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.     

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.     

Reactivity of five N-acetylgalactosamine-recognizing lectins with preimplantation embryos, early postimplantation embryos, and teratocarcinoma cells of the mouse

The expression of receptors for N-acetylgalactosamine-recognizing lectins, namely Helix pomatia agglutinin (HPA), Sophora japonica agglutinin (SJA), Bauhinia purpurea agglutinin (BPA), Vicia villosa agglutinin (VVA), and Wistaria floribunda agglutinin (WFA) was studied in early mouse embryos and teratocarcinoma cells. Each of these lectins as well as Dolichos biflorus agglutinin (DBA) bound differently to early embryonic cells, with the exception of VVA and WFA which showed indistinguishable reactivities. SJA reacted intensely with visceral endoderm, but hardly at all with parietal and primitive endoderm. Therefore, SJA will be useful for analyzing the mechanism of visceral-endoderm formation. Furthermore, the inner cell mass (ICM) of early blastocysts reacted intensely with DBA, while the ICM of late blastocysts reacted only faintly with this lectin. Primary endoderm derived from the ICM reacted faintly with SJA, HPA, and DBA, and these reactivities increased again during the differentiation of the endoderm. Therefore, these three lectins could be used in the analysis of early stages during the differentiation of endoderm from the ICM. The results illustrate the highly complex nature of developmentally regulated alterations of cell-surface carbohydrates during the early stages of embryogenesis.     

Histochemical investigations on lectin binding in normal and irradiated mouse embryos

Lectin binding in normal and irradiated embryonic mouse tissues on day 10 of gestation was studied by peroxidase techniques. Specific binding ofDolichos biorus lectin (DBA) was detected in the mesodermal blood vessels and in the otic vesicles. The amount of DBA as well as that ofsoybean agglutinin (SBA) andpeanut agglutinin (PNA) increased after exposure to low doses of radiation (0.25, 0.50 and 0.75 Gy). The modifying influence of ionizing radiation was observed in the pituitary region, in the otic vesicles and in the blood vessel endothelium. The greatest effect appeared in the pituitary region at 0.75 Gy, while in the otic vesicles it appeared at 0.50 Gy. A dose-effect relationship was established for the DBA lectin affinity of the vascular endothelium. In comparison to DBA, SBA and PNA displayed more extensive staining after irradiation. The reactivity of these lectins appeared especially pronounced on the blood vessels within the central nervous system and in the luminal surface of the ependymal cells. It is of interest that maximal binding for PNA was observed at 0.25 Gy and for SBA at 0.50 Gy at the junctions between neuroepithelial cells.     

Dolichos biflorus agglutinin (DBA) reacts selectively with mast cells in human connective tissues

Dolichos biflorus agglutinin (DBA) binds to N-acetyl-D-galactosamine (GalNAc) residues in glycoconjugates and agglutinates erythrocytes carrying blood group antigen A. In cryostat sections of various tissues from blood group-specified humans, fluorochrome-coupled DBA bound preferentially to fusiform connective tissue cells and to certain epithelial cells. The connective tissue cells were identified as mast cells by their typical metachromasia in consecutive staining with toluidine blue. Double labeling with DBA and conjugated avidin revealed two distinct populations of mast cells. In several tissues the DBA-reactive cells likewise displayed uniform avidin reactivity. In intestinal mucosa, however, morphologically distinct DBA-binding mast cells were found, which were labeled with the avidin conjugates only in specially fixed paraffin sections. DBA did not bind to vascular endothelial cells, which could be identified by double staining with antibodies to factor VIII-related antigen. Labeling with Helix pomatia agglutinin (HPA), another blood group A-reactive lectin, resulted in distinct blood group-dependent fluorescence of the endothelia. Sophora japonica agglutinin (SJA), a blood group B-reactive lectin, labeled vascular endothelial cells in tissues from blood group A, AB, and B donors. HPA and SJA reacted with small mast cells in the gastrointestinal mucosa but failed to label large mast cells in any of the tissues. These results indicate that the blood group reactivity of lectins, as determined by erythroagglutination, is not necessarily consistent with their reactivity with blood group determinants in tissue sections. Moreover, DBA conjugates appear to be a reliable probe for detection of mast cells in various human connective tissues.     

