Modulation of thymocyte membrane potential by concanavalin A.

Binding of convanavalin A, a potent mitogenic lectin, to thymocyte surface membrane causes depolarization of membrane potential. The effect is suppressed by alpha-methyl-D-glucoside, a haptenic inhibitor of this lectin, or by low temperature. Colchicine and cytochalasin B aslso suppress the change. These data indicate that perturbation of thymocyte surface membrane receptors induced by concanavalin A might be linked to change in the functional state of cellular cytoskeletal systems in turn causing depolarization of thymocyte surface membrane. The initial event generated by receptor-ligand interaction on the outer surface could be translated into cellular interior action only under highly fluid conditions of membrane lipid. Depolarization of thymocyte plasma membrane may be involved in the triggering mechanism of metabolic burst associated with blastoid transformation.     

Electrophoretic mobility of the thymocyte during endocytosis of concanavalin A

The electrophoretic mobility of the mouse thymocyte is increased by 11% when concanavalin A is used to induce cell cap formation with subsequent endocytosis. The mere adsorption of concanavalin A to the thymocyte membrane does not modify its electrophoretic mobility.     

De novo purine and pyrimidine biosynthesis during thymocyte concanavalin A stimulation

Measurement of the incorporation of [¹⁴C]formate and [¹⁴C]HCO₃⁻ enable us to characterize the activity of the synthetic pathways of purine and pyrimidine nucleotides in vitro throughout the process of the conA stimulation of rat thymocyte populations enriched for immunocompetent cells by isopycnic centrifugation. Our results show that de novo synthesis compensates for the total absence of exogenous nitrogenous bases and nucleosides in the culture medium. The magnitude of the proliferative response in media supplemented with dialysed fetal calf serum (FCS) was found to be the same as that observed when complete FCS was used in the culture medium. The induction of de novo synthesis (1) contributes to the expansion of the free nucleotide pool (the quantity of ATP measured in the perchloric acid (PCA)-soluble material of a same number of cells is increased by a factor of 10); (2) supplies the nucleotides necessary for nucleic acid synthesis (the total number of cells is increased by a factor of 3 after 4 days culture period). The activity of the metabolic pathways involved appears to be solicited by the dynamic requirements for thymocyte stimulation. For each step in the cellular activation a steady state of adenylic nucleotide synthesis and condensation into nucleic acids is established.     

A model of thymocyte proliferation.

A steady-state model, based upon an analytical approach to the interpretation pf thymic radioautographic data is presented. In the model the process of cellular proliferation is represented by a feedback amplifier. It is shown by computer simulation that a model based on this conception, in which cellular flow through G₁ compartments is treated as a stochastic process and that through the S, G2 and M compartments by a sequential unidirectional process, is in reasonable agreement with the experimental data.     

Acid proteases from species of Mucor. III. Interaction with concanavalin A and concanavalin A Sepharose.

The reaction of Mucor miehei protease with concanavalin A was followed by a turbidimetric assay in the pH range 5-8. At pH 4.0, no turbidity developed but binding of the enzyme to concanavalin A could be demonstrated by gel filtration. Two fractions of apparent molecular weight 65000 and 52000 were isolated, the 65000 molecular weight species apparently representing a protomer of concanavalin A (24000) bound to the enzyme. An analysis of the circular dichroism spectrum of this complex suggested that protomer binding results in a conformational change in the enzyme which is associated with a 30% increase in proteolytic activity. At pH 6.0, the enzyme was strongly bound to columns of concanavalin A Sepharose but could be removed by including alpha-methyl D-glucoside and NaC1 in the elution buffer. Some column degradation occurred at room temperature but was not detectable at 4 degrees C where rapid elution of the enzyme resulted in a greater than 90% yield of highly active protein. Periodate-oxidized Mucor miehei protease and Mucor renin did not react with concanavalin A and were not bound to the affinity column.     

Effects of the mitogen concanavalin A on pathways of thymocyte energy metabolism.

The lectin concanavalin A (Con A) acts as a mitogen that preferentially activates T-cells. It stimulates the energy metabolism of thymocytes within seconds of exposure. We studied short-term effects (<30 min) of Con A on a conceptually simplified model system of rat thymocyte energy metabolism in the concentration range of 0-2 microg Con A per 107 cells, using metabolic control analysis. The model system consisted of three blocks of reactions, linked by the common intermediate mitochondrial membrane potential (Delta[psi]m): the substrate oxidation reactions, which produce the linking intermediate, and the proton conductance (or leak) and ATP turnover pathways which consume Delta[psi]m. Firstly, we used top-down elasticity analysis to establish which subsystems are targeted by Con A. Secondly, we quantitatively analysed the steady-state regulation of the system variables by Con A: how do the subsystem fluxes respond to Con A individually and as a whole? Our results indicate that: (1) steady-state respiration and Delta[psi]m increase as Con A concentration is raised, but at higher concentrations the increase in respiration is less and Delta[psi]m falls; (2) Con A independently changes the kinetics of the reactions that produce and consume Delta[psi]m: the Delta[psi]m-producing reactions are inhibited, and the reactions involved in ATP turnover are stimulated; and (3) the overall effects of Con A are mostly mediated by effects on ATP turnover.     

