Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids

The binding of low density lipoprotein (LDL) to the platelet cell membrane could facilitate the transfer of phospholipids from LDL to the platelets. A polyclonal antibody against the platelet glycoproteins IIb/IIIa inhibited the high affinity binding of 125I-LDL by up to 80%. The transfer of pyrene (py)-labeled sphingomyelin (SM), phosphatidylcholine and phosphatidylethanolamine from LDL to the platelets was unaffected by the antibody. The lectin wheat germ agglutinin (WGA) reduced the binding of 125I-LDL to the platelets by approximately 80%. In contrast, the lectin stimulated the transfer of SM from LDL into the platelets by about three-fold. WGA also specifically augmented the transfer of py-SM between lipid vesicles and the platelets, the stimulation being abolished in the presence of N-acetylglucosamine. Dextran sulfate (DS) increased the specific binding of 125I-LDL to the platelets by up to 2.8-fold. On the other hand, the import of LDL-derived py-phospholipids was unaffected by DS. Together, the results indicate that the phospholipid transfer from LDL to the platelets is independent of the high affinity LDL binding to the platelets and is specifically stimulated by WGA. Thus, the interactions of platelets with LDL phospholipids differ markedly from those with the apoprotein components of the lipoproteins.     

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     

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

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

2-Arachidonoylglycerol enhances platelet formation from human megakaryoblasts

Platelets modulate vascular system integrity, and their loss is critical in haematological pathologies and after chemotherapy. Therefore, identification of molecules enhancing platelet production would be useful to counteract thrombocytopenia. We have previously shown that 2-arachidonoylglycerol (2-AG) acts as a true agonist of platelets, as well as it commits erythroid precursors toward the megakaryocytic lineage. Against this background, we sought to further interrogate the role of 2-AG in megakaryocyte/platelet physiology by investigating terminal differentiation, and subsequent thrombopoiesis. To this end, we used MEG-01 cells, a human megakaryoblastic cell line able to produce in vitro platelet-like particles.

2-AG increased the number of cells showing ruffled surface and enhanced surface expression of specific megakaryocyte/platelet surface antigens, typical hallmarks of terminal megakaryocytic differentiation and platelet production. Changes in cytoskeleton modeling also occurred in differentiated megakaryocytes and blebbing platelets. 2-AG acted by binding to CB₁ and CB₂ receptors, because specific antagonists reverted its effect. Platelets were split off from megakaryocytes and were functional: they contained the platelet-specific surface markers CD61 and CD49, whose levels increased following stimulation with a natural agonist like collagen. Given the importance of 2-AG for driving megakaryopoiesis and thrombopoiesis, not surprisingly we found that its hydrolytic enzymes were tightly controlled by classical inducers of megakaryocyte differentiation.

In conclusion 2-AG, by triggering megakaryocyte maturation and platelet release, may have clinical efficacy to counteract thrombocytopenia-related diseases.     

Glycoconjugates in normal human kidney

Summary The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ($\text{p}\text{I}$) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.     

Cell type-specific binding of Ricinus lectin to murine cerebellar cell surfaces in vitro

Summary The binding of several plant lectins, Concanavalin A (ConA), Lens culinarisA (LCA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin 120 (RCA120) to cell surfaces of developing mouse cerebellar cells was assayed by the use of fluorescein isothiocyanate (FITC)-conjugated compounds. Freshly dissociated, live single-cell suspensions from 6-day-old mouse cerebellum contain 93% ConA, 99% LCA, 98% WGA, and 59% RCA 120-positive cells with ring fluorescence. Of the RCA 120-positive cells, 4% express a high and 55% a lower or very low number of lectin receptors. Flow cytometric analysis of fluorescent lectin binding yields results qualitatively similar to those obtained by scoring positive and negative cells in the fluorescence microscope.In monolayer cultures of 6-day-old mouse cerebellum practically all cells express receptors for ConA, LCA, and WGA, whereas RCA 120 binding sites are absent from neurons with small cell bodies (granule, basket and stellate cells) and present in large number on neurons with large cell bodies (Purkinje and possibly Golgi Type-II cells) and fibroblasts. RCA 120 receptors are weakly expressed on astro-and oligodendroglia. Cell type-specific expression of RCA 120 receptors is constant throughout all ages studied (embryonic day 13 to postnatal day 9). At early embryonic ages the proportion of highly fluorescent neurons with large cell bodies is significantly increased.     

