Inhibitory effect of Concanavalin A on the cell-to-cell adhesion during early development of the starfish, Asterina pectinifera

The inhibitory effect of Concanavalin A (ConA) on the cell-to-cell adhesion was studied in starfish embryos. ConA reversibly blocked the formation of intercellular adhesion in embryos denuded of fertilization membrane as well as in normal embryos, without affecting cell division and thereby inhibiting the morphogenetic movement of blastulation. A large dose of ConA dissociated both denuded and normal embryos to single cells at blastula and gastrula stage. Succinyl ConA (Suc-ConA) has the same effect on cell-to-cell adhesion, though critical concentration was slightly higher than that of ConA. These effects of ConA or Suc-ConA were prevented by α-methyl- -mannoside (αMM). Study of the binding of fluorescein-conjugated ConA to the cell surface showed that ConA receptors were present in the surface of fertilized egg and cells at all stages examined. These findings suggest that ConA receptors play an important role in cell-to-cell adhesion during the early morphogenesis of starfish.     

Cell surface mobility of a bacterial R-form glycolipid, mR595, after binding to rat cells, and its effect on the mobility of concanavalin A receptors

Binding of small amounts of glycolipid mR595 to rat cells, followed by sequential incubation of cells at 37 °C with rabbit anti-glycolipid mR595 and fluorescein-conjugated sheep anti-rabbit γ-globulin antisera results in the localization of fluorescence at one pole of the cell surface (capping). Binding of higher amounts of glycolipid mR595 to cells not only inhibits formation of glycolipid caps but those of the ConA receptor-fluorescent ConA complex as well. Glycolipid mR595 binding does not alter [³H]ConA binding to cells but cell agglutination by ConA is inhibited in a competitive fashion. Binding of small amounts of ConA to cells does not affect glycolipid capping. Colchicine and cytochalasin B (CB) treatment of cells inhibits glycolipid cap formation.     

Effects of CKS-17, a synthetic retroviral envelope peptide,on cell-mediated immunity in vivo: Immunosuppression,immunogenicity, and relation to immunosuppressive tumor products

Summary CKS-17 is a heptadecapeptide corresponding to a region highly conserved in retroviral transmembrane proteins such as p15E. Because a relationship had previously been determined between p15E and immunosuppressive tumor cell products, we examined the effect of CKS-17, control peptides and conjugates thereof on the expression of cell-mediated immunity (delayed-type hypersensitivity, DTH) in mice. Conjugates of CKS-17 inhibited DTH reactions to sheep erythrocytes in the feet of mice. The degree of inhibition was dose-dependent. Unconjugated CKS-17 had almost no effect, and control peptide conjugates had no inhibitory effect. Immunization of mice with CKS-17 conjugates, but not with control conjugates, rendered them resistant to the depression of DTH reactions, not only by CKS-17 conjugates, but also by products of cultured tumor cells. CKS-17 conjugates, but not control conjugates, also depressed the cellular inflammatory reactions induced in mouse footpads by concanavalin A (ConA) and immunized mice against the depression of ConA reactions by products of cultured tumor cells. Injections of globulin from sera of mice immunized with CKS-17 conjugates conferred upon normal recipients resistance to the depression of footpad reactions to ConA by products of cultured tumor cells. Globulin from sera of normal mice or control immunized mice did not confer such resistance. Thus conjugates of a synthetic peptide not only mimic the immunosuppressive effects of tumor products in vivo, but can also immunize mice against those effects.     

A comparative study of four cytochemical detection methods of concanavalin A binding sites on the cell membrane

Four different electron cytochemical methods to detect concanavalin A (ConA) binding sites on the plasma membrane of mouse fibroblasts were compared in this study. The ConA binding sites were made visible either by adding ConA, followed by horseradish peroxidase (HRP) or hemocyanin (HC), or by marking the sites with complexes of ConA with ferritin (Fer) or with micro-peroxidase (MP). HC and Fer are directly visible in the electron microscope; HRP and MP are detected by their electron-dense reaction product with diaminobenzidin and H₂O₂. Differences in sensitivity of the ConA binding sites for the different markers were found and resulted in a tentative interpretation of the labelling reactions. All experiments suggested that normal and transformed murine fibroblasts both have plasma membranes in which the binding sites can move equally well and can be induced to form clusters. These results are discussed in relation with the hypothesis that differences in clustering of ConA sites between normal and transformed cells are responsible for differences in the agglutinability by ConA of these cells.     

