Studies of lectin binding to the human cervix uteri: I. Normal cervix

Summary Lectins ofBauhinia purpurea (BPA),Canavalin ensiformis (Con A),Griffonia simplicifolia I (GS I),Griffonia simplicifolia II (GS II),Maclura pomifera (MPA),Arachis hypogaea (PNA),Glycine max (SBA),Ulex europaeus I (UEA I) andTriticum vulgaris (WGA) were used to evaluate cell surface carbohydrates in formalin-fixed paraffin-embedded tissue sections of normal human cervix uteri. Consistent patterns of staining of the squamous epithelium were obtained in all 30 cases with BPA, GS II, MPA, PNA, SBA and WGA. A variable distribution of lectin binding was seen in squamous epithelium with Con A, GS I and UEA I. The patterns of GS I and GS II binding reflected squamous epithelial maturation. Columnar epithelium did not stain with GS II, stained variably with Con A, and stained consistently with the remaining seven lectins in all cases. No association between lectin binding and blood group or phase of the menstrual cycle was found. These findings may be used as a baseline for evaluation of lectin binding in both preinvasive and invasive lesions of the cervix uteri.     

Trypanosoma rhodesiense Bloodstream Trypomastigote and Culture Procyclic Cell Surface Carbohydrates1, 2, 3

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRATat 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diaminobenzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A < PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WGA, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M. The expression of lectin binding cell surface saccharides of T. rhodesiense WRATat 1 is related to the parasite stage. Sugars resembling α-D-mannose are on the surface of bloodstream trypomastigotes and culture procyclics; n-acetyl-D-galactosamine and D-galactose residues are on bloodstream forms; and n-acetyl-D-glucosamine-like sugars are on procyclic stages.     

Changes in lectin-binding pattern in the digestive tract of Xenopus laevis during metamorphosis. I. Gastric region.

The distribution of structural and secretory glycoconjugates in the gastric region of metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) histochemical staining method using seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin I, UEA-I; and wheat germ agglutinin, WGA). Throughout the larval period to stage 60, the epithelium consisting of surface cells and gland cells was stained in various patterns with all lectins examined, whereas the thin layer of connective tissue was positive only for RCA-I. At the beginning of metamorphic climax, the connective tissue became stained with Con A, SBA, and WGA, and its staining pattern varied with different lectins. The region just beneath the surface cells was strongly stained only with RCA-I. With the progression of development, both the epithelium and the connective tissue gradually changed their staining patterns. The surface cells, the gland cells, and the connective tissue conspicuously changed their staining patterns, respectively, for Con A and WGA; for Con A, PNA, RCA-I, SBA, and WGA; and for Con A, RCA-I, and WGA. At the completion of metamorphosis (stage 66), mucous neck cells became clearly identifiable in the epithelium, and their cytoplasm was strongly stained with DBA, PNA, RCA-I, and SBA. These results indicate that lectin histochemistry can provide good criteria for distinguishing among three epithelial cell types, namely, surface cells, gland cells, and mucous neck cells, and between adult and larval cells of each type.     

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.     

Eosinophilic globule cells in mouse MFH-like sarcomas: lectin histochemistry.

Lectin binding patterns in ten mouse malignant fibrous histiocytoma (MFH)-like sarcomas containing eosinophilic globule (EG) cells and in granular metrial gland (GMG) cells of mouse placenta were stained with nine lectins (Con A, LCA, WGA, DBA, SBA, e-PHA, PNA, RCA-I and UEA-I) by an avidin-biotin-peroxidase-complex method. EG cells stained strongly with DBA, SBA and PNA which are specific for N-acetyl-D-galactosamine and/or D-galactose. DBA and SBA bound throughout the cytoplasm including the globules; PNA reacted preferentially at the cell surface. There was no evidence that these three lectins were reactive for immature EG cells. WGA, RCA-I and e-PHA also gave a slightly to moderately positive reaction to globules of EG cells. The results indicate that the globules contain abundant O-linked sequences of sugars, but also a few N-linked residues. MFH tumor cells showed a variable degree of binding with Con A, RCA-I, and WGA, but did not react with DBA, SBA and PNA. On the other hand, GMG cells exhibited specific affinities for DBA, SBA and PNA with staining patterns similar to those of EG cells. These findings suggest that EG and GMG cells may be of the same cellular lineage.     

