Activation of lymphocytes by concanavalin A requires calcium ions.

Mouse spleen cells were exposed to a short pulse of the mitogenic lectin concanavalin A (con A). After removal of con A mitogenesis was measured by the incorporation of tritiated thymidine into DNA. It was found: (a) the number of cells responding to con A was proportional to the time of exposure to con A; (b) exposure of cells to con A in the absence of extracellular calcium failed to initiate mitogenesis; (c) for a mitogenic effect an extracellular calcium concentration greater than 10(-5)M was required during the time that the cells were exposed to con A.     

Change in acid phosphatase and N-acetyl-beta-glucosaminidase activity in mouse lymphocytes induced by concanavalin A

The treatment with concanavalin A (5 micrograms/ml) of mouse lymphocytes containing 70-72% of T cells entails an increase in the activity of acid phosphatase and a decrease in the activity of N-acetyl-beta-glucosaminidase. These changes were detectable 15 h after lymphocyte incubation with Con A. After 24 h of incubation acid phosphatase activity rose 2-fold whereas that of N-acetyl-beta-glucosaminidase dropped 45-50%. Possible mechanisms of these changes are discussed.     

Inhibitory effect of active oxygen scavengers on the endocytosis of concanavalin A by lymphocytes

It has been shown that of active oxygen species regulate the activity of the processes of Con A endocytosis in rat thymus cells.     

Early plasma-membrane-potential changes during stimulation of lymphocytes by concanavalin A

1. We have monitored the plasma-membrane potential of lymphocytes by measuring the accumulation of the lipophilic cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). 2. The mitogen concanavalin A causes a decrease in TPMP+ accumulation by pig lymphocytes corresponding to a 3 mV depolarization with 2 1/2 min. Concanavalin A does not alter 86Rb+ uptake in the first 30 min. 3. In contrast concanavalin A increased TPMP+ accumulation and the rate of Rb+ uptake in mouse thymocytes. This is consistent with a previous proposal that the mitogen induces a hyperpolarization of mouse thymocytes as a result of stimulation of a Ca2+-dependent K+ channel. 4. Studies with the calcium ionophore A23187 and quinine (an inhibitor of the Ca2+-dependent K+ channel) suggest that the channel is partially closed in mouse resting thymocytes but is almost fully active in pig resting cells. Thus concanavalin A hyperpolarizes mouse thymocytes by activating the Ca2+-dependent K+ channel but cannot do so in pig lymphocytes because the channel is already maximally activated. 5. The 3mV depolarization of pig cells cannot be explained by a decrease in electrogenic K+ permeability.     

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.     

Stimulation of fatty acid synthesis in vitro by gonadotrophin-induced testicular ribonucleic acid

RNA from testes of hypophysectomized rats treated with follicle-stimulating hormone and luteinizing hormone markedly stimulates in vitro the incorporation of acetate and malonate (as CoA derivatives) into polyunsaturated fatty acids. The system in vitro contains the components necessary for both protein and fatty acid synthesis. That the RNA is a hormone-induced messenger type that causes enzyme synthesis that then causes fatty acid synthesis is supported by the following observations: (1) the stimulation of RNA synthesis by follicle-stimulating hormone and luteinizing hormone is decreased by injection of the animals with actinomycin D; (2) puromycin in the system in vitro decreases the synthesis of polyunsaturated fatty acids; (3) the activity of the RNA preparation is destroyed by digestion with ribonuclease; in fact, the digest is inhibitory, which is a characteristic of messenger-RNA-mediated protein synthesis; (4) protein that might be denatured enzyme is virtually absent from the effective RNA preparations.     

The effect of essential fatty acid deficiency upon fatty acid uptake by the brain.

