Ectogalactosyltransferase studies in fibroblasts and concanavalin A- stimulated lymphocytes

In this communication, we have demonstrated that hydrolysis of the nucleotide sugar can cause errors in the detection of an ectoglycosyltransferase. Spleen cell suspensions can incorporate radioactivity when incubated with labeled UDP-galactose, but all the activity is due to decomposition of the nucleotide sugar and uptake of the free sugar. The fibroblast cell lines can incroporate carbohydrate directly from UDP-galactose. Several criteria are presented with can be used to demonstrate that a nucleotide sugar is the direct carbohydrate donor.     

Human monocyte activation by supernatants from concanavalin A (con A) stimulated lymphocytes

Human peripheral lymphocytes were stimulated with Concanavalin A (Con A) in the absence of serum. Supernatants were collected from control and mitogen stimulated lymphocyte cultures and fractions pooled according to the elution before, together with or after human serum albumin which was added as a marker. Only one fraction derived from Con A stimulated lymphocyte culture Supernatants which eluted immediately after human serum albumin had a significant effect on the metabolism and structure of human monocytes in vitro. Monocytes separated by human serum albumin and incubated with this fraction for 20 hr had an increase in nuclear RNA synthesis. Monocytes attached to cover slips in Leighton tubes showed an increase in the percentage of phagocytizing cells and phagocytic activity. Electron microscopy demonstrated highly phagocytic cells containing numerous Golgi associated granules and strands of nondilated rough surfaced endoplasmic reticulum in presence of the active fraction.     

DNA-dependent ATPases in Bacillus subtilis mutants and in competent cells

Three DNA-dependent ATPases (gamma phosphohydrolases) can be isolated from Bacillus subtilis cells. We studied these enzymes in a number of mutants deficient in recombination or repair functions (rec, uvr) and in competent cells. The recA mutant studied had lower ATPase II activity, while competent cells had higher ATPase I activity, in comparison with the parental strain not brought to competence.     

Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A.

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38 degrees C, or the cellular uptake of isotope during a similar incubation was measured. The influx rate of untreated thymocytes was: 2.3-10(-12) moles cm-2-s-1 and efflux rate: 1.8-10(-12) moles cm-2-s-1. When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 mug/ml, but concentrations as low as 0.75 mug/ml were effective. Hydrocortisone resistant thymocytes responded at least was well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times. Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl-:43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present. Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A. The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, alpha-methyl-D-mannoside.     

Proliferation of human peripheral lymphocytes : Characteristics of cells once stimulated or re-stimulated by concanavalin A

About 20% of human blood lymphocytes will subsequently synthesize DNA after culturing with concanavalin A (conA) for 24 h. The stimulated cells go through one but only one round of DNA synthesis unless restimulated. We used the techniques of velocity sedimentation and auto-radiographic grain counts to demonstrate that stimulated cells do divide and return to a mitogen dependent, arrested G 1 state. Once-stimulated lymphocyte populations show the same time course for restimulation as do cultures which are being stimulated for the first time, but require at most one-fifth as much conA added to the medium in order to be maximally stimulated. We have shown by density transfer experiments that all of the progeny of cells which require up to 10 μg/ml of conA for the first DNA replication respond to 2 μg/ml with a second round of replication.     

Visualization of protein kinases in lymphocytes stimulated to proliferate with concanavalin A or inhibited with a phorbol ester

Stimulation of lymphocytes with a mitogenic lectin such as concanavalin A (ConA) results in differentiation and cell division. Among the changes which occur after stimulation are increases in phosphorylation of proteins and in protein kinase activity. We used a high-resolution, nondenaturing gel system to separate and visualize protein kinases in situ. We have clearly identified both autophosphorylating and substrate-dependent kinases. One band of cyclic AMP-dependent kinase activity was significantly enhanced in lectin-stimulated cells. In contrast, treatment of the cells with phorbol ester under conditions which depress stimulation caused a decrease in the activity of one kinase.     

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.     

Effects of hexachlorocyclohexane isomers on concanavalin A 'capping' in bovine lymphocytes.

The effects of alpha, beta, gamma, and delta isomers of hexachlorocyclohexane on concanavalin A 'capping' in bovine lymphocytes were evaluated, gamma and delta hexochlorocyclohexane inhibited 'capping' whereas the alpha and beta isomers were without effect. In addition, gamma-hexachlorocyclohexane has been shown to antagonize the maintenance of preformed 'caps' and cause the rapid dispersal of the concanavalin A-receptor complexes over the surface of cells by a temperature-dependent mechanism. The possible role of a gamma-hexachlorocyclohexane-sensitive process in the organization of microflow patterns in the lectin-activated lymphocyte membrane is discussed.     

Effect of concanavalin A on membrane-bound enzymes from mouse lymphocytes.

The ionic influence and ouabain sensitivity of lymphocyte mg-2+-atpase and Mg-2+-(Na+ +K+)-activated ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5'-nucleotidase and Mg2+-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of (Na+ +K+)-ATPase was located inside the membrane. Concanavalin A induced an early stimulation of Mg2+-APTase and (Na+ +K+)-ATPase both on intact cells and purified plasma membranes. In contrast, 5'-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg2+-ATPase activity was 3-5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20%). (Na+ +K+)-ATPase activity was undectectable in thymocytes. However, in spleen lymphocytes (Na+ +K+)-ATPase activity can be detected and was 30% increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg2+-ATPase, in lymphocyte stimulation.     

Phospholipid metabolism of stimulated lymphocytes. Comparison of the activation of acyl-CoA:lysolecithin acyltransferase with the binding of concanavalin A to thymocytes.

