The effect of temperature of concanavalin A-mediated agglutination of cells with rigid receptors

The agglutination of a yeast, Candida albicans, by concanavalin A has been described. The agglutination was cell-number dependent. Prolonged incubation (60 min) was needed to reach maximum agglutination at 37° C. The rate but not the extent of agglutination was temperature dependent. The dimeric forms of concanavalin A, obtained either at low pH or after succinylation, agglutinated the yeast cells as well as the tetramer. Temperature changes affected the agglutination of yeast cells by dimers and by tetramers to the same extent.     

Mitogenic stimulation and the redistribution of concanavalin A receptors on lymphocytes

When mouse spleen (Ig⁻) cells undergo maximal mitogenic stimulation by optimal concentrations of concanavalin A (conA), the Ig⁻ cells form caps of conA very slowly, with 50% of maximum cap formation occurring after about 10 h and maximal capping after about 24 h. Anti-conA antibody added after optimal conA accelerates the rate of cap formation and effectively blocks mitogenic stimulation (< 10%) by optimal conA concentrations when the rate of capping is increased more than about 2-fold. The effect of anti-conA antibody in accelerating cap formation by optimal conA is antagonized by cytochalasin D (CD), which substantially restores the mitogenic action of optimal conA. Thus there is an inverse relationship between rate of cap formation and extent of mitogenic stimulation. Further experiments showed that if anti-conA antibody, α-methyl mannoside or EGTA were added at increasing intervals after the addition of conA, these inhibitors block the stimulation of the cells with very similar time courses. Addition of appropriate concentrations of an inhibitor at the same time as optimal conA blocks mitogenic stimulation completely, but has negligible effects after 24 h. The extent of stimulation which occurs after the addition of inhibitor at intermediate times closely follows the extent of cap formation at the same time. The simplest interpretation of these results is that mitogenic action by optimal conA can be blocked by (i) accelerated capping of uncapped cells; or (ii) by the removal of either conA or calcium before, but not after, cap formation has occurred. These results suggest that the rate of cap formation by conA, and the presence of external calcium (>10⁻⁴ M) in the medium for some unspecified period before cap formation occurs are both significant factors in generating the primary mitogenic signals which commit the cells to DNA synthesis.     

A comparison of ligand-induced redistribution of surface immunoglobulins, alloantigens, and concanavalin A receptors on mouse lymphoid cells

Redistribution of surface immunoglobulins (Ig), H-2^b, Thy-1.2 and TL. 1,2,3 alloantigens, and concanavalin A (Con A) receptors on mouse thymus, lymph node and spleen cells into “caps” induced by bivalent antibodies or ligands was compared by immunofluorescence. Surface Ig was capped rapidly following attachment of anti-Ig antibody at 37°. Capping of alloantigens and Con A receptors occurred very slowly following attachment of alloantibody or Con A, but much more rapidly after addition of a secondary bivalent antibody. An inverse relationship between the number of surface component sites per cell and the extent of capping of that component was observed. Capping of alloantigens sparsely represented on the cell surface was not inhibited by high concentrations of alloantibody, in contrast to capping of alloantigens present in greater quantities. These results suggest that factors in addition to molecular cross-linking may be involved in ligand-induced redistribution of cell surface components.     

The effect of temperature on concanavalin A-mediated agglutination of cells with rigid receptors.

The agglutination of a yeast, Candida albicans, by concanavalin A has been described. The agglutination was cell-number dependent. Prolonged incubation (60 min) was needed to reach maximum agglutination at 37 degrees C. The rate but not the extent of agglutination was temperature dependent. The dimeric forms of concanavalin A, obtained either at low pH or after succinylation, agglutinated the yeast cells as well as the tetramer. Temperature changes affected the agglutination of yeast cells by dimers and by tetramers to the same extent.     

