A novel application of thermo-responsive polymer to affinity precipitation of polysaccharide

The lectin receptors concanavalin A (ConA) and wheat germ lectin (WGL) were successfully conjugated to a thermo-responsive hydrogel polymer (poly-N-isopropylacrylamide) (PNIPAAm) as an affinity ligand. The coupling efficiencies of the ConA and WGL to PNIPAAm were 5.1% and 44%, respectively. These lectin receptor-polymer conjugates were then tested for their efficiency in purification of various polysaccharides or polysaccharide-containing compounds such as beta-glucan. Results indicated that these conjugates separated various polysaccharides from a complex mixture. The use of a thermo-responsive polymer in low-temperature purification of potentially heat-labile glycoproteins is advantageous. Additionally, other affinity ligands could be coupled to the polymer for separation of the respective bioactive molecules.     

Cell surface changes in transformed human lymphocytes : I. ConA and E-PHA induced unique changes in surface topography

Binding studies with six purified plant lectins were used to investigate membrane alterations that occur in lymphocyte transformation. Normal human peripheral blood lymphocytes transformed with E-Phytohemagglutinin (E-PHA) or concanavalin-A (Con-A) generally possessed increased numbers of lectin receptors. When this increase was corrected for the expanded surface area of transformed lymphocytes, it appeared that E-PHA and ConA each produced a unique and complex reorganization of cell surface topography. Surface alterations occurred independently of DNA synthesis, cell proliferation, and microtubule or microfilament function. Puromycin inhibited emergence of new lectin receptors on cells transformed with E-PHA, but not with ConA. Lymphocytes incubated with either lectin showed increased incorporation of [¹⁴C]galactose into trypsin-sensitive cell surface glycoproteins. This incorporation was abolished by puromycin in cells stimulated by E-PHA but not by ConA. These studies demonstrate that although both lectins induce similar morphological alterations in human lymphocytes, at the molecular level the structural changes induced in the cell membrane by these two lectins differ considerably. Furthermore, these structural alterations are mediated via different mechanisms in the two groups of cells. De novo protein synthesis is required for cell surface reorganization in PHA-stimulated cells, but not in cells stimulated by ConA. The effect of ConA appears to be to enhance attachment of saccharide structures to pre-synthesized membrane proteins.     

BSA–boronic acid conjugate as lectin mimetics

We report bovine serum albumin (BSA)–boronic acid (BA) conjugates as lectin mimetics and their glyco-capturing capacity. The BSA–BA conjugates were synthesized by amidation of carboxylic acid groups in BSA with aminophenyl boronic acid in the presence of EDC, and were characterized by Alizarin Red S (ARS) assay and SDS–PAGE gel. The BSA–BA conjugates were immobilized onto maleimide-functionalized silica beads and their sugar capturing capacity and specificity were confirmed by ARS displacement assay. Further, surface plasmon resonance (SPR) analysis of the glyco-capturing activity of the BSA–BA conjugates was conducted by immobilizing BSA–BA onto SPR gold chip. Overall, we demonstrated a BSA–BA-based lectin mimetics for glyco-capturing applications. These lectin mimetics are expected to provide an important tool for glycomics and biosensor research and applications.     

Effects of jackbean lectin (ConA) on the feeding behaviour and kinetics of intoxication of the pea aphid, Acyrthosiphon pisum

