Addition Of Concanavalin Research Articles

The effect of time of addition of glutamine or nucleosides on proliferation of rat cervical lymph-node T-lymphocytes after stimulation by concanavalin A

1. Addition of concanavalin A to T-cell lymphocytes from rat cervical lymph nodes increases the activity of glutaminase within 1 h and those of carbamoyl-phosphate synthase II and aspartate transcarbamoylase within 3 h. There was a similar time course for the effects of concanavalin A on rates of glutamine utilization, which was increased within 1 h, and on pyrimidine nucleotide synthesis, which was increased by 40% at 2 h and by 100% at 3 h. 2. A delay in the addition of glutamine to the culture medium after addition of concanavalin A caused a decrease in [3H]thymidine incorporation only after 4-6 h. In the absence of glutamine, delay in addition of guanosine or inosine caused a decrease in [3H]thymidine incorporation only after 6-8 h after the addition of concanavalin A. 3. In contrast, a delay in addition of adenosine or uridine to the culture medium had an immediate effect (i.e. within 2 h) on the rate of incorporation of [3H]thymidine. It is suggested that adenosine and uridine have specific effects on proliferation via specific receptors for these nucleosides in the membrane.

Regulation of mitogen- and TCGF-induced lymphocyte blastogenesis by prostaglandins and supernatant from equine embryos and endometrium

Immunosuppressive substances which interfere with lymphocyte blastogenesis are released in vitro by embryos and endometrium from mares in early pregnancy. Immunosuppression was not evident when tissues were cultured in the presence of indomethacin (a prostaglandin-synthesis inhibitor). Various prostaglandins ( PGs) were added to equine lymphocytes and lymphocyte proliferation was measured after the addition of concanavalin A (Con A) or phytohaemagglutinin A ( PHA). PGE 2 and PGF 2α inhibited Con A-induced blastogenesis down to final concentrations of 1·8 × 10 −9 M and 1·3 × 10 −6 M, respectively. Other PGs tested (6-keto- PGF 1α and 13,14-dihydro-15-keto- PGF 2α) did not affect Con A-induced blastogenesis. PHA-induced blastogenesis was inhibited by concentrations down to 1·8 × 10 −9 M PGE 2, 3·3 × 10 −7 M PGF 2α and 2·8 × 10 −4 M 6-keto- PGF 1α. At all concentrations, 13,14-dihydro-15-keto- PGF 2α only slightly reduced PHA-induced blastogenesis. Therefore, PGE 2 was the only prostaglandin tested which, at physiological concentrations, significantly inhibited incorporation of [ 3H] thymidine. The mechanism of PGE 2-mediated suppression was studied by adding PGE 2 and T cell growth factors ( TCGF) to TCGF-responsive lymphocytes. PGE 2 reduced the TCGF-mediated blastogenic response in a dose-dependent manner. Furthermore, culture supernatant frpm embryos and endometrium from 14-day pregnant mares inhibited lymphocyte blastogenesis induced by TCGF. These results show that PGE 2 interferes with lymphocyte blastogenesis by TCGF-dependent mechanisms. It is suggested that the PGE 2 present in the uterus of the early pregnant mare may be one of the factors involved in immunosuppression at the time of maternal recognition of pregnancy.

Polysaccharide-Coated Oil Droplets in Oil-in-Water Emulsions as Targetable Carriers for Lipophilic Drugs

Surface of oil droplets in an oil-in-water (o/w) emulsion were coated with naturally occurring polysaccharides (such as mannan, amylopectin, and pullulan) which were, in part, bearing a cholesterol moiety. The mean size of the colloidal droplets was not altered much, even by coating with the polysaccharide derivatives, while the surface charge of the droplet decreased upon coating. Mannan and amylopectin derivative-coated droplets aggregated upon addition of Concanavalin A. These observations suggest that the terminal sugar moiety of the specific polysaccharides on the surface of colloidal droplets can be recognized by lectin. After intravenous injection of the emulsions into guinea pigs, kinetics of the blood clearance and the tissue distribution of the polysaccharide-coated oil droplets, which contain [14C]coenzyme Q,0 as the marker, were investigated. In the initial rapid phase of blood clearance of the radioactivity, the polysaccharide-coated droplets were cleared from the blood stream slower than the uncoated ones. The lung uptake of the mannan derivative-coated droplet emulsion at 30min after intravenous injection was −15 times higher than that of the conventional emulsion without the polysaccharide coat.

