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.     

Histochemical demonstration of different types of poly-N-acetyllactosamine structures in human thyroid neoplasms using lectins and endo-β-galactosidase digestion

Summary Blood-group-related antigens expressed in papillary carcinomas and other types of neoplasm of the human thyroid glands have been shown to be carried by poly-N-acetyllactosamines containing a linear domain susceptible to endo-β-galactosidase digestion. To make clear more precisely the backbone poly-N-acetyllactosamine structures, labelled lectins specific to different types of these structures and specific to core structures with β1-6GlcNAc branching of N- and O-linked glycoproteins were employed in conjunction with prior endo-β-galactosidase digestion on formalin-fixed, paraffin-embedded neoplasms of the human thyroid glands. In papillary carcinomas,Datura stramonium agglutinin (DSA) and succinyl wheat germ agglutinin (Suc-WGA) reacted most consistently and frequently with papillary carcinomas from all the individuals examined. Pokeweed mitogen (PWM) likewise stained the cells of papillary carcinomas from all the individuals examined, but in some individuals the number of lectin-reactive cells were very small.Lycoperscion esculentum aggultinin (LEA),Solanum tuberosum agglutinin (STA),Phaseolus vulgaris agglutinin L (PHA-L) andArtocarpus integrifolia agglutinin (jacalin) similarly bound to the cancer cells from most of the individuals, and in these cases the number of reactive cells was usually much more restricted than was the case with DSA or PWM. In adenoma and other types of carcinoma, such as follicular carcinomas, these lectins specific to poly-N-acetyllactosamine exhibited slight or no reactivity with the cells, whereas PHA-L and jacalin similarly bound to the cells of adenomas and carcinomas from most of the individuals examined. Prior digestion with endo-β-galactosidase completely eliminated or markedly reduced the reactivity with PWM and LEA in papillary carcinomas. Reactivity with DSA, Suc-WGA, STA, PHA-L and jacalin was slightly reduced or not at all affected by enzyme digestion. These results confirmed that poly-N-acetyllactosamine species found in papillary carcinomas are quite different from those in other types of thyroid neoplasm, suggesting that at least three different types of poly-N-acetyllactosamine, that is, linear unbranched short and long sequences and highly branched ones are produced in these cells.     

Relationship between lectin binding properties and the expression of blood group ABH antigens in vascular endothelia and red blood cells from 18 primate species

Summary The reactivity was examined of horseradish peroxidase labelledUlex europaeus agglutinin-I (UEA-I) andGriffonia simplicifolia agglutinin I-B₄ (GSAI-B₄) with red blood cells and vascular endothelium in formalin-fixed, paraffin embedded tissues from 18 primate species. The expression of blood group ABH antigens in these cells as well as secretions from other tissues was also examined by the indirect immunoperoxidase method using monoclonal anti-ABH antibodies as primary antibodies. In Prosimians and New World monkeys which lack ABH antigens on both red blood cells and endothelial cells, but produce these antigens in other tissue secretions, GSAI-B₄ always reacted with both red blood cells and endothelial cells. In Old World monkeys, which express blood group antigens on endothelial cells but not on red blood cells, neither GSAI-B₄ nor UEA-I reactivity were observed, except the endothelial cells from blood group B or O individuals occasionally reacted with GSAI-B₄ or UEA-I, respectively. Although UEA-I reactivity was not observed in the endothelial cells of gibbon, it reacted with these cells from chimpanzees. In these two anthropoid apes, both endothelial cells and red blood cells expressed ABH antigens as in humans. These results suggest the close evolutionary relationship between the expression of blood group ABH antigens and lectin binding properties of red blood cells and endothelial cells in primate species.     

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.     

Lectin binding sites on head structures of the spermatid and spermatozoon of the mosquito Culex quinquefasciatus (Diptera,Culicidae)

The presence of intranuclear and acrosomal lectin binding sites in spermatids and spermatozoa of the mosquito Culex quinquefasciatus was analysed. Direct and indirect lectin-gold techniques were used on LR White-embedded cells. The nuclear compartment was the structure most intensely labelled. Early spermatid nucleus showed moderate labelling for peanut agglutinin (PNA), Griffonia simplicifolia IB4 (GS-IB4) and Ricinus communis agglutinin (RCA), and light labelling for the other lectins tested. The sperm nucleus was intensely labelled by all lectins. The acrosome, an enzyme-containing structure, was labelled by some lectins. The anterior acrosomal region was labelled by PNA, while the proximal acrosomal region was labelled by PNA and G. simplicifolia II (GS II) lectins, and showed the presence of fucose residues with the use of Ulex europaeus I (UEA-I) lectin. The spermatozoa stored in the spermatheca showed the same pattern of labelling as that observed in spermatozoa localized in testis and seminal vesicles for all lectins tested. Carbohydrate residues in the nuclear compartment may be involved with the process of chromatin condensation. In the acrosomal region these residues may play a role in the process of spermoocyte interaction.     

