A histochemical study of the distribution of lectin binding sites in the developing oocytes of the lancelet Branchiostoma belcheri

The distribution of carbohydrate moieties in lancelet (Branchiostoma belcheri) oocytes has been studied at different stages of development, using a peroxidase-labeled lectin incubation technique, the PAS-reaction and Alcian Blue staining. Binding sites of 5 lectins, indicating the presence of different sugar moieties (Wheat germ agglutinin (WGA) for N-acetylglucosamine, Concanavalin A (Con A) for glucose/mannose, Helix pomatia agglutinin (HPA) for N-acetyl-D-galactosamine, Ricinus communis agglutinin (RCA-I) for galactose and Ulex europaeus agglutinin (UEA-I) for fucose), were identified and were shown to undergo considerable variation during oocyte development. In the previtellogenic stage, HPA, RCA-I and UEA-I were not identified on the oocyte surface, but WGA and Con A gave strongly positive reactions at this site. In the cytoplasm, 4 lectins (Con A, HPA, RCA-I and UEA-I) gave a weak or moderate reaction, and Con A was also observed in the perinuclear region. In vitellogenic oocytes, these 4 lectins were found to also bind to the nuclear envelope, karyoplasm and nucleolus, and, with the exception of Con A, could also be found in the nuclei of more mature stages. The cytoplasmic yolk granules and Golgi vesicles of the vitellogenic oocyte, were moderately positive for Con A, HPA, RCA-I and UEA-I, but HPA, RCA-I and UEA-I were only weakly bound at the oocyte surface. In mature oocytes, all 5 lectins bound moderately or strongly to yolk granules and cell surface. HPA, RCA-I and UEA-I bound moderately or strongly to various nuclear compartments. Thus, carbohydrate content varied with the development and maturation of the oocytes, and the PAS results were in agreement with the lectin-binding results. Charged carbohydrate residues were observed in the egg envelope and Golgi bodies.These results suggest that the appearence of Con A-, HPA-, RCA-I- and UEA-I-binding glycoconjugates in the nuclei of developing oocytes show a varying pattern indicating different phases of nuclear activity which correlate with different carbohydrate synthetic activities of the oocyte.     

Lectin binding sites on CD34+ human haematopoietic stem cells and lymphocytes from peripheral blood: an ultrastructural post-embedding study

This study was performed to obtain a better insight into the glycosylation pattern of human CD34⁺ haematopoietic stem cells and lymphocytes from peripheral blood using an ultrastructural post-embedding technique. Lectins applied were derived from Canavalia ensiformis (Con A), Triticum vulgare (WGA), Lycopersicon esculentum (LEA), Limulus polyphemus (LPA), Ulex europaeus-I (UEA-I), Bauhinia purpurea (BPA), Glycine max (SBA), Helix pomatia (HPA), Arachis hypogaea (PNA) and Erythrina cristagalli (ECA). Our results showed almost identical staining patterns with both CD34⁺ cells and mature lymphocytes from peripheral blood. Con A displayed a prominent reactivity with the nuclear envelope and a weak staining of the plasma membrane. As demonstrated by an elaborate lectin double-labelling technique, WGA revealed an opposite staining pattern. Following neuraminidase treatment of sections, BPA, PNA and SBA exhibited a prominent staining of the plasma membrane in CD34⁺ cells and lymphocytes as well. Membrane reactivity with HPA was restricted to the majority of lymphocytes, presumably T-lymphocytes. Infrequently occurring dense cytoplasmic (lysosomal) bodies were reactive with a variety of lectins, and a weak diffuse nuclear labelling was observable with LPA, UEA-I, WGA and Con A. It is tempting to speculate that carbohydrate moieties on plasma membranes may be involved in the complex mechanisms characterizing cell-to-cell interactions (adhesion) and particularly in the so-called phenomenon of homing. This revised version was published online in November 2006 with corrections to the Cover Date.     

