of these findings are discussed.     

Quantitative estimation of the surface carbohydrates on the infection structures of rust fungi with enzymes and lectins

Lectins of Triticum vulgaris (WGA), Concanavalia ensiformis (ConA), Phaseolus vulgaris (PHA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Griffonia simplicifolia (GSA II) and the enzymes endo-(13)--D-glucanase, exo-(13)--D-glucanase and laminarinase were tested for binding to the infection structures of Puccinia coronata and Uromyces appendiculatus. The enzymes and lectins were labeled with fluorescein and the fluorescence was measured with a microscope photometer. GSA II and ConA bound to all parts of the two rust fungi to a certain extent. The germ tubes of P. coronata bound at least two times more WGA than did the germ tubes of U. appendiculatus. The appressoria of both rust fungi additionally bound exo-(13)--glucanase, endo-(13)--glucanase and laminarinase. The substomatal vesicle and the infection hypha of both rust fungi mainly bound the glucanases. Furthermore, the substomatal vesicle of U. appendiculatus bound PHA. No obvious binding with LTA, RCA I and PNA was observed. Binding generally could be inhibited by appropriate haptens. Binding to uredospores generally appeared unspecific. The results indicate that the germ tubes have chitin on their outer surfaces, the appressoria chitin and glucans and the substomatal vesicles and infection hyphae mainly glucans. Compared to P. coronata, U. appendiculatus has more terminal linked glucose residues or the glucan has more (13)--linkages. Also, U. appendiculatus has N-acetylgalactosamine or a similar sugar on the surface of the substomatal vesicle.Abbreviations ConA Concanavalia ensiformis agglutinin - FITC fluorescein isothiocyanate - GSA II Griffonia simplicifolic agglutimin II - LTA Lotus tetragonolobus agglutinin - PBS phosphate buffered saline - PNA Peanut agglutinin - RCA I Ricinus communis agglutinin I - PHA Phaseolus vulgaris agglutinin - WGA Wheat germ agglutinin     

Enzymatic degradation and quantitative lectin labeling for characterizing glycoconjugates which act as lectin acceptors in cat submandibular gland

Summary Sites of binding of eight different lectins (LTA, UEA I, WGA, SBA, DBA, CON A, PNA, RCA I) to cat submandibular gland were studied after exposure of tissue sections to sialidase, -fucosidase, -galactosidase, -mannosidase, -N-acetylglucosaminidase. All lectins were affected by enzymatic predigestion and the labeling of individual lectins was highly dependent upon the glycosidase used to pretreat the sections. Glycoconjugates of demilunar, acinar and ductal cells exhibited a different composition of terminal sequences. For example, fucose proved to form the disaccharide fucose-galactose in demilunar and acinar cells, whereas it was present with the sequence fucose-N-acetyl-d-glucosamine in striated duct cells. Sialic acid participated both to the terminal sequence sialic acid-galactose and sialic acid-N-acetyl-d-galactosamine either in demilunar or in ductal cells. Lectin labeling combined with glycosidase digestion was also helpful in verifying the influence of neighbouring oligosaccharides on the affinity of lectins for the respective sugars.     

Ultrastructural distribution of lectin-binding sites on gastric superficial mucus-secreting epithelial cells

