Limulus Polyphemus Agglutinin Research Articles

Sialoglycoproteins in Morphological Distinct Stages of Mucor polymorphosporus and their Influence on Phagocytosis by Human Blood Phagocytes

The possible role of sialic acids in host cells-fungi interaction and their association with glycoproteins were evaluated using a clinical isolate of the dimorphic fungus Mucor polymorphosporus. Lectin-binding assays with spores and yeast cells denoted the presence of surface sialoglycoconjugates containing 2,3- and 2,6-linked sialylglycosyl groups. Western blotting with peroxidase-labeled Limulus polyphemus agglutinin revealed the occurrence of different sialoglycoprotein types in both cell lysates and cell wall protein extracts of mycelia, spores, and yeasts of M. polymorphosporus. Sialic acids contributed to the surface negative charge of spores and yeast forms as evaluated by adherence to a cationic substrate. Sialidase-treated spores were less resistant to phagocytosis by human neutrophils and monocytes from healthy individuals than control (untreated) fungal suspensions. The results suggest that sialic acids are terminal units of various glycoproteins of M. polymorphosporus, contributing to negative charge of yeasts and spore cells and protecting infectious propagules from destruction by host cells.

Splicing Determines the Glycosylation State of Ameloblastin

In developing porcine enamel, the space between enamel rods selectively binds lectins and ameloblastin (Ambn) N-terminal antibodies. We tested the hypothesis that ameloblastin N-terminal cleavage products are glycosylated. Assorted Ambn cleavage products showed positive lectin staining by peanut agglutinin (PNA), Maclura pomifera agglutinin (MPA), and Limulus polyphemus agglutinin (LPA), suggesting the presence of an O-linked glycosylation containing galactose (Gal), N-acetylgalactosamine (GalNAc), and sialic acid. Edman sequencing of the lectin-positive bands gave the Ambn N-terminal sequence: VPAFPRQPGTXGVASLXLE. The blank cycles for Pro(11) and Ser(17) confirmed that these residues are hydroxylated and phosphorylated, respectively. The O-glycosylation site was determined by Edman sequencing of pronase-digested Ambn, which gave HPPPLPXQPS, indicating that Ser(86) is the site of the O-linked glycosylation. This modification is within the 15-amino-acid segment (73-YEYSLPVHPPPLPSQ-87) deleted by splicing in the mRNA encoding the 380-amino-acid Ambn isoform. We conclude that only the N-terminal Ambn products derived from the 395-Ambn isoform are glycosylated.

Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates.

Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent.

Cell surface composition of promastigote and opisthomorph forms of Herpetomonas roitmani (Kinetoplastida : Trypanosomatidae).

Cell surface saccharide composition and surface charge of promastigote (PRO) and opisthomorph (OPM) forms of Herpetomonas roitmani were analyzed using labeled lectins and flow cytometry and cell electrophoresis. The FITC signals for concanavalin A, Helix pomatia agglutinin and wheat germ agglutinin were stronger in PRO forms, whereas for Limulus polyphemus agglutinin (LPA) and Wisteria floribunda agglutinin they were stronger in OPM forms. Prior treatment of the cells with neuraminidase decreased the FITC signal for LPA in OPM but not in PRO forms. Furthermore OPMs displayed a high negative charge (-15.45+/-1.10 mV) than PROs (-9.47+/-1.01 mV). Neuraminidase and phospholipase C treatment of the parasites significantly reduced the surface charge, especially in OPM forms. TLC analysis of the acidic components of H. roitmani showed the presence of N-acetyl-neuraminic acid. The results presented in this work indicate that changes in exposed cell surface components occur between PRO and OPM forms of H. roitmani obtained by growing the cells under different conditions.

