Rapid qualitative changes in mRNA populations in cultured human lymphocytes: comparison of the effects of cycloheximide and concanavalin A

1984 ◽  
Vol 62 (9) ◽  
pp. 859-864 ◽  
Author(s):  
Donald R. Forsdyke
Keyword(s):  
Concanavalin A ◽  
Human Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Lymphocyte Activation ◽  
Relative Mass ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Mrna Species ◽  
Reticulocyte Lysate ◽  
Qualitative Changes

To examine the hypothesis that the stimulation of cultured lymphocytes by lectins involves the inactivation of a protein repressor of putative "activation genes," the effects of a protein synthesis inhibitor (cycloheximide) and a lectin (concanavalin A) were compared. Qualitative changes in mRNA populations were assessed by translating RNA prepared from cycloheximide- or lectin-treated cultures in a rabbit reticulocyte lysate. [35S]Methionine-labelled translation products were analysed by two-dimensional polyacrylamide gel electrophoresis. Cycloheximide increased the radioactive labelling of cultured lymphocytes with the RNA precursor [3H]uridine, as previously reported. This was observed during the first 3 h of culture; thereafter, cycloheximide was inhibitory. The period of increased labelling with [3H]uridine coincided with a period of great increase in mRNA corresponding to an acidic protein of a relative mass of approximately 55 000. This mRNA was not detected in RNA prepared from control cultures, but was one of the most abundant mRNA species detected in RNA prepared from cycloheximide-treated cultures. Increases in certain less abundant mRNA species were also noted. However, the mRNAs were not observed in RNA prepared from lectin-treated cultures. If an increase in these mRNAs is important for lymphocyte activation, then the increase must be to an extent not detected by our current methods.

Modulation of Arachidonic Acid Metabolism in Human Endothelial Cells by Glucocorticoids

1986 ◽  
Vol 55 (03) ◽  
pp. 369-374 ◽  
Author(s):  
Raffaele De Caterina ◽  
Babette B Weksler
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Arachidonic Acid Metabolism ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Prolonged Incubation ◽  
Dependent Inhibition ◽  
Human Thrombin ◽  
Umbilical Vein Endothelial Cells

SummaryTo learn whether glucocorticoids inhibit prostaglandin (PG) production in vascular endothelial cells, we investigated the effects of glucocorticoids on PG synthesis by cultured human umbilical vein endothelial cells (EC). Pretreatment of EC with dexamethasone (DX, 10-9 to 5 x 10-5 M) caused a dose-dependent inhibition of PGI2 production when PG synthesis from endogenous arachidonate was stimulated by human thrombin (0.25-2 U/ml) or ionophore A 23187 (1-5 μM). The inhibition was detectable at 10-7 M DX and maximal at 10-5 M (4.0 ± 0.7 vs. control: 7.7 ± 1.9 ng/ml, mean ± S.D., P <0.01). The production of PGE2 and the release of radiolabelled arachidonate (AA) from prelabelled cells were similarly inhibited. Prolonged incubation of EC with glucocorticoids was required to inhibit PG production or arachidonate release: ranging from 8% inhibition at 5 h to 44% at 38 h. In contrast, prostaglandin formation from exogenous AA was not altered by DX treatment. When thrombin or ionophore-stimulated EC were restimulated with exogenous AA (25 μM), DX-treated cells released more PGI2 than control cells (5.7 ± 0.5 vs. 4.1 ± 0.6 ng/ml, P <0.01). Both the decrease in PGI2 production after thrombin/ionophore and the increase after re-stimulation with AA were blunted in the presence of the protein synthesis inhibitor cycloheximide (0.1-0.2 μg/ml). Thus, incubation of EC with glucocorticoids inhibits PG production at the step of phospholipase activation. The time requirement for these steroid effects and their blunting by cycloheximide are consistent with the induction of regulatory proteins, possibly lipocortins, in endothelial cells.

scholarly journals TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) Is Upregulated in Lymphocytes Stimulated with Concanavalin A

2021 ◽  
Vol 22 (14) ◽  
pp. 7436
Author(s):  
Helga Simon-Molas ◽  
Xavier Vallvé-Martínez ◽  
Irene Caldera-Quevedo ◽  
Pere Fontova ◽  
Claudia Arnedo-Pac ◽  
...  
Keyword(s):  
Concanavalin A ◽  
Carbon Flux ◽  
Human Lymphocytes ◽  
Tumor Development ◽  
Pentose Phosphate ◽  
Akt Signaling Pathway ◽  
G6pdh Activity ◽  
Physiological Conditions ◽  
Glucose 6 Phosphate Dehydrogenase

