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.     

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.     

Expression of blood group A and B antigens in nuclear heterochromatin in mucous cells of human cervical glands.

Using a post-embedding immunogold labeling procedure, we found that monoclonal antibody against A (MAb-A) or B antigen (MAb-B) reacted with nuclear heterochromatin regions, as well as secretory granules, in mucous cells of human cervical glands. Systematic and critical observation of specimens from 24 individuals of different blood groups revealed that the labeling pattern with MAb with strictly dependent on the blood group (A,B, or O) of the donors, i.e., MAb-A reacted with the heterochromatin from blood group A and AB but not with B and O individuals. Labeling with MAb-B was also specific for the heterochromatin from blood group B donors. On the other hand, MAb against H antigen did not react with the heterochromatin from any individuals examined, despite the fact that H antigens were detected by the MAb in secretory granules. Such specific reactions provide evidence that certain types of blood group-related antigens exist in the nuclear heterochromatin in mucous cells of human cervical glands. In contrast to the secretory granules in which ABH antigens were recognized by blood group-specific lectin, heterochromatin regions had little or no affinity for these lectins. Furthermore, the secretory status of individuals affected the staining intensity with MAb in secretory granules but not in the heterochromatin. These results suggest that the blood group substances found in the heterochromatin may have different molecular properties from those in the secretory granules, although both have the same determinant structures of ABH antigens.     

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.     

Early events of secretory granule formation in the rat parotid acinar cell under the influence of isoproterenol. An ultrastructural and lectin cytochemical study

The events involved in the maturation process of acinar secretory granules of rat parotid gland were investigated ultrastructurally and cytochemically by using a battery of four lectins [Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Glycine max agglutinin (SBA), Arachys hypogaea agglutinin (PNA)]. In order to facilitate the study, parotid glands were chronically stimulated with isoproterenol to induce secretion. Specimens were embedded in the Lowicryl K4M resin. The trans-Golgi network (TGN) derived secretory granules, which we refer to as immature secretory granules, were found to be intermediate structures in the biogenesis process of the secretory granules in the rat parotid acinar cell. These early structures do not seem to be the immediate precursor of the mature secretory granules: in fact, a subsequent interaction process between these early immature granule forms and TGN elements seems to occur, leading, finally, to the mature granules. These findings could explain the origin of the polymorphic subpopulations of the secretory granules in the normal acinar cells of the rat parotid gland. The lectin staining patterns were characteristic of each lectin. Immature and mature secretory granules were labelled with WGA, SBA, PNA, and lightly with UEA-I. Cis and intermediate cisternae of the Golgi apparatus were labelled with WGA, and trans cisternae with WGA and SBA.     

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.     

Histochemical demonstration of O-glycosidically linked, type 3 based ABH antigens in human pancreas using lectin staining and glycosidase digestion procedures

Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo- and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with alpha-N-acetylgalactosaminidase and alpha-L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with alpha-galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B4 reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo-alpha-N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from non-secretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(beta 1-3)-N-acetyl-D-galactosamine alpha 1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.     

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.     

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

Effects of alpha-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. beta-Galactosidase digestion following alpha-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 beta-galactosidase digestion as well as sequential digestion with alpha- and beta-galactosidase. However, when alpha-L-fucosidase digestion procedure was inserted between alpha- and beta-galactosidase digestion, UEA-I staining imparted by alpha-galactosidase digestion was markedly decreased in intensity and GSA-II reactivity was appeared in GSAI-B4 positive acinar cells. Furthermore, after sequential digestion with alpha-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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the binding specificity ofAnguilla anguilla agglutinin (AAA) in comparison toUlex europaeus agglutinin I (UEA-I)

Using immunochemical and immunohistochemical methods, the binding site ofAnguilla anguilla agglutinin (AAA) was characterized and compared with the related fucose-specific lectin fromUlex europaeus (UEA-I). In solid-phase enzyme-linked immunoassays, the two lectins recognized Fuc1-2Gal-HSA. AAA additionally cross-reacted with neoglycolipids bearing lacto-N-fucopentaose (LNFP) I [H type 1] and II [Le^a] and lactodifucotetraose (LDFT) as glycan moieties. UEA-I, on the other hand, bound to a LDFT-derived neoglycolipid but not to the other neoglycolipids tested. Binding of AAA to gastric mucin was competitively neutralized by Le^a-specific monoclonal antibodies. UEA-I binding, on the other hand, was reduced after co-incubation with H type 2- and Le^y-specific monoclonal antibodies. According to our results, AAA reacts with fucosylated type 1 chain antigens, whereas UEA-I binds only to the 1-2-fucosylated LDFT-derived neoglycolipid. In immunohistochemical studies, the reactivity of AAA and UEA-I in normal pyloric mucosa from individuals with known Lewis and secretor status was analysed. AAA showed a broad reaction in the superficial pyloric mucosa from secretors and non-secretors, but AAA reactivity was more pronounced in Le^(a+b-) individuals. On the other hand, UEA-I stained the superficial pyloric mucosa only from secretor individuals. A staining of deep mucous glands by the lectins was found in all specimens. Both reacted with most human carcinomas of different origin. Slight differences in their binding pattern were observed and may be explained by the different fine-specificities of the lectins.     