Receptors to Dolichos biflorus Agglutinin

Dolichos biflorus agglutinin (DBA), a lectin specific to N-acetylgalactosamine residue, identifies cell surface markers on teratocarcinoma cells. These receptors are found in very limited types of adult tissues. In the present investigation, mouse embryos collected on days 1 (2-cell) to 3 (early blastocyst) were shown to be stained with FITC-conjugated DBA. Embryos homozygous for t ^w32 were also positively stained. These results suggest that receptors for DBA on preimplantation embryos include components distinct from Forssman, F9, and SSEA-1 antigens.     

Pinocytosis and locomotion in amoebae XIV. Demonstration of two different receptor sites on the cell surface ofAmoeba proteus

Summary Two different receptor sites, located on the cell surface ofAmoeba proteus were detected by using fluorescent analog cytochemistry (FAC) and electron microscopy (EM). Bovine serum albumin labeled with fluoresceine-isothiocyanate (FITC-BSA) and unlabeled ferritin bind, in a pH-dependent manner, as cations at the outer filaments of the mucous layer. The anionic receptor sites show a high affinity for Ca-ions which suppress the binding capacity of FITC-BSA and ferritin at low pH-values. The cation receptors obviously play an important role in the initiation of pinocytosis as demonstrated by the internalization, intracellular translocation and sequestration of the FITC-BSA. FITC- or ferritin-labeled concanavalin A (FITC-Con A, ferritin-Con A) bind predominantly in a pH-independent manner at the tips of the outer filaments and the basal zone of the mucous layer. The binding capacity of FITC-Con A is not influenced by external Ca-ions. Other lectins such asDolichos bifloris agglutinin (DBA), peanut agglutinin (PNA),Ricinus communis agglutinin I (RCA I), soybean agglutinin (SBA),Ulex europaeus agglutinin I (UEA I) and wheat germ agglutinin (WGA) are not specifically bound to the cell surface. So far, no experimental evidence has been gathered for the definitive function of a Con-A receptor in the mucos layer ofAmoeba proteus.Abbreviations BSA bovine serum albumin - Con A concanavalin A - CTC chlorotetracycline - DBA Dolichos bifloris agglutinin - DTE dithioeritritol - FITC fluorosceine-isothiocyanate - IEP iso electric point - PIPES 1-4-piperazine-diethane sulfonic acid - PNA peanut agglutinin - RCA I Ricinus communis agglutinin I - SBA soybean agglutinin - Uac uranylacetat - UEA I Ulex europaeus agglutinin I - WGA wheat germ agglutinin     

Egg jelly of the newt, Cynops pyrrhogaster contains a factor essential for sperm binding to the vitelline envelope

The acrosome reaction of newt sperm is induced at the surface of egg jelly and the acrosome-reacted sperm acquire the ability to bind to the vitelline envelope. However, because the substance that induces the acrosome reaction has not been identified, the mechanism by which the acrosome-reacted sperm bind to the vitelline envelope remains unclear. We found here that a Dolichos biforus agglutinin (DBA) specifically mimicked the acrosome reaction immediately upon its addition in the presence of milimolar level Ca(2+). Fluorescein isothiocyanate-labeled DBA bound specifically to the acrosomal cap of the intact sperm in the presence of a Ca(2+)-chelating agent, EDTA, suggesting that binding of DBA to the native receptor for the egg jelly substance on the acrosomal region took the place of the egg jelly substance-induced acrosome reaction. In contrast, the sperm that had been acrosome reacted by DBA treatment did not bind to the vitelline envelope of the egg whose jelly layers were removed. Subsequent addition of jelly extract caused the sperm binding to vitelline envelope, indicating that the egg jelly of the newt contains substances that are involved in not only inducing the acrosome reaction but also binding to the vitelline envelope. This is the first demonstration of the involvement of egg jelly substance in the binding of acrosome-reacted sperm to the vitelline envelope.     