Relevance of sialoglycoconjugates in murine thymocytes during maturation and selection in the thymus

Differentiation of most T lymphocytes is characterized not only by the variable expression of CD4/CD8 coreceptor molecules and increased surface density of the T cell antigen receptor, but also by changes in the glycosylation pattern of cell surface glycolipids or glycoproteins. In this work we evaluated the changes in the sialylation pattern in thymus sections from normal and dexamethasone treated mice. We used sialic acid specific lectins, such as Sambucus nigra agglutinin (SNA, NeuAcα2,6-Gal specific) and Maackia amurensis agglutinin (MAA, NeuAcα2,3-Gal specific). Our results indicate that the sialylation pattern was modified during the maturation process of thymic cells. The immature CD4-CD8– and CD4+CD8+ cortical thymocytes were recognized by SNA, whereas the mature single positive (CD4+ or CD8+) medullary cells, preferentially bound MAA lectin. However, in the corticomedullary region we found not only SNA+ cells, but also MAA+ cells. In the thymus of dexamethasone treated mice, the clusters of thymocytes undergoing apoptosis in the cortex were characteristically stained by SNA. These results suggest that in the initial stages of the differentiation pathway, a great number of thymocytes express an α2,6 linked sialic acid on their surface and as they progress to more mature stages there is a change in the sialylation pattern to α2,3 linked sialic acids probably due to a regulated expression of different sialyltransferases, which could be modulated by the thymic microenviroment. Abbreviations: SNA (Sambucus Nigra agglutinin); MAA (Maackia amurensis agglutination); NeuAc (N-acetyl-neuraminic acid); Gal (Galactose); GalNac (N-acetyl-galactosamine); GlcNAc (N-acetyl-glucosamine); Ser (Serine); Thr (Threonine); CMP (Cytidine monophosphate); LPA (Limulus polyphemus agglutinin); DAB (3,3' diaminobenzidine tetrahydrochloryde); HE (Hematoxilin-eosin); FITC (Fluorescein isothiocyante)

Lectin histochemistry in the aged dog brain.

Formalin-fixed and paraffin-embedded canine brains were examined histochemically using 15 selected lectins. Concanavalin A (Con A), Lens culinaris agglutinin, Lycopersicon esculentum agglutinin (LEL) and Limulus polyphemus agglutinin (LPA) labeled neurons in an age-dependent manner. These and some other lectins [Dolichos biflorus agglutinin (DBA), Vicia villosa agglutinin (VVA), Ricinus communis agglutinin 120 (RCA-I), Bandeiraea simplicifolia agglutinin (BSL-I), and Phaseolus vulagaris agglutinin-L (PHA-L)] also age-dependently labeled glial cells. These results indicate that monosaccharide composition and biochemical metabolism in brain cells change with age and that these lectins may be useful as histochemical markers for investigating senile changes in the canine brain. However, no significant correlation was found between ApopTag-positive and lectin-positive cells. Amyloid plaques were positive for Con A, DBA, Glycine maximus agglutinin (SBA), LEL, PHA-L, Limax flavus agglutinin (LFA) and VVA. Among these lectins, VVA, SBA and LFA intensely stained amyloid both in blood vessel walls and senile plaque cores. Therefore, the sugar residues recognized by these lectins likely play specific roles in beta-amyloid deposition in the aged dog brain.

Anionogenic groups and surface sialoglycoconjugate structures of yeast forms of the human pathogen Paracoccidioides brasiliensis.

