Mechanisms of Adhesion Among Cells of the Early Chick Blastoderm

Cells from the extraembryonic endoderm of the gastrulating chick embryo contain a β-d-galactoside-binding lectin inhibited by thiodigalactoside (TDG). TDG inhibits the aggregation of freshly prepared cells. In these fresh cell suspensions, adhesion is also inhibited when purified lectin is added to the aggregation assay. If these cells are incubated at 22° C their adhesion decreases. Associated with this is an increase in lectin activity in the cell supernatants. In these incubated cells aggregation is stimulated by TDG and desialyzed fetuin. These data suggest that the lectin may have a role to play in cellular adhesion. Under some experimental conditions extraembryonic endoderm cells from rosettes with trypsinized glutaraldehyde-fixed rabbit erythrocytes. This phenomenon is inhibited, to a certain extent, by TDG.     

Characterization of myogenic cell membrane: spontaneous formation of heterokaryotic myotubes between two different kinds of myoblasts

In a previous study, it has been shown that presumptive mouse C2 myoblast cells are strongly resistant to HVJ (hemaglutinating virus of Japan, Sendai virus)-mediated cell fusion, but do become capable of fusion upon differentiation. Quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) also become more sensitive to HVJ-mediated cell fusion during differentiation. Investigations were undertaken to see whether heterokaryotic myotubes were formed spontaneously by co-culture of two different kinds of myogenic cells, QM-RSV cells and C2 cells. When both cells were committed to myotube formation, they spontaneously fused without HVJ on co-culture. On the other hand, when both or one of the cells were in the presumptive state, heterokaryons were not formed by co-culturing. Furthermore, committed QM-RSV cells did not fuse with non-myogenic cells. These results indicate that the membranes of myogenic cells change to become capable of fusion for myotube formation during differentiation.     

Reduced virulence of Yersinia enterocolitica by copper-induced injury.

A sublethal concentration of copper (0.75 mg/liter) caused substantial injury (87 to 95%) of Yersinia enterocolitica serotype O:8 cells in 72 h at 4 degrees C without producing extensive cell death. Copper-injured cells had a higher 50% lethal dose in mice (2,700 CFU) than uninjured cells (150 CFU). This reduced virulence correlated with more rapid clearance of the injured cells from the blood of mice after intravenous inoculation. A possible role of the liver in this process was shown by significant cell accumulation in mouse livers when copper-injured Y. enterocolitica cells were administered, compared with uninjured bacteria. In vitro studies with isolated mouse liver membranes showed higher titers of aggregation with copper-injured cells than control cells. The in vitro aggregation reaction and blood clearance activity in vivo were abolished by sugars that are known to interact with a hepatic lectin. Our data suggest that copper-induced injury reduces the virulence of Y. enterocolitica and that the liver may be involved in nonimmune rapid clearance of the injured cells, probably by interaction with a hepatic lectin(s).     

Reduced virulence of Yersinia enterocolitica by copper-induced injury

A sublethal concentration of copper (0.75 mg/liter) caused substantial injury (87 to 95%) of Yersinia enterocolitica serotype O:8 cells in 72 h at 4 degrees C without producing extensive cell death. Copper-injured cells had a higher 50% lethal dose in mice (2,700 CFU) than uninjured cells (150 CFU). This reduced virulence correlated with more rapid clearance of the injured cells from the blood of mice after intravenous inoculation. A possible role of the liver in this process was shown by significant cell accumulation in mouse livers when copper-injured Y. enterocolitica cells were administered, compared with uninjured bacteria. In vitro studies with isolated mouse liver membranes showed higher titers of aggregation with copper-injured cells than control cells. The in vitro aggregation reaction and blood clearance activity in vivo were abolished by sugars that are known to interact with a hepatic lectin. Our data suggest that copper-induced injury reduces the virulence of Y. enterocolitica and that the liver may be involved in nonimmune rapid clearance of the injured cells, probably by interaction with a hepatic lectin(s).     