Young adult rats, either control or essential fatty acid deficient, were administered either [3-H] oleic acid or [3-H] arachidonic acid by stomach tube. In addition, a group of control rats was given [3-H] palmitic acid. The rats were killed at various times therafter, and the radioactivity of the lipids of brain and plasma was examined. In confirmation of previous work, the blood lipid label was found to rise rapidly and then fall, wheras the activity of brain lipids increased slowly and did not show a decline through the 24-h period studied. Analysis of the brain uptake data according to first-order kinetics confirmed the impressions gained from visual inspection of the data. The initial rate of uptake of arachidonic acid was about 4.5 times that of oleic acid in control animals and in deficient animals. Essential fatty acid deficiency, however, did not induce an altered rate of uptake for either oleic acid or arachidonic acid. The rate of uptake of palmitic acid by control rats was not significantly different from that of oleic acid. Even though the initial rates of incorporation of oleic and arachidonic acids were not changed during essential fatty acid deficiency, the final levels of radioactivity obtained in brain lipids were higher in deficient rats with both fatty acids. The plateau value obtained with oleic acid was 1.5 times higher in deficient animals, while the plateau value for arachidonic acid was 1.7 times higher. An experiment in which deficient animals were allowed access to a control diet for 12 or 24 h prior to the labeling experiment suggested that the higher levels of radioactivity found in brain lipids of deficient animals was not due to an isotope dilution effect. Such animals still displayed the labeling pattern of deficient animals with arachidonic acid, while the results with oleic acid varied somewhat. Our results suggest that essential fatty acid deficiency does not alter the ability of the brain to take up the fatty acids studied. However, the fatty acids, especially arachidonic, are retained in the brain to a greater extent in the deficient animals.     

Stimulation of fatty acid release in glioblastoma cells by caffeine

Caffeine, a well-known behavior stimulant, was found to activate the fatty acid release mechanism in glioblastoma cells. The enhancement of the liberation of fatty acids from esterified complex lipids by caffeine was time dependent. Cell density had a strong influence on the responsiveness to caffeine. The mobilization of unsaturated fatty acids were preferentially stimulated by caffeine. Therefore, caffeine may alter membrane structure and enhance eicosanoid biosynthesis.     

Medium-chain fatty acid synthesis by goat mammary-gland fatty acid synthetase. The effect of limited proteolysis.

Fatty acid synthetase from goat mammary gland was subjected to limited proteolysis by trypsin and elastase. Both proteolytic enzymes selectively cleaved the chain-terminating thioester hydrolase component from the enzyme complex, leaving all other partial activities intact in the core peptides. Trypsin, but not elastase, caused extensive degradation of the released thioester hydrolase. The released thioester hydrolase could be purified to homogeneity by gel filtration. The molecular weight was estimated as 29 000 and the enzyme showed only significant hydrolytic activity toward long-chain acyl-CoA esters. The core peptides retained the ability to synthesize medium-chain acyl-CoA esters in the presence of 2,6-di-O-methyl-alpha-cyclodextrin. The results conclusively show that the terminating thioester hydrolase of goat mammary-gland fatty acid synthetase is not involved in termination of medium-chain-length fatty acid synthesis by this enzyme.     

Enigmatic effect of cellular ATP on fatty acid biosynthesis. Stimulation by moderate decrease and inhibition by increase of cellular ATP

The cellular ATP concentration was tested for its effect on fatty acid biosynthesis from glucose in hepatocytes. ATP was manipulated by adding increasing concentrations of cycloheximide, amytal, atractyloside, 2,4-dinitrophenol or adenosine. A slight decrease in cellular ATP coincided with a stimulation of fatty acid biosynthesis whereas a further lowering of cellular ATP resulted in a gradual inhibition. Increasing the cellular ATP level by titration with adenosine had the opposite effect. These results are in line with the suggestion that fatty acid biosynthesis from glucose is an energy-yielding process which is stimulated by a moderate drop in cellular ATP.     

Stimulation of prostaglandin E production by concanavalin A in isolated Graafian follicles

Addition of Concanavalin A (Con A) to isolated rat Graafian follicles induced prostaglandin E (PGE) production after 2 h of incubation. PGE synthesis continued throughout 24 h culture period. Cyclic AMP accumulation was noted after 6 h of incubation with Con A. .Aspirin, indomethacin and flufenamate prevented both the stimulation of PGE production and of cyclic AMP accumulation by Con A; antibodies to PGE prevented the cyclic AMP production. These studies indicate that the interaction of Con A with the follicle results in PGE production. It seems that besides the known pathway for the induction of PGE synthesis in the ovarian follicle, via elevation of intracellular cyclic AMP, an additional pathway, via an external signal which is independent of cyclic AMP exists.