The surface anionogenic groups and sialoglycoconjugate structures of Paracoccidioides brasiliensis yeast forms were analysed by cell microelectrophoresis, binding assays with lectins and viral particles, ultrastructural cytochemistry, enzymic digestion and flow cytofluorimetry. P. brasiliensis yeast forms, particularly the budding primordia, reacted strongly with cationized ferritin. Binding assays showed that the reaction with sialic-acid-specific Limax flavus lectin (LFA) was distributed over the entire P. brasiliensis cell wall. Treatment of yeast forms with neuraminidase significantly reduced their negative surface charge and LFA labelling, which suggests that sialic acid residues are major anionogenic groups exposed on the P. brasiliensis surface. Furthermore, after neuraminidase treatment, labelling with Arachis hypogaea (peanut) agglutinin increased due to unmasking of subterminal beta-D-galactopyranosyl residues. The sialic acid linkages to galactose are alpha 2,6 and alpha 2,3 as assessed, respectively, by fungal attachment to M1/5 and M1/5 HS8 strains of influenza A virus and binding of Sambucus niger and Maackia amurensis agglutinins. The alpha 2,6 linkage clearly predominated in both experiments. Flow cytofluorimetry analysis revealed the heterogenicity of P. brasiliensis yeast cell populations, which comprised young and mature budding yeasts. Both express binding sites to LFA and Limulus polyphemus agglutinin.

Lectin‐Binding Sites Involved in Paramecium primaurelia Mating Pair Formation

ABSTRACT. Mating pair formation in Paramecium primaurelia was shown to be inhibited by incubating mating‐competent mating type (mt) I and mt U cells with Limulus polyphemus agglutinin (LPA) or wheat germ agglutinin (WGA). Preincubation of LPA and WGA with their specific binding‐monosaccharides, N‐acetylneuraminic acid (NeuAc) and N‐acetylglucosamine (GlcNAc), respectively, prevented the lectin effect on pair formation. Addition either of NeuAc or GlcNAc resulted in a reversal of cell pairing inhibition by LPA or WGA, respectively. Both NeuAc and GlcNAc monosaccharides inhibited pair formation when their concentration exceeded a threshold value. The pattern of the relative distribution of NeuAc and GlcNAc molecules on the cell surface was analyzed using fluorescence resonance energy transfer techniques combined with imaging systems. Mt I1 cells showed a high lectin‐binding site density localized just on the surface region engaged in conjugation. The pattern of mt I cells, consisting of a quite homogeneous lectin‐binding site density spread on the cell surface, was also common to nonmating‐competent cells and to immature cells. These findings suggest that in P. primaurelia pair formation involves both NeuAc and GlcNAc residues and that the development of mating‐competence is related to a modification in NeuAc and GlcNAc relative distribution on the cell surface of mt 11 cells.

Lectin-histochemical reactivity of sialic acid in breast cancer and its relationship to prognosis using limulus polyphemus agglutinin

Studies of circulating sialic acid have revealed its relationship with a variety of malignant tumors. It is not vet clear whether sialic acid could be used as a prognostic marker of breast cancer, and few studies have examined sialic acid expression in the cell membrane and cytoplasm of breast cancer cells by means of the lectin-histochemical technique. In the present study, we used biotinylated limulus polyphemus agglutinin (LPA), a special binding lectin of sialic acid, to stain sialic acid in breast cancer cells. Of the 104 cases of breast cancer examined, 59 (56.7%) positive cases were observed. There was a significant correlation between the LPA staining and the clinicopathologic features of all patients, including pathological stage and lymph node metastasis. Among the 100 patients who underwent curative operation, the mean disease-free survival rate of the 45 patients who were LPA-negative was significantly higher than that of the 55 LPA-positive patients (p<0.05). These results suggest that the positive expression of sialic acid in breast cancer could be used as a marker of malignancy potential, as well as a poor survival factor, and the biotinylated LPA assay may provide a convenient and useful method to predict the prognosis of breast cancer.

A lectin histochemical study on carbohydrate moieties of the gonadotropin-like substance in the epithelial cells of Hatschek's pit ofBranchiostoma belcheri

The present light microscopic lectin, histochemical study suggests for the first time that the vertebrate gonadotropin-like substance in the basal part of the epithelial cells of Hatschek's pit is a sialic acid-containing glycoprotein. The binding intensity of the epithelial cells in Hatschek's pit to 6 lectins (Limulus polyphemus agglutinin (LPA), Wheat germ agglutinin (WGA),Helix pomatia agglutinin (HPA), Concanavalin A (Con A),Ulex europaeus agglutinin I (UEA I) andRicinus communis agglutinin I (RCA I)) indicate that the carbohydrate composition of the gonadotrophic glycoprotein is similar to that of mammals and fish, and that N-acetyl-D-galactosamine, sialic acid, glucosamine, D-mannose and L-fucose are components of the carbohydrate portion.