Cell surface microvilli and cell agglutinability.

Detached cells of some transformed mouse fibroblast lines have a villous surface whereas similarly treated cells of other lines are relatively smooth. These differences in surface morphology of detached cells are not reflected in their agglutinability with ConA and they cannot unambigously be explained from their morphology in situ. Treatments of normal and transformed Swiss mouse fibroblasts that induce marked changes in agglutinability with ConA do not cause equivalent changes in surface morphology. It is, therefore, unlikely that agglutinability of mouse fibroblasts by ConA is determined by the number of microvilli on the cell surface.     

Different cyclic changes in the surface membrane of normal and malignant transformed cells

Transformed fibroblasts in interphase and normal fibroblasts in mitosis were agglutinated by Con A and the lectin from wheat germ, whereas normal fibroblasts in interphase and transformed fibroblasts in mitosis were not agglutinated by these lectins. The percentage of fluorescent cells at non-saturation concentrations of fluorescent ConA was also higher with transformed interphase and normal mitotic cells, than with normal interphase and transformed mitotic cells. Under the same conditions, a similar number of radioactively labeled ConA molecules were bound to normal and transformed cells in interphase and mitosis. Our results indicate different cyclic changes in the surface membrane of normal and transformed fibroblasts, so that regarding interaction with these lectins, normal mitotic cells resemble transformed interphase cells and transformed mitotic resemble normal interphase cells. The data suggest that there is a reversed cyclic change in the mobility of specific surface membrane sites in normal and transformed cells.     

A study of metabolic cooperation with established myoblast cell lines.

The present study was undertaken to test the action of ConA on the distribution of intramembranous particles (IMPs) and on the reassembly of junctional contacts in isolated and reaggregated embryonic neuronal and glial cells. The lectin ConA causes all embryonic cells to aggregate in unorganized cell patterns. ConA does not alter the distribution of IMPs but it inhibits the formation of the zonula occludens (ZO) by preventing the alignment and fusion of IMPs or by inducing them to become arranged in bizarre arrays. The possible relationship between ConA receptor sites and the IMPs is discussed. From a morphological viewpoint the aggregation of embryonic cells influenced by lectin is distinctly different from the normal processes of cell adhesion, cell sorting and establishment of intercellular contacts.     

Allogeneic tumor-specific cytotoxic T lymphocytes

Summary We report the development of cytotoxic T lymphocytes specific for an allogeneic brain tumor in a rat model. DA strain cytotoxic T cell precursors stimulated by an allogeneic tumor (9L gliosarcoma) from the Fischer rat could generate a population of cytotoxic T lymphocytes that lysed the allogeneic 9L tumor but failed to lyse other targets, including Fischer concanavalin-A(ConA)-stimulated lymphoid blast targets. DA T cells depleted of reactivity to the Fischer haplotype (DA-f) retained reactivity to the 9L tumor, demonstrating that T cell precursors with specificity for normal Fischer alloantigens were not required for the generation of a response to the 9L Fischer tumor. The preferential lysis of the tumor target did not simply reflect a higher density of Fischer target antigens on the tumor than that found on normal Fischer ConA blast targets. First, the relative densities of class I antigen on the 9L tumor and normal Fischer ConA blasts were comparable. Second, cytotoxic T cells could not be generated from DA-f precursors when Fischer ConA blasts were used as stimulators. If DA-f T cells were simply responding to the higher density of Fischer antigen found on 9L tumor, it would have been expected that the ConA blasts expressing comparable levels of antigen to that found on the tumor would have generated cytotoxicity for both the 9L and ConA targets. We conclude that the cytotoxic T cells are specific for a determinant expressed only by the tumor. Such tumor-specific cytotoxic T cells could be useful in vivo for adoptive immunotherapy of brain tumors.     