Mannose‐binding lectins were shown to be useful in creating transgenic plants resistant to insects, including many phloem‐feeding Hemiptera. Before these plants can be used extensively, it is important to understand how these lectins exert their toxic effects on the target organisms. We investigated the feeding alterations induced by presenting the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), with a diet containing the lectin from Canavalia ensiformis (ConA). A series of behavioural experiments were carried out to detect potential sensory mediation of lectin activity. Choice tests performed with a 400 µg ml^?1 ConA diet (3.7 µm of native tetramer) showed that A. pisum quickly rejected the ConA diet, but that this reaction was not typical of a sensory‐mediated phagodeterrent effect. In addition, the aphids did not develop a conditioned taste aversion to the lectin. Diet uptake was evaluated using a radioactive tracer (¹⁴C‐methylated inulin), and showed depression of ingestion only after 16 h at 200 µg ml^?1 or after 8 h at 400 µg ml^?1 ConA. This effect was reversible under our test conditions. No evidence was obtained for early detection of the lectin, even by intoxicated aphids. An electrical penetration graph technique was adapted to artificial diets and provided short‐term continuous analysis on feeding/probing events. At the 400 µg ml^?1 level, adults were affected and had reduced ingestion durations as early as in the first 4 h of contact, but experienced an adaptation to the behavioural alterations induced by lectin feeding. Overall, feeding deterrency following exposure to mannose lectins appeared to be a consequence of intoxication, and not due to a sensory mediated process.     

The effect of amphotericin B on lectin-induced aggregation of cell surface receptors

The mobility of concanavalin A (ConA) and ricin receptors from NS20 neuroblastoma and C₆ glioma cells was studied using an electrophoretic technique. Cells attached to a solid support were exposed to an electrical field (12V cm⁻¹) at room temperature. The distribution of lectin receptors on the cell surface was revealed by fluorescent conjugates of lectins and microscopic observation of the fixed cells. This technique allowed the estimation of the mobilities of lectin receptors either in free or liganded form, depending on the time at which the cells are labeled with lectins (either after or before electrophoresis). In line with previous observations [1] it is shown that in their free form ConA and ricin receptors are mobile all over the cell surface. Ligand binding induced an apparent receptor immobilization. Immobilization of ricin receptors from C₆ glioma cells could be induced either by the multivalent or the monovalent form of the lectin indicating that cross-linking of receptors by the ligand did not play a predominant role in the process of receptor immobilization. Amphotericin B but not ionophores like valinomycin or gramicidin blocked ligand-induced receptor immobilization. It is concluded from this observation that the effect of amphotericin B is not related to its ionophoretic properties but more likely to its capacity to interact with membrane cholesterol. When cells were incubated at 37 °C extensive patching of lectin receptors could be observed. This process was also inhibited by amphotericin B. A model is proposed to account for a role of cholesterol in ligand-induced receptor immobilization and patching.     

Thermodynamic, kinetic, and electron microscopy studies of concanavalin A and Dioclea grandiflora lectin cross-linked with synthetic divalent carbohydrates

The jack bean lectin concanavalin A (ConA) and the Dioclea grandiflora lectin (DGL) are highly homologous Man/Glc-specific members of the Diocleinae subtribe. Both lectins bind, cross-link, and precipitate with carbohydrates possessing multiple terminal nonreducing Man residues. The present study investigates the binding and cross-linking interactions of ConA and DGL with a series of synthetic divalent carbohydrates that possess spacer groups with increasing flexibility and length between terminal alpha-mannopyranoside residues. Isothermal titration microcalorimetry was used to determine the thermodynamics of binding of the two lectins to the divalent analogs, and kinetic light scattering and electron microscopy studies were used to characterize the cross-linking interactions of the lectins with the carbohydrates. The results demonstrated that divalent analogs with flexible spacer groups between the two terminal Man residues possess higher affinities for the two lectins as compared with those with inflexible spacer groups. Furthermore, despite their high degree of homology, ConA and DGL exhibit differences in their kinetics of cross-linking and precipitation with the divalent analogs. Electron microscopy shows the loss of organized cross-linked lattices of the two lectins with analogs possessing increased distance between the terminal Man residues. The loss of lattice patterns with the analogs is distinct for each lectin. These results have important implications for the interactions of lectins with multivalent carbohydrate receptors in biological systems.     