Changes in glycoconjugate-lectin binding characteristics of in vitro and in vivo mouse blastocysts.

To investigate the influence of embryo culture, on blastocysts cultured from the zygote stage, on blastocyst cell surface glycoconjugates as an indicator of cellular metabolism. Eleven fluorescein isothiocyanate (FITC)-labelled lectins, which recognize different oligosaccharide structures, were applied to zona pellucida intact and zona pellucida free blastocysts which had developed either in vivo or in vitro. The differential binding patterns for the zona pellucida, trophectoderm and inner cell mass were determined using fluorescence microscopy. With the exception of wheat germ agglutinin (WGA) and Limax flavus agglutinin (LFA) the remaining nine lectins bound less intensely to the zona pellucida of the in vitro blastocysts compared with the in vivo blastocysts. The trophectoderm from both groups bound seven lectins with equal intensity, of the remaining four Lotus tetragonolbus agglutinin (LTA), Bandeiraea simplicifolia 4 (BSB4), Phaseolus vulgaris agglutinin P (PHA-P) and Limulus polyphemus agglutinin (LPA) demonstrated differential binding. The inner cell mass from the two blastocyst groups bound only three lectins identically, peanut agglutinin (PNA), Bandeiraea simplicifolia 4 (BSB4) and Dolichos biflorus agglutinin (DBA). Oviductal glycoproteins contribute to the zona pellucida glycoprotein content during early development, however embryo culture from the zygote stage may not provide adequate substrates for normal inner cell mass glycoconjugate biosynthesis.

Occurrence of N-acetyl- and N-O-diacetyl-neuraminic acid derivatives in wild and mutant Crithidia fasciculata.

The cell-surface expression of sialic acids in wild-type Crithidia fasciculata and three drug-resistant mutants (FU(R)11, TR3, and TFRR1) was analyzed using fluorescein-labeled Limulus polyphemus agglutinin (LPA) binding, glycosidase of known sugar specificity, and thin-layer chromatography (TLC). Gas-liquid chromatography-mass spectrometry (GC-MS) analysis using both electron-impact (EI-MS) and chemical ionization (CI-MS) by isobutane with selected ion monitoring (SIM) was also used. The surface location of sialic acid was inferred from LPA binding to whole cells abrogated by previous treatment with neuraminidase. An exception occurred with the TFRR1 strain, which after incubation with neuraminidase showed increased reactivity with the fluorescent lectin. Both N-acetyl- and N-O-diacetyl-neuraminic acids were identified in the flagellates by TLC, with a clear predominance being noted for the former derivative. However, the content of N-O-diacetyl-neuraminic acid was preferentially found in the TFRR1 strain. The GC-MS analysis of the acidic component of the TFRR1 mutant strain confirmed the occurrence of N-acetyl-neuraminic acid (Neu5Ac) by the presence of the diagnostic ions (m/z values: 684 and 594 for CI-MS and 478, 298, and 317 for EI-MS) and also by comparison with the standard Neu5Ac retention time. GC-MS analysis also showed fragments (m/z values: 654 and 564 for CI-MS and 594, 478, 298, and 317 for EI-MS) expected for the 7-O- and 9-O-acetyl-N-acetyl-neuraminic acids (Neu5,7Ac2 and Neu 5,9Ac2, respectively).

Crossreactivity of IgE antibody against Dermatophagoides farinae with Limulus polyphemus agglutinin.