Reversible inhibition of cAMP-induced axon formation in neuroblastoma cells by concanavalin a and vinblastine : Relationship to microtubules and cytotoxicity

The formation of axons induced by dibutyryl-adenosine 3′,5′-cyclic monophosphate (db-cAMP) in neuroblastoma cells was inhibited by concanavalin A (ConA) and vinblastine. These compounds also caused the retraction of existing axons. After removal of ConA or vinblastine, addition of db-cAMP again resulted in axon formation. The cytotoxicity of ConA and vinblastine for neuroblastoma cells was reduced when cell multiplication was inhibited by db-cAMP. Linearly growing normal fibroblasts were also more sensitive to the cytotoxic effect of ConA than confluent non-multiplying fibroblasts. The effects of ConA and vinblastine were additive both in their effects on axon formation and cytotoxicity. Wheat germ agglutinin (WGA) and lumicolchicine did not affect axon formation or reduce cell viability. It is suggested that ConA bound to the cell surface can interfere with the assembly of cytoplasmic microtubules involved in axon formation and cell division.     

Cyclic AMP specifically blocks proliferation of rat 3T3 cells transformed by polyomavirus.

Elevated exogenous and intracellular levels of cyclic AMP could totally block proliferation of polyomavirus (PyV) transformants derived from rat 3T3 cells without affecting proliferation of normal cells or simian virus 40 (SV40)-induced transformants. Concanavalin A (ConA) had the opposite effect; it could totally block proliferation of both normal cells and SV40 transformants but reduced proliferation of PyV transformants only twofold. Adenylate cyclase was threefold less active in membranes of PyV transformants, and the number of ConA receptors was similar to that of normal cells. Proliferating PyV transformants contained threefold less cyclic AMP than did proliferating SV40 transformants. The sensitivity to cyclic AMP did not correlate with the degree of transformation: cells transformed by Rous sarcoma virus and tumor cells derived from SV40 transformants were not sensitive to cyclic AMP. The differential effect of cyclic AMP and ConA on proliferation was probably due to the activity of an intact middle t protein. The presence of both large T and small t together with middle t was also required for cyclic AMP sensitivity.     

Lectin binding patterns in normal, metaplastic, and neoplastic gastric mucosa.

We investigated lectin binding patterns on tissue specimens of normal and metaplastic gastric surface mucosae, gastric adenomas, and intestinal and diffuse-type gastric carcinomas. Compared with normal gastric mucosa, metaplastic mucosa exhibited an increase of ConA binding and decreases of WGA, PNA, UEA-1, and DBA binding in the cytoplasm, and decreases of ConA, PNA, and UEA-1 binding at the luminal surface. Intestinal carcinomas were similar to metaplastic gastric surface mucosa in ConA, WGA, and UEA-1 binding in the cytoplasm, while diffuse-type carcinomas were similar to normal gastric mucosa in WGA and UEA-1 binding in the cytoplasm. Adenomas were similar to intestinal carcinomas in ConA and UEA-1 binding in the cytoplasm, but were different from intestinal carcinomas in Con A and UEA-1 binding at the luminal surface. For UEA-1, normal and metaplastic gastric surface mucosae did not show a significant difference between the blood type A, AB, B group and the O group. Intestinal and diffuse carcinomas and adenomas also did not show such a difference between the blood groups. For DBA, normal gastric surface mucosa showed a significant difference between the blood type B, O group and the A, AB group. Normal gastric mucosa of the blood type A, AB group was frequently positive for DBA binding in the cytoplasm and at the luminal surface. Metaplastic mucosa did not show a significant difference between the blood groups. Intestinal and diffuse-type carcinomas and adenomas also did not show a difference between the blood groups. DBA binding in the cytoplasm of intestinal carcinomas and adenomas was more frequently positive than that of normal and metaplastic mucosae, except for normal gastric mucosa of the blood type A, AB group. Compared with diffuse-type carcinomas, intestinal carcinomas were accompanied by a significant increase of ConA binding and decreases of WGA and PNA binding in the cytoplasm.     

Comparative study of mitogenic responses in man and Japanese monkeys (Macaca fuscata): responses of T-cell subsets, accessory cell dependency, and interleukin-2 receptor expression

Responses of peripheral blood mononuclear cells to phytohemagglutinin-P (PHA-P), concanavalin-A (ConA), and pokeweed mitogen (PWM) were compared in man and Japanese monkeys. Both CD8+ and CD8- T subsets showed greater responses to ConA than to PHA-P in the Japanese monkey. Addition of macrophages to each T subset produced more effective augmentation of ConA response in the Japanese monkey than in man, and ConA induced more interleukin-2-receptor-positive blast cells than PHA-P did in Japanese monkeys.     