Electron microscopic demonstration of lectin binding sites in the taste buds of the European catfish Silurus glanis (Teleostei)

Summary Taste buds in the European catfish Silurus glanis were examined with electron microscopic lectin histochemistry. For detection of carbohydrate residues in sensory cells and adjacent epithelial cells, gold-, ferritin-and biotin-labeled lectins were used. A post-embedding procedure carried out on tissue sections embedded in LR-White was applied to differentiate between the sensory cells: The lectins from Helix pomatia (HPA) and Triticum vulgare (WGA) bound to N- acetyl-galactosamine and to N-acetylglucosamine residues occurring especially in vesicles of dark sensory cells. This indicates a secretory function of these cells. Most light sensory cells — with some exceptions, probably immature cells —, are HPA-negative. The mucus of the receptor field and at the top of the adjacent epithelial cells was strongly HPA-positive. Pre-embedding studies were performed in order to obtain information about the reaction of the mucus with lectins under supravital conditions. The mucus of the taste bud receptor field exhibited intensive binding to WGA, but not to the other lectins tested. Most lectins bound predominantly to the surface mucus of the nonsensory epithelium and to the marginal cells close to the receptor field. The strong lectin binding to mucins and the relatively weak lectin binding to cell surface membranes in pre-embedding studies suggest that the mucus possibly serves as a barrier which is passed selectively only by a small amount of lectins or lectincarbohydrate complexes. Lectin-carbohydrate interactions may play a role in recognition phenomena on the plasmalemmata of the taste bud sensory cells. Recognition processes directed to bacteria or viruses should be considered as well.Parts of this investigation were presented at the XI. Annual Meeting of the Association for Chemoreception Sciences (AChemS XI), held at Sarasota, Fl, April 12–14, 1989 (Witt and Reutter 1989)     

Heterogeneity of the blood group ABH antigens and variation in the expression of these antigens of secretory granules in human cervical glands

Summary Cytochemical localization of blood group ABH antigens was examined in secretory cells of human cervical glands by application of a post-embedding lectin-gold as well as immuno-gold labeling procedure using monoclonal antibodies. Blood group specific lectins such as Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Griffonia simplicifolia agglutinin I-B₄ (GSAI-B₄) and Ulex europaeus agglutinin-I (UEA-I) reacted with secretory granules but not with other cytoplasmic organellae such as nucleus and cell membrane. The reactivity of secretory granules with these lectins showed strict dependence on the blood group and secretor status of tissue donors. The binding patterns with these lectins were not homogeneous, but exhibited marked cellular and subcellular heterogeneity. Thus, for example, in blood group A individuals, some granules were stained strongly with DBA and others were weakly or not at all with the lectin. Such a heterogenous labeling with the lectin was observed even in the same cells. Similar results were obtained with UEA-I and GSAI-B₄ staining in blood group O and B secretor individuals, respectively. Monoclonal antibodies likewise reacted specifically with the granules but they occasionally bound to some nucleus. The labeling pattern of the antibodies with the granules was essentially the same as those of lectins. However, difference was also observed between monoclonal antibody and lectin staining, that is, monoclonal anti-A antibody reacted weakly but consistently with granules from blood group A nonsecretors but DBA (HPA) did not; staining with UEA-I was observed in granules from the secretor individuals of any blood groups whereas monoclonal anti-H antibody reacted with granules from blood group O and some A secretor individuals but not from B and AB secretor individuals; GSAI-B₄ reacted uniformly with granules throughout the cells whereas monoclonal anti-B antibody bound to limited number of granules in the same cells. This was confirmed by the double labeling experiments with the lectin and the antibody. These results suggest that the different types of antigens as to the binding ability for monoclonal antibodies and lectins are expressed on different granules in the same cell.     

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

Summary 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 cisternac 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.     

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.     

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

Summary 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 forWistaria floribunda (WFA),Helix pomatia (HPA) andDolichos biflorus (DBA) agglutinins could be seen in the kidneys studied, whereas the binding sites forUlex 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.     