Human T-lymphocytes synthesize and secrete a protease resistant proteoglycan in a delayed, serum-dependent response to Concanavalin A

In the presence of radioactive precursors, the addition of Concanavalin A (Con A) to cultures of mononuclear cells (MC) from human blood resulted in marked accumulation in the medium of a protease-resistant proteoglycan (PG) with chondroitin sulfate (CS) chains of an average molecular weight of 28 KDa. This PG was shown to be elaborated primarily by T-cells. The addition of serum was necessary for maximal stimulation with Con A and under these conditions PG accumulation in the medium rose slowly during the first few days of culture and much more rapidly between days 6–8.

Rapid induction of thymosin beta 4 in concanavalin A-stimulated thymocytes by translational control.

The expression of thymosin beta 4, an ubiquitous peptide of high cellular content, was studied in concanavalin A-stimulated rat thymocytes within the first 3 h after activation of the cells. An early 6.3-fold increase of the peptide occurred after 1 h of stimulation amounting to 0.4% of the total cellular protein. This increase coincided with that of thymosin beta 4 biosynthesis measured by [35S]methionine incorporation. The share of thymosin beta 4 synthesis in total protein synthesis 1 h after addition of concanavalin A amounts to 1% but no elevation of the corresponding mRNA was observed. These data suggest that a translational control mechanism is involved in this rapid induction. Consequently, actinomycin D did not inhibit thymosin beta 4 induction in contrast to cycloheximide. The peaks of maximal thymosin beta 4 levels and biosynthesis were followed by rapid decreases of these parameters suggesting a function of thymosin beta 4 in the early phase of T cell activation.

Gametocytogenesis induction by ammonium compounds in cultured Plasmodium falciparum

Asexual parasites of a strain that seldom or never produce gametocytes in in vitro culture began gametocytogenesis after 24 h treatment with RPMI 1640 medium containing concanavalin A (final concentration, 10 μg ml −1) and ammonium carbonate (final concentration, 15 m m ml −1) or ammonium bicarbonate (final concentration, 15 m m ml −1). Gametocytogenesis was consistently observed from the 3rd day after the treatment. Concanavalin A enhanced gametocytogenesis induction by ammonium carbonate or ammonium bicarbonate, although concanavalin A does not itself have gametocytogenesis induction activity. Whereas no gametocytogenesis was observed after addition of concanavalin A and ammonium acetate (final concentration, 5–25 m m ml −1) or ammonium chloride (final concentration, 5–15 m m −1). Addition of ammonium compounds resulted in decrease of parasitemia, regardless of gametocytogenesis.

Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures.

The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined. Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of concanavalin A to the culture medium induced a morphologic alteration of the fibroblastic cells to spherical chondrocytes and increased by 3- to 4-fold incorporation of [35S]sulfate and [3H]glucosamine into large chondroitin sulfate proteoglycan that was characteristically found in cartilage. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, as chemical analyses showed a 4-fold increase in the accumulation of macromolecules containing hexuronic acid in concanavalin A-maintained cultures. Furthermore, the effect of concanavalin A on [35S]sulfate incorporation into proteoglycans was greater than that of various growth factors or hormones. However, concanavalin A had smaller effects on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant glycosaminoglycans. Since other lectins tested, such as wheat germ agglutinin, lentil lectin, and phytohemagglutinin, had little effect on [35S]sulfate incorporation into proteoglycans, the concanavalin A action on chondrocytes seems specific. Although concanavalin A decreased [3H]thymidine incorporation in chondrocytes, the stimulation of proteoglycan synthesis could be observed in chondrocytes exposed to the inhibitor of DNA synthesis, cytosine arabinoside. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.