Difference in the ability of blood group-specific lectins and monoclonal antibodies to recognize the ABH antigens in human tissues

Summary Twelve different kinds of blood group-specific lectins have been used along with monoclonal anti-A,-B and-H antibodies for detecting the corresponding antigens in selected human tissues. Although most of the lectins recognized the antigens in the tissue sections examined, they displayed marked differences in their recognition patterns in certain tissues.Helix asparsa agglutinin (HAA),Helix pomatia agglutinin (HPA) and monoclonal anti-A antibody recognized A antigens in the mucous cells of salivary glands from blood group A or AB nonsecretor as well as secretor individuals, whereasDolichos biflorus agglutinin (DBA).Griffonia simplicifolia agglutinin-I (GSA-I),Sophora japonica agglutinin (SJA) andVicia villosa agglutinin (VVA) did not bind to them from nonsecretors. A antigens in endothelial cells, lateral membrane of pancreatic acinar cells and small mucous-like cells of submandibular glands from some individuals were likewise recognized by HAA and HPA but not by other blood group A-specific lections. In contrast, both HAA and HPA did not recognize the A antigens in mucous cells of Brunner's glands while other A-specific lectins and monoclonal anti-A antibody reacted specifically with the antigens. Such a difference was not observed with lectins specific for blood group B. However, the B antigens in Brunner's glands were recognized by these lectins but not with monoclonal anti-B antibody. The difference in labelling ability was also noted among the blood group H-specific lectins and monoclonal anti-H antibody in endothelial cells of blood vessels.Ulex europaeus agglutinin-I reacted with these cells irrespective of ABO and the secretor status of the individuals, whileAnguilla anguilla agglutinin and monoclonal anti-H antibody reacted only with those cells from blood group O individuals. No reaction was observed withLotus tetragonolobus agglutinin in these tissue sites. These results suggest a great diversity of blood group antigens in different human tissues.     

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.     

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)     

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 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     

Distribution of saccharides in pig lymph-node high-endothelial venules and associated lymphocytes visualized using fluorescent lectins and confocal microscopy

Summary The distribution of saccharides in pig lymph nodes, particularly on high-endothelial venule (HEV) endothelium and on lymphocytes in these vessels, was studied by examining the binding of fluorescent conjugates of 18 different lectins. Eight of the lectins, particularly with glycan specificity restricted to mannose and polyacetyllactosamine determinants, were found to bind with a high affinity to these structures. Competitive inhibition experiments revealed that polylactosamine-containing glycans were present on endothelia and lymphocytes using lectins from Lycopersicon esculentum and Solanum tuberosum, the latter lectin reacting with lymphocytes only when apparently adherent to the luminal endothelium. The absence on pig endothelium of the Ulex europaeus binding, shown by human endothelia due to the presence of certain fucose epitopes, was confirmed. Pig lymph-node endothelium, however, bound the fucose-specific lectin of Tetragonolobus purpureas, indicating the presence of fucose on pig endothelia in a different conformation to that seen on human endothelia. The results suggested that pig lymph-node HEV endothelium expressed a core fucosylated tri- or tetra-antennary complex glycan with polylactosamine extensions and expressing an Le^y determinant.Abbreviations used BS-I Bandeiraea simplicifolia BS-I - BS-I-B B. simplicifolia isolectin B₄ - BS-II B. simplicifolia, lectin II - FACS fluorescence-activated cell sorter - FITC fluorescein isothiocyanate - HEV high-endothelial venule - LN lymph node - MLR mixed lymphocyte reaction - PBS phosphate-buffered saline - PPME phosphomannan - UEA-I Ulex europeaus lectin I     

N-Acetyl-D-glucosamine binding lectins. A model system for the study of binding specificity