Lectin binding in the anterior segment of the bovine eye

Summary Eleven different fluorescent lectin-conjugates were used to reveal the location of carbohydrate residues in frozen sections of the anterior segment of bovine eyes. The lectins were specific for the following five major carbohydrate groups: (1) glucose/mannose group (Concanavalin A (Con A)); (2)N-acetylglucosamine group (wheat germ agglutinin (WGA)); (3) galactose/N-acetylgalactosamine group (Dolichos biflorus agglutinin (DBA),Helix pomatia agglutinin (HPA),Helix aspersa agglutinin (HAA),Psophocarpus tetragonolobus agglutinin (PTA),Griffonia simplicifolia agglutinin-I-B₄ (GSA-I-B₄),Artocarpus integrifolia agglutinin (JAC), peanut agglutinin (PNA) andRicinus communis agglutinin (RCA-I)); (4)l-fucose group (Ukex europaeus agglutinin (UEA-I)); (5) sialic acid group (wheat germ agglutinin (WGA)). All the studied lectins except UEA-I reacted widely with different structures and the results suggest that there are distinct patterns of expression of carbohydrate residues in the anterior segment of the bovine eye. UEA-I bound only to epithelial structures. Some of the lectins reacted very intensely with apical cell surfaces of conjunctival and corneal epithelia suggesting a different glycosylation at the glycocalyx of the epithelia. Also, the binding patterns of conjunctival and corneal epithelia differed with some of the lectins: PNA and RCA-I did not bind at all, and GSA-I-B₄ bound only very weakly to the epithelium of the cornea, whereas they bound to the epithelium of the conjunctiva. In addition, HPA, HAA, PNA and WGA did not bind to the corneal basement membrane, but bound to the conjunctiva and vascular basement membranes. This suggests that corneal basement membrane is somehow different from other basement membranes. Lectins with the same carbohydrate specificity (DBA, HPA, HAA and PTA) reacted with the sections almost identically, but some differences were noticed: DBA did not bind to the basement membrane of the conjunctiva and the sclera and did bind to the basement membrane of the cornea, whereas other lectins with same carbohydrate specificities reacted vice versa. Also, the binding of PTA to the trabecular meshwork was negligible, whereas other lectins with the same carbohydrate specificities reacted with the trabecular meshwork. GSA-I-B₄ reacted avidly with the endothelium of blood vessels and did not bind to the stroma, so that it made blood vessels very prominent and it might be used as an endothelial marker. This lectin also reacted avidly with the corneal endothelium. Therefore, GSA-I-B₄ appears to be a specific marker in bovine tissues for both blood vessel and corneal endothelium cells.     

Cytochemical localization of blood group substances in human salivary glands using lectin-gold complexes

We investigated localization of blood group antigens and their related substances in human labial salivary and submandibular glands by application of a post-embedding cytochemical staining procedure using lectin- or glycoprotein-gold complexes. Surgical tissue was obtained from 10 patients. Blood group-specific lectins, such as Dolichos biflorus agglutinin or Helix pomatia agglutinin (group A-specific), Griffonia simplicifolia agglutinin-I B4 (group B-specific), and Ulex europaeus agglutinin I (group H-specific) could recognize A, B, and H antigens, respectively, only in mature secretory granules (mature SG), which were found preferentially in cells in the late phase of the maturation cycle. In immature secretory granules (immature SG), which were found in cells in the early or middle phase of the maturation cycle, no binding with these lectins was observed. The Golgi complexes and endoplasmic reticula also were not labeled with these lectins. In blood group O and B secretors, blood group antigens were uniformly distributed throughout all the mature SG examined. However, in blood group A secretors, the distribution was heterogeneous, i.e., in some granules only H antigen was demonstrated, whereas in others both A antigens and a small amount of H antigens were detected. Among the blood group-nonspecific lectins, wheat germ agglutinin (WGA) was found to bind more preferentially to immature SG than to mature SG. This was demonstrated irrespective of the blood group and secretor status of the tissue donor, except that in blood group A secretors WGA bound strongly to some mature SG which possessed A antigen. We discuss the significance of cellular and subcellular mosaic distribution of blood group antigens in connection with morphological differences of secretory granules and the maturation cycle of mucous cells.     

