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As was demonstrated elsewhere (L. V. Teslenko et al., 1987), the epidermal growth factor (EGF) can recycle after internalization by A431 cells in membrane-bound state. In the present study, direct evidence on recycling of EGF-receptor complexes is presented using a covalently crosslinking reagent. The recycling was shown to occur via peripheral endosomes as well as through para-Golgi endosomes. It was found that among EGF degradation inhibitors tested only primaquine (300 microM) was able to decrease significantly the rate of recycling. The lowering of the temperature to 17 degrees C led to blocking the EGF degradation as well as to inhibiting the recycling. The data obtained suggested that the recycling of EGF-receptor complexes is relatively independent of their degradation.

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The role of intracellular processing of epidermal growth factor (EGF) in the induction of proliferation of quiescent Swiss 3T3 cells was studied using various inhibitors. The number of amines (dansylcadaverine, chloroquine, cystamine, 5-methoxytryptamine) dimethylurea and monensin were shown to block the mitogenic effect of EGF. The majority of these substances while used in concentrations sufficient to inhibit the proliferation do not significantly influence 125I-EGF binding and internalization. The level of EGF degradation was reduced only by chloroquine. The inhibitory effect of amines and monensin on the generation of proliferative signal was supposed to take place at the stages of EGF processing in "specialized" endosomes and in Golgi apparatus.

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The influence of epidermal growth factor (EGF) and insulin on uridine phosphorilation was investigated in cell cultures Swiss 3T3 and 3T6, arrested in the medium with serum content of 0.5%. It is shown that following 5-10 minutes the addition of EGF into the culture medium in concentrations from 0.15 to 51 nM results in the increase in the rate of uridine phosphorilation which reaches the maximum value, similar to that of the stimulating effect of 10% serum. Insulin in the 4-85 nM concentrations also enhanced the rate of uridine phosphorilation and exerted a potential influence on EGF effect only in high concentrations. The investigation of the dynamics of binding and internalization of 125I-EGF showed that the number of EGF molecules on membrane increase during 10 minutes after addition of EGF into the medium and then begin to decrease. The binding of EGF with not more than 2% of the total number of cell receptors was shown by approximal estimation to be enough for stimulation of uridine phosphorilation. A conclusion is drawn that the presense of single growth factor EGF in enough for maximum stimulation of early reaction of cells on the proliferation stimulus realized at the posttranscriptional level, while both the additional factors and higher concentrations of EGF are necessary for the maximum induction of DNA synthesis.

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Several amines were shown to inhibit growth stimulation in quiescent confluent and sparse cultures of Swiss 3T3 and 3T6 cells by changing for a fresh medium containing 10-20% serum. Proliferation was inhibited by dansylcadaverine (0.1 mM), chloroquine (0.05 mM), 5-methoxytryptamine (0.1 mM), cystamine (0.1 mM), dimethylurea (100 mM), methylurea (100 mM), and in some experiments--by urea (100 mM). The inhibitory activity was not associated with a direct influence of amines on DNA synthesis or thymidine phosphorylation. The findings suggest that amines may influence the process of clustering of growth factor-receptor complexes, or the fusion of plasma membrane and intracellular vesicles containing some components required for cell proliferation.

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The measurements of intralysosomal pH under the action of the number of amines earlier reported to block the process of the initiation of cell proliferation (Nikolsky et al., 1984) were made on Swiss 3T3 cells. The intralysosomal pH (pH1) value was estimated by parameters of fluorescence of fluorescein-labeled dextran in single intact cells. The pHl value was equal to 4.7 +/- 0.2 for both actively growing and quiescent cells. The pH gradient between lysosomes and the cytoplasm was completely destroyed by monensin and partially by carbonylcyamide-m-chlorophenylhydrasone. Methylamine and chloroquine rapidly enhanced the pHl, value to 6.4-7.0. Dansylcadaverine, 5-methoxytryptamine and dimethylurea did not affect pHl value. Intracellular accumulation of dansylcadaverine was shown to be due to the existence of acidic compartments into the cell and highly decreased in the presence of monensin. A conclusion is made that the inhibition of mitogenic signal by amines cannot be unequivocally accounted for by increasing the pH in organelles involved in the intracellular processing of growth factors.

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The dependence of L-alanine uptake by 3T6 and CHO-K1 cells on Na+ electrochemical gradient has been studied. The Na+ chemical gradient was changed by a short-term (partial or complete) replacement of Na+ for choline. The membrane potential change was achieved by addition of potassium ionophore--valinomycin (10 microM) into the medium. It is determined that the value of Km for alanine uptake by 3T6 cells increases from 2 mM, with 140 mM Na+ in the medium, up to 30 mM, if the replacement of Na+ for choline is complete. Similar results are obtained for CHO cells. The membrane potential increase under the influence of valinomycin leads to the increase in the value of Vmax of the uptake. The data obtained are interpreted on the basis of the well known scheme of Na+ alanine complex transfer, where Na+ increases the affinity of the carrier to the amino acid, and the membrane potential increases the carrier mobility.

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The transport of L-alanine in cultured 3T3, 3T6 and CHO cells has been studied. The estimated Michaelis kinetic constants KM and Vmax for 3T3, 3T6 and CHO cells were 0.6 mM and 3 mM/min, 2.5 mM and 8.3. mM/min, 0.7 mM and 8.4 mM/min, respectively. Alanine transport was inhibited by glycine, serine, alpha-aminoisobutyric acid, methionine, and in a lesser degree by leucine and valine. Glycine depressed alanine transport with the constant of inhibition (Ki) similar to the constant of glycine transport (Km). Alanine transport was strictly diminished in a Na+-dependent medium at the expense of the increase of Km for transport. It is concluded that in 3T3, 3T6 and CHO cells, alanine is transported by the Na+-dependent "A" system.

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In connection with a study of the chemical evolution of abiogenically synthesized organic compounds on primitive Earth and the physical conditions of other planets, this paper reports the experimental results obtained by the photolysis of solutions of aliphatic amino acids (glycine, alanine, valine, leucine, n. leucine) and peptides in the atmosphere of the air, N2, Ar and CO2 in the presence of the most simple photocatalyzers-cations of sulphates. The evidence shows that the photochemical conversion of NH2 acids depends on the content of the atmosphere. The decay of NH2-group is most active in air. N2 and Ar exert no significant influence on deamination, whereas in the atmosphere of CO2 the formation of ammonia in valine, for example, was only 29 per cent of its total amount during photolysis in the air. Cu2+ and Fe2+ catalyzed while Al3+ inhibited the ammonia excretion. The formation of acetaldehyde during alanine photolysis was actually independent from the atmosphere of N2 and was inhibited in Ar and CO2. Oxydative processes inducing the formation of glyoxalic acid and formaldehyde were sharply inhibited in Ar, N2 and CO2. Under the influence of ultraviolet light of the decay of NH2-acids is also accompanied by the formation of new NH2-acids. The photosensitizing effect of cations induces a rupture of -CO-NH-bonds in peptides and, provided heavy radiation doses, prevents the formation of new NH2-acids. The longer the dipeptide chain, the more significant the quantum yield of its decomposition. The photolysis of dipeptides, leading to their decay, does not necessarily induce a hydrolytic rupture of -CO-NH-bonds resulting in the formation of three amino acids. The results obtained permit approaching problems concerning the effect of the gas content of the atmosphere and various cations (photocatalyzers) on photolytic conversion of abiogenically synthesized and biogenically significant substances, amino acids for example, at the action of ultraviolet light.

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