Lectin histochemical study on the infraorbital gland of the Japanese serow (Capricornis crispus)

Histology and lectin histochemistry were performed in the infraorbital gland of the Japanese serow. The gland is composed of glandular tissues and a pouch filled with the secretion. The tissues consist of an inner layer of sebaceous glands and an outer layer of apocrine glands. The male sebaceous layer is made up of the ordinary type, whereas the female's layer consists of the ordinary and modified types. In the apocrine gland stained with Arachis hypogaea (PNA), nine different patterns of glandular tubules were distinguished on the basis of staining of the cytoplasm, the Golgi area of secretory cells and secretion. Secretory modes of apocrine secretion and exocytosis were included in these stainings. Myoepithelial cells stained constantly with Glycine max (SBA) except when only the Golgi area of secretory cells was positive. The modified sebaceous gland was stained with PNA, SBA, Ricinus communis I (RCA), Triticum vulgaris (WGA), Canavalia ensiformis (Con A) and Ulex europaeus I (UEA), while the ordinary type was positive in PNA, RCA, SBA, WGA and Con A. The secretion in the pouch was stained with PNA, RCA, SBA, Dolichos biflorus (DBA), WGA and Con A. These findings suggest that the modified sebaceous gland contains large amounts of glycoconjugates and the apocrine gland shows a cyclic secretory process of apocrine secretion and exocytosis.     

Different binding to squamous and columnar epithelium of the uterine cervix as a marker of epithelial differentiation

Biotinylated lectins were used to investigate the expression of carbohydrate residues on columnar and squamous epithelium of the uterine cervix. Con A, WGA, RCA I, PNA, UEA I, DBA and SBA were used. In the native exocervical and in metaplastic squamous epithelium of the transformation zone, one group of lectins (Con A, WGA, RCA I and PNA) stained the cell periphery of all epithelial layers. A second group (UEA I, DBA and SBA) colored the cell periphery of the suprabasal cells. The basal layer was always negative. All lectins labeled the apical border and occasionally the cytoplasm of the endocervical columnar epithelium. Lectin-binding of metaplastic and native squamous epithelium could possibly be used as a marker of epithelial differentiation in normal and abnormal conditions.     

Lectin binding to rat spermatogenic cells: Effects of different fixation methods and proteolytic enzyme treatment

Summary The binding of a panel of eight different fluorescein-conjugated lectins to rat spermatogenic cells was investigated. Particular attention was paid to the effects of different fixation methods and proteolytic enzyme digestion on the staining pattern.Concanavalin A (Con A), wheatgerm agglutinin (WGA), succinylated WGA (s-WGA) and agglutinin from gorse (UEA I) stained the cytoplasm of most germ cells as well as the spermatid acrosome. In contrast, peanut agglutinin (PNA), castor bean agglutinin (RCAI) and soy bean agglutinin (SBA) mainly stained the acrosome. The staining pattern varied depending on the fixation method used. PNA was particularly sensitive to formalin fixation, while SBA, DBA and UEA I showed decreased binding and Con A, WGA, s-WGA and RCA I were insensitive to this type of fixation. Pepsin treatment of the sections before lectin staining caused marked changes in the staining pattern; staining with PNA in formalin-fixed tissue sections was particularly improved but there was also enhanced staining with SBA and horse gram agglutinin (DBA). On the other hand, in Bouin- and particularly in acetone-fixed tissue sections, pepsin treatment decreased the staining with several of the lectins, for example WGA and UEA I.     

Lectin histochemistry of the hyaline layer in the asteroid,pisaster ochraceus

Six different lectins were used to study the carbohydrate nature of the hyaline layer (HL), the external extracellular matrix of the starfish embryo. Thin sections of embryos fixed in the late gastrula stage were incubated with five fluoresceinated lectins: Con A, WGA, RCA, UEA-I, and SBA. All but UEA-I labelled the HL, suggesting that the following sugars are present: mannose and/or glucose, glcNAc and/or Neu5Ac, galactose, and galNAc. The different lectins produced variable degrees of labelling, with WGA, RCA, and SBA producing more intense labelling than Con A. Binding of lectins by the HL was studied at the ultrastructural level by exposing ultrathin sections to the following lectin-gold conjugates: Con A, WGA, PNA, SBA, and LFA. Lectin binding was observed over the various regions of the HL, recognized by Crawford and Abed (J. Morphol. 176:235–246, '86), i.e., the intervillus layer, the supporting layer and the coarse outer meshwork. Local differences in labelling patterns were observed among the various lectins, with SBA labelling all regions intensely, WGA and PNA labelling the supporting layer predominantly, and Con A labelling the HL only lightly. No labelling was observed with LFA. These lectin-labelling patterns in the HL demonstrate the presence of different glycoconjugates in different regions of the HL, suggesting that the layers differ biochemically. The existence of biochemical differences strengthens the idea that each layer may have different functions in the developing starfish embryo.     