Heterogeneity of the blood group ABH antigens and variation in the expression of these antigens of secretory granules in human cervical glands. An electron microscopic observation using lectins and monoclonal antibodies

Cytochemical localization of blood group ABH antigens was examined in secretory cells of human cervical glands by application of a post-embedding lectin-gold as well as immuno-gold labeling procedure using monoclonal antibodies. Blood group specific lectins such as Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) and Ulex europaeus agglutinin-I (UEA-I) reacted with secretory granules but not with other cytoplasmic organellae such as nucleus and cell membrane. The reactivity of secretory granules with these lectins showed strict dependence on the blood group and secretor status of tissue donors. The binding patterns with these lectins were not homogeneous, but exhibited marked cellular and subcellular heterogeneity. Thus, for example, in blood group A individuals, some granules were stained strongly with DBA and others were weakly or not at all with the lectin. Such a heterogenous labeling with the lectin was observed even in the same cells. Similar results were obtained with UEA-I and GSAI-B4 staining in blood group O and B secretor individuals, respectively. Monoclonal antibodies likewise reacted specifically with the granules but they occasionally bound to some nucleus. The labeling pattern of the antibodies with the granules was essentially the same as those of lectins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopic demonstration of lectin binding sites in the taste buds of the European catfish Silurus glanis (Teleostei)

Taste buds in the European catfish Silurus glanis were examined with electron microscopic lectin histochemistry. For detection of carbohydrate residues in sensory cells and adjacent epithelial cells, gold-, ferritin- and biotin-labeled lectins were used. A post-embedding procedure carried out on tissue sections embedded in LR-White was applied to differentiate between the sensory cells: The lectins from Helix pomatia (HPA) and Triticum vulgare (WGA) bound to N-acetyl-galactosamine and to N-acetylglucosamine residues occurring especially in vesicles of dark sensory cells. This indicates a secretory function of these cells. Most light sensory cells--with some exceptions, probably immature cells--, are HPA-negative. The mucus of the receptor field and at the top of the adjacent epithelial cells was strongly HPA-positive. Pre-embedding studies were performed in order to obtain information about the reaction of the mucus with lectins under supravital conditions. The mucus of the taste bud receptor field exhibited intensive binding to WGA, but not to the other lectins tested. Most lectins bound predominantly to the surface mucus of the nonsensory epithelium and to the marginal cells close to the receptor field. The strong lectin binding to mucins and the relatively weak lectin binding to cell surface membranes in pre-embedding studies suggest that the mucus possibly serves as a barrier which is passed selectively only by a small amount of lectins or lectin-carbohydrate complexes. Lectin-carbohydrate interactions may play a role in recognition phenomena on the plasmalemmata of the taste bud sensory cells. Recognition processes directed to bacteria or viruses should be considered as well.     

Lectin histochemistry of the gastric mucosa in normal and Helicobacter pylori infected guinea-pigs