Concanavalin A affects β-tubulin mRNA expression during neuritic processes of mouse neuroblastoma N18TG2 cells in a different manner from colchicine

Addition of concanavalin A (Con A) to mouse neuroblastoma N18TG2 cells cultured with dibutyryl-cAMP which can stimulate neurite outgrowth, stopped the neuritic processes effectively. The extended neurites showed a gradual retraction for at least 8 hrs after addition of Con A, while addition of colchicine caused rapid retraction of the neurites. Immunocytochemistry showed that the addition of Con A did not disorganize the microtubules but the addition of colchicine did. The increase in β-tubulin mRNA expression which was observed after cell culture and after stimulation by dB-cAMP was suppressed by the addition of Con A. Con A did not affect the β-tubulin mRNA expression when the cells had already been cultured, while colchicine drastically decreased it. Thus, Con A appeared to affect the β-tubulin mRNA expression in a different manner from colchicine, probably through inhibition of cell movement.

Effect of Cysteine Ethylester Hydrochloride (Cystanin®) on Host Defense Mechanisms (V): Potentiation of Nitroblue Tetrazolium Reduction and Chemiluminescence in Macrophages or Leukocytes of Mice or Rats

L-Cysteine ethylester hydrochloride (Cystanin®, ethylcysteine) at doses of 3-30 mg/kg, p.o., potentiated the reduction of nitroblue tetrazolium (NBT) by mouse peritoneal macrophages ex vivo. In in vitro experiments, this drug (30 μM) augmented NBT reduction of mouse peritoneal macrophages induced by opsonized zymosan (OZ). At the same concentration, this drug accelerated the enhancement of the OZ-induced NBT reduction by the addition of concanavalin A, N-formyl-L-methionyl-L-leucyl-L-phenylalanine or phorbol myristate acetate. This enhancing effect of ethylcysteine was completely diminished by the addition of SOD, sodium azide and catalase. In ex vivo experiments, the OZ-induced chemiluminescence of rat peritoneal macrophages and white blood cells was enhanced by the administration of ethylcysteine at doses of 3-10 mg/kg (i.p.) and 3-30 mg/kg (p.o.). In addition, this drug significantly enhanced the lumisphere-induced chemiluminescence of rat peritoneal leukocytes at 30 mg/kg (i.p.), but not the OZ-induced chemiluminescence. In in vitro experiments, this drug (30 μM) did not enhance the OZ-induced chemiluminescence response of rat peritoneal macrophages. These results suggest that ethylcysteine may enhance the intracellular generation of antimicrobial oxidants in macrophages and leukocytes.

Age-associated changes in antibody production of murine spleen cells.

In the present work, we studied age-associated changes in murine immune functions. We estimated total antibodies and autoantibodies to single-stranded DNA (ss-DNA), double-stranded DNA (ds-DNA) histone and collagen in sera and culture supernatants of spleen cells from young and aged BALB/c, C57BL/6 and MRL/MpJ-(+/+) (MRL/n) mice. In MRL/n mice, the IgM class of the total antibody level in serum increased gradually to the maximum at 3 months of age, and then started to decrease. In contrast, the IgG class started to rise with age after the age of 9 months. Serum levels of IgM and IgG autoantibodies to DNA were dominant in MRL/n mice, and the IgG class started to increase in earlier stages of life than in BALB/c and C57BL/6 mice. Anti-DNA autoantibodies were produced in lipopolysaccharide (LPS)-stimulated cultures of spleen cells from BALB/c, C57BL/6 and MRL/n mice. The stimulatory effect of LPS on autoantibody production was significantly reduced by the addition of concanavalin A (Con A) to the LPS-stimulated cultures. The Con A-induced suppressive activity increased with the donor age in MRL/n mouse spleen cells. On the other hand, total antibody production in LPS-stimulated cultures was not affected by the addition of Con A to the cultures. These results may suggest that IgG autoantibody-producing B cells increase with age in MRL/n mouse spleen cells, and that the suppressive activity on autoantibody production is selectively augmented.