Lectin cytochemistry in the gastrointestinal tract with special reference to glycosylation in the Golgi apparatus of Brunner's gland cells.

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.     

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

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

Comparative study of blood group-recognizing lectins toward ABO blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides

Binding specificities of ABO blood group-recognizing lectins toward blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides were studied by lectin-blotting analysis, enzyme linked immunosorbent assay and lectin-conjugated agarose column chromatography. Human serum albumin conjugated with A- and B-trisaccharides was clearly recognized by Helix pomatia (HPA), Phaseolus lunatus, Dolichos biflorus agglutinins, and Griffonia simplicifolia I agglutinin B(4), respectively. Almost the same results were obtained for human group A and B ovarian cyst and A-active hog gastric mucins, but Glycine max agglutinin only reacted to the group A hog mucin. When human plasma von Willebrand factor (vWF), having Asn-linked blood group antigens, was tested, HPA was highly sensitive to blood group A antigen on the vWF. Ulex europaeus agglutinin I (UEA-I) preferentially bound to the vWF from blood group O plasma. Within the GalNAc-recognizing lectins examined, a biantennary complex-type oligosaccharide having the blood group A structure retarded on an HPA-agarose column, and the affinity was diminished after digestion with alpha-N-acetylgalactosaminidase. This product bound to UEA-I agarose column. These results indicate that HPA and UEA-I are most sensitive for detection of glycoproteins possessing small amounts of blood group A and H antigens and also useful for fractionation of complex-type oligosaccharides with blood group A and H antigens, respectively.     

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 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.     

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.     

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.     

Characteristics of the distribution of lectin receptors in intrahepatic cholangiocellular carcinoma

Summary The receptors of peanut agglutinin (PNA),Dolichos biflorus agglutinin (DBA) andUlex europaeus agglutinin I (UEA-I) were localized in intrahepatic cholangiocellular carcinoma, hepatocellular carcinoma, intrahepatic bile ducts and normal, cirrhotic and pericarcinomatous liver using the avidin—biotin—peroxidase complex method. It was found that epithelial cells of normal bile ducts had many UEA-I receptors, fewer DBA receptors and no PNA receptors. The positive rates of PNA, UEA-I and DBA receptors in 18 cases of intrahepatic cholangiocellular carcinoma were 88.9%, 61.1% and 33.3% respectively, which were significantly higher than those in hepatocellular carcinoma (16.0%, 4.0% and 4.0% respectively). Hepatocytes in normal, cirrhotic and pericarcinomatous liver had no receptors for these three lectins. It is suggested that lectin receptor distribution in intrahepatic cholangiocellular carcinoma is obviously different from that in normal bile duct cells and in hepatocellular carcinoma, and might be used as an auxiliary index in its clinical diagnosis.     

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.     

Comparison of the carbohydrate-binding specificities of seven N-acetyl-D-galactosamine-recognizing lectins

Seven plant lectins, Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin (GSA, isolectin A4), Helix pomatia agglutinin (HPA), soybean (Glycine max) agglutinin (SBA), Salvia sclarea agglutinin (SSA), Vicia villosa agglutinin (VVA, isolectin B4) and Wistaria floribunda agglutinin (WFA), known to be specific for N-acetyl-D-galactosamine-(GalNAc) bearing glycoconjugates, have been compared by the binding of their radiolabelled derivatives, to eight well-characterized synthetic oligosaccharides immobilized via a spacer on an inert silica matrix (Synsorb). The eight oligosaccharides included the Forssman, the blood group A and the T antigens, as well as alpha GalNAc coupled directly to the support (Tn antigen) and also structures with GalNAc linked alpha or beta to positions 3 or 4 of an unsubstituted Gal. The binding studies clearly distinguished the lectins into alpha GalNAc-specific agglutinins like DBA, GSA and SSA, and lectins which recognize alpha- as well as beta-linked GalNAc residues like HPA, VVA, WFA and SBA. HPA was the only lectin which bound to the beta Gal1----3 alpha GalNAc-Synsorb adsorbent (T antigen) indicating that it also recognizes internal GalNAc residues. Among the alpha GalNAc-specific lectins, DBA strongly recognized blood group A structures while GSA displayed weaker recognition, and SSA bound only slightly to this affinity matrix. In addition, DBA and SSA were able to distinguish between GalNAc linked alpha 1----3 and GalNAc linked alpha 1----4, to the support, the latter being a much weaker ligand. These results were corroborated by the binding of the lectins to biological substrates as determined by their hemagglutination titers with native and enzyme-treated red blood cells carrying known GalNAc determinants, e.g. blood group A, and the Cad and Tn antigens. For SSA, the binding to the alpha GalNAc matrix was inhibited by a number of glycopeptides and glycoproteins confirming the strong preference of this lectin for alpha GalNAc-Ser/Thr-bearing glycoproteins.