Thrombopoietic-mesenchymal interaction that may facilitate both endochondral ossification and platelet maturation via CCN2

CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.     

Effect of lectins on the invasion of Ichthyophthirius theront to channel catfish tissue.

This study determined the effects of lectin binding to theronts of Ichthyophthirius multifiliis on theront immobilization, invasion, trophont development and survival in channel catfish Ictalurus punctatus excised fins in vitro. Soybean agglutinin (SBA), lentil agglutinin (LCA), gorse agglutinin (UEA-I) and wheat germ agglutinin (WGA) were used to treat theronts. Percentages of theronts immobilized by 4 lectins ranged from 12.0 to 19.4% at a concentration of 1000 microg ml(-1). These lectins bound more than half of the theronts at a concentration of 50 microg ml(-1). More theronts were labeled by SBA and WGA than by lectin LCA at concentrations of 50 and 100 microg ml(-1), respectively. The binding of these lectins to theronts indicated that monosaccharides (D-galactose, L-fucose, D-mannose and D-glucose) and amino sugar derivatives (N-acetylgalactosamine and N-acetylglucosamine) were present on the surface of theronts. Invasion was reduced significantly for theronts treated with LCA, UEA-I and WGA. No difference in invasion was found between control and SBA bound theronts (p > 0.05). The binding of lectin LCA, UEA-I and WGA to theronts significantly reduced the development of trophonts (p < 0.05). The mean volumes of trophonts labeled with these 3 lectins were smaller than volumes in control trophonts from 8 to 48 h after exposure. Survival was lower in trophonts labeled with lectins than in control trophonts at 48 h after exposure.     

Megakaryocyte precursors (promegakaryoblasts and megakaryoblasts) in the normal human bone marrow. An immunohistochemical and morphometric study on routinely processed trephine biopsies

On routinely processed trephine biopsies of the normal human bone marrow derived from 15 patients, immunostaining with a monoclonal antibody against platelet glycoprotein IIIa (Y2/51) and morphometric measurements were performed for the determination of megakaryocyte precursor cells. Based on cell sizes and on comparison with (1) specimens stained by the periodic acid-Schiff reaction and (2) smears, the smallest elements clearly identifiable as belonging to the megakaryocyte series were classified as promegakaryoblasts. Promegakaryoblasts had a frequency of 1.7/sq mm and 140/cu mm of bone marrow and constituted about 8% of the total positively stained megakaryocytic elements; they were characterized by a size of 41.5 sq microns, a diameter of 7.7 microns, a high nuclear-cytoplasmic ratio (0.32) and a nearly circular outline of their nuclear and cellular perimeters.     

The role of blood platelets in nucleoside metabolism: regulation of megakaryocyte development and platelet production

In higher vertebrates, different types of blood cells develop from common precursors. Mammals are unique in possessing two types of blood cells — erythrocytes and platelets — which lack nuclei. Although platelets display consistent and easily-recognisble morphological and ultrastructural characteristics and show exreme metabolic and functional versatility, they are not true cells, being produced by fragmentation of giant polyploid precursors called megakaryocytes. At present, the physiological mechanisms which regulate megakaryocyte development and platelet production are not well understood.

Platelets are actively involved in metabolism of purine derivatives and a significant platelet role in pyrimidine metabolism has also been demonstrated (see previous papers). Here an attempt is made to integrate information about platelet involvement in nucleic precursor metabolism with current concepts of haematopoiesis, particularly megakaryocyte development and platelet production.

It is concluded (i) that megakaryocytic cels are immediate descendents of haematopoietic stem cells which have become polyploid as a result of genetic damage or metabolic imbalances, (ii) megakaryocytes and platelets are the ultimate regulators of stem cell development because they control the availability of thymidine and (iii) that the production of megakaryocytes and platelets is a physiological safety mechanism which prevents fixation of genetic damage and protects other cells from potentially cytotoxic and genotoxic stimuli.     

