Ulex Europeus Agglutinin Research Articles

Histochemical study of colonic cancer in experimental colitis of rats.

A reliable test for premalignant lesions in the development of colonic cancer in chronic ulcerative colitis has been needed. Thus, we designed this cytochemical study, using a model of experimental colitis and colonic tumors induced in Wistar male rats by the feeding of dextran sulfate sodium. The colitis had histologic similarities to ulcerative colitis in man. The percent frequency of polypoid lesions (dysplasia or dysplasia with carcinoma in situ) in the cecum and ascending colon was about 25% at three months and 90% at six months of dextran sulfate feeding. The cytochemical findings by high-iron diamine-Alcian blue staining and Ulex europeus agglutinin binding were chronologically paralleled by histological changes in the colonic mucosa, and the binding pattern of peanut agglutinin was not different between normal and dextran-treated animals. Moreover, some cytochemical changes that occurred during the inflammatory responses were not present in dysplastic or malignant lesions. Thus, the histochemical tests were not useful for detecting of premalignant lesions earlier than by conventional histology. Nevertheless, the dextran sulfate model of colitis in the rat appears suitable for study of cancer development in ulcerative colitis.

Evidence for the presence of complex high-molecular mass N-linked oligosaccharides in intranuclear glycoproteins from HeLa cells.

Nonhistone proteins were extracted in 0.4 M NaCl from membrane-depleted nuclei of HeLa cells grown in the presence or the absence of [5,6-3H]fucose. Control experiments strongly suggest that most extracted proteins were indeed nuclear components. Several proteins, present in the 0.4 M NaCl nuclear extract, with M(r) ranging from 35,000 to 115,000 were identified on Western blots as fucosylated glycoproteins owing to their binding to the fucose-specific lectin, Ulex europeus agglutinin I. Results of experiments involving mild alkaline treatment and peptide N-glycosidase F digestion showed that the carbohydrate moieties of these fucosylated nuclear glycoproteins were N-linked to the polypeptide backbone. Analysis of the N-glycans revealed the presence of two populations of sialylated oligosaccharides on the basis of their relative molecular masses. The sensitivity of the high-M(r) oligosaccharides to endo-beta-galactosidase and their incorporation of [3H]glucosamine suggest that they could contain repeating N-acetyllactosamine units. [3H]Fucose incorporated into nuclei was confined to the nucleoli, as judged by autoradiography of sections cut through cells grown in the presence of [3H]fucose. Electron microscopy autoradiography showed that the fibrillar centers were never labeled, while silver grains were observed on the dense and the granular components of nucleoli. Taking into account of these data most nuclear fucosylated glycoproteins extracted in 0.4 M NaCl might be nucleolar ribonucleoproteins.

Cellular heterogeneity in human transitional cell carcinoma: an analysis of optical properties and lectin binding.

Flow cytometry was used to measure the binding of a panel of ten fluorescein isothiocyanate(FITC)-conjugated lectins to fifteen samples of normal and neoplastic human urothelium. Concurrent measurement of light scattering and fluorescence permitted the quantification of lectin binding to cellular subpopulations defined by their light-scattering properties. In normal urothelium, we previously demonstrated levels of lectin binding to the cellular subpopulations derived from the superficial and intermediate cell layers which were higher than levels which bound to the subpopulation derived from the basal cell layer (Ward et al., 1987). This difference was most marked with Maclura pomifera agglutinin (MPA), Ricinus communis agglutinin (RCA) and Ulex europeus agglutinin (UEA). We now report a similar correlation between the degree of differentiation of a cellular subpopulation and the level of lectin binding in human transitional cell carcinomas (TCCs). Morphological differentiation in human TCCs is accompanied by alterations in cell-surface carbohydrates which are similar to those which accompany cellular differentiation in the corresponding normal tissue. No systematic difference in lectin binding was observed between the corresponding subpopulations of normal and neoplastic urothelial cells.

Anatomical and immunohistological demonstration of the primary neural connections of the vomeronasal organ in the dog.

Macro- and microdissection methods together with conventional histology and lectin immunohistochemistry have been used to identify the course of the vomeronasal nerves and their site of termination (accessory olfactory bulb; AOB) in the dog. The AOB in this species is small and variable in size, situated on the medial surface of the main olfactory bulb, and has an anatomical structure unlike that described for other mammals. The vomeronasal nerves and their terminal glomeruli in the AOB are easily identifiable by selective immunohistochemical staining using Ulex europeus agglutinin I.