Pore formation is not associated with macroscopic redistribution of P2X7 receptors

The present study examines whether changes in P2X7 purinergic receptor density precede formation of the cytolytic pore characteristic of this receptor. We fused P2X7 receptors with enhanced green fluorescent protein (EGFP) at the amino or carboxy termini (EGFP-P2X7 and P2X7-EGFP). Electrophysiological characterization in Xenopus oocytes revealed wild-type responses to ATP for GFP-tagged receptors. However, differences in sensitivity to ATP were apparent with the P2X7-EGFP receptor displaying a threefold reduction in ATP sensitivity compared with control. Ethidium ion uptake was used to measure cytolytic pore formation. Comparison of tagged receptors with wild type in HEK-293 and COS-7 cells showed there was no significant difference in ethidium ion uptake, suggesting that fusions with EGFP did not interfere with cytolytic pore formation. Confocal microscopy confirmed that tagged receptors localized to the plasmalemma. Simultaneous monitoring of EGFP and ethidium ion fluorescence revealed that changes in receptor distribution do not precede pore formation. We conclude that it is unlikely that large scale changes in P2X7 receptor density precede pore formation.     

Focal adhesion kinase pp125FAK is associated with both intercellular junctions and matrix adhesion sites in vivo

Previous studies have characterized pp125^FAK as a focal adhesion (FA)-associated non-receptor tyrosine kinase. However, there are few data available on the expression and localization of this kinase in tissues. In this study we show that in human tissues the highest expression of pp125^FAK is found in some developing epithelia, where pp125^FAK is associated with either intercellular junctions or with sites of adhesion to the basement membrane, whereas the same adult tissues show only a faint reactivity. Connective tissue cells do not show any reactivity for pp125^FAK in vivo, but developing arterial smooth muscle expresses pp125^FAK at high levels. The expression pattern in malignant tissues is variable, but most carcinomas do not express this kinase. In primary cultures of human amnion epithelial cells pp125^FAK first becomes associated with the polarized adhesion lamellae, but is subsequently translocated to the forming adherens junctions (AJs). Later upon culturing pp125^FAK becomes associated with prominent FAs, as in cultured cell lines. Taken together, our results suggest that the association of pp125^FAK with FAs in cultured cells is principally due to a process of adaptation, whereas in vivo pp125^FAK mainly functions as a regulatory component of intercellular AJs and cell-matrix adhesions of developing epithelia and also in developing arterial smooth muscle.     

Inhibition of yeast mitochondrial ATPase by concanavalin A

Concanavalin A binds to and inhibits enzyme activity of the energy transducing ATPase from yeast mitochondria. Activity loss is completely reversed by glucose or α-methyl-d-mannose. Concanavalin A reacts with the F₁ portion of the ATPase complex, suggesting that this enzyme unit may be a glycoprotein. A major concanavalin A binding site is associated with the largest subunit of the F₁ enzyme.     

Migration of cell surface concanavalin A receptors in pulsed electric fields.

Concanavalin A (con A) receptors on the surface of cultured Xenopus myoblasts redistributed in response to monopolar, pulsed electric fields. The prefield uniform distribution of the receptors became asymmetrical, and was polarized toward the cathodal pole, in the same way as in DC fields. The extent of asymmetry depended on the duration of field exposure, pulse width (or alternatively, interpulse interval), frequency, and intensity. This relationship was most conveniently expressed by using duty cycle, a quantity determined by both pulse width and frequency. Pulses of average intensity 1.5 V/cm induced detectable asymmetry within 5 min. At the lowest average field intensity used, 0.8 V/cm, significant asymmetry was detected at 150 min. For pulses of high duty cycle (greater than 25%), steady state was reached after 30 min exposure and the steady state asymmetry was dependent on average field intensity. For low duty cycle fields, the time required to reach steady state was prolonged (greater than 50 min). Before reaching a steady state, effectiveness of the pulses, as compared with DC fields of equivalent intensity, was a function of duty cycle. A low duty cycle field (fixed number of pulses at low frequency or long interpulse interval) was less effective than high duty cycle fields or DC.     