Mitogenic responses of peripheral blood mononuclear cells of vervet monkeys (Cercopithecus aethiops): Apparent role of adherent cells

Peripheral blood mononuclear cells (PBM) from normal vervet monkeys (Cercopithecus aethiops) were examined for blastogenic responses to concanavalin A (ConA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM). The mitogen stimulated PBM in a dose-dependent manner. Response to ConA was apparently higher than for the other two mitogens. Cell density and mitogen concentration were critical parameters for optimal lymphocyte proliferation, an observation in line with that reported for other mammalian species. Depletion of an adherent cell population probably of monocyte/macrophage lineage from vervet PBM gave higher proliferative responses to both ConA and PHA, but the response without adherent cells to ConA was greater than the response without adherent cells to PHA. This latter finding is in contrast to what has been reported in many other species.     

Capping of concanavalin a receptors and their association with microfilaments in monolayer grown human fibroblastoid cells

Summary The distribution of ConA binding sites on the surface of normal human fibroblastoid cells grown in monolayer culture was carefully examined. Low concentrations of ConA (between 0.5–5.0 g/ml) were found to induce the ConA receptors to form a single, large cap structure. High concentrations of ConA (between 50–100 g/ml) inhibit cap formation at temperatures above 20° C. Pretreatment of the cells in the cold or with colchicine allows cap formation to occur with high concentrations of ConA. The ConA caps appear to be preferentially localized near the nucleus. Using a double immunofluorescence technique, we have observed actin and myosin molecules concentrated underneath the surface receptor cap in the perinuclear region of the cells. These findings suggest that the binding of ConA to fibroblastoid cells may trigger the transmembrane association of cytoplasmic microfilaments with surface membrane receptors as previously proposed for lymphocytes and other round cells grown in suspension culture.Abbreviations IgG immunoglobulin - Fl fluorescein - Rh rhodamine - PBS 0.1 M phosphate buffer (pH:7.4) plus 0.15 M NaCl - ConA Concanavalin A This work was supported by U.S. Public Health Service Grants AI 15258-01 and CA 22453     

c-fos and c-myc gene activation, ionic signals, and DNA synthesis in thymocytes

Among the earliest responses to mitogens that have been detected in normal quiescent cells are ionic changes: we have described rapid increases in the cytosolic free Ca2+ concentration ([Ca]i) and in the intracellular pH (pHi) in mitogen-stimulated thymocytes and fibroblasts (Hesketh, T. R., Moore, J. P., Morris, J. D. H., Taylor, M. V., Rogers, J., Smith, G. A., and Metcalfe, J. C. (1985) Nature 313, 482-484). Here we investigate the relationship between these ionic signals and the subsequent expression of the c-fos and c-myc proto-oncogenes in murine thymocytes. We show that the plant lectin concanavalin A (ConA), the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the Ca2+-ionophore A23187 each causes a rapid increase in both c-fos and c-myc mRNAs. The activation of both genes is completely dependent on the extracellular Ca2+ concentration ([Ca]o) for A23187 and independent of [Ca]o for TPA. Activation of c-myc, but not c-fos, by ConA is partially dependent on [Ca]o. The pHi increases generated by ConA or TPA are not necessary for expression of mRNA from either gene in response to these mitogens. Exogenous 8-bromo-cyclic AMP (but not 8-bromo-cyclic GMP) inhibits the c-myc responses to ConA and TPA. The data also show that neither early c-fos nor c-myc expression is sufficient to commit the cells to DNA synthesis.     