Synthetic glycoconjugates prepared by the direct reductive amination of di-N-acetylchitobiose and tetra-N-acetyl-chitotetraose to poly-l-lysine with sodium cyanoborohydride have been used to explore the binding specificities of the lectins wheat germ agglutinin and Bandeiraea simplicifolia II. These conjugates are effective precipitating antigens for these lectins, and hapten inhibition experiments, employing the per-N-acetylated oligomers of chitin as inhibitors, demonstrate that wheat germ agglutinin and Bandeiraea simplicifolia II lectin have binding sites complementary to three and two contiguous β 1,4-linked N-acetyl-d-glucosamine residues, respectively, in agreement with conclusions reached using other methods. Conjugates prepared by this technique should be useful for examining the binding specificities of other lectins, and the results of a study of the effect of chain length of the hapten on the affinity of the lectin for these conjugates should provide guidance in selection of the hapten most appropriate for these studies.     

Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Summary Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. Paul's Scarlet). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 10⁵M^–1 or 9.8 × 10⁵M^–1 with a maximum of approximately 10⁸ lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.Abbreviations MPR medium, minimal organic medium (Nothnagel andLyon 1986) - APA Abrus precatorius agglutinin - CSA Cytisus sessilifolius agglutinin - ECA Erythrina cristagalli agglutinin - GS-I Griffonia simplicifolia agglutinin - LcH Lens culinarus agglutinin - PNA Arachis hypogaea agglutinin - SBA Glycine max agglutinin - VAA Viscum album agglutinin - VFA Vicia faba agglutinin - WGA Triticum vulgaris agglutinin - Con A Canavalia ensiformis agglutinin - HPA Helix pomatia agglutinin - TPA Tetragonolobus purpureas agglutinin - RCA Ricinus communis agglutinin - DBA Dolichos biflorus agglutinin - SJA Sophora japonica agglutinin - BPA Bauhinia purpurea agglutinin - FITC fluorescein isothiocyanate - Ga1NAc N-acetylgalactosamine - FDA fluorescein diacetate - 2-O-Me-D-Fuc 2-O-methyl-D-fucose Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree.     

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.     

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.     

Simultaneous expression of keratan sulphate epitope (a sulphated poly-N-acetyllactosamine) and blood group ABH antigens in papillary carcinomas of the human thyroid gland

Summary The monoclonal antibody 5-D-4 recognizes heavily sulphated forms of keratan sulphate epitope. It reacted strongly with the cell surfaces of most thyroid papillary carcinomas from all the individuals examined, independently of the blood group of the patients. Cells of follicular variants of papillary carcinomas were also labelled by 5-D-4. In contrast, no labelling with this antibody was observed in other types of thyroid neoplasms, or in normal tissues. The reactivity of 5-D-4 with papillary carcinomas was markedly reduced or abolished by prior digestion with endo-β-galactosidase keratanase II, or N-glycosidase F. Although keratanase digestion had no effect on 5-D-4 labelling, it revealed the binding sites ofGriffonia simplicifolia agglutinin II (GSA-II), which recognizes terminalN-acetylglucosamine in a limited number of carcinoma cells from some individuals. Blood group ABH antigens, which are simultaneously expressed together with keratan sulphate epitope in cancer cells, were eliminated by digestion with endo-β-galactosidase and N-glycosidase F, but were resistant to keratanase and keratanase II treatment. These results indicate that keratan sulphate oligosaccharides are cancer-associated and are probably oncofoetal antigens, as are the blood group antigens in human thyroid glands. The results suggests that poly-N-acetyllactosamine, which is ubiquitously and consistently produced in papillary carcinomas, is modified in two different ways: sulphation on the 6-position of at least some units of either galactose, orN-acetylglucosamine or both, and decoration of non-reducing termini with the blood group antigens. Along with the endo-β-galactosidase-GSA-II labelling procedure, labelling with 5-D-4 may be a useful diagnostic means for distinguishing papillary carcinoma from other types of thyroid neoplasms.     

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.     