Selective separation of microorganisms by lectins: yeast and concanavalin A as a model system

Specific aggregation and separation of microorganisms was investigated using yeasts and concanavalin A as a model system. Cells of Saccharomyces cerevisiae were specifically aggregated and so separated from those of Schizosaccharomyces pombe. Optimum aggregation with over 99% of cells aggregated was achieved by adjustment to pH value and applied agitation. Dimeric lectin structure caused a far higher degree of aggregation than did tetrameric. Degree of aggregation was also strongly influenced by the ratio of lectin/cell concentrations, optimum aggregation occurring in the middle range of ratios. A high ratio of lectin to cells inhibited aggregation, occupation of most of the available receptors preventing intercellular bonding by divalent lectins. Detachment and reuse of concanavalin A was demonstrated using switching from moderate to low pH value. Potential uses for species-specific-separation of microorganisms are discussed. (c) 1992 John Wiley & Sons, Inc.     

A monoclonal antibody reactive with a 40-kDa molecule on fetal thymocytes and tumor cells blocks proliferation and stimulates aggregation and apoptosis.

E710.2.3 is a murine thymic lymphoma cell line with an immature phenotype (CD4-CD8-) that proliferates in response to thymocytes or PMA when cultured at low density and proliferates spontaneously when grown at high density. To identify functional molecules on this cell line, we screened for mAbs that could block its proliferation. A hamster mAb, DMF10.62.3, inhibited the spontaneous, thymocyte-induced, and PMA-stimulated proliferation of E710.2.3 in vitro and induced these cells to undergo apoptosis. The mAb also caused homotypic aggregation of E710.2.3, which was inhibited by cytochalasin B, trifluoperazine, a combination of sodium azide and 2-deoxyglucose, EDTA, incubation at 4 degrees C, or treatment with paraformaldehyde. The DMF10 62.3 mAb stained a number of immortalized murine and human cell lines and, where tested, blocked their proliferation and caused death to varying extents by apoptosis. The molecule recognized by the mAb DMF10.62.3 was expressed on day 14 fetal thymus Thy1.2-positive cells. However, it was not detected on adult murine thymocytes, splenocytes, or bone marrow cells or on splenic LPS-activated B cells or Con A-activated T cells. The Ab immunoprecipitated a 40-kDa molecule from E710.2.3 that was not glycosylphosphatidylinositol linked. The data suggest that the molecule recognized by DMF62.3 is a novel cell surface molecule that may be involved in cell proliferation and/or cell death.     

Lectin staining of rat testis and epididymis after ligation of excurrent ducts at different levels

Seven rhodamine-conjugated lectins (PNA, RCA I, SBA, Con A, WGA, UEA I, and DBA; see Table 1) were utilized in studying the staining pattern of glycoproteins in rat testis and epididymis after ligation of ductuli efferentes (DE), corpus epididymidis (CE), and vas deferens (VD) for various time periods. Ductuli efferentes ligation caused a widening of seminferous tubules and detachment of spermatids with formation of multinuclear cells. These cells acquired a strong affinity for all lectins. Corpus epididymidis ligation also caused degeneration of spermatids with increased lectin staining in some tubules, but after 7 days another cell population close to the periphery of seminiferous tubules showed an increased nuclear affinity for some lectins followed by a clear degeneration and strong cytoplasmic staining with all lectins. Vas deferens ligation caused no degenerative changes in testicular spermatids. However, the peripheral cell population showed degenerative changes similar to those found after CE ligation. In both cases this was coincident with the formation of spermatic granulomas at the site of ligation. Ductuli efferentes ligation caused a gradual decrease of intratubular content in caput epididymidis, while the contrary was true after CE ligation. The latter was associated with intratubular accumulation of lectin-positive swollen cells and sperm aggregates as well as an increased lectin staining of narrow cells in initial segment and light cells in distal caput. After VD ligation an increased staining of light cells was initially found in distal cauda and distal caput, but, concomitant with distension of the tubules this reaction decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional sialylated O-glycan to platelet aggregation on Aggrus (T1alpha/Podoplanin) molecules expressed in Chinese hamster ovary cells

Aggrus, also called T1alpha and podoplanin, is a novel platelet aggregation-inducing factor that is expressed in various carcinoma cells. Aggrus/T1alpha/podoplanin is known to be expressed in lung type I alveolar cells or lymphatic endothelial cells. However, its physiological role has not been clarified. To assess the attribution of glycosylation to Aggrus platelet aggregation activity, recombinant molecules were stably expressed in a series of Chinese hamster ovary (CHO) cell mutants, N-glycan-deficient Lec1, CMP-sialic acid transporter-deficient Lec2, and UDP-galactose transporter-deficient Lec8. A new anti-human Aggrus monoclonal antibody, YM-1, was established to detect the expression of human Aggrus on these CHO cell mutants. Aggrus on Lec1 cells induced platelet aggregation, but those on Lec2 and Lec8 cells did not. Further, the glycans on Aggrus were analyzed by lectin blotting. Aggrus expressed in CHO and Lec1 cells showed Wheat-germ agglutinin, Jacalin, and Vicia villosa lectin bindings. Lectin blotting results indicated that sialylated core 1 structures, sialic acid plus Galbeta1,3GalNAc-Ser/Thr, were critical for the platelet aggregation activity. This oligosaccharide structure is known as tumor-associated antigen, which is potentially related to the metastasis process of cancer cells.     