Distribution of cell surface saccharides on pancreatic cells. II. Lectin-labeling patterns on mature guinea pig and rat pancreatic cells

The surface saccharide composition of collagenase-dispersed pancreatic cells from adult guinea pig and rat glands was examined by using eight lectins and their ferritin conjugates: Concanavalin A (ConA); Lens culinaris (LCL); Lotus tetragonolobus (LTL); Ricinus communis agglutinins I and II (RCA I, RCA II); Soybean agglutinin (SBA); Ulex europeus lectin (UEL); and wheat germ agglutinin (WGA). Binding studies of iodinated lectins and lectin-ferritin conjugates both revealed one population of saturable, high-affinity receptor sites on the total cell population (approximately 95% acinar cells). Electron microscopy, however, revealed differences in lectin-ferritin binding to the plasmalemma of acinar, centroacinar, and endocrine cells. Whereas acinar cells bound heavily all lectin conjugates, endocrine and centroacinar cells were densely labeled only by ConA, LCL, WGA, and RCA I, and possessed few receptors for LTL, UEL, and SBA. Endocrine and centroacinar cells could be differentiated from each other by using RCA II, which binds to centroacinar cells but not to endocrine cells. Some RCA II receptors appeared to be glycolipids because they were extracted by ethanol and chloroform-methanol in contrast to WGA receptors which resisted solvent treatment but were partly removed by papain digestion. RCA I receptors were affected by neither treatment. The apparent absence of receptors for SBA on endocrine and centroacinar cells, and for RCA II on endocrine cells, was reversed by neuraminidase digestion, which suggested masking of lectin receptors by sialic acid. The absence of LTL and UEL receptors on endocrine and centroacinar cells was not reversed by neuraminidase. We suggest that the differential lectin-binding patterns observed on acinar, centroacinar, and endocrine cells from the adult pancreas surface-carbohydrate-developmental programs expressed during morphogenesis and cytodifferentiation of the gland.     

The effect of cytochalasin B and colchicine on concanavalin A induced vacuolation in mouse peritoneal macrophages

Macrophages bind concanavalin A (ConA) and interiorize the ConA-receptor conjugates in minute ConA-bearing pinosomes that undergo intracellular fusion processes to yield medium size (2–5 μ) and large size (>5 μ) vacuoles. The number and size of the vacuoles depends on ConA concentration and on the composition of the incubation medium. Cytochalasin B (CB) reduces ConA internalization by 30% and the surface bound lectin redistributes to form numerous clumps of fluorescence. Macrophage ConA-induced vacuolation is inhibited by CB both when the incubation is carried out in Dulbecco's modified Eagle's medium (90% inhibition) or in phosphate buffered saline (80% inhibition). Colchicine reduces [³H]ConA internalization by 20% and has no detectable effect on the surface distribution of fluorescein-ConA conjugates. Incubation of macrophages with colchicine prior to or during ConA induction of vacuole formation results in an enhancement of cell vacuolation; the number of the developing large size vacuoles exceeds 4–6-fold that obtained in the absence of the alkaloid. The results suggest the involvement of elements of the cytoskeleton (microfilaments-microtubuli) in regulation of the sequence of events leading to ConA-induced vacuolation and that CB and colchicine have opposite effects on the process.     

Glycoconjugate distribution in the human fundic mucosa revealed by lectin- and glycoprotein-gold cytochemistry

The glycoconjugates of the human fundic mucosa were characterized at the ultrastructural level by means of direct (Helix pomatia agglutinin-gold complex) and indirect lectin techniques (Concanavalin A and horseradish peroxidase-gold complex; wheat germ agglutinin and ovomucoid-gold complex). Surface mucous cells and mucous neck cells secreted O-glycoproteins with N-acetylgalactosamine and N-acetylglucosamine residues at the non reducing terminus of the saccharidic chain. The secretory granules of the mucous neck cells showed condensed areas slightly reactive to ConA. The results obtained in the chief cells suggest that these cells secrete N-glycoproteins rich in mannose and/or glucose residues. "Transitional cells", presenting both morphological characteristics and lectin binding pattern intermediate to the mucous neck and chief cells have been observed. The surface of the intracellular canaliculi of the parietal cell was labelled by HPA, WGA and ConA. In the neck region of the gastric glands, immature parietal cells containing abundant mucous granules reactive to HPA, WGA and ConA were observed. The present results further corroborate the existence of a common cell precursor for surface mucous, mucous neck and parietal cells. In a further step, mucous neck cells gradually differentiate into chief cells the transitional cells being an intermediate stage.     