Calf thymocytes were isolated and incubated with concanavalin A. The effect of the mitogen on the enzyme activity of membrane-bound lysolecithin acyltransferase (acyl-CoA:1-acylglycero-3-phosphorylcholine-O-acyltransferase, EC 2.3.1.23) was determined as also the binding of 125I-labelled concanavalin A to intact cells and isolated membranes. The lysolecithin acyltransferase was found to be activated three times in microsomal membranes. The activation occurred directly after binding of concanavalin A and was temperature independent, since similar activities were found in cells treated with concanavalin A at 0 and 37 degrees C. The acyltransferase activation using increasing concentrations of concanavalin A revealed a different behaviour, as compared to the binding of concanavalin A. While the binding of concanavalin A to intact cells expressed a normal hyperbolic saturation function the activation process of the acyltransferase described a sigmoidal relationship. Correspondingly, the interaction coefficients for both functions were different (Sips coefficient for binding = 1.0 and Hill coefficient of the enzyme activation = 1.8). These results indicate that the acyltransferase activation is due to a cooperative interaction between the ligand-receptor complex and the enzyme.     

Surface and intracellular localization of concanavalin A in human lymphocytes

Normal human lymphocytes were treated at +4 °C with concanavalin A and subsequently washed. The cells were then incubated for various periods at +37 °C in a culture medium and the fate of the agglutinin was followed by an electron microscopic stain specific for concanavalin A. At zero incubation time, a positive reaction was noted on the entire cell membrane and on numerous pinocytic vesicles; 80% of the lymphocytes were stained. Within 15 min of incubation, the cell surface appeared discontinuously labelled while all the intracytoplasmic vesicles were strongly positive. After 2 h of incubation, the cell surface was almost free of label and positive vesicles were found to be concentrated at one pole of the cell. After 3 h of incubation, no label was found on the cell membrane; instead, large vesicles communicating with the cell membrane contained labelled material.     

Serum factors affecting the incorporation of [3H]uridine by lymphocytes stimulated by concanavalin A. Studies of the role of complement

1. Rat lymph-node cells cultured in serum and medium 199 were activated to transform and proliferate by concanavalin A. Initial cell activation was assessed by measuring the enhanced radioactive labelling of cells with [(3)H]uridine produced by concanavalin A during the first 6h of culture. 2. In medium containing serum the degree of activation was dependent on the ratio of concanavalin A to non-diffusible serum macromolecules; however, cells could be activated to a normal extent in a medium containing only diffusible molecules. This indicates that certain serum macromolecules buffer cell receptor sites against reaction with concanavalin A. 3. At high concanavalin A concentrations labelling was depressed below that of control cultures without concanavalin A. This inhibition of labelling (i) occurred in calf serum, but not in homologous serum, (ii) was removed by pretreatment of serum with various complement inhibitors, and (iii) first appeared after 1h of culture following an initial phase of cell activation by concanavalin A. Cells pre-labelled with [(3)H]uridine slowly released the label into the culture medium; this rate of release was suddenly accelerated after 1h of culture with concanavalin A if complement was present. The results suggest that inhibition of labelling requires the sequential binding of concanavalin A and then complement to the cell surface. 4. Results from experiments in which calf serum was mixed in various proportions with calf serum which had been preheated to inactivate complement, suggest (i) a requirement for complement in stoicheiometric quantities dependent on the number of cells being inhibited, and (ii) that preheated serum can inactivate complement in unheated serum. 5. The proliferative response over 3 days of culture was assessed by measuring the enhanced labelling of cells with [(3)H]thymidine produced by concanavalin A. In preheated calf serum two types of inhibition were noted. (i) A progressive inhibition at high concanavalin A concentrations so that the optimum response was shifted to lower concanavalin A concentrations as the duration of culture was extended; it is suggested that this reflects the secretion of complement by cultured cells. (ii) An inhibition of the optimum response appearing late in the culture period at high cell concentrations; it is suggested that this is due to the exhaustion of medium nutrients in most actively growing cultures.     

Stimulation of lymphocytes by concanavalin A. Temperature-dependent effect of fatty acid replacements

The membrane fatty acyl composition of lymphocytes was altered by growth in lipid-depleted serum containing fatty acid supplements, as well as avidin to block endogenous synthesis of fatty acids. Under these growth conditions over 50% of the total fatty acid in membrane phospholipid were derived from the added fatty acid. Enrichment of lymphocyte membranes with oleate (cis C18:1) or elaidate (trans C18:1) shifted the optimum temperature for mitogenic stimulation by concanavalin A as measured by [3H]thymidine incorporation. These results suggest that the fluidity of the membrane lipid phase plays a role in the process of lymphocyte stimulation by lectins.     

Protein phosphorylation in intact lymphocytes stimulated by Concanavalin A

The phosphorylation of proteins in intact mouse spleen lymphocytes was monitored following mitogenic activation. Little change in the autoradiographic patterns of phosphorylated protein fractionated by polyacrylamide gel electrophoresis occurred during the first 8 h after Concanavalin A (conA) treatment. The intensity of ³²P incorporation into two proteins of 135 000 and 150 000 mol. wt began to increase, relative to control cells, 10 h after conA treatment and was maximal at 50 h. This increased phosphorylation followed the rise in RNA synthesis but preceded the onset of DNA synthesis. In addition to this temporal link between enhanced phosphorylation of these proteins and the initiation of DNA synthesis, various agents which inhibited the onset of S phase also blocked the phosphorylation of both proteins. Such treatments included the displacement of conA from its surface receptors by α-methyl-mannoside (αMM), the omission of serum from the culture medium, and the presence of indomethacin. The similar time courses of phosphorylation and responses to various proliferation inhibitors supports the idea that the 135 000 and 150 000 mol. wt proteins have a common physiological function. These proteins may be involved in the progression of stimulated lymphocytes toward S phase, and their phosphorylation may be an important regulatory event in this sequence.