Helicobacter pylori attaches via lectins, carbohydrate binding proteins, to the carbohydrate residues of gastric mucins. Guinea-pigs are a suitable model for a H. pylori infection and thus the carbohydrate composition of normal and H. pylori infected gastric mucosa was investigated by lectin histochemistry. The stomach of all infected animals showed signs of an active chronic gastritis in their mucosa, whereas no inflammation was present in the control animals. The corpus–fundus regions of the controls showed heterogeneous WGA, SNA-I, UEA-I and HPA binding in almost all parts of the gastric glands. While these lectins labelled the superficial mucous cells and chief cells heterogeneously, the staining of the parietal cells was limited to WGA and PHA-L. Mucous neck cells reacted heterogeneously with UEA-I, HPA, WGA and PHA-L. In the antrum, the superficial mucous cells and glands were stained by WGA, UEA-I, HPA, SNA-I or PHA-L. WGA, UEA-I, SNA-I and HPA labelled the surface lining cells strongly. The mucoid glands reacted heterogeneously with WGA, UEA-I, HPA, SNA-I and PHA-L. In both regions, the H. pylori infected animals showed similar lectin binding pattern as the controls. No significant differences in the lectin binding pattern and thus in the carbohydrate composition between normal and H. pylori infected mucosa could be detected, hence H. pylori does not induce any changes in the glycosylation of the mucosa of the guinea-pig. This unaltered glycosylation is of particular relevance for the sialic acid binding lectin SNA-I as H. pylori uses sialic acid binding adhesin for its attachment to the mucosa. As sialic acid binding sites are already expressed in the normal mucosa H. pylori can immediately attach via its sialic acid binding adhesin to the mucosa making the guinea-pig particularly useful as a model organism.This work is dedicated to Professor B. Tillmann on the occasion of his 65th birthday     

Enzyme characterisation of the circulating haemocytes of the carpet shell clam,Ruditapes decussatus(Mollusca:bivalvia)

The occurrence of a number of lysosomal enzymes (Proteases, glycosidases, phosphatases, and esterases) inRuditapes decussatushaemocytes was demonstrated by cytochemical and colorimetric techniques. The levels of 18 enzymes tested monthly varied through the study period (18 months), although they did not conform to a seasonal pattern of variation. No important effect of clam age on enzyme activity levels of haemocytes was detected. In those cytochemical assays in which distinction between granulocytes and hyalinocytes was possible, lysosomal enzymes were only found in granulocytes. Phosphatase was detected inside cytoplasmic granules of granulocytes, suggesting the granules to be lysosomes. NADPH oxidase was not detected in clam haemocytes, which is consistent with the absence of oxidative metabolism coupled with phagocytosis in haemocytes of this clam species. Levels of lysozyme detected inside haemocytes were higher than in serum.     

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.     

Histochemical study of glycoconjugates in the epididymis of the hamster (Mesocricetus auratus)

Summary The glycoconjugates of hamster epididymis were investigated with conventional and lectin histochemistry. A zone of the caput epididymis, with particular histochemical characteristics, has been differentiated. β-Elimination in combination with lectins was used to establish the presence and distribution of N- and O-linked glycoconjugates. The epithelium, spermatozoa and the intertubular matrix were rich in glycoconjugates. The Golgi apparatus and stereocilia of the principal cells were intensely positive with HPA, PNA and SBA lectins. β-limination indicated that these cells contained abundant O-linked glycoconjugates. Apical and clear cells presented a common lectin affinity; their reactivities towards WGA and UEA-I were very positive. These cells probably contain abundant N-glycoconjugates. The spermatozoa were stained by periodic acid-Schiff (PAS) and by all the lectins (especially in the acrosome), except by those with an affinity for α-l-fucosyl residues; the most intense reaction was found with HPA, WGA, PNA and SBA. Changes in the sperm lectin binding along the ductus were observed: sperm flagellum abruptly acquired WGA and PNA labelling from the posterior caput, and HPA reactivity was negative only in the zone between the caput and the corpus.     

Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor

Biochemical and histochemical studies have shown the presence of various carbohydrates in enamel. Using lectin-gold cytochemistry, we have examined the distribution of glycoconjugates containing N-acetyl-D-galactosamine (GalNAc) and/or N-acetyl-glucosamine (GlcNAc)/N-acetyl-neuraminic acid (NeuNAc) residues in rat incisor ameloblasts and in forming and maturing enamel embedded in Lowicryl K4M, LR Gold, and LR White resins. The enamel proteins that contain these carbohydrate moieties were further characterized by lectin blotting. All three resins allowed, albeit to a variable degree, detection of the binding sites for Helix pomatia agglutinin (HPA) and wheat germ agglutinin (WGA) GalNAc, and GlcNAc/NeuNAc, respectively. In general, Lowicryl K4M permitted more intense reactions with both lectins. Lectin binding was observed over the rough endoplasmic reticulum (weak labeling with WGA), the Golgi apparatus, lysosomes, secretory granules, and the enamel matrix. These compartments were shown by double labeling with WGA and anti-amelogenin antibody, and by previous immunocytochemical studies, to contain enamel proteins. Furthermore, WGA binding was more concentrated at the growth sites of enamel. Lectin blotting showed that several proteins in the amelogenin group were glycosylated and contained the sugars GalNAc and GlcNAc/NeuNAc. Fewer proteins were stained by HPA than by WGA, and the staining pattern suggested that the extracellular proteins recognized by these two lectins are processed differently. The HPA-reactive proteins were lost by or during the early maturation stage, whereas many of the WGA-reactive proteins persisted into the mid maturation stage. The heterogeneous staining of certain protein bands observed with WGA suggests that they contain more than one component. Two distinct glycoproteins containing GlcNAc/NeuNAc also appeared during the maturation stage. These results are consistent with the notion that ameloblasts produce an extracellular matrix composed mainly of glycosylated amelogenins which are differently processed throughout amelogenesis.     

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.     

Comparative lectin histochemistry on taste buds in foliate,circumvallate and fungiform papillae of the rabbit tongue

Summary Taste buds (TB) in the foliate, circumvallate and fungiform papillae of the rabbit tongue were examined with lectin histochemistry by means of light (LM) and electron (EM) microscopy. Biotin- and gold-labeled lectins were used for the detection of carbohydrate residues in TB cells and subcutaneous salivary glands. At the LM level, the lectins of soybean (SBA) and peanut (PNA) react with material of the foliate and circumvallate taste pores only after pretreatment of the section with neuraminidase. This indicates that the terminal trisaccharide sequences are as follows: Sialic acid-Gal-GalNAc in O-glycosylated glycoproteins or Sialic acid-Gal-GlcNAc in N-glycosylated glycoproteins. In fungiform taste buds the lectins of Dolichos biflorus (DBA) and Helix pomatia (HPA), also specific to GalNAc residues, are reactive without preincubation with neuraminidase. Wheat germ agglutinin (WGA), specific to GlcNAc, reacts with TBs of all papillae; and the lectin from Ulex europaeus (UEA I), specific to fucose, binds to individual TB cells. The presence of sialic acid may protect mucus or other glycoproteins in TB cells and inside the taste pore from premature enzymatic degradation. In a post-embedding EM procedure on LR-White-embedded tissue sections, only gold-labeled HPA was found to bind especially on membrane surfaces of the microvilli which protrude into the taste pore; however HPA did not bind to the electron-dense mucus inside the taste pore. The mucus situated in the trough and at the top of the adjacent epithelial cells also is strongly HPA-positive, but is of different origin and composition than that found in the taste pore. These results demonstrate distinct carbohydrate histochemical differences between fungiform and circumvallate/foliate taste buds. The different configuration of galactosyl residues and the occurrence of mannose in circumvallate and foliate TBs leads to the suggestion that the lectin reactivities of TBs are not only due to the presence of mucins, but also to N-linked glycoproteins, possibly with a hormone-like, paraneuronal function. A possible relationship to v. Ebner glands in these papillae is discussed.     