Precipitation of Soluble Affinity Complexes by a Second Affinity Interaction: A Model Study

Lactate dehydrogenase was purified by affinity precipitation. The enzyme bound to Blue Dextran (the Cibacron blue residues) and was precipitated by addition of concanavalin A. The lectin functions as a crosslinking agent, building up large flocs of dextran that subsequently precipitate, thus co-precipitating the affinity-bound lactate dehydrogenase.

Characterization of the inducible polyamine transporter in bovine lymphocytes.

The polyamine uptake system in bovine lymphocytes was activated by concanavalin A. The system was common to putrescine, spermidine and spermine. The Kt values for uptake activities of putrescine, spermidine and spermine were 3.7 microM, 0.38 microM and 0.23 microM in that order. The uptake activity was inhibited by carbonyl cyanide m-chlorophenylhydrazone, gramicidin D or valinomycin in the presence of 20 mM K+ suggesting that polyamine uptake depends on the membrane potential. The uptake activity appeared 10 h after addition of concanavalin A, and the maximum was reached at 28 h indicating that induction of the polyamine transporter precedes the initiation of DNA synthesis. Addition of polyamine antimetabolites, such as alpha-difluoromethylornithine and ethylglyoxal bis(guanylhydrazone), to the medium enhanced at least eightfold the induction of the polyamine transporter. The induction was repressed by addition of 50 microM spermidine or spermine, but not putrescine. We propose here that the induction of the membrane-potential-dependent polyamine transporter is regulated by the intracellular level of spermidine and spermine.

Phorbol myristate acetate potentiates superoxide release and membrane depolarization without affecting an increase in cytoplasmic free calcium in human granulocytes stimulated by the chemotactic peptide, lectins and the calcium ionophore

We investigated the inter-relationships of superoxide (O 2 - release, membrane depolarization and an increase in cytoplasmic free Ca 2+, [Ca 2+] i, in human granulocytes stimulated by various agonists. When concanavalin A or the Ca 2+ ionophore ionomycin was used as stimulus, an increase in [Ca 2+] i clearly preceded the onset of membrane depolarization, which was followed by O 2 - release. On the other hand, when N-formylmethionylleucylphenylalanine or wheat-germ agglutin was used as stimulus, no demonstrable lag was seen in any of the responses. O 2 release and membrane depolarization stimulated by all these agonists were markedly potentiated in parallel by pretreatment of cells wiht a low concentration of phorbol myristate acetate (0.25 ng/ml), whereas an increase in [Ca 2+] i was not affected or minimally potentiated. The lag time between addition of the stimulus (concanavalin A or ionomycin) and onset of membrane depolarization or O 2 - release was significantly reduced by pretreatment of cells with phorbol myristate acetate, whereas the lag time between addition of concanavalin A and onset of the increase in [Ca 2+] i was not affected. The dose-response curves for triggering of O 2 - release and membrane depolarization by each of receptor-mediated agonists in phorbol myristate acetate-pretreated or control cells were identical. These findings suggest that; (a) an increase in [Ca 2+] i stimulates membrane depolarization indirectly; (b) a low concentration of phorbol myristate acetate potentiates membrane depolarization and O 2 - release by acting primarily at the post-receptor level, in particular, at the level distal to an increase in [Ca 2+] i, but not by augmenting an increase in [Ca 2+] i; and (c) the system provoking membrane depolarization and the system activating NADPH oxidase share a common pathway, which may be susceptible to a low concentration of phorbol myristate acetate.

Possible participation of calmodulin in stimulation of leucine transport by concanavalin a in human lymphocytes

Stimulation of leucine uptake by addition of concanavalin A, mediated by increase of intracellular free Ca2+ concentration [( Ca2+]), in lymphocytes (Mitsumoto, Y., Sato, K. and Mohri, T. (1988) Biochim. Biophys. Acta 968, 353-358) was abolished by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and chlorpromazine, which inhibited membrane hyperpolarization induced by the mitogen. Quinine (0.5-1 mM) completely inhibited the concanavalin A-induced hyperpolarization and extensively inhibited the induced stimulation of leucine uptake. Based on these results, we suggest that the stimulation of leucine uptake by concanavalin A is largely due to activation of the Ca2+-dependent K+ channel which reinforces negative potential of the plasma membrane and is regulated by calmodulin.