Carbohydrate exposure of human promyelocytic HL60 cells and histiocytic U937 cells during phagocytic differentiation assessed with fluoresceinated lectins

The display of carbohydrate structures was measured in promyelocytic HL60 cells and in histiocytic U937 cells induced to differentiate to phagocytic cellsin vitro during three to seven days of cultivation in the presence of dimethylsulfoxide (DMSO). It was assessed by micro-or spectrofluorometric quantification of the binding of fluorescent lectins. Changes in the cell size and the association and uptake of IgG-or complementopsonized yeast cells (Saccharomyces cerevisiae) were used as signs of phagocyte differentiation.The binding of wheat germ agglutinin (WGA), concanavalin A (Con A),Ricinus communis agglutinin-I (RCA-I) andUlex europaeus agglutinin-I (UEA-I) varied due to the presence of DMSO during cultivation, and without DMSO also on the number of days in culture and the type of cell.Abbreviations DMSO dimethylsulfoxide - PMA phorbol 12-myristate 13-acetate - KRG Krebs-Ringer phosphate buffer with glucose - WGA wheat germ agglutinin - Con A concanavalin A - RCA-I Ricinus communis agglutinin-I - UEA-I Ulex europaeus agglutinin-I     

Interactions of galactose-binding lectins with plant protoplasts

Summary Protoplasts isolated from cell suspension cultures of carrot (Daucus carota L.) and leaves of tobacco (Nicotiana tabacum L.) were treated with three lectins specific for galactosyl residues. After incubation with RCA I (Ricinus communis agglutinin, molecular weight 120,000) conjugated to ferritin or fluorescein, freshly isolated protoplasts displayed heavy labeling of their surfaces. Moreover, they agglutinated rapidly when exposed to low concentrations of RCA I. In parallel studies, PNA (peanut agglutinin) also bound extensively to the protoplast plasma membranes whileBandeiraea simplicifolia lectin I attached relatively weakly. When protoplasts were cultured for two days and then incubated with conjugates of RCA I and PNA, additional binding sites were revealed on the regenerating walls.The results indicate that galactosyl residues are distributed densely over the surface of plant protoplasts. They also allow inferences to be made regarding the positions and linkages of the galactose groups being recognized by the lectins. Moreover, they open up the question whether the galactosyl moieties detected in the wall derive from those labeled on the plasma membrane. To conclude, we make comparisons with binding by concanavalin A, and predict that galactose-recognizing lectins will join and in certain respects prove superior to concanavalin A as probes of the plant cell surface.     

Effects of α-galactosidase digestion on lectin staining in human pancreas

Summary Effects of -galactosidase (from green coffee beans) digestion on lectin staining were examined in formalin-fixed, paraffin-embedded human pancreatic tissues from individuals of blood-group B and AB. Digestion with the enzyme resulted in almost complete loss of Griffonia simplicifolia agglutinin I-B₄(GSAI-B₄) staining in the acinar cells with concomitant appearance of Ulex europaeus agglutinin-I(UEA-I) staining in the corresponding cells. In addition, reactivity with soybean agglutinin(SBA) was also imparted by the enzyme digestion in GSAI-B₄ positive acinar cells. -Galactosidase digestion following -galactosidase digestion neither reduced the reactivity with SBA nor induced the reactivity with Griffonia simplicifolia agglutinin-II(GSA-II) in GSAI-B₄ positive cells, while in UEA-I positive cells, both reduction of SBA reactivity and appearance of GSA-II reactivity occurred after simple -galactosidase digestion as well as sequential digestion with - and -galactosidase. However, when -l-fucosidase digestion procedure was inserted between - and -galactosidase digestion, UEA-I staining imparted by -galactosidase digestion was markedly decreased in intensity and GSA-II reactivity was appeared in GSAI-B₄ positive acinar cells. Furthermore, after sequential digestion with -galactosidase and fucosidase, reactivity with peanut agglutinin(PNA) was revealed in GSAI-B₄ positive acinar cells as well as UEA-I positive cells in secretors. In non-secretors, strong PNA staining was usually observed in the acinar cells throughout the glands without enzyme digestion. These results confirmed that the -galactosidase induced GSA-II reactivity and the fucosidase induced PNA reactivity are due to precursors of different kinds of blood-group determinants and suggest that at least two kinds of B antigen determinants, i.e. Gal(1-3)[Fuc(1-2)]Gal(1-3,4)GlcNac and Gal(1-3)-[Fuc(1-2)]Gal(1-3)GalNAc are produced in GSAI-B₄ positive acinar cells. The synthesis of the latter type of B antigen is assumed to be controlled under the secretory gene in human pancreas.Abbreviation GalNAc N-acetyl-d-galactosamine - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Fuc l-fucose - NeuNAc N-acetylneuraminic acid (sialic acid)