Glycoconjugates in normal human kidney. A histochemical study using 13 biotinylated lectins

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), Arachis hypogaea (PNA), Sophora japonica (SJA), 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.     

Changes in lectin-binding pattern in the digestive tract of Xenopus laevis during metamorphosis. II. Small intestine.

The binding of seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin, UEA-I; and wheat germ agglutinin, WGA) to the small intestine in metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) method. The staining pattern of the epithelium with all lectins except for UEA-I and Con A changed gradually during metamorphic climax; the main component of the epithelium, absorptive cells, gradually became positive for DBA, PNA, and SBA and the scattered goblet cells for RCA-I and WGA. On the other hand, the change of the staining pattern in the connective tissue occurred only for Con A, RCA-I, and WGA, and this change took place rapidly at the beginning of climax (stage 60). Increased staining for Con A and WGA at stage 60 was observed only in a group of connective tissue cells close to the epithelium and in the basement membrane. As metamorphosis progressed, this localization of the staining intensity became less clear. At the completion of metamorphosis (stage 66), the absorptive cells were stained with all lectins except for UEA-I, whereas the goblet cells stained only with RCA-I and WGA. These results indicate that lectin histochemistry can distinguish between larval and adult cells of both two epithelial types (absorptive and goblet cells). The technique may also identify a group of connective tissue cells, close to the epithelium, that possibly induce the metamorphic epithelial changes.     

Lectin histochemistry of human skeletal muscle

Biotinyl derivatives of seven plant lectins-concanavalin A (Con A), peanut agglutinin (PNA), Ricinus communis agglutinin I (RCA I), Ulex europeus agglutinin I (UEA I), soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), and wheat germ agglutinin (WGA)-were bound to cryostat sections of biopsied normal human muscle and visualized with avidin-horseradish peroxidase conjugates. A distinct staining pattern was observed with each lectin. The most general staining was observed with Con A, RCA I, and WGA, which permitted strong visualization of the plasmalemma-basement membrane unit, tubular profiles in the interior of muscle fibers, blood vessels, and connective tissue. PNA gave virtually no intracellular staining, while SBA and UEA I selectively stained blood vessels. DBA was unique in providing good visualization of myonuclei. In each case, lectin staining could be blocked by appropriate sugar inhibitors. Neuraminidase pretreatment of the cryostat sections altered the pattern of staining by all lectins except UEA I and Con A; staining with RCA I became stronger and that with WGA became less intense, while staining with PNA, SBA and DBA became stronger and more generalized, resembling that of RCA I. These effects of neuraminidase pretreatment are in conformity with the known structure of the oligosaccharide chains of membrane glycoproteins and specificities of the lectins involved.     

Lectin binding to injured corneal endothelium mimics patterns observed during development

Summary Fluorochrome conjugated lectins were used to observe cell surface changes in the corneal endothelium during wound repair in the adult rat and during normal fetal development. Fluorescence microscopy of non-injured adult corneal endothelia incubated in wheat-germ agglutinin (WGA), Concanavalin A (Con A), and Ricinus communis agglutinin I (RCA), revealed that these lectins bound to cell surfaces. Conversely, binding was not observed for either Griffonia simplicifolia I (GS-I), soybean agglutinin (SBA) or Ulex europaeus agglutinin (UEA). Twenty-four hours after a circular freeze injury, endothelial cells surrounding the wound demonstrated decreased binding for WGA and Con A, whereas, RCA binding appeared reduced but centrally clustered on the apical cell surface. Furthermore, SBA now bound to endothelial cells adjacent to the wound area, but not to cells near the tissue periphery. Neither GS-I nor UEA exhibited any binding to injured tissue. By 48 h post-injury, the wound area repopulates and endothelial cells begin reestablishing the monolayer. These cells now exhibit increased binding for WGA, especially along regions of cell-to-cell contact, whereas, Con A, RCA and SBA binding patterns remain unchanged. Seventy-two hours after injury, the monolayer is well organized with WGA, Con A and RCA binding patterns becoming similar to those observed for non-injured tissue. However, at this time, SBA binding decreases dramatically. By 1 week post-injury, binding patterns for WGA, ConA and RCA closely resemble their non-injured counterparts while SBA continues to demonstrate low levels of binding. In early stages of its development, the endothelium actively proliferates and morphologically resembles adult tissue during wound repair. The 16-day fetal tissue is mitotically active, does not exhibit a well defined monolayer, and demonstrates weak fluorescence binding for WGA, Con A and RCA. Conversely, SBA binding is readily detected on many cell surfaces. By 19 days in utero, the endothelial monolayers becomes organized and cell proliferation greatly diminishes. WGA, Con A and RCA now exhibit binding similar to that seen in the adult tissue. SBA binding is not detected at this time. Thus, changes in lectin binding during wound repair of the adult rat corneal endothelium mimic changes in lectin binding seen during the development of the tissue.Supported by grant EY-06435 from The National Institutes of Health     