Normal human gastric epithelial cells were examined by electron microscopy using each of five biotinylated lectins [Ulex europaeus agglutinin I (UEA-I), peanut agglutinin (PNA), wheat germ agglutinin (WGA), soybean agglutinin (SBA) andDolichos biflorus agglutinin (DBA)] as a probe. We employed 35 gastric surgical specimens removed from complicated peptic disease. The lectin-binding sites were revealed with streptavidin-colloidal gold complex. All specimens were embedded in Spurr and LR White resins. In superficial foveolar epithelial cells, the lectins used were generally positive in all cell types (mainly UEA-1 and PNA) on the Golgi region and mucus cytoplasmic vacuoles, with many variations among cells in the same case. On the other hand, extracellular mucus was negative for WGA. Labelling with PNA revealed a biphasic pattern (peripheral positivity) on mucous droplets in surface and foveolar cells. Thecis side of the Golgi apparatus was labelled with SBA and PNA and rough endoplasmic reticulum with SBA (only five cases). Lectin-binding variability could be related to heterogeneous composition of gastric mucus. Our results with SBA suggest initiation ofO-glycosylation at the Golgi apparatus; however a role of the rough endoplasmic reticulum cannot be excluded (N-glycosylation). We propose the following sequence of sugar addition to the carbohydrate side-chains of gastric glycoproteins: (1) GaNAc (Golgi apparatuscis-side), (2) GlcNAc (Golgi apparatus intermediate face), (3) GalNac or Gal, -l-fucose (Golgi apparatustrans-side).Supported by a grant from Junta de Andalucía (Consolidación de Grupos de Investigación. Ref. 541A.6.60.609.018311)     

Diabetes mellitus associated changes in glomerular glycocompounds: A fluorescence microscopical study

Summary Renal biopsy specimens showing histological alterations typical of advanced diabetic glomerulopathy were studied for changes in glomerular glycoconjugates, using fluorochrome-coupled lectins as probes. All samples studied showed a marked reduction in the binding ofTriticum vulgaris (WGA) lectin in the glomerular basement membranes. On the other hand, new glomerular binding sites for the lectins ofDolichos biflorus (DBA),Helix pomatia (HPA) andArachis hypogaea (PNA), recognizing galactosyl moieties of glycoconjugates and giving no reaction in normal glomeruli, were seen in all samples studied. In addition,Wistaria floribunda lectin (WFA), recognizing galactosyl and.N-acetylgalactosaminyl configurations in glycoconjugates, gave a typical linear binding along the glomerular basement membranes, differing markedly from its reaction with normal kidney.Ulex europaeus (UEA I) showed reduced binding in the glomeruli of diabetic nephropathy.The results show that changes in glomerular glycoconjugates may appear in diabetic nephropathy, suggesting a disturbance in the turnover of the non-reducing terminal saccharide residues. In addition, the results show that lectins are useful probes for studying these changes further.     

Use of a biosensor to determine the binding kinetics of five lectins for Galactosyl-N-acetylgalactosamine

The dietary lectins, edible mushroom (ABL) and Jacalin (JAC) inhibit the proliferation of colonic cancer cells, whereas Amaranth (ACL) and peanut (PNA) stimulate their proliferation. All these lectins share as their preferred ligand the Thomsen-Friedenreich (TF) antigen galactosyl 1,3 N-Acetylgalactosamine (Gal1,3GalNAc), but differ in their finer specificities for modifications of this determinant and in their specificities for cancerous epithelia. We have investigated, using a resonant mirror biosensor, the kinetics of binding of these lectins, and Maclura pomifera lectin (MPL), which is similar to JAC, to two different Gal-GalNac bearing glycoproteins, antarctic fish antifreeze glycoprotein (AFG) and asialofetuin. JAC had the highest affinity for AFG [K _d 0.027 M] due to a fast association rate constant [k _ass 610,000 (Ms)^–1]. The other lectins had considerably lower affinities, with K _d ranging from 0.16 M (ABL) to 5.7 M (PNA), largely due to slower k _ass [ABL 74,000 (Ms)^–1 to PNA 2700 (Ms)^–1]. Similarly, JAC had a much higher affinity for asialofetuin [K _d 0.083 M] than the other lectins [K _d 1.0 M–4.5 M]. Affinities were also calculated from the extent of binding at equlibrium and were generally similar to those calculated from the kinetic parameters indicating the true nature of these values.     