Monoclonal antibody GOM-2 binds to blood group B-Le(y) active glycolipid antigens on human gastric cancer cells, KATO-III.

The antigen structure of a mouse monoclonal antibody, GOM-2, established by immunization with KATO-III human gastric cancer cells, was examined. GOM-2 reactive glycolipids were prepared from KATO-III cells and treated with endoglycoceramidase. Structural studies of ten GOM-2 reactive oligosaccharides by a combination of glycosidase digestions, methylation, and affinity chromatography on an Ulex europeus agglutinin I (UEA-I) column revealed that nine of them had a Y-related B-active difucosylated determinant (B-Le(y)) and one had a B-active determinant. Affinity chromatography of the purified and modified oligosaccharides on an immobilized GOM-2 column demonstrated that GOM-2 has a novel binding specificity: it binds tightly to the biantennary structure carrying the B-Le(y) determinant at the termini or the branched structure carrying the B-Le(y) structure at two nonreducing termini.

Differences in cellular proteins between high and low metastatic potential sublines from the methylcholanthrene-induced rat fibrosarcoma cell line.

The purpose of the present study was to determine with two-dimensional electrophoresis whether two sublines (high and low metastatic potential to lymph nodes) of rat fibrosarcoma induced by methylcholanthrene have differences in cellular proteins. The high metastatic potential subline had a specific protein in the protein map of the whole cell lysate. Its isoelectric point was 6.4 and the molecular weight was 18,000. This specific protein appeared in maps of the precipitate of 1,000 g centrifugation and in the supernatant from treatment with 0.5% Nonidet P-40 (NP-40). But this specific protein was not stained by five lectins, concanavalin A, wheat germ agglutinin, Ulex europeus agglutinin-1, Dolichis biflorus agglutinin, and Ricinus communis agglutinin. Apparently, this high metastatic potential subline has a specific protein in the plasma membrane which has no glycochain.

The histogenesis of inflammatory fibroid polyps of the gastrointestinal tract

Inflammatory fibroid polyps are lesions occurring in the submucosa of the gastrointestinal tract. These lesions have been identified by a variety of names, indicative of their uncertain histogenesis. Three cases were studied by light microscopy and cytochemistry and, in one case, by electronmicroscopy. The most characteristic feature consisted of concentric fibrovascular structures. Peroxidase reaction for muscle actin and Ulex europeus agglutinin was positive in the two main cellular components. Electronmicroscopy revealed that the two principal cell constituents were endothelial and myocytic cells. These observations support the conclusion that inflammatory fibroid polyps are lesions of vascular origin.

Lectin histochemistry of developing submandibular glands of fetal Syrian golden hamsters

Lectin binding was studied in the developing submandibular glands of fetal Syrian golden hamsters (Mesocricetus auratus) from gestational day 12 to 16 (the day of birth). The fetuses were fixed, embedded in paraffin, sectioned and stained with nine lectin-horseradish peroxidase conjugates: concanavalin A (Con A), wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Maclura pomifera agglutinin (MPA). Griffonia simplicifolia agglutinin I-B4 (GSA I-B4), peanut agglutinin (PNA). Ulex europeus agglutinin I (UEA I) and Limulus polyphemus agglutinin (LPA). The developing glands showed dramatic morphological alterations on a daily basis, accompanied by progressive changes in lectin staining. On day 12 the primitive gland showed only trace lectin staining with WGA, HPA, MPA, PNA and UEA I, but by day 13, strong staining with these lectins, as well as with DBA, was seen at the ductal lumenal surface, after the formation of the ductal lumens. Secretory granules first appeared in cells of the primitive acini on day 14: the secretion products were stained strongly with WGA. DBA, HPA, MPA, PNA and UEA I. On day 15, the secretion products were also stained moderately with GSA I-B4. Secretory differentiation was further developed on day 16, but the staining intensity of the mucins with the different lectins varied among the secretory cells. LPA failed to stain any part of the gland throughout the observation period, and Con A stained only glycogen.

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.

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.