Deglycosylation is necessary but not sufficient for activation of proconcanavalin A.

Concanavalin A (ConA), one of the most studied plant lectins, is formed in jack bean (Canavalia ensiformis) seeds. ConA is synthesized as an inactive glycoprotein precursor proConA. Different processing events such as endoproteolytic cleavages, ligation of peptides and deglycosylation of the precursor are required to generate the different polypeptides constitutive of mature ConA. Among these events, deglycosylation of the prolectin appears as a key step in the lectin activation. The detection of deglycosylated proConA in immature jack bean seeds indicates that endoproteolytic cleavages are not prerequisite for its deglycosylation. Both the structure of the lectin precursor N-glycans Man8-9GlcNAc2 and the capacity of Endo H to cleave these oligosaccharide from native proConA in vitro favoured Endo H-type glycosidases as candidates for proConA deglycosylation in planta. Evidence for pH-dependent changes in the prolectin folding were obtained from analysis of the N-glycan accessibility and activation of the deglycosylated lectin precursor in acidic conditions. These data are consistent with the observation that both deglycosylation and acidification of the pH are the minimum requirements to convert the inactive precursor into an active lectin.     

Lectin-mediated stimulation of DNA synthesis in cultures of embryonic neural retina cells

Plant lectins and other agents which are mitogenic for lymphocytes and fibroblasts were tested for their effects on DNA synthesis in primary monolayer cultures of neural retina cells from 10-day chick embryos. Concanavalin A (ConA), phytohemagglutinin (PHA), wheat germ agglutinin (WGA), and anti-retina cell antiserum significantly stimulated [³H]TdR incorporation; the maximum increase was reached 15 h after exposure of the cultures to these agents. Cells stimulated by ConA to synthesize DNA subsequently divided. The divalent succinyl derivative of ConA had a considerably lesser effect than the native tetramer, suggesting that cross-linking of cell surface components may be an important aspect of the changes that lead to the stimulation of DNA synthesis in these cells.Using [¹²⁵I]ConA, the average number of ConA-binding sites per 10-day retina cell was estimated to be 1.7 × 10⁶ (under the culture conditions employed); binding of the lectin to 25–50% of these sites was sufficient to elicit the maximal stimulation of DNA synthesis. Continuous association of the lectin with the cell surface for up to 8 h was essential for the maximal effect, since removal of the lectin from the cell surface (with α-methyl mannose) prior to this time reduced or prevented the stimulation of DNA synthesis.The stimulation by ConA of DNA synthesis in these cultures was dependent on the cell density and was reduced or absent at lower than optimal densities. Examination of this effect suggested that the frequency of intercellular contacts or specific cell associations play a role in the responsiveness of these cells to stimulation of DNA synthesis by ConA.     

Rearrangement of concanavalin A receptor sites on cells tagged with dinitrofluorobenzene: Evidence for the chemical induction of a change usually associated with malignant transformation

Normal human peripheral blood granulocytes which are tagged with 1-fluoro-2,4-dinitrobenzene (DNFB) are agglutinated by concanavalin A (ConA) in a way which resembles the pattern of reactivity displayed by leukemic cells. The present study further defines this reaction. The binding of ConA to untagged and DNP-tagged granulocytes, treated with DNFB at a ratio of 10¹¹ molecules/cell, was quantified by isotopic dilution experiments employing [³H]ConA. Similar amounts of the lectin were bound to untagged and DNP-tagged cells following incubation for 5 min at 4 °C or 30 min at 24 °C: 1.1 × 10⁵ molecules/cell, 4.6 × 10²/μ² of surface area, and 1.6 × 10³/μg of protein. The binding of [³H]ConA to both untagged and DNP-tagged cells was inhibited to the same degree by α-methylglucopyranoside (α-MG). Fixation with either glutaraldehyde or formaldehyde, which immobilizes ConA receptor sites, completely inhibited the agglutination of both untagged and DNP-tagged cells although lectin binding was unchanged. This suggests that the inhibition of agglutination was not due to the blocking of ConA-binding sites by aldehyde groups but rather to the immobilization of lectin receptors. We conclude that dinitrophenylation of normal granulocytes facilitates the rearrangement of lectin receptors in a way which resembles the ConA-induced clustering of sites which have been observed with malignant and transformed cells.     