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.     

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.     

Binding of fluorescein isothiocyanate conjugated lectins to rat spermatogenic cells in tissue sections. Enhancement of lectin fluorescence obtained by fixation in Bouin's fluid

The testes from three months old Sprague-Dawley rats were fixed in Bouin's fluid or neutral buffered 10% formalin, embedded in paraffin, sectioned and after deparaffination stained with the following fluorescein isothiocyanate coupled lectins: PNA, WGA, Con A, RCA, SBA, DBA and UEA. The results show that there are considerable differences in the staining pattern of various spermatogenic cells between different lectins. The fixation in Bouin's fluid enhanced the staining of all the lectins compared to formalin fixation in which only a weak staining could be seen in the acrosomes of spermatids after WGA or PNA staining. PNA and WGA stained specifically the acrosome of the developing spermatids, which was seen from the beginning of the acrosome formation and lasted up to late spermiogenesis. However, the staining with PNA decreased in the late spermatids whereas the intensity of the staining remained unchanged with WGA. Con A did not stain the acrosome but stained unspecifically the cytoplasm of all spermatogenic cells. RCA stained faintly the acrosome throughout the spermatid differentiation. DBA and UEA stained specifically the chromosomes of B spermatogonia. DBA also faintly stained the cell membranes of early spermatids. SBA did not show any specific staining of the spermatogenic cells. Based on this it is suggested that the carbohydrates and glycoproteins which are known to be present in the acrosome are formed already in the beginning of the acrosome formation. The decrease in the PNA staining in late spermatids possibly reflects the fact that the receptor molecules are not synthesized in late spermatids but are formed in earlier developmental stages and are thereafter preserved in the acrosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin-binding pattern of neuroepithelial and respiratory epithelial cells in the mouse nasal cavity

Summary Sections from the nasal cavity of 12-day-old Swiss albino mice (NMRI strain) were subjected to lectin histochemistry. A panel of biotinylated lectins (Con A, WGA, s-WGA, PNA, SBA, DBA and UEA I) and a horseradish peroxidase-conjugated lectin (GSA II) showed marked differences in binding to the respiratory and the neuroepithelial cells. SBA (affinity for galactose andN-acetylgalactosamine), PNA (galactose) and WGA (sialic acids andN-acetylglucosamine) labelled the receptor neurons in the olfactory and vomeronasal epithelium. DBA (N-acetylgalactosamine) labelled a subgroup of about 5% of the olfactory receptor neurons, but most neurons in the vomeronasal organ. UEA I (fucose) and s-WGA (N-acetylglucosamine) intensely labelled the entire nerve cell population in the vomeronasal organ, but in the olfactory epithelium the labelling with these lectins was stratified. In the respiratory epithelium the ciliated cells were labelled with WGA and s-WGA, while the secretory cells bound most of the lectins. Thus different sugars are exposed on the surface of the different types of epithelia in the nasal cavity, providing a basis for selectivity in microbial attacks on these areas.     