Differences in lectin binding patterns of normal endometrium and endometrial adenocarcinoma, with special reference to staining with Ulex europeus agglutinin 1 and peanut agglutinin

Differences in glycoconjugate composition between proliferative endometrium and endometrial adenocarcinoma were investigated by histochemical techniques using seven different lectins as probes. For light microscopy, the avidin-biotin-peroxidase complex (ABC) method was used. Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) stained almost all glandular cells in both proliferative (normal) and malignant endometria. Ulex europeus agglutinin 1 (UEA-1) strongly stained cancer cells, especially well-differentiated adenocarcinoma, but it scarcely stained normal endometrium. Peanut agglutinin (PNA) binding sites were observed only along the apical surface of normal endometrial glands, while the cytoplasm of endometrial adenocarcinoma cells was often positive for PNA. Soybean agglutinin (SBA) faintly reacted with proliferative endometrium and occasionally with malignant cells. Dolichos biflorus agglutinin (DBA) slightly stained proliferative and malignant endometria. By electron microscopic examinations using horseradish peroxidase (HRP)-labeled lectins, we observed that UEA-1, PNA, and Con A stained the Golgi membranes and plasma membrane of carcinoma cells. In addition, the Con A reaction was positive in the endoplasmic reticulum and nuclear envelope. These results revealed the differences in oligosaccharide chains between normal and malignant endometria, suggesting that UEA-1 and PNA, in particular, may be useful indicators of malignancy of the endometrium.

Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. I. Growth conditions and morphology of murine thymic epithelial and mesenchymal cells.

We report here the successful selective cultivation of murine thymic mesenchymal reticular cells (MTMC) and murine thymic epithelial cells (MTEC) grown on extracellular matrix in the presence of defined medium. The selective growth of these two cell types was based on 1) conditions of tissue disruption and 2) differential growth requirements. Both cell types were dependent on transferrin, high density lipoproteins, insulin, hydrocortisone, and epidermal growth factor, whereas MTMC was dependent also on selenium and 3,5,3'-triiodothyronine. The elimination of single factors or extracellular matrix resulted in specific and different changes in the growth pattern of each cell subpopulation. Cells of both types exhibited the ultrastructural features of high metabolic activity. The epithelial nature of MTEC cultures was defined by bundles of tonofilaments and desmosomes and by positive staining to keratins and negative to vimentin. In addition MTEC were positively stained with mAb to thymic medullary epithelial cells and by Ulex europeus agglutinin, and were able to form Hassall's corpuscles, suggesting their medullary origin. MTEC were also H-2 and Ia positive. In contrast MTMC were positive for vimentin and periodic acid-Schiff, low positive for H-2, and negative for keratin and Ia. Both cells did not contain nonspecific esterase, nor did they phagocytize latex beads. With the use of all these criteria we classified MTEC as epithelial cells from the medullary compartment of the thymus and MTMC as reticular cells of mesenchymal origin.

Distribution of thymocytes expressing gamma delta receptors in the murine thymus during development.

We have examined the appearance of thymocytes expressing gamma delta TCR within the developing thymus by using immunohistochemical techniques and flow cytometry in conjunction with the mAb 3A10, which recognizes a determinant associated with the constant region of the delta-chain. gamma delta+ Cells were first detected at day 16 of gestation, attained maximal levels at day 17 of gestation, and declined thereafter. By using the Ulex europeus agglutinin to identify medullary epithelial cells in situ, we observed a striking colocalization of gamma delta+ thymocytes and U. europeus agglutinin-positive medullary epithelial cells during late fetal and neonatal periods of development. In the thymuses of adult mice, gamma delta+ thymocytes were scattered throughout cortical and medullary areas of the thymus and most concentrated in the subcapsular areas of the thymus. Ultrastructural immunohistochemistry confirmed the close association between medullary thymic epithelial cells and gamma delta+ thymocytes in the neonatal thymus and also showed that some TCR-gamma delta molecules were patched to areas of contact with medullary epithelial cells. In contrast to the cellular distribution of either CD3 molecules or the TCR-alpha beta, where extensive intracellular labeling of thymocytes has been observed, cytoplasmic accumulation of delta-chain was not detected.