Membrane characteristics of old and Rauscher leukemia virus infected mouse red blood cells

Some membrane characteristics of normal and Rauscher leukemia virus (RLV)-infected mouse red blood cells (RBC) were compared, both with regard to total populations and young and old groups of cells. Osmotic fragility, density distribution of cells and agglutinability by poly- -lysine (pLys), concanavalin A (ConA), phytohemagglutinin (PHA) and soybean agglutinin (SBA), were examined. RBC from RLV-infected mice were agglutinated at a higher rate and to a higher degree than normal mice RBC by pLys and by the lectins PHA and ConA. These RBC were generally osmotically more resistant and contained a young cell population of unusually high specific gravity. Comparison of RBC from RLV-infected mice with old RBC from normal mice showed some common membrane characteristics. Similarly to old RBC, RBC from RLV-infected mice have a high specific gravity and high agglutinability by pLys. However, they differ in that the RBC from RLV-infected mice are osmotically more resistant and are agglutinated by ConA; they are also agglutinated at a higher rate by PHA.     

Modulation of the cell cycle during reversible growth inhibition of 3T3 and SV40-3T3 cells with succinylated concanavalin A

Succinylated concanavalin A (ConA), a non-toxic, non-agglutinating derivative of the jack bean lectin ConA inhibits the growth of both normal and SV40-transformed 3T3 cells. The quantitative but not the qualitative growth inhibitory effect of succinyl-ConA can be modulated by the composition of the growth medium. Succinyl-ConA inhibited cells show a low rate of DNA synthesis and accumulate in the G0/G1 phase of the cell cycle. Upon removal of the succinyl-ConA, inhibited cells re-enter the cell cycle synchronously.     

Recombinant interleukin 2 regulates levels of c-myc mRNA in a cloned murine T lymphocyte.

The cellular oncogene c-myc has been implicated in the regulation of growth of normal and neoplastic cells. Recently, it was suggested that c-myc gene expression may control the G0----G1-phase transition in normal lymphocytes that were stimulated to enter the cell cycle by the lectin concanavalin A (ConA). Here we describe the effects of purified recombinant interleukin 2 (rIL2) and of ConA on levels of c-myc mRNA in the noncytolytic murine T-cell clone L2. In contrast to resting (G0) primary cultures of lymphocytes, quiescent L2 cells have a higher RNA content than resting splenocytes and express receptors for interleukin 2 (IL2). Resting L2 cells are therefore best regarded as early G1-phase cells. Purified rIL2 was found to stimulate the rapid accumulation of c-myc mRNA in L2 cells. Levels of c-myc mRNA became maximal within 1 h and declined gradually thereafter. In contrast, ConA induced slower accumulation of c-myc mRNA in L2 cells, with increased levels of c-myc mRNA becoming detectable 4 to 8 h after stimulation. Experiments with the protein synthesis inhibitor cycloheximide demonstrated that the increase in levels of c-myc mRNA that were induced by ConA was a direct effect of this lectin and not secondary to IL2 production. Cyclosporin A, an immunosuppressive agent, markedly reduced the accumulation of c-myc mRNA that was induced by ConA but only slightly diminished the accumulation of c-myc mRNA that was induced by rIL2. Taken together, these data provide evidence that (i) c-myc gene expression can be regulated by at least two distinct pathways in T lymphocytes, only one of which is sensitive to cyclosporine A, and (ii) the accumulation of c-myc mRNA can be induced in T cells by IL2 during the G1 phase of the cell cycle.     

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.     

Cellular heterogeneity in normal human urothelium: Quantitative studies of lectin binding

Summary A panel of 10 FITC-labelled lectins (MPA, PNA, ConA, DBA, SBA, RCA-120, WGA, UEA, GS-I, GS-II) was applied to cryosections of seven specimens of normal urothelium. Seven of the lectins (MPA, ConA, RCA, WGA, UEA, GS-I and GS-II) showed a pattern of increasing fluorescence intensity from basal to superficial cells of the urothelium whereas PNA, DBA and SBA showed more uniform binding throughout the urothelium. Urothelial cell suspensions labelled with FITC-lectins were studied by flow cytometry to quantify the variation in binding to different cells types. Three cellular subpopulations were identified in normal urothelium on the basis of their optical properties. Fluorescence intensity due to specific lectin binding was then measured separately for each subpopulation. Although there was some variation among individual cases, a general pattern emerged in this small series. WGA, RCA, and GS-II bind in large quantities to all urothelial cells while PNA, SBA, ConA and DBA show little binding. MPA, RCA, UEA and GS-I showed the most marked increase in fluorescence intensity from basal to superficial cells as observed microscopically and quantified by flow cytometry.