Low- and high-affinity concanavalin A binding to thymocyte plasma membrane vesicles

Binding of concanavalin A to isolated thymocyte membrane vesicles occurs through (a) numerous (～6 × 10⁶/cell equivalent) low-affinity sites (K_a = 1.3 × 10⁵ M^?1) and (b) fewer (～0.4 × 10⁶/cell equipment) specific receptors (K_a = 6.8 × 10⁶ M^?1) defined as 55,000 D glycoprotein and its multimers. Specific binding is positively-cooperative, with a Hill coefficient of～1.8. Low concentrations of glutaraldehyde selectively crosslink the 55,000 D glycoprotein with replacement of positively-cooperative sites by high-affinity sites. It is proposed that concanavalin A-binding induces multimerization of the 55,000 D glycoprotein.     

Change of concanavalin A induced agglutinability during preimplantation mouse development

Mouse embryos during early cleavage (zygote to eight-cell stage) were agglutinable with a low concentration (10 μg/ml) of concanavalin A (ConA). This agglutinability was reduced during the first mitotic division. Morulae were agglutinable with a slightly higher concentration (100 μg/ml), whereas blastocysts were not agglutinable even with ConA at a concentration of 5000 μg/ml; however, isolated inner cell masses agglutinated readily at 10 μg/ml of ConA. Embryos grown in vitro behaved as did those isolated directly from the genital tract. Treatment with proteolytic enzymes did not induce agglutinability of mouse blastocyst. The change in agglutinability of trophoblastic cells reflects dramatic changes in the cell surface.     

Protein-tyrosine phosphorylations induced by concanavalin A and N-formyl-methionyl-leucyl-phenylalanine in human neutrophils.

The ability of the lectin concanavalin A (ConA) and N-formyl-methionyl-leucyl-phenylalanine (fMLF) to induce protein-tyrosine phosphorylation in human neutrophils was examined by immunoblot analysis. ConA caused an increase in tyrosine phosphorylation of protein bands with apparent molecular masses of 120, 80, 76, 66 and 40 kDa; on the other hand, fMLF caused an increase in those of only 80-kDa and 40-kDa proteins. These protein-tyrosine phosphorylations were time- and dose-dependent. The tyrosine phosphorylation of 40-kDa protein induced by fMLF was suppressed but that by ConA was not suppressed by pertussis toxin pretreatment. At the same time, pertussis toxin pretreatment also inhibited lysozyme release and aggregation of neutrophils induced by fMLF but did not inhibit those responses induced by ConA. These results suggest that the tyrosine phosphorylation of 40-kDa protein may be involved in a part of neutrophil activation and be regulated via pleiotropic signal transduction pathways. In addition, immunoblot analysis employing antibodies against microtubule-associated protein 2 (MAP2) kinase suggested that this tyrosine-phosphorylated 40-kDa protein might be the MAP2 kinase.     

Cooperative binding of concanavalin A to thymocyte membrane vesicles augments uptake of alpha aminoisobutyrate.

(1) Membrane vesicles from rabbit thymocytes accumulate alpha-aminoisobutyrate in the presence of 0.1 M NaCl. Uptake is 1/2 maximal after about 2 min and reaches a plateau value (61 pmoles/mg protein) after 30 min. (2) Up to 25 mug concanavalin A/ml, binding of the lectin describes a sigmoid curve indicative of a cooperative process. (3) At lectin concentrations up to 8 mug/ml, lectin binding enhances the uptake of alpha-aminoisobutyrate (maximally 30%).     