Dolichos biflorus agglutinin (DBA) reveals a similar basal cell differentiation in normal and psoriatic epidermis

Binding of N-acetyl galactosamine (GalNAc)-specific Dolichos biflorus agglutinin (DBA) conjugates to frozen sections of normal epidermis and of psoriatic uninvolved and lesional skin was studied in fluorescence microscopy. The DBA conjugates bound only to single basal cell layer in normal and uninvolved psoriatic epidermis from patients with different blood group status. In the lesional area of psoriatic skin a similar reaction with a single basal cell layer was revealed. Other lectin-conjugates applied, presenting also GalNAc specificity, reacted with most cell layers of normal and both uninvolved and lesional psoriatic epidermis and gave an attenuated reaction with the middle epidermal layers. The results show that the basal cell characteristics are confined only to the cells along the basal membrane also in psoriatic epidermis, although cells in three lowest layers may be able to proliferate.     

Lectin-binding patterns on the plasma membranes of dissociated rat liver cells

Carbohydrate moieties on the surface of dissociated rat liver cells were examined electron microscopically using ferritin- or horseradish peroxidase (HRP)-conjugated lectins as probes. Rat liver was fixed by perfusion with 0.7% glutaraldehyde via the portal vein and dissociated into single cells with gentle homogenization. Concanavalin A (Con A), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) bound almost evenly to the entire cell surface of hepatocytes as well as of endothelial cells. Ulex europaeus agglutinin I (UEA-I) and peanut agglutinin (PNA) revealed no binding to any region. Dolichos biflorus agglutinin (DBA) was found to bind exclusively to the sinusoidal surface of hepatocytes and to endothelial cell surfaces. Soybean agglutinin (SBA)-binding was restricted to the endothelial cell surfaces and part of the sinusoidal microvilli of hepatocytes. Regional differences in lectin-binding pattern were visualized between the sinusoidal and the lateral or bile-canalicular surfaces of the hepatocytes. A polarity may exist on the hepatocyte cell surfaces in terms of the distribution pattern of the carbohydrate moieties, especially those of N-acetylgalactosamine.     

Exposed and hidden lectin-binding epitopes at the surface of<Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

The presence of surface- and subsurface-located lectin-binding epitopes of Borrelia burgdorferi was examined by electron microscopy using a variety of gold-labeled lectins. Concanavalin A reacted predominantly with extracellular material adjacent to the spirochetes. Wheat germ agglutinin bound weakly to the surface of borreliae; however, alterations of the outer membrane by preincubation in 100 ppm Triton X-100 or boiling uncovered numerous periplasmic sites recognized by the lectin. The periplasmic flagella liberated by some cells after detergent treatment were labeled with concanavalin A, wheat germ agglutinin and Ulex europaeus agglutinin UEA-I. No surface-exposed or periplasmic epitopes for the lectins from Glycine max, Dolichos biflorus or Helix pomatia were detected.     

Dolichos biflorus agglutinin (DBA) reveals a similar basal cell differentiation in normal and psoriatic epidermis

Summary Binding of N-acetyl galactosamine (GalNAc)-specific Dolichos biflorus agglutinin (DBA) conjugates to frozen sections of normal epidermis and of psoriatic uninvolved and lesional skin was studied in fluorescence microscopy. The DBA conjugates bound only to single basal cell layer in normal and uninvolved psoriatic epidermis from patients with different blood group status. In the lesional area of psoriatic skin a similar reaction with a single basal cell layer was revealed. Other lectin-conjugates applied, presenting also GalNAc specificity, reacted with most cell layers of normal and both uninvolved and lesional psoriatic epidermis and gave an attenuated reaction with the middle epidermal layers. The results show that the basal cell characteristics are confined only to the cells along the basal membrane also in psoriatic epidermis, although cells in three lowest layers may be able to proliferate.     

Lectins influence chondrogenesis and osteogenesis in limb bud mesenchymal cells

The role of cell surface glycoproteins in cell behavior can be characterized by their interactions with plant lectins. This study was designed to identify the effects of lectins on chondrogenesis and osteogenesis in limb bud mesenchymal cells in vitro. Limb bud mesenchymal cells from mouse embryos were cultured in high-density micromass culture. Wheat germ agglutinin (WGA), concanavalin A (ConA), peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Ricinus communis agglutinin (RCA) were added separately to the culture media. Cells were cultured for 5 or 9 days, and cell viability was assayed by neutral red on day 5. The micromasses were stained with alcian blue, alizarin red S and Von Kossa stains, and alkaline phosphatase assays were also done. Dolichos biflorus agglutinin induced an increase in chondrogenesis, calcium precipitation and proteoglycan production. ConA and PNA did not affect chondrocyte differentiation but induced chondrocytes to produce more proteoglycan. Wheat germ agglutinin reduced chondrification and ossification but induced mesenchymal cells to store lipid droplets. Ricinus communis agglutinin 1 was toxic and significantly reduced cell survival. In conclusion, DBA was the most effective inducer of ossification and chondrification. Wheat germ agglutinin induced adipogenesis instead. These assays showed that lectins play important roles in limb bud development.     