Action of interferon on cell membrane of mouse lymphocytes : Inhibition of ligand-induced redistribution of surface receptors

Mouse interferon at 50–100 U/ml or higher concentrations inhibited cap formation of surface components on mouse lymphocytes induced by anti-lymphocyte serum, concanavalin A (ConA), phytohemagglutinin (PHA) and soybean agglutinin (SBA). Interferon from species other than mouse were not effective, suggesting that interferon itself in the preparations used was responsible for the observed effect. The inhibition was observed immediately after addition of interferon, without preincubation of cells with it. The effect was reversible, disappearing after a short lag when interferon was removed. The effect may thus be a reflection of a rapid change in cell membrane upon binding of interferon molecules. On the other hand, the cap formation by anti-immunoglobulin, anti-thy 1, 2, anti-H-2 sera and wheat germ agglutinin (WGA) were not influenced by interferon at all, indicating that the inhibition by interferon was not a general phenomenon. The cap formations susceptible to interferon are those that are enhanced by pretreatment of cells with colchicine and are accompanied by marked uropod formation, but those insusceptible to interferon occur readily, becoming sharp spots which are then shed. It is suggested that interferon may modify the microtubule-containing structure, it it may selectively affect those membrane components that are anchored to it.     

Inhibition of the enzymatic activity of thrombin by concanavalin A

Concanavalin A, a carbohydrate lectin derived from the jack bean, prolongs the thrombin clotting time of human plasma or purified fibrinogen. Prolongation is due to delay in peptide release from fibrinogen. The rate of fibrin monomer polymerization is not affected. Hydrolysis of protamine sulfate by thrombin is inhibited by concanavalin A. All inhibitory effects are prevented by α-methyl-D-mannoside. Concanavalin A does not delay clotting of fibrinogen by reptilase (releases fibrinopeptide A only) or by Ancistrodon contortrix contortrix (releases fibrinopeptide B initially followed by a small amount of A). It is concluded that concanavalin A binds to a carbohydrate on the thrombin molecule thus inhibiting its enzymatic activity.     

Inhibition of modulation of thymus-leukemia antigens by concanavalin A.

When incubated at 37 °C in medium containing antibodies specific for thymus-leukemia (TL) antigens, viable cells bearing these antigens become resistant to the cytolytic effects of guinea pig complement, a process termed antigenic modulation. Antibody-induced membrane redistribution of the TL antigens, detected by indirect immunofluorescence, occurs with a similar pace. When high concentrations of concanavalin A (Con A) were included with antibodies in the incubation medium, TL antigenic modulation as well as antigen patching and capping were markedly inhibited, similar to effects of Con A on membrane immunoglobulin redistribution with murine spleen cells. Colchicine antagonized the inhibition by Con A suggesting the involvement of microtubules. In parallel experiments high concentrations of Con A failed to alter the quantity of TL antigen expression or its rate of change with time during incubation in cognate antisera. These results support the hypotheses that (a) generalized alterations in membrane receptor mobility may be induced by ligand binding to the cell membrane, and (b) under certain conditions stable interactions occur between normally independent cell surface antigens.     

Height is associated with more self-serving beliefs about wealth redistribution

People vary widely in their attitudes towards how much their government should redistribute wealth. Evolutionary theory may shed light on why this variation occurs. Numerous studies have established an association between upper body strength and attitudes towards equality and wealth redistribution in males, showing that physically stronger men are more likely to hold self-serving beliefs on these issues. This effect is typically weaker or absent in women. A question that has received little attention is whether there are similar associations between other aspects of formidability and attitudes towards wealth redistribution. One such aspect is height. I tested this prediction using data from the European Social Survey, in a sample of 27031 people from 20 European countries. Results show that taller people are more likely to have self-serving attitudes towards government redistribution of wealth. The result was robust to numerous control variables and alternative model specifications, but the direct effects of height were small. Taller individuals were less supportive of government wealth redistribution overall, but were especially averse if they were also wealthier. Post-hoc analyses suggested that for lower income deciles, the association was reversed. For these people, there was a positive association between height and support for wealth redistribution. However, effects were equally strong in males and females, and so are not fully consistent with current evolutionary psychological theories of resource distribution.     