Reversal by Ascorbic Acid of Suppression by Endogenous Histamine of Rat Lymphocyte Blastogenesis

Addition of concanavalin A (Con A) to a culture of spleen cells of ODS-od/od rats, which cannot synthesize ascorbic acid, increased the activity of histidine decarboxylase (HDC) in the culture, leading to accumulation of histamine (Hm) in the medium. Treatment of the culture with cimetidine, a type 2 Hm antagonist, enhanced Con A-dependent lymphocyte blastogenesis even in the absence of any exogenously added Hm. Addition of low doses of histaminase increased Con A-dependent lymphocyte transformation. At higher doses, it abrogated the reaction. At concentrations of more than 10(-10) M, exogenously added Hm suppressed the Con A-dependent uptake of [3H]thymidine by the lymphocytes, but it significantly augmented the response at 10(-14) M. The addition of ascorbic acid (10(-8)-10(-5) M) to the culture suppressed the Con A-mediated HDC induction and inhibited Hm biosynthesis. Concomitantly added ascorbic acid at the concentrations of 10(-8)-10(-4) M increased the uptake of [3H]thymidine dependent on Con A or phytohemagglutinin by the lymphocytes. These results suggest that mitogen-dependent lymphocyte blastogenesis is activated by Hm produced by the spleen cells per se. However, when culture was prolonged, high concentrations of Hm suppressed the reaction. Ascorbic acid enhances mitogen-dependent lymphocyte blastogenesis through inhibition of HDC induction, leading to attenuation of immunosuppressive Hm production by the spleen cells.

Role of ornithine decarboxylase on glycolytic enzyme induction during thymocyte proliferation.

Resting rat thymocytes were stimulated to cell cycle entry and proliferation by the addition of concanavalin A and interleukin 2 to the culture medium. Maximal rats of DNA and RNA synthesis and of glycolysis with a 20-fold increase in glucose uptake were observed 48 h after stimulation. Glycolytic enzyme levels increased 3-8-fold, also reaching their maxima 48 h after mitogenic stimulation. Actinomycin D (1.3-1.5 ng/ml) completely inhibited DNA and RNA synthesis in 24- and 48-h cultured cells but showed no inhibitory effect on glycolytic enzyme induction or on enhanced glycolysis. These results suggest the presence of sufficient mRNAs for the synthesis of glycolytic enzymes even though transcription is abolished by actinomycin D. Ornithine decarboxylase activity increased rapidly up to 41-fold within 6 h after stimulation with concanavalin A and interleukin 2. This rapid increase could not be prevented by actinomycin D; however, ornithine decarboxylase activity of thymocytes was affected after 24- and 48-h culture periods. Inhibition of ornithine decarboxylase by 5 mM difluoromethylornithine completely abolished glycolytic enzyme induction. This inhibitory effect could be reversed by exogeneous putrescine. The results obtained indicate the importance of early ornithine decarboxylase activation and polyamine biosynthesis for the induction of glycolytic enzymes in proliferating thymocytes.

Relationship among activation of the Na +/H + antiporter, ornithine decarboxylase induction, and DNA synthesis

The relationship among activation of the Na +/H + antiporter, ornithine decarboxylase, and DNA synthesis was examined with bovine small lymphocytes stimulated by concanavalin A (Con A). The Na +/H + antiport activity was activated immediately after addition of concanavalin A; the maximum was reached 1 h after Con A addition and the activation continued at least 6 h. With increasing concanavalin A concentrations, the activities of the Na +/H + antiporter, ornithine decarboxylase, and DNA synthesis increased in a parallel manner. In the presence of HCO − 3 in the medium, the internal alkalinization of lymphocytes was not induced by Con A. Ornithine decarboxylase and DNA synthetic activities were not inhibited by 5-( N-ethyl- N-isopropyl) amiloride (EIPA), a specific inhibitor of the Na +/H + antiporter. In contrast, in the absence of HCO − 3 in the medium, the internal pH was alkalinized approximately 0.06 pH units by Con A. EIPA did inhibit the alkalinization of the internal pH or DNA synthesis significantly. Ornithine decarboxylase activity was not inhibited by EIPA. These results indicate that the activation of a Na +/H + antiporter is not a trigger for cell proliferation, but its activation is important probably through the maintenance of the internal pH optimum, especially in HCO − 3-free medium.