Characterization of the sugar-binding specificity of the toxic lectins isolated from Abrus pulchellus seeds

The sugar-binding specificity of the toxic lectins from Abrus pulchellus seeds was investigated by combination of affinity chromatography of glycopeptides and oligosaccharides of well-defined structures on a lectin-Sepharose column and measurement of the kinetic interactions in real time towards immobilized glycoproteins. The lectins showed strong affinity for a series of bi- and triantennary N-acetyllactosamine type glycans. The related asialo-oligosaccharides interact more strongly with the lectins. The best recognized structures were asialo-glycopeptides from fetuin. Accordingly, the kinetic interaction with immobilized asialofetuin was by far the most pronounced. Human and bovine lactotransferrins and human serotransferrin interacted to a lesser extent. The interaction with asialofetuin was inhibited by galactose in a dose dependent manner. Lactose, N-acetyllactosamine and lacto-N-biose exhibited similar degree of inhibition while N-acetylgalactosamine was a poor inhibitor. These results suggested that the carbohydrate-binding site of the Abrus pulchellus lectins was specific for galactose and possess a remarkable affinity for the sequences lactose [-D-Gal-(14)-D-Glc], N-acetyllactosamine [-D-Gal-(14)-D-GlcNAc] and lacto-N-biose [-D-Gal-(13)-D-GlcNAc].     

Light microscopic characterization of glycoconjugates in secretory cells of the carp (Cyprinus carpio) gill epithelium

Summary Secretory products of granular and mucous cells in the gill epithelium of the carp, Cyprinus carpio, were distinguished by their cytochemical reactions with peroxidase-labelled lectins and with the galactose oxidase (GO)-Schiff reagents. Secretory products of granular cells reacted with lectins from Triticum vulgaris (WGA), Arachis hypogaea (PNA), Dolichos biflorus (DBA), Glycine max (SAB), and Lotus tetragonolobus (LTA). They also reacted with GO-Schiff reagents. After sialic acid cleavage with HCl, new binding sites for DBA and SBA appeared, suggesting the terminal sequence sialic acid-N-acetylgalactosamine (SA-GalNAc) for the secretion of this cell type. In mucous cells, binding sites for WGA, DBA, and SBA and, after acid hydrolysis, binding sites for PNA and a positive GO-Schiff reaction were detected. The terminal trisaccharide sialic acid-galactose (1-3)-N-acetylgalactosamine (SA-Gal-GalNAc) is proposed for the secretion of mucuous cells. These cytochemical differences are discussed in light of the involvement of both cell types in fish mucus elaboration.     

Binding interactions of glycoproteins with lectins

Summary Since plant lectins were used to help define differences between normal and transformed cell surfaces (reviewed in References^1–4), they have been employed in many other situations where their sugar-recognition specificities could be used to advantage. One of these applications has been the purification and characterization of enzymes and other proteins; this work is reviewed here in order to define some of the variables that affect binding of glycoproteins to lectins, as well as to demonstrate how this technique has been profitably exploited for isolation of purified glycoproteins, and for their better understanding.Abbreviations ConA concanavalin A - WGA wheat germ agglutinin - RCA₆₀ (RCA_II, mol. wt. 60,000) and RCA₁₂₀ (RCA, mol. wt. 120,000) Ricinus communis agglutinin - LCA Lens culinaris agglutinin - LTA Lotos tetragonolobus agglutinin - SBA soybean agglutinin - PNA peanut agglutinin - Me--Glc methyl--glucoside - Me--Man methyl--mannoside - Gal galactose - GlcNac N-acetylglucosamine     