Specific binding of peanut agglutinin and soybean agglutinin to chondroitinase ABC-digested cartilage proteoglycans: Histochemical, ultrastructural cytochemical, and biochemical characterization

Summary The binding of peanut agglutinin (PNA) and soybean agglutinin (SBA) to cartilage proteoglycans was investigated by histochemical, ultrastructural cytochemical, and biochemical methods. Following aldehyde fixation, specimens of rat epiphyseal cartilage were examined by horseradish peroxidase-labelled lectin cytochemistry with and without prior digestion in chondroitinase ABC. At the light microscope level neither PNA nor SBA exhibited any affinity for cartilage matrix, but became strongly bound following chondroitinase treatment. Similarly, at the ultrastructural level, extracellular matrix granules, presumed to be proteoglycan monomer(s), lacked PNA affinity in undigested specimens, and stained very weakly with SBA. Both PNA and SBA weakly to moderately stained thetrans cisternae of the Golgi-flattened cisternae in chondrocytes. The chondrocyte plasmalemma lacked PNA staining, but reacted weakly with SBA. Following chondroitinase digestion, PNA and SBA stained matrix granules, and the cell surface of chondrocytes intensely, whereas the Golgitrans cisternae, the Golgi-derived vacuoles, and multivesicular bodies demonstrated weak to moderate reactivity. Proteoglycan aggregates purified from rat chondrosarcoma and bovine nasal cartilage bound PNA and SBA avidly after digestion with chondroitinase. Undigested proteoglycans lacked affinity for PNA and reacted very weakly with SBA. These results indicate that both PNA and SBA specifically react with chondroitinase-modified oligosaccharide(s) bound to core proteins of cartilage proteoglycans. This provided a specific histochemical and ultrastructural cytochemical procedure for localizing chondroitin sulphate-containing proteoglycans.     

Binding of fluorescein isothiocyanate conjugated lectins to rat spermatogenic cells in tissue sections

Summary The testes from three months old Spague-Dawley rats were fixed in Bouin's fluid or neutral buffered 10% formalin, embedded in paraffin, sectioned and after deparaffination stained with the following fluorescein isothiocyanate coupled lectins: PNA, WGA, Con A, RCA, SBA, DBA and UEA. The results show that there are considerable differences in the staining pattern of various spermatogenic cells between different lectins. The fixation in Bouin's fluid enhanced the staining of all the lectins compared to formalin fixation in which only a weak staining could be seen in the acrosomes of spermatids after WGA or PNA staining. PNA and WGA stained specifically the acrosome of the developing spermatids, which was seen from the beginning of the acrosome formation and lasted up to late spermiogenesis. However, the staining with PNA decreased in the late spermatids whereas the intensity of the staining remained unchanged with WGA. Con A did not stain the acrosome but stained unspecifically the cytoplasm of all spermatogenic cells. RCA stained faintly the acrosome throughout the spermatid differentiation. DBA and UEA stained specifically the chromosomes of B spermatogonia. DBA also faintly stained the cell membranes of early spermatids. SBA did not show any specific staining of the spermatogenic cells. Based on this it is suggested that the carbohydrates and glycoproteins which are known to be present in the acrosome are formed already in the beginning of the acrosome formation. The decrease in the PNA staining in late spermatids possibly reflects the fact that the receptor molecules are not synthesized in late spermatids but are formed in earlier developmental stages and are thereafter preserved in the acrosome. The enhancement of lectin binding caused by Bouin's fixative might also be applied to other tissues where previous experiments with formalin fixed tissue have failed to show any staining.     

Lectin histochemistry of mammalian endothelium

Summary Lectin-histochemical studies were performed on formalin-fixed, paraffin-embedded tissues from ten mammalian species to demonstrate the pattern of carbohydrate residues in vascular endothelium. Ten different biotinylated lectins were used as probes and avidin-biotin-peroxidase complex (ABC) was used as visualant. Ricinus communis agglutinin-I (RCA-I) and wheat germ agglutinin (WGA) stained vascular endothelium in all species. Peanut agglutinin (PNA) stained vascular endothelium in all species only after preincubation with neuraminidase. Bandeirea simplicifolia agglutinin-I (BS-I) stained vascular endothelium in all species but human, while, Ulex europeus agglutinin-I (UEA-I) stained only human endothelium. Individual differences in staining of human vascular endothelium were noted with BS-I and succinylated-WGA (SWGA). Similarly, individual differences in staining of animal vascular endothelium were noted with soybean agglutinin (SBA) after preincubation with neuraminidase. Finally, Concanavalia ensiformis agglutinin (Con A), Dolichos biflorus agglutinin (DBA) and Lens culinaris agglutinin (LCA) did not stain vascular endothehuman in any of the species studied.