Interaction of concanavalin A with lactogenic receptors in isolated membranes

Specific binding of lactogenic hormones to their receptors in membranes from lactating mouse liver is inhibited by concanavalin A (Con A). Binding to solubilized receptors is not affected by the presence of Con A in the binding reaction. However, these solubilized binding proteins are retained on Con A-Sepharose columns. Prebound hormone-receptor complexes are also retained on Con A-Sepharose. These data indicate that lactogenic receptors have a Con A binding site distinct from the hormone binding site. Moreover, once bound, the hormone is not released by the action of Con A. Somatogenic receptors do not have a Con A binding site and the binding of bovine growth hormone to hepatic membranes is not affected by the presence of Con A in the binding reaction. Inhibition of binding to lactogenic receptors by Con A occurs independently from other membrane perturbing events such as phospholipid methylation.     

Antibody-induced redistribution of normal and tumor associated surface antigens

Redistribution (capping) of normal and tumor-associated surface antigens was studied on murine and human cells by the indirect membrane immunofluorescence (MIF) technique. The capping of H-2 isoantigens was compared on normal mouse T-lymphocytes and on YAC cells, a Moloney leukemia virus (MLV) induced lymphoma. H-2 and Moloney virus induced cell surface antigen (MCSA) capping was compared on three YAC lines with different MCSA concentrations. H-2 and tumor-associated surface antigen capping was compared on two polyoma induced sarcoma lines and five methylcholanthrene induced sarcoma lines. In the human system, IgM-capping was compared on normal lymphocytes and on the Burkitt lymphoma derived Daudi line. Capping of HL-A and the Epstein-Barr virus (EBV) determined membrane antigen (MA) was compared on the Burkitt lymphoma derived line Maku and on EBV-superinfected Daudi cells. H-2 antigens on normal murine cells capped more promptly and on a larger fraction of the cell population on the various tumor cells. Surface associated IgM showed a better capping on normal lymphocytes than on Daudi cells. All tumor associated antigens except MCSA, showed good capping. MCSA was almost completely refractory to capping. Increasing concentrations of MCSA appeared to inhibit the capping of H-2 on the YAC sublines with different concentrations of MCSA. The polyoma induced ascites sarcoma (SEWA) did not cap either with regard to H-2 or the polyoma determined surface antigen.     

The mobility of concanavalin A receptors and surface immunoglobulins on rat hepatocyte plasma membranes

Lateral mobilities of lectin receptors and surface immunoglobulins were measured in plasma membranes of hepatocytes prepared by smearing small pieces of rat liver tissue and then using the fluorescence recovery after photobleaching (FRAP) technique. Smears were treated with various doses of fluorescein isothiocyanate (FITC) conjugated concanavalin A (ConA), succinylated ConA (SConA), wheat germ agglutinin (WGA), and soybean agglutinin (SBA), as well as with rabbit anti-rat IgG (RARa/IgG) and goat anti-rat IgM(Fc) (GARa/IgM(Fc] antisera. 10 micrograms/ml ConA and SConA concentrations and a 55 X dilution of the GARa/IgM(Fc) antiserum were found to be suitable for measuring the lateral mobilities dependent on age. Diffusion constant and mobile fractions of receptor complexes were measured in different age groups of female Fisher rats (from 1 to 26 month-old). The FRAP measurements revealed that at least two major receptor sites can be distinguished in cell membranes of compact tissue (similar to the cultured and isolated cells), forming a mobile and an immobile fraction. The mobile fractions of both the lectin receptors and the surface immunoglobulins tended to decrease with age, while the age differences of the diffusion constants were not statistically significant. The observed alterations could be due to the covalent crosslinking of the mobile receptors to immobile patches and/or to the retardation of free diffusion by the cytoskeleton, dependent on age.     