Identification and characterization of taglin, a mannose 6-phosphate binding, trypsin-activated lectin from Giardia lamblia

We have previously reported the presence of a cell surface associated lectin activity in Giardia lamblia, a human protozoan parasite that is a significant cause of diarrheal disease worldwide [Lev, B., Ward, H., Keusch, G. T., & Pereira, M. E. A. (1986) Science (Washington, D.C.) 232, 71-73]. This lectin is specifically activated in vitro by a host protease, trypsin, which is secreted in vivo at the site of infection. The activated lectin agglutinates cells to which the parasite adheres in vivo and binds specifically to isolated brush border membranes of these cells. These findings suggest that this lectin may be of importance in the host-parasite interaction. We now report the identification of this lectin, which we have named taglin (to denote trypsin-activated Giardia lectin), and describe some of its properties. A monoclonal antibody that inhibits the hemagglutinating activity of taglin recognizes a protein of 28,000/30,000 kdaltons in Western blots of Giardia lysates. This finding was confirmed by direct demonstration of lectin activity with the technique of erythrocyte binding to proteins electroblotted to nitrocellulose, which revealed specific red cell binding to giardial protein bands in the same molecular weight range as those recognized by the monoclonal antibody. This study also elucidates the binding of taglin to terminal phosphomannosyl residues. The involvement of cell surface phosphate in binding of taglin to erythrocytes is shown by the abolition of lectin activity by alkaline phosphatase treatment of the erythrocytes. Taglin also requires divalent cations, Ca2+ or Mn2+, for hemagglutinating activity and is active within a narrow pH range of 6-7.     

Dynamic distribution of an antigen involved in the differentiation of avian myoblasts: II. Possible association of beta1 integrin with myofibril organization

Previous studies have shown that a monoclonal antibody, H-145, inhibits myotube formation of quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) [Hyodo and Kim, 1994: Exp. Cell Res. 212:120-131]. The antigen recognized by H-145 (H-145 antigen), which is a glycoprotein with a molecular mass of about 116 kDa, is related to a step immediately before myoblast fusion. To determine the functional significance of H-145 antigen, we examined its dynamic state during myogenic differentiation of QM-RSV cells. H-145 antigen showed a unique and discrete distribution. In immature myotubes immediately after myoblast fusion, many ring-like structures of H-145 antigen appeared on the ventral surface of the cells, encircling the actin dots detected simultaneously by immunofluorescence and interference reflection microscopy. The core of the ring-like structures was filled with the termini of actin bundles, mainly formed by alpha-actin. Other cytoskeletal-associated proteins, such as vinculin and alpha-actinin, were also associated with these structures. The ring-like structures of H-145 antigen were observed only during a restricted period when myoblasts fused actively, suggesting their relationships to myotube formation and an early stage of myofibril formation. With maturation of the myotubes, most of the H-145 antigen became redistributed in linear arrays on the apical cell surface and was probably associated with the termini of actin bundles to organize myofibrils, suggesting that the antigen was also related to maturation of myotubes. Experiments using monoclonal antibodies against chick beta1 integrin showed that H-145 antigen is beta1 integrin or a very closely related derivation. Thus H-145 antigen (beta1 integrin) is possibly involved in both myoblast fusion and the myofibril organization in myotubes.     