Glycoconjugate Expression During Early Mouse Oculogenesis

The carbohydrate side-chain of glycoconjugates can show a developmentally regulated expression pattern. In order to analyse these changes during the development of the eye, 13 lectins were used to reveal glycoconjugates histochemically in 8.5- to 14-day- old mouse embryos. During this period, eyes develop from the most immature vesiculation of the neural plate neuroepithelium into a primitive stage with all structures present, such as pigment epithelium, not yet differentiated neuroretina and lens. A striking diversity of carbohydrate side-chain expression was observed in the preocular somatoectoderm and neural plate of 8.5- day-old embryos, as indicated by the binding of nine different lectins. Binding sites at the apical poles of neuroepithelium of five of these lectins (PNA, LCA, SBA, LPA and GSA-II) disappeared completely during further development. The binding sites of four other lectins, WGA, MPA, Con A and BPA, remained expressed during the course of development, being indicative for the carbohydrate side-chains -GlcNAc(1-4)Gluc, -Gal(1-3)GalNAc, -D-Man/-D-Gluc and -GalNAc. In contrast, binding sites for GSA-I, RCA-I (-D-Gal), UEA-I (-l- Fuc) and DBA (-GalNAc(1-3)GalNAc) were not present at any developmental stage. The time point of gross changes of lectin binding sites correlates well with the period of neural tube formation. During later development from neuroectoderm to the ocular pigment epithelium, a sharp reduction in all lectin binding sites at the apical cell poles, except for WGA and MPA, was observed. WGA binding sites were present until embryonic day 10, while those for MPA were present until day 9. At the basal cell poles of the pigment epithelium, all lectin binding sites except for WGA were lost after e mbryonic day 11.5. These results indicate that there are sophisticated kinetics of glycoconjugate expression during the course of early embryonic development of ectoderm into its descendent tissues.     

Peptide nucleic acid (PNA): A model structure for the primordial genetic material?

It is proposed that the primordial genetic material could have been peptide nucleic aicds,i.e., DNA analogues having a peptide backbone. PNA momomers based on the amino acid, , -diaminobutyric acid or ornithine are suggested as compounds that could have been formed in the prebiotic soup. Finally, the possibility of a PNA/RNA world is presented, in which PNA constitutes the stable genetic material, while RNA which may be polymerized using the PNA as template accounts for enzymatic activities including PNA replication.     

Lectin-histochemical analysis of glycans in ovine and bovine near-term placental binucleate cells

Chorionic binucleate cells (BNC) occur in several ruminants including cow, deer, goat and sheep. They migrate through the chorionic tight junction to fuse with uterine epithelial cells and discharge their granules into maternal connective tissue. We have compared the BNC of near-term, resin-embedded, ovine and bovine placentae using 15 biotinylated lectins and an avidinperoxidase revealing system. There was pronounced conservation of saccharides between the two species. Several sub-types of N-glycan were present, with highly branched structures being abundant, as shown by Galanthus nivalis, Pisum sativum and Phaseolus vulgaris (leuko) agglutinins. Among the non-reducing terminal saccharides conserved were GalNAc1,3(Fuc1,2)-Gal1,4GlcNAc1-, GalNAc1,6Gal1-, Gal1,4GlcNAc-and Gal1,3GalNAc1- shown by Dolichos biflorus, Wisteria floribunda, Erythrina cristagalli, and Maclura pomifera agglutinins, respectively. Arachis hypogaea and Glycine max agglutinins tended to bind to bovine BNC at different stages of maturity, while fucosyl residues detectable by Tetragonolobus purpureus and Ulex europaeus-1 agglutinins were not observed in either species. The only major difference related to sialyl residues, with 2,3-linked sialic acid being present in bovine (Maackia amurensis, Limax flavus) and 2,6 sialic acid being present in ovine (Sambucus nigra agglutinin) cells. This conservation of glycan may be related to glycosylation of peptide hormones in the granules, and may thus be important in the targeting of these hormones to their receptors.     

Characterization by lectin binding of the sugar moiety of glycocompounds stored in inherited diseases

Summary Kidney and liver samples from two cases of Fabry's disease and spleen and liver samples from Gaucher and Niemann—Pick diseases were tested for binding to lectins such as peanut agglutinin (PNA),Bandeiraea simplicifolia, (BSA),canavalia ensiformis (Con A), soybean agglutinin (SBA) and wheat germ agglutinin (WGA) labelled with horseradish peroxidase using histochemical techniques. These techniques allowed the localization of compounds with -galactosyl residues in tissues from Fabry's disease. In tissues from the Gaucher and Niemann—Pick cases, the storage material was found to be more complex than expected, and some problems regarding the significance of lectin binding are discussed.     