Characterization of the Adhesive Holdfast of Marine and Freshwater Caulobacters

Caulobacters are prosthecate (stalked) bacteria that elaborate an attachment organelle called a holdfast at the tip of the cellular stalk. We examined the binding of lectins to the holdfasts of 16 marine Caulobacter strains and 10 freshwater species or strains by using a panel of fluorescein-conjugated lectins and fluorescence microscopy. The holdfasts of all the marine isolates bound to only wheat germ agglutinin (WGA) and other lectins that bind N-acetylglucosamine (GlcNac) residues. The freshwater caulobacters showed more variability in holdfast composition. Some bound only to WGA and comparable lectins as the marine strains did. Others bound additional or other lectins, and some did not bind to the lectins tested. The binding of WGA appeared to involve the regions of the holdfast involved with adhesion; a holdfast bound to WGA was significantly less adhesive to glass. Competition experiments with WGA-binding holdfasts and oligomers of GlcNac demonstrated that trimers of GlcNac (the preferred substrate for WGA binding) were more effective than dimers or monomers in preventing WGA binding to holdfasts, suggesting that stretches of contiguous GlcNac residues occur in the WGA-binding holdfasts. In addition, differences between freshwater and marine holdfasts in the strength of WGA binding were noted. The effect of a number of proteolytic and glycolytic enzymes on holdfast integrity was examined; the proteases had no effect for all caulobacters. None of the glycolytic enzymes had an effect on marine caulobacter holdfasts, but chitinase and lysozyme (both attack oligomers of GlcNac) disrupted the holdfasts of those freshwater caulobacters that bound WGA. Despite some similarity to chitin, holdfasts did not bind Calcofluor and no measurable effects on holdfast production were detectable after cell growth in the presence of diflubenzuron or polyoxin D, inhibitors of chitin synthesis in other systems. Finally, the holdfasts of all caulobacters bound to colloidal gold particles, without regard to the coating used to stabilize the gold particles. This binding was stronger or more specific than WGA binding; treatment with colloidal gold particles prevented WGA binding, but the reverse was not the case.     

Localization of binding sites of Ulex europaeus I,Helix pomatia and Griffonia simplicifolia I-B4 lectins and analysis of their backbone structures by several glycosidases and poly-N-acetyllactosamine-specific lectins in human breast carcinomas

Several studies have shown the deletion of blood group A or B antigens and the accumulation of H antigens in human breast carcinomas. Other studies have independently demonstrated that the binding sites of lectins such asHelix pomatia agglutinin (HPA) andGriffonia simplicifolia agglutinin I-B₄ (GSAI-B₄) are highly expressed in these cells. In order to clarify the molecular mechanisms of malignant transformation and metastasis of carcinoma cells, it is important to understand the relationship between such phenotypically distinct events. For this purpose, we examined whether the binding sites of these lectins andUlex europaeus agglutinin I (UEA-I) are expressed concomitantly in the same carcinoma cells and analyzed their backbone structures. The expression of the binding sites of these lectins was observed independently of the blood group (ABO) of the patients and was not affected by the histological type of the carcinomas. Observation of serial sections stained with these lectins revealed that the distribution of HPA binding sites was almost identical to that of GSAI-B₄ in most cases. Furthermore, in some cases, UEA-I binding patterns were similar to those of HPA and GSAI-B₄ but in other cases, mosaic staining patterns with these lectins were also observed, i.e., some cell clusters were stained with both HPA and GSAI-B₄ but not with UEA-I and adjacent cell clusters were stained only with UEA-I. Digestion with endo-β-galactosidase orN-glycosidase F markedly reduced the staining intensity of these lectins. Together with the reduction of staining by these lectins, reactivity withGriffonia simplicifolia agglutinin II appeared in carcinoma cells following endo-β-galactosidase digestion. Among the lectins specific to poly-N-acetyllactosamine,Lycopersicon esculentum agglutinin (LEA) most vividly and consistently stained the cancer cells. Next to LEA, pokeweed mitogen agglutinin was also effective in staining these cells. Carcinoma cells reactive with these lectins corresponded well to those stained with both HPA and GSAI-B₄, and in some cases, with UEA-I. These results demonstrate that the binding sites of UEA-I, HPA, and GSAI-B₄ are expressed concomitantly in the same carcinoma cells and all carry linear and branched poly-N-acetyllactosamine onN-glycans, suggesting that the synthesis of this complex carbohydrate is one of the most important and basic processes leading to the malignant transformation of cells, invasion, and metastasis of carcinoma cells.