Crossreactivity of IgE antibody against Dermatophagoides farinae (Der f) with Limulus polyphemus agglutinin (LPA) was examined using RAST and immunoblot analysis. Of 40 Der f-sensitive asthmatic patients, 28 revealed a positive RAST reaction to LPA, while none of 20 Der f-insensitive hay fever patients showed this reaction. LPA-specific RAST levels of the 40 asthmatic patients correlated with their Der f-specific levels. The RAST reactivity to LPA was competitively inhibited by the addition of either soluble Der f or LPA, but not by the specific inhibitory sugar of sialic acid. LPA could also induce histamine release from leucocytes of Der f-sensitive asthmatic patients. IgE immunoblot analyses showed that the positive RAST sera for LPA had a strong IgE binding activity to the 30 kDa and 80 kDa components of Der f body extract, whereas gel filtration studies showed that the high molecular weight fractions above 150 kDa retained antigenic constituents associated with IgE reactivity to LPA. These results suggest that the antigenic materials of Dermatophagoides mites share some determinants with the haemagglutinin of horseshoe crabs.

Purification and characterization of 240-kDa cGMP-dependent protein kinase substrate of vascular smooth muscle. Close resemblance to inositol 1,4,5-trisphosphate receptor

The 240-kDa, cGMP-dependent protein kinase substrate protein obtained from porcine aortic smooth muscle, whose phosphorylation was closely associated with stimulation of plasma membrane Ca(2+)-pump ATPase (Yoshida, Y., Sun, H.-T., Cai, J.-Q., and Imai, S. (1991) J. Biol. Chem. 266, 19819-19825), was purified to near homogeneity by three successive chromatographic runs with calmodulin-, concanavalin A-, and heparin-Sepharose columns from microsomes solubilized with Triton X-100. The purified protein was found to bind inositol 1,4,5-trisphosphate (InsP3) in a specific, heparin-inhibitable manner with a Kd of 2.0 nM and Bmax of 450 pmol/mg protein (the binding of inositol 1,3,4,5-tetrakisphosphate was much weaker). In sedimentation experiments on a linear sucrose density gradient the InsP3 binding activity was always with the 240-kDa protein. Protein kinase G phosphorylated the InsP3 receptor purified from the rat cerebellum as well as the 240-kDa protein. Sialic acid content of the protein measured with Limulus polyphemus agglutinin was not significantly different from that of the cerebellar InsP3 receptor. Thus, 240-kDa protein closely resembles InsP3 receptor and may be a type of InsP3 receptor. The only difference was the behavior on SDS-polyacrylamide gel electrophoresis. The 240-kDa protein presented itself as two polypeptides with similar but slightly differing M(r) values, both of which were phosphorylated by protein kinase G.

Dimethyl sulfoxide (DMSO) increases expression of sialyl Lewis x antigen and enhances adhesion of human gastric carcinoma (NUGC4) cells to activated endothelial cells.

Dimethyl sulfoxide (DMSO) exerts a number of biological effects including the promotion of cell differentiation in cultured cells. In this study, we examined the effect of DMSO on the adhesion of tumor cells to endothelial cells. In vitro treatment of human gastric adenocarcinoma (NUGC4) cells with DMSO resulted in increased adhesion to interleukin-I (IL-I)-activated human endothelial cells compared with DMSO-untreated NUGC4 cells. In flow cytometry, treating NUGC4 cells with DMSO enhanced the expression of sialyl Lewis x (sialyl Le(x)) and sialyl dimeric Le(x) antigens on their surface. Also, the binding of Limulus polyphemus agglutinin (LPA), which specifically binds to cell-surface sialic acids, was increased by DMSO. The adhesion of DMSO-treated NUGC4 cells to activated endothelial cells was blocked by neuraminidase pre-treatment of tumor cells or by antibody against either endothelial leukocyte adhesion molecule-I (ELAM-I) or sialyl Le(x). Thus, it is suggested that enhanced adhesion following DMSO treatment is mediated by the interaction of sialyl Le(x) expressed on NUGC4 cells with ELAM-I of endothelial cells. The modulation of sialyl Le(x) antigen by DMSO provides a useful system for studying the regulatory mechanism of Lewis-related carbohydrate antigens and also for understanding the metastatic properties of cancer cells.