Effect of Malnutrition and Hormone Treatments on Intestinal Microvillus Membrane Glycosylation in Suckling Rats

Imposition of undernutrition in suckling rats significantly enhanced (p less than 0.001) sialic acid and reduced (p less than 0.001) fucose contents of intestinal microvillus membranes (MVM) compared to the controls. Administration of cortisone or insulin to undernourished (UN) pups reversed the sialylation and fucosylation pattern of the membranes. The binding of wheat germ agglutinin (WGA) to membranes was increased (p less than 0.001) and that of Ulex europeus agglutinin (UEA) was decreased in nutritionally deprived pups. Cortisone and insulin treatments reduced WGA and enhanced UEA bindings to UN membranes. The binding of peanut agglutinin (PNA) to UN membranes was 60% higher compared to that in the control rats. Cortisone and insulin treatments further enhanced PNA binding to the brush borders in UN pups. The incorporation of 14C-GlcNAc, 14C-mannose and 3H-fucose into MVM was reduced by 32-57% in UN suckling rats compared to the controls. The incorporation of labelled sugars into membranes from 21- and 50- to 60-day-old rats was essentially similar. Cortisone administration to UN pups augmented the incorporation of labelled GlcNAc and mannose but had no effect on fucose incorporation. Insulin treatment of UN pups insignificantly elevated GlcNAc incorporation into the membranes. These findings suggest that imposition of UN during early suckling period delays the maturational development of intestinal glycosylation. Cortisone and insulin induced adult-type glycosylation pattern of the brush borders in nutritionally deprived pups.

Intestinal Absorption of Macromolecules and Epithelial Cell Surface Glycosylation in Suckling Rats Nursed on Mothers Fed Low-Protein Diet-I

The effect of feeding 8% protein (low-protein; LP) diet to lactating rats has been studied on the absorption of macromolecules and the glycosylation of enterocytes in suckling animals. The absorption of 125I-labelled bovine serum albumin, gamma-globulin and alpha-lactalbumin was 24-314% more in pups nursed on mothers fed LP diet compared to the pair-fed controls. The observed enhancement in protein absorption was associated with a significant increase in the binding of these proteins to microvillus membranes (MVM). The sialic acid content of brush borders was unaltered but the fucose level was augmented (p less than 0.01) in pups nursed on rats fed LP diet. The binding of 125I-labelled wheat germ agglutinin and Ulex europeus agglutinin I to MVM was in agreement to the data on sialic acid and fucose levels of the membranes. The binding of peanut agglutinin to MVM was 30% low in pups from LP-diet-fed animals. The incorporation of (14C) D-mannose into MVM was essentially unaffected but that of (14C) N-acetylglucosamine was reduced in pups reared on mothers given LP diet. These findings suggest that the quality of maternal nutrition affects the absorption of macromolecules and the glycosylation in developing rat intestine.

The Use of Lectins in Histopathology

Lectins are proteins and glycoproteins extracted predominantly from plants which have the capacity to bind sugars specifically. This property makes them of interest for histopathology since they will bind to saccharides forming parts of glycoproteins and glycolipids of tissue constituents. Lectins have and can be used as reagents for mucin histochemistry, to identify specific cells, in the recognition of glycoprotein alterations in disease states, in studies of infectious diseases, and in the assessment of glycoconjugate alterations occurring with malignancy. They can be used for both light microscopic and ultrastructural localisation and various methods are available. It is important though, to consider the nature of the glycoconjugates under study and select lectins appropriately because of their varying specificities and binding characteristics. A panel of lectins should be used to study a particular configuration. Care should be taken with tissue fixation and processing. It must be remembered that an open and critical mind should be kept concerning interpretation of results. At the present time lectins have a limited value diagnostically, but the binding of Ulex europeus agglutinin to endothelium is certainly of value.

Gliosarcoma: an immunohistochemical study.

Gliosarcomas contain both neuro-ectodermal and mesenchymal elements. Its histogenesis has been much debated and endothelial and adventitial fibroblast origins have been suggested, as has a "histiocytic" origin following the demonstration of antiprotease activity. Eight gliosarcomas have been examined with a panel of ten monoclonal and polyclonal antibodies to investigate the origin of the sarcomatous element. Glial fibrillary acid protein expression showed a sharp distinction between gliomatous and sarcomatous tumour components. Contrary to some previous reports factor 8-related antigen and Ulex europeus agglutinin stained vascular luminal endothelium but no tumour cells. Vimentin and fibronectin expression was extensive and confined largely to sarcomatous areas. Desmin and neurofilament protein could not be demonstrated in any of the cases. Numerous cells, particularly in the sarcoma areas, expressed alpha-1-antitrypsin and -chymotrypsin. A proportion of these stained for the monocyte/macrophage marker MAC 387. Four cases focally exhibited a true storiform pattern and this and the immunohistochemical results suggest analogies with the fibrous histiocytomas. These tumours contain reactive histiocytes but are now thought to be derived from fibroblasts or from pluripotent mesenchymal cells in perivascular adventitia. This resembles the pattern exhibited in the sarcomatous component of gliosarcomas.