Carbohydrate antigens of human megakaryocytes and platelet glycoproteins: a comparative study

Until now, carbohydrate antigens of human megakaryocytes have not been studied very extensively. For this reason, we investigated the staining pattern of 25 lectins and carbohydrate-specific monoclonal antibodies on paraffin-embedded trephine biopsies and acetone-fixed smears from patients with reactive and neoplastic bone marrow lesions. A biotin-streptavidin-alkaline phosphatase assay was used to visualize the binding of lectins or antibodies. Ulex europaeus agglutinin I (UEA-I) stained megakaryocytes in all cases tested. Monoclonal antibodies detecting fucosylated Lewis type 2 chain antigens (19-OLE, 12-4LE and LeuM1) were also reactive. Several lectins detecting backbone and core oligosaccharides [Helix pomatia agglutinin (HPA), peanut agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), soybean agglutinin (SBA)] bound to megakaryocytes only after neuraminidase digestion. Moreover, we investigated human platelet lysates to gain some information about the carbohydrate residues of platelet glycoproteins which are synthesized by megakaryocytes. The carbohydrate expression of platelets showed striking similarities to that of megakaryocytes. Immunoblotting experiments revealed a strong binding of UEA-I, 19-OLE and 12-4LE to a band isographic to glycoprotein (gp) Ib. After desialylation of glycoproteins transblotted to nitrocellulose, ECA and PNA also reacted with a band of this molecular weight. Gp Ib is known to contain a mucin-like peptide core with a great number of potential O-glycosylation sites. Therefore, it is tempting to speculate that carbohydrate residues characterized in this study are involved in the complex biological interactions of gp Ib.     

A Survey of Lectin Binding in Paramecium1

To better understand the general distribution of glycoproteins and the distribution of specific glycoprotein-bound sugar residues in Paramecium, a survey of the binding pattern of selected lectins was carried out in P. tetraurelia, P. caudatum, and P. multimicronucleatum. Lectins studied were concanavalin A (Con A), Griffonia simplicifolia agglutinins I and II (GS I and GS II), wheat germ agglutinin (WGA), Ulex europaeus (UEA I), peanut agglutinin (PNA), Ricinis communis toxin (RCA₆₀) and agglutinin (RCA₁₂₀), soybean agglutinin (SBA), Bauhinia purpurea agglutinin (BPA), Dolichos biflorus agglutinin (DBA), and Maclura pomifera agglutinin (MPA). Those giving the most distinctive patterns were Con A, GS II, WGA, UEA I, and PNA. No significant differences were found between the three species. Concanavalin A, a mannose/glucose-binding lectin, diffusely labeled the cell surface and cytoplasm and, unexpectedly, the nuclear envelopes. Events of nuclear division, and nuclear size and number were thus revealed. Both WGA and GS II, which are N-acetylglucosamine-binding lectins, labeled trichocyst tips, the cell surface, and the oral region, revealing stages of stomatogenesis. The lectin WGA, in addition, labeled the compartments of the phagosome-lysosome system. The lectin PNA, an N-acetyl galactosamine/galactose-binding protein, was very specific for digestive vacuoles. Finally, UEA I, a fucose-binding lectin, brightly labeled trichocysts, both their tips and body outlines. We conclude that a judicious choice of lectins can be used to localize glycoproteins and specific sugar residues as well as to study certain events of nuclear division, cellular morphogenesis, trichocyst discharge, and events in the digestive cycle of Paramecium.     

Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures

Summary Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) -naphthylphosphate and Fast Blue BB; (4) -glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and -glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, and capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections.     

Characterisation of N-glycans bound to IGFBP-3 in sera from healthy adults

Human IGFBP-3 contains three potential N-linked glycosylation sites. Published data concerning the type and saccharide composition of the N-glycans is scarce. The aim of this study was to characterise N-glycans covalently attached to IGFBP-3 from sera of healthy adults (men and women). In order to do that a panel of eight lectins covering broad saccharide specificity was used: agarose-immobilised SNA (Sambucus nigra agglutinin), Con A (lectin from Canavalia ensiformis), RCA I (Ricinus communis agglutinin I), PHA-E (Phaseolus vulgaris erythroagglutinin), PHA-L (P. vulgaris leukoagglutinin), succinylated WGA (wheat germ agglutinin), ECL (Erythrina cristagalli lectin) and UEA (Ulex europaeus agglutinin). IGFBP-3 interacted with SNA, Con A, RCA I, PHA-E and, to a much lesser extent, with PHA-L. These results indicate that human IGFBP-3 bears mostly biantennary complex type N-glycans with a very high content of α-2,6-linked Sia at their termini. Hybrid type and high-mannose type N-glycans are present, as well as a bisecting GlcNAc residue, which may be core fucosylated. N-glycosylation of IGFBP-3 follows the N-glycosylation pattern of major serum proteins. This study represents a ground for the future research of glycosylation pattern of IGFBP-3 from the circulation of men and women diagnosed with different illnesses.     

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.     

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.     