A dual staining method for identifying mucins of different gastric epithelial mucous cells

Summary A dual staining method has been developed to identify two types of mucous secreting cells in the gastric mucosa of human and rat in one and the same tissue section. Sections were stained first using the galactose oxidase-cold thionin Schiff (GOCTS) procedure and then with paradoxical Concanavalin A staining (PCS). Surface mucous cell mucin stained blue with GOCTS, whereas gland mucous cell mucin stained brown with PCS. This method enabled us to differentiate these two types of mucins not only in gastric epithelial cell cytoplasm but also in the extracellular space. Sugar residues detected by GOCTS were explored by employing four species of lectins, which were peanut andAllomyrina dichotoma agglutinins for -galactose andVicia villosa andWistaria floribunda agglutinins for -N-acetylgalactosamine. The effect of oxidation with galactose oxidase was also examined on the affinities of reactive sites for these lectins. The results indicated that, in the human stomach, the sugar residues responsible for this reactivity were most likely -N-acetylgalactosamine and -galactose in specimens lacking secretion of blood group determinants and -N-acetylgalactosamine in those showing the secretion. In the rat stomach, on the other hand, sugar residues responsible for GOCTS were not elucidated by these lectins.     

Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.     

Use of sialylated or sulfated derivatives and acrylamide copolymers of Galβ1,3GalNAcα- and GalNAcα- to determine the specificities of blood group T- and Tn-specific lectins and the copolymers to measure anti-T and anti-Tn antibody levels in cancer patients

Sialylated or sulfated derivatives and acrylamide copolymers of blood group T-(Gal1,3GalNAc-) and Tn-(GalNAc) haptens were studied for their interaction with the lectins of peanut (PNA),Agaricus bisporus-(ABA),Helix pomatia-(HPA) andVicia villosa B₄-(VVA), using asialo Cowper's gland mucin (ACGM), which contains both T and Tn epitopes, as the coating substrate in enzyme linked lectin assay. Both T and Tn copolymers (40 haptens) showed high affinity and strict specificity; although the T-copolymer at 0.05–0.07 µm concentration caused 50% inhibition of interaction of either PNA or ABA with ACGM, there was little inhibition of the HPA and VVA interactions at over 100 times that concentration. The Tn-copolymer at 0.02–0.05 µm inhibited HPA or VVA interaction with ACGM by 50% but gave virtually no inhibition of PNA and ABA binding. Sialyl, sulfate or methyl group substitution on C-6 of GalNAc of the T-haptene did not prevent interaction with PNA but almost abolished interaction with ABA. In contrast, sialyl or sulfate group on C-6 and sulfate on C-3 of Gal in Gal1,3GalNAc- inhibited almost completely the interaction of PNA with ACGM but had only a slight effect on the interaction of ABA; C-6 substitution with either sialic acid or sulfate on GalNAc- almost abolished the interaction of both HPA and VVA with ACGM. Preliminary studies revealed a significant depression in the serum level of anti-T (two to three-fold decrease) and anti-Tn ( two-fold decrease) antibodies in breast cancer compared with normal control subjects when the acrylamide T- and Tn-copolymers were used as coating substrates in enzyme linked immunoassays.Abbreviations PNA peanut agglutinin - ABA agaricus bisporus agglutinin - HPA helix pomatia agglutinin - VVA (B4),vicia villosa agglutinin - ACGM Asialo Copwer's gland mucin - CA carcinoma - BSA bovine serum albumin - HRP Horseradish peroxidase - ABTS 2,2-azino-di (3-ethyl-benzthiazoline sulfonate) - ELISA enzyme-linked immunosorbent assay - Al allyl - Bn benzyl - AA acrylamide - CP copolymer     

Differential response of the epidermal growth factor receptor tyrosine kinase activity to several plant and mammalian lectins