Establishment of human mucosal microvascular endothelial cells from inferior turbinate in culture

Human microvascular endothelial cells were isolated and cultured from the mucosa of inferior turbinates. Using dish-coated collagen and a medium composed of a 1:1 mixture of sarcoma 180-conditioned medium and Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum and 75 μg/mL endothelial cell growth factors prepared from bovine pituitary glands), these cells grew rapidly to confluence and survived serial passages until the 16th population doubling level. The cells were identified as endothelial cells by their morphology, immunostaining of factor VIII antigen, and cytochemical staining with Ulex europeus agglutinin. Furthermore, Weibel-Palade bodies and numerous pinocytotic vesicles were confirmed by electron microscopy. Proliferation experiments demonstrated the need for either endothelial cell growth factor or tumor-conditioned medium. An exogenous matrix was also required for these cells in tissue culture. A tubule-like morphology appeared in the original monolayer of human microvascular endothelial cells after 1 month in the same plate, indicating that these cells have the ability to form tubules in the presence of sarcoma 180-conditioned medium.

Ulex europeus agglutinin I binds exclusively to primary olfactory neurons in the rat nervous system

Lectin binding histochemistry was used to investigate the distribution of binding sites for the fucose-selective lectin Ulex europeus agglutinin I in the rat nervous system. It was found that this lectin bound exclusively to a surface membrane component, probably a glycoprotein, on primary olfactory and vomeronasal sensory neurons, and to no other structure in the nervous system. A similar pattern of Ulex europeus agglutinin I binding was found in adult, immature and embryonic rats. Binding was also demonstrated on olfactory axons regenerating through a peripheral nerve graft. Three other lectins, including another fucose-selective lectin, and theN-acetyl galactosaminebinding soybean lectin failed to show similar exclusive binding. The presence of a specific surface glycoconjugate solely on olfactory neurons suggests that the molecule may play some role in development or maintenance of organization of this neuronal pathway.

Bis-(β-Chloroethyl)sulfide (BCES)-induced changes in epidermal cell homeostasis in vitro

A rat cutaneous keratinocyte culture system was developed to study the effects of the vesicant bis-(β-chloroethyl)sulfice (BCES) on the homeostasis of cell proliferation and differentiation. Lectins were used to reveal cell surface carbohydrate changes as the keratinocytes differentiate. In the newborn rat epidermis, the isolectin, Griffonia simplicifolia I-B 4 (GS I-B 4), binds to basal cell surfaces. Ulex europeus agglutinin I (UEA) binds to the surfaces of spinous and lower granular cells and is therefore considered an indicator of keratinocyte differentiation. A fluorometric assay was developed which determines the ratio of bound UEA to bound GS I-B 4 (the UEA B 4 ratio) in primary monolayer cultures of rat cutaneous keratinocytes maintained in low Ca 2+ medium. The UEA B 4 ratio was found to be a representation of the relative sizes of the differentiating and proliferating cell compartments in the monolayer cultures, respectively (W. W. Ku and I. A. Bernstein, 1988, Exp. Cell Res., 175, 298–316) . Monolayer cultures exposed for 1 hr to BCES at Day 1 exhibited a dose-related increase in the UEA B 4 ratio at Day 7 when compared to solvent controls. The results from the analysis of lectin binding sites showed a decrease in GS I-B 4 binding with little or no change in UEA binding as a result of BCES exposure, contributing to the increase in the UEA B 4 ratio. BCES-exposed monolayers also showed early perturbations in replicative DNA synthesis as revealed by autoradiography. Subsequent to the perturbations in replicative DNA synthesis was an inability of BCES-exposed cultures to produce cells into the monolayer through mitosis. In addition to an increase in the UEA B 4 ratio, BCES-exposed monolayers also showed a dose-related loss of DNA, with the appearance of enlarged cells at Day 7. These enlarged cells failed to show evidence of DNA synthesis, with groups of these cells showing intense UEA staining with only faint GS I-B 4 staining. Overall, exposure to low concentrations of BCES appeared to disrupt the normal homeostasis of cell proliferation and differentiation in this monolayer culture system. This disruption was primarily through a reduction in the fraction of germinative (basal) cells with concomitant retention of some early differentiated cells, presumably early spinous or spinous cells.