Expression of carbohydrate residues in plasma membrane glycoproteins during the differentiation of amphibian epidermal cells

Summary Expression of various sugar residues on the plasma membrane of frog (Rana perezi) epidermal cells at different stages of differentiation has been monitored with the use of a battery of HRP-conjugated lectins. In paraffin-embedded tissue, mannose residues (stained by Concanavalin A) were detected at the keratinocyte cell surface in all epidermal strata. However,Lens culinaris agglutinin (LCA), also specific for mannose, specifically stained the plasma membrane of cells from the stratum germinativum. Expression of N-acetyl-glucosamine (GlcNAc), labelled with wheat germ agglutinin (WGA), was maximum at the cell surface of basal cells and progressively decreased through the stratum spinosum. Galactose (Gal) and N-acetyl-galactosamine (GalNAc) residues, labelled withGriffonia simplicifolia I (GS I) andGlycine max (SBA) agglutinins, respectively, were expressed according to the degree of differentiation in amphibian epidermal cells. Sialic acid-containing glycoproteins, labelled withLimax flavus agglutinin (LFA), were found in the outermost plasma membrane of the replacement cell layer and stratum corneum. Glycoproteins responsible for the observed lectin-binding patterns have been identified by staining on nitrocellulose filters after electrophoresis of solubilized plasma membrane fractions and Western blotting. Changes at the level of glycosylation of plasma membrane glycoproteins as epidermal cells differentiate are discussed on the basis of a progressive addition of Gal residues. Integral membrane proteins have been solubilized with the non-denaturing detergent CHAPS and glycoproteins containing terminal Gal residues, that are expressed according to the degree of differentiation in frog epidermis, have been partially purified by affinity chromatography on a GS I-Sepharose 4 B column. The purified fraction was composed by four acidic glycoproteins with isoelectric points between 4.6 and 5.2 and, in SDS-gels gave five major protein bands with approximate molecular weights of 148, 140, 102, 60, and 52 kDa in SDS-gels. The 102 and 52 kDa bands correspond to the a and subunits of amphibian epidermal Na⁺,K⁺-ATPase as demonstrated by specific staining with a polyclonal antibody against the catalytic subunit of pig kidney proton pump and staining with lectins GS I, GS II, and WGA. Possible relationships between higher molecular weight proteins and the constituents of intramembranous particles from the outermost plasma membranes of the replacement cell layer and the stratum corneum are also discussed.Abbreviations BSA bovine serum albumin - CHAPS (3-[(cholamidopropyl) dimethyl-ammonio] 1-propanesulfonate) - Con A Canavalia ensiformis agglutinin - DTT dithiothreitol - Gal galactose - GalNAc N-acetyl-D-galactosamine - GlcNAc N-acetyl-D-glucosamine - GS I Griffonia simplicifolia agglutinin I - GS II Griffonia simplicifolia agglutinin II - HRP horseradish peroxidase - LFA Limax flavus agglutinin - LCA Lens culinaris agglutinin - NDPAGIF non-denaturing polyacrylamide gel isoelectric focusing - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase - PBS phosphate buffered saline - PMSF phenyl methyl sulphonyl fluoride - RCL replacement cell layer - SBA soybean agglutinin (Glycine max) - SB stratum basal - SDS sodium dodecyl sulphate - SG stratum granulosum - SS stratum spinosum - UEA I Ulex europaeus agglutinin I - WGA wheat germ (Triticum vulgaris) agglutinin     

Lectin histochemical studies on the olfactory epithelium and vomeronasal organ in the Japanese striped snake,Elaphe quadrivirgata

The olfactory epithelium and the vomeronasal organ of the Japanese striped snake were examined by lectin histochemistry. Of the 21 lectins used in the study, all lectins except succinylated‐wheat germ agglutinin (s‐WGA) showed similar binding patterns in the vomeronasal receptor cells and the olfactory receptor cells with varying intensities. The binding patterns of s‐WGA varied among individuals in the vomeronasal and olfactory receptor cells, respectively. Four lectins, Bandeiraea simplicifolia lectin‐II (BSL‐II), Dolichos biflorus agglutinin (DBA), Sophora japonica agglutinin (SJA), and Erythrina cristagalli lectin (ECL) stained secretory granules and the organelles in the olfactory supporting cells and did not stain them in the vomeronasal supporting cells. These results suggest that the glycoconjugate moieties are similar in the vomeronasal and olfactory receptor cells of the Japanese striped snake. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.