Distribution and nature of saccharides of plasma membranes of rat ascites hepatoma cells as detected by ferritin-conjugated lectins and dialysed iron

Electron microscopic cytochemical studies were made on saccharides involved in the plasma membranes of rat ascites hepatoma cells (AH7974F) using ferritin-conjugated lectins and dialysed iron (DI). In the rat hepatoma cells, saccharide receptors for each of the three lectins used (concanavalin A (ConA), wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA)) were shown to be distributed homogeneously throughout the plasma membranes. When the cells were agglutinated, however, the saccharide receptors for each lectin appeared to form clusters on the plasma membranes. The cluster formation induced by one lectin was found to lead to a changed distribution of saccharide receptors for another lectin. None of the cluster formation types induced by lectins yield any noticeable effects upon the distribution of DI reactive acidic saccharides on the plasma membranes.     

Coupling of peptides to protein carriers by mixed anhydride procedure

Carboxyl groups of succinylated bovine serum albumin were activated by isobutylchloroformate in dimethylformamide solution. Subsequent reaction of the mixed anhydride with amino groups of the added peptide provided rapid and efficient coupling of peptide to protein. For different peptides the yield of coupling was equal to 40-100%. These values corresponded to 20-50 mol peptide bound/mol protein. Immunization of rabbits with these conjugates produced antisera to peptides with titers of 1:1000-1:3000 (estimated by enzyme-linked immunosorbent assay).     

The role of microvilli in the agglutination of cells by concanavalin A

Morphological correlates of lectin agglutinability were examined in eight cell lines of varying sensitivity to agglutination by concanavalin A (ConA). The number of microvilli on the surface of cells growing in monolayers was positively correlated with agglutinability. However, when cells were brought into suspension, they all developed numerous microvilli which persisted when the cells were treated with ConA regardless of whether or not they were agglutinated by the lectin. Treatment of cells with dibutyryl cyclic AMP (db-cAMP) and theophylline caused a parallel decrease in agglutinability and numbers of microvilli in monolayer cultures, but suspended cells from control and treated cultures were identical in appearance in the absence or presence of ConA. The surface morphology of cells agglutinated by ConA was very similar to that of cells that spontaneously agglutinated in the absence of the lectin, and surface bound ConA was rapidly withdrawn from microvilli on all cell types. Neither the morphology of cells nor the surface distribution of ConA can explain observed differences in agglutinability.     

Differential effects of lectins mediating erythrocyte attachment and ingestion by macrophages