Preparation and some properties of a derivative of wheat germ agglutinin with altered biological activity

An inactive derivative of wheat germ agglutinin, which is a strong activator of blood platelets, was prepared by selective chemical modification of the lectin with cyanogen bromide at acid pH. The derivative was then used as a probe to learn about the initial events in platelet stimulation by physiological agents. Amino acid analysis of the modified lectin confirmed specific cleavage of a methionine residue. Gel filtration studies indicated a molecular weight for the lectin derivative similar to the unmodified lectin. In gel electrophoresis in the presence of sodium dodecyl sulfate, reduced samples of the derivative showed two bands and the main component migrated slightly faster than the native lectin. The derivative retained the capacity to precipitate an antibody to the lectin although at least one of the antigenic sites was lost due to chemical modification. The derivative did not compete with the unmodified lectin for binding to platelets. Unlike the parent lectin, the derivative did not aggregate platelets even at a ten fold higher concentration. Under similar conditions, there were about 1.0 X 10(5) binding sites/platelet for the lectin derivative with an apparent dissociation constant of 1.7 microM compared to 5 X 10(5) sites/cell and a dissociation constant of 0.4 microM for the native lectin. Overnight incubation of platelets or red cells with the derivative in microtiter plates showed about 2-5% agglutinating activity for the derivative compared to the unmodified lectin. Incubation of platelets with the lectin derivative inhibited platelet aggregation by thrombin while aggregation induced by a number of other agents was not significantly affected. This inhibitory effect of the lectin derivative on thrombin-induced platelet aggregation could be readily reversed with GlcNAc. The lectin derivative may be a useful tool to explore the structure-function relationship of cell surface components.     

Concanavalin a and wheat germ agglutinin receptors on dictyostelium: Their visualization by scanning electron microscopy with microspheres

The cellular slime mold, Dictyostelium discoideum, is a convenient model for studying cellular interactions during development. Evidence that specific cell surface components are involved in cellular interactions during its development has been obtained by Gerisch and co-workers (1, 2) using immunological techniques. Smart and Hynes (3) have shown that a cell surface protein can be iodinated on cells in aggregation phase, but not in vegetative phase, by the lactoperoxidase procedure. Recently, McMahon et al. (4), and Hoffman and McMahon have demonstrated, by SDS gel electrophoresis, considerable differences in cell surface proteins and glycoproteins of plasma membranes isolated from cells at different stages of development. Plant lectins have also been used to monitor changes in cell surface properties of D. discoideum cells during development. Weeks and co-workers (5, 6) have detected differences in the binding and agglutination of cells by concanavalin A (Con A). Gillette and Filosa (7) have shown that Con A inhibits cell aggregation and prematurely induces cyclic AMP phosphodiesterase. Capping of Con A receptors has also been reported (8). Reitherman et al. (9) have recently reported that agglutination of cells by several plant lectins and the slime mold agglutination, discoidin, changes during development. Such studies indicate that differences in surface properties exist for cells at various stages of development. However, owing to the uncertainties in the factors which contribute to lectin-induced cell agglutination (10), the molecular basis for these observations remain to be determined. In this study, we have used microspheres (11-14) coupled to either Con A or wheat germ agglutinin (WGA) as visual markers to study by scanning electron microscopy the topographical distribution of lectin receptors on D. discoideum cells fixed at different stages of development. We also describe the effect of labeling on the distribution of lectin receptors and on the morphology of the cell surface.     

Sugar-attached upconversion lanthanide nanoparticles: A novel tool for high-throughput lectin assay

To create a novel high-throughput lectin assay (HTPLA) method based on the emission of a luminophore by highly penetrable near-infrared excitation, sugar-attached upconversion lanthanide nanoparticles (LNPs) were synthesized as a tool to highlight the aggregates caused by the sugar-mediated specific bridging between LNP and lectin. The emissions from a mannose-coated LNP in the aggregates with a mannose-binding lectin were much stronger than those from the non-aggregated samples, being sensitive enough for HTPLA. A galactose-coated LNP was also applicable to a macrophage aggregation assay for the sugar specificity of its surface lectin.     

A novel C-type lectin regulating cell growth, cell adhesion and cell differentiation of the multipotent epithelium in budding tunicates

We have isolated two Ca(2+)-dependent, galactose-binding polypeptides from the budding tunicate, Polyandrocarpa misakiensis. Based on their partial amino acid sequences, full-length cDNAs were cloned. One of them was identical with a tunicate C-type lectin (TC14-2) reported previously. The other was a novel C-type lectin, referred to as TC14-3. In living animals, they appeared to be coupled. This complex of lectins, when applied in vitro to tunicate multipotent cells of epithelial origin, blocked cell proliferation and induced cell aggregation. The aggregates expressed a homolog of the integrin alpha-chain and other differentiation markers specific for epithelial cells. Recombinant TC14-3 could reproduce all the activities of native lectins by itself, which was accelerated by recombinant TC14-2. The inhibitory activity of TC14-3 on cell growth was completely abolished by the addition of 50 microM D-galactose. Anti-TC14-3 monoclonal antibody showed that the antigen was expressed constitutively by the multipotent epithelial and mesenchymal cells. These results provide evidence that in P. misakiensis a C-type lectin plays a novel, cytostatic role in regulating cell growth, cell adhesion and cell differentiation during asexual reproduction.     