Stimulation of thymocyte proliferation by phosphorothioate DNA oligonucleotides

DNA is a complex macromolecule the immunological properties of which depend on short sequence motifs called CpG motifs or immunostimulatory sequences (ISS). These sequences are mitogenic for B cells and can stimulate macrophage cytokine production. While these sequences do not directly activate T cells, they can augment effects of stimulation via the TCR. Furthermore, ISS can affect T cells because of macrophage production of IL-12 and IFN-alpha/beta. In these studies, we further evaluated the immune effects of DNA on T cells, testing the possibility that certain T cell populations can respond directly to this stimulus. We therefore tested the in vitro responses of thymocytes to a series of phosphodiester (Po) and phosphorothioate (Ps) oligonucleotides (ODNs) varying in sequence. In in vitro cultures, phosphorothioate ODNs (sODNs) containing CpG motifs induced significant proliferation of murine thymocytes, although phosphodiester compounds lacked activity. The magnitude of stimulation varied with sequences flanking the CpG motifs, as both dA and dT sequences enhanced the stimulatory capacity of the CpG motif. Furthermore, CpG sODNs were strong costimulators of anti-CD3-mediated thymocyte activation, increasing proliferation compared to anti-CD3 in the absence of DNA. This activation was only partially inhibited by cyclosporine A and was not dependent on a calcium influx. Together, these results indicate that phosphorothioate oligonucleotides containing CpG motifs can directly induce thymocyte proliferation as well as augment TCR activation. These observations thus extend the range of actions of CpG DNA and suggest additional mechanisms for its function as an immunomodulatory agent or adjuvant.     

Accelerated calcium ion uptake in murine thymocytes induced by concanavalin A.

The mechanism of enhancement of Ca2+ uptake by the T cell mitogen concanavalin A (Con A) was studied in murine thymocytes. Native Con A enhanced the rate of Ca2+ uptake as much as 9-fold, an increase being observed within five minutes after Con A addition. The effect of Con A was reversed completely by alpha-methyl mannopyranoside (alpha-MM). Increased Ca2+ uptake was observed with increasing concentrations of Con A, between 2 and 400 microgram/ml, indicating that the stimulation of Ca2+ uptake is not restricted to mitogenic lectin concentrations (0.5-2 microgram/ml). Succinyl Con A exhibits only a slight effect in the same concentration ranges as native Con A. Ca2+ uptake, both in the absence and presence of Con A, is strongly dependent on energy metabolism and is carrier mediated. The augmentation of Ca2+ uptake by native Con A is due to an enhanced Vmax. Uptake of the anion, CrO42-, by thymocytes, found to be a non-saturable process, was also enhanced by Con A. The effect of Con A on CrO42- permeability appears to be independent of its effect on Ca2+ uptake.     

The shift of an increase in phosphofructokinase activity from protein synthesis-dependent to -independent mode during concanavalin A induced lymphocyte proliferation

Phosphofructokinase activity increased dramatically in cultured mouse spleen lymphocytes 8 hours after concanavalin A stimulation and preceded the onset of DNA synthesis by 8 hours. The increase in enzyme activity and [³H]-thymidine incorporation were mitogen-concentration dependent. Enzyme activity increased 12-fold over control level at 48 hours when DNA synthesis peaked. The protein synthesis inhibitor, cycloheximide, blocked the rise in phosphofructokinase only when given prior to the increase in enzyme activity. Once the increase began, later addition of cycloheximide became progressively less inhibitory. These observations suggest that the period of increase in phosphofructokinase activity involves the activation of preexisting enzyme molecules.     

Lymphocyte commitment to DNA replication induced by concanavalin A

Commitment of lymphocytes to DNA replication induced by ConA was investigated by adding α-MM (a saccharide that competes with the lectin's lymphocyte binding sites) at various times to lymphocyte cultures that had been treated with a wide range of ConA concentrations. After 24 h of culture, α-MM addition has essentially no effect on cells treated with optimal concentrations of ConA, results in a marked blastogenic response of cells treated with supra-optimal (non-mitogenic) concentrations of ConA, and severely inhibits blastogenesis of cells treated with suboptimal concentrations of the lectin. Furthermore, the time of commitment is progressively shortened as the concentration of mitogen is increased. Thus, cells were committed to DNA replication as early as 5 h after incubation with supra-optimal concentrations of ConA. Induction of commitment does not occur when cells are incubated with ConA at 4 °C. Extensive crosslinking of membrane sites appears to be associated with the induction of early commitment by ConA, since the bivalent succinyl ConA failed to exhibit this effect. These findings indicate the need for re-evaluation of previous studies utilizing competing saccharides to determine kinetics of cell commitment to DNA replication.     

Ability of human leukocytic pyrogen to enhance phytohemagglutinin induced murine thymocyte proliferation

Human leukocytic pyrogen, a monokine produced by stimulated human mononuclear phagocytes, will enhance the murine thymocyte proliferation response to phytohemagglutinin (lymphocyte activating factor (LAF) activity). During all steps of purification of human LP, pyrogenicity and LAF activity are coincidental suggesting a single identity for the two monokines. The LAF assay for human LP is highly sensitive and can detect human LP at a concentration of 10^?12M. Further experiments suggests that human LP and LAF activities could be destroyed by heating to 70 °C. Furthermore, while in vivo pyrogenicity of human LP can be blocked by ibuprofen, the in vitro LAF activity of the same molecule is unaffected by ibuprofen. Immune rabbit serum directed against human LP could also block in vitro LAF activity either by preincubation with LP or by blocking during culture.