A Yolk-granule Component Acts as an Adhesive Material for Dissociated Gastrula Cells of the Newt, Cynops pyrrhogaster: (amphibia/gastrula/cell adhesion/cell migration/yolk-granule component).

Yolk granules were collected from full-grown ovarian oocytes of the newt, Cynops pyrrhogaster, and dissolved in 3% acetic acid or 8 M urea solution. Culture dishes were then coated with either of the yolk-granule solutions. The yolk-coated surfaces acted as adhesive substrata for dissociated gastrula cells, which showed active locomotion when spread on the surfaces. Divalent cation was required for cell adhesion and spreading on the yolk-coated surface. Trypsin and glycosidase digestions of dissociated cells or the yolk-coated surfaces inhibited cell adhesion and spreading. Addition of concanavalin A to the culture medium inhibited cell spreading on the yolk-coated surfaces, while high concentration (50 mM) of the saccharides, D-galactose, D-lactose and D-mannose, had a slightly inhibitory effect on cell spreading. Two yolk components (30-kD and 108-kD proteins) were isolated from yolk granules and applied to the culture dish surfaces. Surfaces coated with the 30-kD protein showed strong adhesiveness for dissociated gastrula cells.

Spontaneous human T-cell cytotoxicity against murine hybridomas expressing the OKT3 monoclonal antibody: Comparison with natural killer cell activity

Human peripheral blood lymphocytes (PBL) exhibited spontaneous cytotoxicity against OKT3 monoclonal antibody (mAb)-expressing murine hybridoma cells (OKT3 hybridomas). In contrast, other murine hybridomas expressing OKT4, OKT8, anti-HLA DR, and anti-HLA A, B, and C mAb were not lysed. PBL showed much lower levels of cytotoxicity (3 folds) against OKT3 hybridomas as compared with NK activity against the K562 targets. Lymph node (LN) cells exhibited the inverse relationship of cytotoxicity levels. The addition of OKT3 mAb to the effector cells totally blocked both the binding and the lysis of OKT3 hybridoma targets, indicating that the CD3 antigen on the effector cells may be involved in recognition of the targets. The addition of concanavalin (Con A) also inhibited the cytotoxicity of OKT3 hybridomas. OKT4 mAb-expressing hybridomas became susceptible to lysis after chemical attachment of OKT3 mAb with CrCl 3. The kinetics of lysis of OKT3 hybridomas resembled that of NK activity. Both cytotoxicities were detectable after 1 to 2 hr and reached plateau levels by 4 to 6 hr. Effector cells responsible for lysis of OKT3 hybridomas expressed T3, T8, and Leu 7 antigens, but lacked T4 and Leu 11b antigens, and were sensitive to the treatment with L-leucine methyl ester. These results indicate that T3 +, T8 +, Leu 7 + and T4 −, and Leu 11 − granular lymphocytes have a spontaneous cytotoxic activity against OKT3 hybridomas which is different from classic NK activity. These findings may provide a method for the assessment of T-cell cytotoxicity mediated presumably by in vivo generated cytotoxic T lymphocytes in blood and the other immune organs.

Comparison of the stimulation of glycosaminoglycan synthesis in cultures of mononuclear cells from blood and of fibroblastic cells

A small molecular weight factor, derived from bovine bone (‘matrigenin’), stimulated glycosaminoglycan and proteoglycan synthesis of cultured human fibroblastic cells but not of mononuclear cells from human blood. However, proteoglycan synthesis and secretion by the mononuclear cells was stimulated by the addition of concanavalin A. The proteoglycan from the concanavalin-A-stimulated mononuclear cells was of smaller molecular weight than the proteoglycan from the ‘matrigenin’-stimulated fibroblastic cells. The major increase in proteoglycan synthesis and secretion occurred much later during the culture period for concanavalin-A-stimulated mononuclear cells than for ‘matrigenin’-stimulated fibroblastic cells.