Interaction of HIV-1 with dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-expressing cells is influenced by gp120 envelope modifications associated with disease progression

Dendritic cells can enhance the replication of HIV-1 in CD4(+) lymphocytes through the interaction of the gp120 envelope protein with such molecules as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin. The variable loops of gp120 have previously been shown to modulate the interaction of HIV-1 with its principal receptor CD4 and its various coreceptors, namely CCR5 and CXCR4. Here, we utilized a panel of molecular cloned viruses to identify whether gp120 modifications can influence the virus interaction with immature dendritic cells or a cell line expressing dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (Raji-DC-SIGN). The viruses encompass the R5, R5X4 and X4 phenotypes, and are based upon V1V2 and V3 sequences from a patient with disease progression. We found that dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin enhancement of virus replication can be modulated by the V1V2 length, the overall V3 charge and N-linked glycosylation patterns; similar results were observed with immature dendritic cells. Viruses with higher V3 charges are more readily transferred to CD4(+) lymphocytes when the V1V2 region is longer and contains an additional N-linked glycosylation site, whereas transfer of viruses with lower V3 charges is greater when the V1V2 region is shorter. Viruses differing in the V1V2 and V3 regions also demonstrated differential capture by Raji-DC-SIGN cells in the presence of mannan. These results indicate that the interaction between HIV-1 and immature dendritic cells via such molecules as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin may have a role in selecting viruses undergoing transmission and evolution during disease progression.     

Sequential exocytosis of insulin granules is associated with redistribution of SNAP25

We have investigated sequential exocytosis in beta cells of intact pancreatic islets with the use of two-photon excitation imaging of a polar fluorescent tracer, sulforhodamine B, and a fusion protein comprising enhanced cyan fluorescent protein (ECFP) and the SNARE protein SNAP25 (synaptosome-associated protein of 25 kD) transfected with an adenoviral vector. Sequential exocytosis was found to account for <10% of exocytic events in beta cells stimulated either with glucose under various conditions or by photolysis of a caged-Ca2+ compound. Multigranular exocytosis, in which granule-to-granule fusion occurs before exocytosis, was rarely found. We detected redistribution of ECFP-SNAP25 from the plasma membrane into the membrane of the fused granule occurred in a large proportion (54%) of sequential exocytic events but in only a small fraction (5%) of solitary fusion events. Removal of cholesterol in the plasma membrane by methyl-beta-cyclodextrin facilitated both redistribution of ECFP-SNAP25 and sequential exocytosis by threefold. These observations support the hypothesis that SNAP25 is a plasma membrane factor that is responsible for sequential exocytosis.     

Concanavalin A surface receptors and cytoplasmic actin in cell adhesion.

A double immunofluorescence staining technique to locate concanavalin A (Con A) surface receptors and cytoplasmic actin in the same cell was applied to monolayer cultures of rat foetal fibroblasts during cell detachment induced by trypsin and during cell attachment to glass substratum. Con A receptors were demonstrated by fluorescein-isothiocyanate-labelled Con A (FITC-Con A) and actin by specific anti-actin antibody (AAA) traced with rhodamine-labelled goat anti-human globulin (R-AHG). Untreated, control cells had an elongated shape, Con A receptors restricted to cell margins and prominent actin filaments. After 2 min treatment with 0.001% trypsin the cells became angular with Con A receptors in clusters and actin in a diffuse or aggreagate staining pattern. Progressive cell rounding followed and this was accompanied by the development of long, thin, arborized cell processes, studded with Con A receptors and containing fine actin filaments. Complete cell rounding preceded cell detachment. The sites of detached cells were marked by fine aggregates containing Con A receptors and actin. In cells attaching to a glass substratum, actin was present in a diffusely stained or aggregate pattern in round cells, in filaments restricted to cell margins in partially spread cells and in numerous filaments in fully spread cells. Con A receptors were present in clusters in round cells, in clusters or caps in partially spread cells and in cell margins in fully spread cells. Binding of FITC-Con A to partially spread cells resulted in dissolution of the few, newly formed, actin filaments. We believe our observations are consistent with the idea that actin filaments, formed during cell attachment, contribute towards the maintenance of cell adhesion by helping in the preservation of cell shape and by anchorage of Con A receptors at points of cell attachment to the substratum.