Detection of glycosylated sites in embryonic rabbit kidney by lectin chemistry

The reciprocal cell biological interaction between mesenchymal and epithelial tissue plays a critical role during nephrogenesis. It is unknown to date whether the tissues interact during nephron induction by pure diffusion of substances or whether cellular contacts via gap junctions or focal adhesion molecules are involved. In neonatal rabbit kidney the interface between both tissues shows unique features. It consists of a distinct space, which is filled with specific extracellular matrix consisting of glycosylated proteins such as fibronectin, laminin, collagen, and proteoglycans. In the present experiments we tested by histochemistry whether it is possible to detect additional glycosylated proteins using Soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Ulex europaeus I agglutinin (UEA I), and Peanut agglutinin (PNA) as molecular markers. All tested lectins showed distinct labeling patterns in embryonic renal tissue. Within the collecting duct ampulla, DBA and UEA I revealed intensive cellular reaction. In contrast, PNA and SBA reacted at the basal aspect of the collecting duct ampulla tip in addition to a cellular reaction. To identify the individual molecules labeled by the lectins, embryonic tissue was fractionated and separated by electrophoretic methods. For the first time, we were able to show by two-dimensional electrophoresis and subsequent western blot experiments that lectins bind to a series of individual protein spots, which have not been identified to date.     

Characterization of murine thymocyte subpopulations reacting with Dolichos bifloris agglutinin

Reactivity of murine thymocytes with the Dolichos bifloris agglutinin (DBA) was examined using flow cytometry. This agglutinin, which has nominal specificity for alpha-linked N-acetyl-D-galactosamine residues, labels 1-4% of unfractionated BALB/c thymocytes. Among thymocyte subpopulations identified on the basis of CD4/CD8 labeling, DBA bound to a substantial percentage of the CD4-8- thymocyte subpopulation. In addition, small populations of thymocytes expressing CD4 and/or CD8 were also labeled with DBA, but with less intensity then seen on many CD4-8- cells. Within the CD4-8- thymocyte population, labeling with DBA was positively correlated with reactivity with J11d antibody, the expression of IL-2 receptor and high levels of the homing receptor molecule, MEL-14. Reactivity with DBA was negatively correlated with the expression of Lyt-1, V beta 8 TCR determinants, and CD3.     

Dolichos biflorus agglutinin receptors in mouse muscle. II. Biochemical properties in relation to molecular forms of acetylcholinesterase

A biochemical analysis has been performed on the relationship between the receptors for Dolichos biflorus agglutinin (DBA) and collagen tailed acetylcholinesterase (16S AChE) in mouse skeletal muscle. The molecular forms of AChE were separated by differential salt extraction and by gradient centrifugation. DBA binding activity was measured using a microtiter plate binding assay and affinity chromatography. The 16S form of AChE was bound to DBA, whereas globular forms of AChE were not. However, only a small proportion of 16S AChE was capable of binding to DBA, and most of the DBA binding capacity in muscle extracts was not associated with the 16S AChE. The possible association with the neuromuscular synapse of DBA binding molecules other than 16S AChE is discussed with respect to our previous histochemical study on DBA binding sites in mouse muscle.     

Tissue interactions in the induction of anterior pituitary: role of the ventral diencephalon, mesenchyme, and notochord.