Biosignalling via lectins may involve modulation of protein kinase activities. This aspect of the biological action of mammalian and plant lectins has been investigated for their effect on the activity of the isolated epidermal growth factor receptor (EGFR). The constitutive tyrosine kinase activity of the epidermal growth factor receptor from rat liver, isolated by calmodulin-affinity chromatography, was activated by concanavalin A (ConA), and wheat germ agglutinin (WGA) to a similar extent as the measured enhancement induced by EGF. In contrast, two mannose-specific lectins, the mannan-binding protein (MBP) and serum amyloid P component (SAP), isolated from human serum, have inhibitory effects, both in the absence and presence of EGF. The differential effects of these lectins were tested using as phosphorylatable substrates a co-polymer of glutamic acid-tyrosine, as well as calmodulin. However, two galactoside-specific lectins, the laminin-binding -galactoside-binding 14 kDa lectin, isolated from bovine heart (14K-BHL), and the /-galactoside-binding lectin, isolated from mistletoe (Viscum album L.) leaves (VAA), do not inhibit the EGFR tyrosine kinase activity. The sugar dependence of the lectin-mediated action was studied by inhibition assays. Mannose and a mannose-containing neoglycoprotein prevent the activating effect of ConA, and N-acetyl-D-glucosamine partially prevents the activation produced by WGA. However, mannose and mannose-containing neoglycoprotein were ineffective to reduce the inhibitory effect of MBP or SAP. Although the response to binding of ConA and WGA was different to that of MBP or SAP with respect to the tyrosine kinase activity of the EGFR, it should be noted that the four lectins inhibited the binding of [¹²⁵I]EGF to its receptor with similar efficiency.Abbreviations EGF epidermal growth factor - EGFR epidermal growth factor receptor - ConA concanavalin A - MBP mannan-binding protein - SAP serum amyloid P component - WGA wheat germ agglutinin - 14K-BHL bovine heart 14 kDa lectin - VAA Viscum album L. (mistletoe) agglutinin - EGTA [ethylenebis(oxyethylenenitrilo)]-tetraacetic acid; poly(Glu:Tyr)-co-polymer of L-glutamic acid and L-tyrosine - Hepes 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - Tris tris(hydroxymethyl)-aminomethane - DSS suberic acid bis(N-hydroxy-succinimide ester) - PMSF phenylmethanesulfonyl fluoride - Man mannose - Gal galactose - BSA bovine serum albumin - Man-BSA neoglycoprotein containing -D-mannose - Lac-BSA neoglycoprotein containing -lactose - Gal-BSA neoglycoprotein containing galactose     

Lectin histochemistry of mammalian Brunner's glands

Summary Lectin histochemical study was performed on twenty-eight specimens of formalin-fixed paraffin embedded tissues of proximal duodenum from human, cat, dog and Rhesus (macaque) monkey to demonstrate the pattern of carbohydrate residues in submucosal glands of Brunner as compared to that of the duodenal absorptive and goblet cells. Ten different biotinylated lectins were used as probes, and avidin-biotin-peroxidase (ABC) or avidin-gold-silver (AGS) complexes were used as visualants. Brunner's gland cells of the four species studied exhibited a similar lectin-binding pattern which differ from other duodenal cells. The epithelium of Brunner's gland stained intensely with Ricinus communis agglutinin-I (RCA-I), succinylated-WGA (S-WGA) and wheat-germ agglutinin (WGA), moderately with Bandeirea simplicifolia agglutinin-I (BS-I), Concanavalia ensiformis agglutinin (Con A) peanut agglutinin (PNA) and Ulex europaeus agglutinin-I (UEA-I) and occasionally with Dolichos biflorus agglutinin (DBA), Lens culinaris agglutinin (LCA) and soybean agglutinin (SBA). Desialylation with neuraminidase resulted in only a slight elevation in binding intensities of PNA, DBA and SBA, indicating that glycoconjugates of the Brunner's gland cells are rich in asialo-oligosaccharides, which differs from duodenal epithelial cells. In addition, these histochemical reagents were useful in localizing Brunner's gland elements in the duodenal mucosa.     