Cell surface carbohydrates modulate neutrophil adherence to alveolar type II cells in vitro.

We characterized the influence of phosphorylated sugars and cell surface sialic acids on the adherence of human polymorphonuclear leukocyte (PMN) to rat alveolar type II cell (ATII cells) and human-derived A549 cell monolayers in vitro. Percent adherence of radiolabeled polymorphonuclear leukocytes was assessed after incubating cells with the carbohydrates, enzymes, or lectins to be tested. Lactose-1-phosphate (Lact1P) and maltose-1-phosphate (Malt1P) (10 mM) inhibited adherence of PMN to ATII cells and A549 cells. Maximal inhibition followed treatment of both PMN and rat ATII cells and amounted to 85 +/- 7% with Lact1P and 92 +/- 3% with Malt1P. Inhibition was concentration dependent. Incubation of PMN with mannose-6-phosphate reduced adherence to rat ATII cells and A549 cells by 36 +/- 11 and 39 +/- 8%, respectively. Maximal concentrations of sugars did not alter cellular viability. Neuraminidase-induced desialilation of ATII cells increased adherence of PMN by 36 +/- 7% to rat ATII cells and by 86 +/- 18% to A549 cells. Masking of terminal sialic acids on rat ATH cells with Limulus polyphemus agglutinin (100 micrograms/ml) increased adherence by 50 +/- 2%. These results indicate that cell surface carbohydrates are involved in the regulation of the adhesive interaction between PMN and ATII cells in vitro.

Identification of sialic acids on the cell surface of hyphae and yeast forms of the human pathogen Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, when grown in a synthetic medium, expresses at the cell surface of both yeast and mycelial forms acidic glycoconjugates containing N-acetylneuraminic acid units. Sialic acids were extracted using mild hydrolytic conditions, and were identified by thin-layer and gas chromatography, standard colorimetry, reaction with periodate-resorcinol and mass spectrometry. Their surface location was inferred from fluorescent-lectin (Limulus polyphemus agglutinin) binding to whole cells abrogated by previous treatment with neuraminidase. Expression of sialic acids on virulent yeast forms of P. brasiliensis (3.7 x 10(6) residues per cell) may inhibit fungal phagocytosis during early infection, when the immunological response is still being built up.

Identification of sialic acids on the cell surface of hyphae and yeast forms of the human pathogen Paracoccidioides brasiliensis

Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, when grown in a synthetic medium, expresses at the cell surface of both yeast and mycelial forms acidic glycoconjugates containing N-acetylneuraminic acid units. Sialic acids were extracted using mild hydrolytic conditions, and were identified by thin-layer and gas chromatography, standard colorimetry, reaction with periodate-resorcinol and mass spectrometry. Their surface location was inferred from fluorescent-lectin (Limulus polyphemus agglutinin) binding to whole cells abrogated by previous treatment with neuraminidase. Expression of sialic acids on virulent yeast forms of P. brasiliensis (3.7 x 10(6) residues per cell) may inhibit fungal phagocytosis during early infection, when the immunological response is still being built up.

Glycosylation and deglycosylation of proteasomes (prosomes) from calf-liver cells: High abundance of neuraminic acid

Proteasomes (prosomes) of calf-liver cells were probed with three different biotinylated lectins: Limulus polyphemus agglutinin (LPA), specific for neuraminic acid; Solanum tuberosum agglutinin (STA), specific for GlcNac; and concanavalin A (Con A), specific for Man/Glc. While only one proteasomal protein reacted with STA, most of the proteasomal proteins reacted with LPA and several with Con A. Deglycosylation with N-glycosidase F showed that the detected glycan residues were asparagine-linked. Finally we demonstrate an alternative method for the isolation of proteasomes based on the affinity of certain proteasomal proteins to Con A.

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.