Lectin-mediated interaction of erythrocytes and macrophages was brought about in two steps. Step I involved macrophage treatment with lectin, and step II is the incubation of lectin-treated macrophages with mouse erythrocytes. The extent and nature of lectin-mediated macrophage erythrocyte interaction was studied using concanavalin A (ConA), wheat germ (WGA), soybean (SBA) and waxbean (WBA) agglutinins. The parameters affecting the interaction were studied in detail with the first two lectins.Under comparable conditions of lectin interaction with macrophages (step I), WGA mediates rosette formation involving interaction with several times the number of erythrocytes than those interacting with ConA-treated macrophages. The interaction mediated by WGA reaches, at 37 °C, a saturation value after 30 min of step II, whereas that mediated by ConA is still linear and exhibits half the amount of attached erythrocytes at 60 min. ConA-mediated attachment of erythrocytes is highly temperature-dependent being at 37 °C twice that observed at 24 °C. The temperature dependence of attachment is not affected by changes of either ConA concentration (5–40 μg/ml) or the temperature in step I. An optimum is observed, however, when the temperature of incubation in step I ranges between 14–18 °C. WGA-mediated attachment of erythrocytes is markedly less temperature-sensitive, exhibiting 70% of optimal attachment already at 8 °C. Only when the attachment phase follows incubation with a low concentration of WGA (2 μg/ml) high temperature sensitivity is exhibited. At 37 °C, however, the number of attached erythrocytes is the same for macrophages treated with WGA at concentrations of 2, 5, 10 and 40 μg/ml.ConA-mediated erythrocyte-macrophage interaction does not lead to erythrophagocytosis. When mediated by WGA, the attachment step is followed by a temperature-dependent ingestion step, i.e. 10% and 50% of the erythrocytes that attach to macrophages during the 60 min incubation at 24 °C and 37 °C, respectively, are ingested. There is a lag period of 10–20 min between attachment and ingestion implicating involvement of additional cellular processes preceding engulfment. Electron microscope images of areas of interaction of attached erythrocytes with macrophages indicate a significantly tighter binding (a thinner gap at membrane-membrane apposition areas) in the case of WGA-mediated rosette formation as compared with that established in ConA-mediated rosettes. Attachment via WGA is followed by a rapid change in the relative position of the attached erythrocytes on the macrophage, from a primary attachment at the distal peripheral regions of the cell, to a perinuclear position. In contrast, erythrocytes attached via ConA remain at the primary attachment point (at 37 °C) for extended periods. This differential behaviour does not stem from effects of ConA on macrophages, since when yeast cells were attached to ConA treated macrophages, the yeast cells showed the same movement as that exhibited by erythrocyte when attached via WGA.The different interaction patterns of erythrocytes with macrophages coated with ConA and WGA can be fitted into the following working hypothesis: the number of WGA-binding sites on the plasma membrane of macrophages is at least three times that of ConA-binding sites. Stable cell-cell interactions involve multibridge formation at the contact area of the two cells and this involves a delicate balance between number of lectin-receptor conjugates and their aggregation state within the membrane phase. A certain amount of clustering is a prerequisite for attachment, while a high degree of clustering reduces the chance of fruitful interactions. The engulfment step depends on the ability of membrane areas adjacent to primary contact area to establish additional stable bridges in the entire circumference of the attached cell. ConA-receptor conjugates appear to be less abundant and more aggregated within the membrane plane, preventing the completion of fruitful circumferential interaction of the adjacent membrane. WGA-receptor conjugates, being more abundant and apparently less aggregated are available at membrane areas needed for cell enclosure and provide the additional bridging without which engulfment does not take place. Change in relative position of attached erythrocytes seems to be a step in the manifold events occurring from attachment to ingestion.     

Design of metalloporphyrin-carbohydrate conjugates for a new superoxide dismutase mimic with cellular recognition

Metalloporphyrin-carbohydrate conjugates have been synthesized as superoxide dismutase (SOD) mimics with cellular recognition. To synthesize the conjugates, aliphatic primary amino groups for conjugation were introduced, with the cationic pyridyl groups for the SOD activity of porphyrin preserved. The reductive amination between introduced amino groups and the reducing end of lactose was then carried out. The resulting conjugates consisting of manganese (Mn)-porphyrin surrounded by several lactose molecules possessed significant SOD activity and low cytotoxicity. Compared with metalloporphyrins having no lactose molecule, the recognition of the resulting conjugates by human hepatoma HepG2 cells increased. The cellular recognition was inhibited by competitors of beta-galactose. These results suggest that the Mn-porphyrin-lactose conjugates recognized the hepatic lectin on the cell surface.     