Rathke's pouch, the epithelial primordium of the anterior pituitary, differentiates in close topographical and functional association with the ventral diencephalon. It is still not known whether the ventral diencephalon acts as the initial inducer of pituitary development. The roles of the adjacent mesenchyme and notochord, two other tissues located in close proximity to Rathke's pouch, in this process are even less clear. In this report we describe an in vitro experimental system that reproduces the earliest steps of anterior pituitary development. We provide evidence that the ventral diencephalon from 2- to 4-day-old chick embryos is able to function as an inducer of pituitary development and can convert early chick embryonic head ectoderm, which is not involved normally in pituitary development, into typical anterior pituitary tissue. This induction is contact-dependent. In our experimental system, there is a requirement for the supporting action of mesenchyme, which is independent of the mesenchyme source. Transplantation of the notochord into the lateral head region of a six-somite chick embryo induces an epithelial invagination, suggesting that the notochord induces the outpouching of the roof of the stomodeal ectoderm that results in formation of Rathke's pouch and causes the close contact between this ectoderm and the ventral diencephalon. Finally, we demonstrate that the ventral diencephalon from e9.5-e11.5 mouse embryos is also an efficient inducer of anterior pituitary differentiation in chick embryonic lateral head ectoderm, suggesting that the mechanism of anterior pituitary induction is conserved between mammals and birds, using the same, or similar, signaling pathways.     

Lectin-binding patterns on the plasma membranes of dissociated rat liver cells

Summary Carbohydrate moieties on the surface of dissociated rat liver cells were examined electron microscopically using ferritin-or horseradish peroxidase (HRP)-conjugated lectins as probes. Rat liver was fixed by perfusion with 0.7% glutaraldehyde via the portal vein and dissociated into single cells with gentle homogenization. Concanavalin A (ConA), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) bound almost evenly to the entire cell surface of hepatocytes as well as of endothelial cells. Ulex europaeus agglutinin I (UEA-I) and peanut agglutinin (PNA) revealed no binding to any region. Dolichos biflorus agglutinin (DBA) was found to bind exclusively to the sinusoidal surface of hepatocytes and to endothelial cell surfaces. Soybean agglutinin (SBA)-binding was restricted to the endothelial cell surfaces and part of the sinusoidal microvilli of hepatocytes. Regional differences in lectin-binding pattern were visualized between the sinusoidal and the lateral or bile-canalicular surfaces of the hepatocytes. A polarity may exist on the hepatocyte cell surfaces in terms of the distribution pattern of the carbohydrate moieties, especiàlly those of N-acetylgalactosamine.     

Fluorochrome-coupled lectins reveal distinct cellular domains in human epidermis

The distribution of saccharide moieties in human interfollicular epidermis was studied with fluorochrome-coupled lectins. In frozen sections Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin I (RCAI), and wheat germ agglutinin (WGA) stained intensively both dermis and viable epidermal cell layers, whereas peanut agglutinin (PNA) bound only to living epidermal cell layers. Ulex europaeus agglutinin I (UEAI) bound to dermal endothelial cells and upper cell layers of the epidermis but left the basal cell layer unstained. Dolichos biflorus agglutinin (DBA) bound only to basal epidermal cells, whereas both soybean agglutinin (SBA) and Helix pomatia agglutinin (HPA) showed strong binding to the spinous and granular cell layers. On routinely processed paraffin sections, a distinctly different staining pattern was seen with many lectins, and to reveal the binding of some lectins a pretreatment with protease was required. All keratin-positive cells in human epidermal cell suspensions, obtained with the suction blister method, bound PNA, whereas only a fraction of the keratinocytes bound either DBA or UEAI. Such a difference in lectin binding pattern was also seen in epidermal cell cultures both immediately after attachment and in organized cell colonies. This suggests that in addition to basal cells, more differentiated epidermal cells from the spinous cell layer are also able to adhere and spread in culture conditions. Gel electrophoretic analysis of the lectin-binding glycoproteins in detergent extracts of metabolically labeled primary keratinocyte cultures revealed that the lectins recognized both distinct and shared glycoproteins. A much different lectin binding pattern was seen in embryonic human skin: fetal epidermis did not show any binding of DBA, whereas UEAI showed diffuse binding to all cell layers but gave a bright staining of dermal endothelial cells. This was in contrast to staining results obtained with a monoclonal cytokeratin antibody, which showed the presence of a distinct basal cell layer in fetal epidermis also. The results indicate that expression of saccharide moieties in human epidermal keratinocytes is related to the stage of cellular differentiation, different cell layers expressing different terminal saccharide moieties. The results also suggest that the emergence of a mature cell surface glycoconjugate pattern in human epidermis is preceded by the acquisition of cell layer-specific, differential keratin expression.