Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63 Ag8 cells

Summary Using the suspension cell line P3X63 Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (M_r 40 kD) and Semliki Forest virus core protein (M_r 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0°C. Subsequent resealing of the pores was completed after a 5-min incubation at 37°C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37°C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K⁺, Mg²⁺, and Ca²⁺) and resealing (in the presence of K⁺, Mg²⁺, and Ca²⁺), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure.     

Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells

P2X4 and P2X7 receptors are ATP-gated ion channels that are co-expressed in alveolar epithelial type I cells. Both receptors are localized to the plasma membrane and partly associated with lipid rafts. Here we report on our study in an alveolar epithelial cell line of the molecular organization of P2X7R and P2X4R receptors and the effect of their knockdown. Native gel electrophoresis reveals three P2X7R complexes of ~430, ~580 and ~760 kDa. The latter two correspond exactly in size to signals of Cav-1, the structural protein of caveolae. Interestingly knockdown of P2rx7 affects protein levels, the intracellular distribution and the supramolecular organization of Cav-1 as well as of P2X4R, which is mainly detected in a complex of ~430 kDa. Our data suggest upregulation of P2X4R as a compensatory mechanism of P2X7R depletion.     

Glucose-evoked recovery of hepatic thyroxine 5'-deiodinase independent of de novo protein synthesis in fasted rat

The glucose-evoked recovery of Type I thyroxine 5'-deiodinase activity in the hepatic microsomes of fasted rat was not inhibited by either cycloheximide, puromycin or actinomycin D during 3 h after glucose feeding; however, [3H]-leucine uptake by the liver or the hepatic microsomal fraction was significantly inhibited by cycloheximide and puromycin but not by actinomycin D. These results indicate that the glucose-evoked recovery of deiodinase activity may be independent of de novo protein synthesis.     

Effect of colicin E3 on leukemia cells P 388 in vitro

Summary Proliferation of murine leukemia cells P388 is stimulated by less than 1.0 mg/ml colicin E3, while being inhibited by higher concentrations. By 1.6 mg/ml colicin E3, uptake of thymidine into cold TCA-precipitable fraction is decreased by 59% during 24 h, uptake of uridine by 29%.     

Influence of progesterone on protein and RNA synthesis in cultured chick embryo liver cells

Summary Chick embryo liver primary cultures, when added with progesterone, exhibit, in comparison with the controls, a normal growth, a decline of both³H-uridine uptake and incorporation into total RNA, a decline of³H-leucine and¹⁴C serine incorporation into the proteins. Progesterone is not able to stimulate phosvitin synthesis induction.These studies were supported in part by the Italian CNR grants.     

Effect of colicin E3 on leukemia cells P388 in vitro.

Proliferation of murine leukemia cells P388 is stimulated by less than 1.0 mg/ml colicin E3, while being inhibited by higher concentrations. By 1.6 mg/ml colicin E3, uptake of thymidine into cold TCA-precipitable fraction is decreased by 59% during 24 h, uptake of uridine by 29%.     

Influence of progesterone on protein and RNA synthesis in cultured chick embryo liver cells.

Chick embryo liver primary cultures, when added with progesterone, exhibit, in comparison with the controls, a normal growth, a decline of both 3H-uridine uptake and incorporation into total RNA, a decline of 3H-leucine and 14C serine incorporation into the proteins. Progesterone is not able to stimulate phosvitin synthesis induction.     

Inhibition by carbaryl of DNA,RNA and protein synthesis in cultured rat lung cells

Summary The carbamate pesticide carbaryl rapidly inhibited DNA, RNA and protein synthesis in L-2 cells from rat lung. the inhibiton was partly reversible and was not accompanied by inhibiton of transport of tritiated precursors into intracellular pools or destruction of the integrity of the cell membrane.We thank W. H. J. Douglas for his gift of L-2 cells.This work was supported by contract 22140 of the Kentucky Tobacco Research Board.     

Dose-effects of 8-methoxypsoralen and long wave UV-light in 3T3 cells: evaluation of a phototoxic index

Summary 3T3 cells were cultured until confluency and treated with various doses of 8-methoxypsoralen followed by long wave UV light irradiation. The inhibition of³H-thymidine incorporation was dose-dependent for both, psoralen and light. A phototoxic index (PTI) was established demonstrating that a constant correlation between psoralen and UVA light exists for the photoinactivation in living cells.Supported by Deutsche Forschungsgemeinschaft.We thank Miss Anne Schröder for her skillful assistance.     

Dose-effects of 8-methoxypsoralen and long wave UV-light in 3T3 cells: evaluation of phototoxic index.

3T3 cells were cultured until confluency and treated with various doses of 8-methoxypsoralen followed by long wave UV light irradiation. The inhibition of 3H-thymidine incorporation was dose-dependent for both, psoralen and light. A phototoxic index (PTI) was established demonstrating that a constant correlation between psoralen and UVA light exists for the photoinactivation in living cells.     

Effect of protein kinase C activation and depletion on insulin stimulation of glycogen synthesis in cultured hepatoma cells

Summary Insulin stimulation of glycogen synthesis was nearly abolished in hepatoma cells shortly treated with 4 ß-phorbol 12 \-myristate, 13 -acetate (protein kinase C activation) but remained unmodified in cells chronically treated with the phorbol ester (protein kinase C depletion). Thus, although exogenous activation of protein kinase C results in an inhibition of insulin action, protein kinase C depletion has no influence on this process. The results suggest that, in hepatoma cells, no endogenous activation of protein kinase C may occur in response to the signal triggered by insulin.     

Effect of protein kinase C activation and depletion on insulin stimulation of glycogen synthesis in cultured hepatoma cells

Insulin stimulation of glycogen synthesis was nearly abolished in hepatoma cells shortly treated with 4 beta-phorbol 12 beta-myristate, 13 alpha-acetate (protein kinase C activation) but remained unmodified in cells chronically treated with the phorbol ester (protein kinase C depletion). Thus, although exogenous activation of protein kinase C results in an inhibition of insulin action, protein kinase C depletion has no influence on this process. The results suggest that, in hepatoma cells, no endogenous activation of protein kinase C may occur in response to the signal triggered by insulin.     

Stimulation by ACTH of protein synthesis in cultured adrenal cells. Absence of an effect by cyclin AMP]

ACTH stimulates the incorporation of 14C-leucine into proteins of adrenal tumor cells in culture. This stimulation though about 20% over controls is statistically significant. Cyclic AMP did not reproduce the stimulation observed with ACTH.     

Inhibition of protein synthesis in ehrlich ascites tumor cells by irradiation (365 nm) in the presence of skin-photosensitizing furocoumarins

Riassunlo La sintesi proteica nelle cellule del tumore ascitico di Ehrlich viene inhibita dall'irradiazione a 365 nm in presenza di furocumarine fotosensibilizzatrici cutanee (psoralene, xantotossina e 8-metilpsoralene).