Appearance of lectin-binding sites during vascularization of the primordium of the central nervous system in 10 to 12-day-old mouse embryos

Summary In the present study lectin-binding sites were investigated for the lectins Ricinus communis agglutinin (RCA I), wheat germ agglutinin (WGA), soya bean agglutinin (SBA), concanavalin A (Con A), Lotus tetragonolobus(LTA) and Limulus polyphemus agglutinin (LPA) during the initial stages of vasculogenesis of the CNS-anlage in 10 to 12-day-old NMRI mouse embryos. Specific binding sites for the lectins RCA I (sugar specificity: -D-galactose, N-acetylgalactosamine), WGA (sugar specificity: N-acetylglucosamine, sialic acid), and SBA (sugar specificity: N-acetylgalactosamine, -D-galactose) were detected in the newly formed capillaries within the neuroepithelial cell layer. In contrast, binding sites for Con A, LTA and LPA could not be observed at the start of the vascularization of the CNS-anlage. From these results, the conclusion can be drawn that glycoconjugates containing D-galactose, N-acetylgalactosamine and N-acetyl-glucosamine moieties are involved in the early vasculogenesis of the embryonic CNS-anlage of the mouse.     

Lectin binding sites inParamecium tetraurelia cells

Summary Though all three lectins tested (ConA, RCA II, WGA) bound to the entire cell membrane, none bound selectively to the docking site of secretory organelles (trichocysts); the same results were achieved with FITC-conjugates, or, on the EM level, with peroxidase- or gold-labeling. Only WGA triggered the release of trichocysts and none of the lectins tested inhibited AED-induced synchronous exocytosis.When exocytosis was triggered synchronously in the presence of any of these three lectins (FITC-conjugates), the resulting ghosts trapped the FITC-lectins and the cell surface was immediately afterwards studded with regularly spaced dots (corresponding to the ghosts located on the regularly spaced exocytosis sites). These disappeared within about 10 min from the cell surface (thus reflecting ghost internalization with a half life of 3 min) and fluorescent label was then found in 6–10 vacuoles, which are several m in diameter, stain for acid phosphatase and, on the EM level, contain numerous membrane fragments (other-wise not found in this form in digesting vacuoles). We conclude that synchronous massive exocytosis involves lysosomal breakdown rather than reutilization of internalized trichocyst membranes and that these contain lectin binding sites (given the fact free fluorescent probes did not efficiently stain ghosts).Trichocyst contents were analyzed for their lectin binding capacity in situ and on polyacrylamide gels. RCA II yielded intense staining (particularly of tips), while ConA (fluorescence concentrated over bodies) and WGA yielded less staining of trichocyst contents on the light and electron microscopic level. Only ConA- and WGA-staining was inhibitable by an excess of specific sugars, while RCA II binding was not. ConA binding was also confirmed on polyacrylamide gels which also allowed us to assess the rather low degree of glycosylation (1% by comparison with known glycoprotein standards) of the main trichocyst proteins contained in their expandable matrix.Since RCA II binding could be due to its own glycosylation residues we looked for an endogenous lectin. The conjecture was substantiated by the binding of FITC-lactose-albumin (inhibitable by a mixture of glucose-galactose). This preliminary new finding may be important for the elucidation of trichocyst function.Abbreviations AED aminoethyldextran - BSE backscatter electrons - ConA Concanavalin A - DAB 3,3-diaminobenzidine - EM electron microscope - FITC fluorescein-isothiocyanate - kD kiloDalton - ME mercaptoethanol - MIP membrane-intercalate particle - M_r apparent molecular weight - PAGE polyacrylamide-gel-electrophoresis - PAS periodic acid Schiff - pI isoelectric point - POX peroxidase - RCA II Ricinus communis agglutinin II - SDS sodium dodecylsulphate - SEM scanning electron microscope - WGA wheat germ agglutinin