The glycolytic modulator TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) is overexpressed in several types of cancer and has a role in metabolic rewiring during tumor development. However, little is known about the role of this enzyme in proliferative tissues under physiological conditions. In the current work, we analysed the role of TIGAR in primary human lymphocytes stimulated with the mitotic agent Concanavalin A (ConA). We found that TIGAR expression was induced in stimulated lymphocytes through the PI3K/AKT pathway, since Akti-1/2 and LY294002 inhibitors prevented the upregulation of TIGAR in response to ConA. In addition, suppression of TIGAR expression by siRNA decreased the levels of the proliferative marker PCNA and increased cellular ROS levels. In this model, TIGAR was found to support the activity of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentose phosphate pathway (PPP), since the inhibition of TIGAR reduced G6PDH activity and increased autophagy. In conclusion, we demonstrate here that TIGAR is upregulated in stimulated human lymphocytes through the PI3K/AKT signaling pathway, which contributes to the redirection of the carbon flux to the PPP.

scholarly journals Effect of emetine, a protein synthesis inhibitor, on larval tail resorption in the ascidian Halocynthia roretzi

2006 ◽  
Vol 23 (2) ◽  
pp. 43-46
Author(s):  
Kiyotaka Matsumura ◽  
Manami Nagano ◽  
Sachiko Tsukamoto ◽  
Haruko Kato ◽  
Nobuhiro Fusetani
Keyword(s):  
Protein Synthesis ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Halocynthia Roretzi

scholarly journals Specific glycoprotein changes during development of the chick neural retina.

1978 ◽  
Vol 79 (1) ◽  
pp. 132-137 ◽  
Author(s):  
G Mintz ◽  
L Glaser
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Neural Retina ◽  
Cell Type ◽  
Retinal Antigens ◽  
Qualitative Changes

After separation of whole proteins of chick neural retina by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), a number of glycoproteins can be detected by staining the gels with 125I-labeled wheat germ agglutinin (WGA) and other lectins. The glycoprotein patterns show both quantitative and qualitative changes between days 7 and 13 of development. Some of these glycoproteins can be separated by chromatography on columns of insolubilized lectins. These observations suggest that purification of some of these glycoproteins identified by staining with radioactive lectins would yield retinal antigens which may be specific for developmental stage and cell type.

Affinity chromatography purification and partial characterization of phytohemagglutinin-receptor glycoproteins from porcine splenic lymphocytes

1985 ◽  
Vol 63 (9) ◽  
pp. 932-940 ◽  
Author(s):  
Gilles Dupuis ◽  
Jean-Pierre Doucet ◽  
Bânû Bastin ◽  
Jeannine Cardin
Keyword(s):  
Affinity Chromatography ◽  
Competitive Inhibition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Interleukin 2 ◽  
Lymphocyte Activation ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Murine Cell Line ◽  
Lymphocyte Cultures

We describe the isolation of pig spleen lymphocyte glycoproteins that interact with phytohemagglutinin (PHA), the lectin from Phaseolus vulgaris. Purification was achieved by affinity chromatography of a Nonidet P-40 extract of the cells on a PHA – Affi-Gel 10 column. The retained glycoproteins were eluted with an acidic (pH 3.0) glycine buffer and represented 1.9–2.4% of the amount of protein applied to the column. They contained 20 ± 1.3% hexose and 1.7 ± 0.7% fatty acids, on a weight basis. Electrophoretic analyses (sodium dodecyl sulfate – polyacrylamide gel electrophoresis) showed the presence of major Coomassie blue positive bands with apparent molecular masses of 50–55, 75, 95, 130, and 155 kdaltons along with minor bands of 20–40, 42, 45, 60–65, 175, and 200–250 kdaltons. The purified PHA-receptor glycoproteins inhibited, in a dose-dependent manner, the incorporation of [3H]thymidine in pig lymphocytes cultured at a concentration of 106 cells/mL in the presence of PHA. A 50% inhibition was observed when 20 μg/mL of the glycoproteins was added to the lymphocyte cultures containing 0.5 μg/mL of PHA. Scatchard analysis of the binding of 125I-labelled PHA, in the presence of increasing amounts of the purified glycoproteins, showed a suppression of the binding of the lectin to high affinity sites of the cells, as evidenced by a change from biphasic to a linear profile. Results of binding suggested a competitive inhibition by a population of purified glycoproteins with a similar affinity for the lectin. The purified glycoproteins decreased PHA-dependent interleukin 2 (IL-2) production by pig lymphocytes as assayed with a IL-2 dependent murine cell line. It is suggested that the affinity-purified PHA-reactive glycoproteins are inhibitors of PHA-dependent cellular responses because they compete with PHA-receptor sites on the lymphocyte plasma membrane. A mouse antiserum raised against the purified glycoproteins inhibited PHA-induced lymphocyte activation, but did not stimulate lymphocytes when added alone to lymphocyte cultures or in combination with a antimouse antiserum.

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