Binding of the insecticidal lectin Concanavalin A in pea aphid, Acyrthosiphon pisum (Harris) and induced effects on the structure of midgut epithelial cells

Concanavalin A (lectin from Canavalia ensiformis L., ConA) has previously been shown to act as a feeding inhibitor for Acyrthosiphon pisum, the pea aphid. In the present study a range of histochemical and biochemical techniques were used to elucidate the target tissues and binding sites of the lectin in the aphid. Diet uptake was evaluated using a radioactive tracer (¹⁴C-methylated inulin) and demonstrated that adults were capable of ingesting high quantities of the toxin (approx. 1 μg over a 48 h period). Electophoretic analysis and enzyme-linked immuno-sorbent assay of honeydew samples confirmed these results and further demonstrated that only small levels of ConA were excreted. Histofluorescence and immunolocalisation studies on nymphs revealed that the stomach was the primary target for ConA. At concentrations up to 400 μg ml⁻¹, lectin binding only occurred in the stomach region, however, at high concentrations (800 μg ml⁻¹) the whole digestive tract was stained, although there was no evidence of binding in either the oesophagus or rectum. In addition to binding, there was evidence to suggest that ConA was also causing systemic effects in that the lectin appeared to cross the intestinal epithelial barrier. Immunohistochemical and electron microscopy studies revealed that ConA induced severe cellular swelling of the epithelial cells, accompanied by hypersecretion and a progressive detachment of the apical membrane; however, the striated border itself did not appear to be directly affected. Furthermore, there was no lysis of the epithelium, nor loss of integrity of the epithelial cells themselves. Our results suggest that ConA interacts with glycosylated receptors at the surface of the stomach epithetial cells, interfering with normal metabolism and cell function, resulting in a rapid feedback response on feeding behaviour. Whilst our results provide a much greater understanding regarding the modes of action of ConA in insects, they suggest that different lectins, including other mannose binding lectins, have different modes of action at the cellular levels, and thus generalizations should be treated with caution.     

Lectin-induced deciduoma formation in the pseudopregnant rat

Decidual cell induction in the pseudopregnant rat was examined in this study using the lectin concanavalin A (ConA). The histochemical binding of the lectin to the uterine cell surface at the time of deciduomatic induction was also studied. ConA was found to induce significant deciduomata (decidual-like tissue) in the uterine horn when injected intraluminally on day 5 of pseudopregnancy (PSP). ConA-induced deciduomata appeared as a series of discrete nodules in the uterine horn, reminiscent of the anatomical appearance of normal embryo implantation sites. Deciduoma induction by ConA was greatly reduced by pre-absorption of the lectin with its competitive sugar. Lectin histochemistry revealed binding of ConA to the cell surface on day 5 of PSP. Pre-absorption of the lectin with its competitive sugar also significantly reduced surface binding of the lectin, and this finding may be correlated with the greatly reduced ability of the pre-absorbed lectin to induce deciduomata. Possible mechanisms for the induction of deciduomata by lectins are considered.     

Lectin staining of cultured CNS microglia.

Carbohydrate binding proteins, known as lectins, bind to specific sugar groups on most membranes. We used fluorescent and light microscopy to study the interaction of various lectins with the membranes of microglia cultured from neonatal rat or fetal mouse cerebral cortices. Microglia stained intensely with GS-1, RCA, WGA, and ConA and slightly with DBA, UEA, BPA, and SBA. No staining was seen with GS-2, MPA, or PNA. Staining was specific for microglia in the mixed glial cultures and was dose dependent. In addition, microglial lectin binding could be reduced or blocked by competitive inhibition using specific sugars. Treatment of the microglia with agents such as dimethylsulfoxide (DMSO), interleukin-1 (IL-1), interferon (IFN), or lipopolysaccharide (LPS) did not eliminate lectin staining, although the degree of staining was altered. Positive staining of the microglia was also associated with a functional change for at least one lectin, i.e., ConA. Superoxide anion production by microglia was increased in the presence of ConA. Overall, binding of the lectins GS-1, RCA, WGA, and ConA can be used as an identifying tool for microglia in glial cultures, but intensity of staining varies depending on their functional state.