Intercellular adhesion in the cellular slime mold Polysphondylium pallidum inhibited by interaction of asialofetuin or specific univalent antibody with endogenous cell surface lectin.

Previous work has shown that, as amoebae of the cellular slime mold Polysphondylium pallidum become aggregation competent, they accumulate on their cell surface a carbohydrate-binding protein (lectin) named pallidin. These amoebae also possess cell surface receptors, presumed to contain complex oligosaccharides with a high affinity for the endogenous lectin. If lectin-receptor interactions mediate cell-cell contact, then appropriate concentrations of pallidin inhibitors should block cell cohesion. Two potent macromolecular antagonists of the lectin were employed: the desialylated form of the glycoprotein fetuin and the univalent antibody (Fab) prepared against pallidin. We studied the effects of these inhibitors on rotation-mediated aggregation of P. pallidum amoebae under a variety of assay conditions. Amoebae exposed to hypertonic conditions or to antimetabolites (“Permissive conditions”) were selectively blocked from associating by microgram quantities of the lectin inhibitors, whereas cells in isotonic buffer (“nonpermissive condition”) were only slightly affected. A comparison of the morphology of agglutinates formed under the various conditions allows several explanations for the different susceptibilities to inhibition by antipallidin reagents. Although not conclusive, the work supports a model of cell adhesion in this simple eukaryotic system based at least in part on specific interactions between carbohydrate-binding proteins and receptors on adjoining cells.     

Identification and characterization of a novel neural cell adhesion molecule (NCAM)-associated protein from quail myoblasts: relationship to myotube formation and induction of neurite-like protrusions

Abstract We identified a novel neural cell adhesion molecule (NCAM)-associated protein, myo genesis-related and N CAM- a ssociated p rotein (MYONAP), the expression of which increases during the formation of myotubes in quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells). MYONAP shares homology with PL48 in human cytotrophoblasts and KIAA0386 in human brain. Excess expression of MYONAP in presumptive QM-RSV myoblasts induced long protrusions like neurites in cooperation with microtubules. Suppression of MYONAP by antisense cDNA prevented myotubes from forming in spite of the expression of myogenin, creatine kinase, and myosin, and rendered myoblast membranes resistant to fusion. Yeast two-hybrid screening showed that MYONAP interacted with NCAM specifically. Deletion of the NCAM-associated domain resulted in a loss of the function that induces neurite-like protrusions to form and disturbed the elongation of microtubules. The results suggested that MYONAP influenced the functions of microtubules and was involved in the formation of myotubes via its interaction with NCAM.     

Galectin-1, a cell adhesion modulator, induces apoptosis of rat Leydig cells in vitro

Galectin-1 (Gal-1), a beta galactoside-binding lectin, is involved in multiple biological functions, such as cell adhesion, apoptosis, and metastasis. On the basis of its ability to interact with extracellular matrix (ECM) glycoproteins, we investigated the Gal-1 effect on Leydig cells, which express and are influenced by ECM proteins. In this study, Gal-1 was identified in Leydig cell cultures by immunofluorescence. To gain insight into its biological role, Gal-1 was added to purified rat Leydig cells, under both basal and human chorionic gonadotrophin-stimulated conditions. Substantial morphological changes were observed, and cell viability showed an 80% decrease after 24 h culture. As a functional consequence of Gal-1 addition, testosterone production was reduced in a dose-dependent fashion, reaching a minimum of 26% after 24 h compared with basal values. cAMP showed a similar variation after 3 h. Assessment of DNA hypodiploidy and caspase activity determinations indicated that the reduction in viability and in steroidogenesis was caused by apoptosis induced by Gal-1. Besides, addition of Gal-1 caused Leydig cell detachment. Presence of laminin-1 or lactose prevented the effect of Gal-1, suggesting that the carbohydrate recognition domain is involved in inducing apoptosis. These findings demonstrate a novel mechanism, based on Gal-1 and laminin-1 interaction, which could help us better understand the molecular basis of Leydig cell function and survival control.