共查询到20条相似文献,搜索用时 15 毫秒
1.
Herman Polet 《Experimental cell research》1983,148(2):345-362
Concanavalin A (conA) inhibits the degradation of [3H]leucine-labeled cellular proteins of human lymphocytes. The lectin also stimulates the migration of non-histone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H]actinomycin D [(3H]AD). Decreased protein breakdown and increased migration of NHP are parallel events, i.e. both changes occur as a function of the lectin concentration and display a similar time course, suggesting that these events could be related. Similar effects are observed with fluoride, chloroquine and iodoacetate: these agents simultaneously decrease proteolysis and increase the nuclear level of NHP, associated with increased cellular [3H]AD binding. Fractionation of the acidic NHP according to pH 2.5-6.5 shows that proteins with a high degree of degradation in unstimulated cells correspond to proteins with a high degree of migration in conA-stimulated cells. A similar correlation was observed in fluoride-treated lymphocytes. conA, fluoride and iodoacetate decrease cellular [3H]chloroquine [(3H]CQ) accumulation, indicating a lysosomotropic effect. These and previously reported data suggest, but do not prove that conA inhibits degradation of cellular proteins via the lysosomal pathway. Ammonium chloride, methylamine and sodium azide also inhibit proteolysis and increase cellular [3H]AD binding; however, their effects are weak. On the basis of these observations it appears that lysosomal degradation and migration of NHP to the nucleus are linked; however, the mechanism of the linkage is unknown. 相似文献
2.
H Polet 《Experimental cell research》1987,169(1):178-190
Stimulation of resting WI38 cells, prelabeled with [3H]leucine, with fibroblastic growth factor (FGF) or serum, caused increased nuclear translocation of [3H]non-histone proteins [( 3H]NHP) and DNA synthesis, and a parallel decrease of proteolysis. [3H]NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5-6.5 showed that [3H]NHP fractions with high degradation rates in resting cells corresponded to the [3H]NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H]NHP are linked. FGF inhibited cellular uptake of [3H]chloroquine, suggesting that FGF inhibits NHP degradation via lysosomes. The lysosomotropic amine eserine had similar effects as FGF. It is proposed that FGF induces NHP migration to the nucleus by inhibiting their lysosomal degradation. FGF also caused migration of [3H]histones, however, the mechanism is not clear. 相似文献
3.
H Polet 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1992,199(4):417-423
Stimulation of resting normal rat kidney fibroblasts, prelabeled with [3H]leucine, by platelet-derived growth factor (PDGF) caused inhibition of cellular protein degradation and a parallel increased nuclear translocation of 3H-labeled nonhistone proteins (3H-NHP) and DNA synthesis. Nuclear translocation of these proteins was independent of protein synthesis. Fractionation of the nuclear 3H-NHP in a pH gradient of 2.5-6.5 showed that the protein fractions with a high degree of proteolysis in resting cells corresponded to the protein fractions with a high extent of translocation in stimulated cells, suggesting that degradation and translocation of these proteins may be related. PDGF inhibited cellular uptake of [3H]chloroquine, suggesting that PDGF inhibits NHP degradation via the lysosomal pathway. These observations support the hypothesis that PDGF induces NHP translocation to the nucleus by inhibiting lysosomal degradation of these proteins. 相似文献
4.
The uptake of [3H]actinomycin D ([3H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [3H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [3H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [3H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [3H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [3H]AD to pure DNA in the absence of histones. The degree of [3H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD. 相似文献
5.
The uptake of [3H]actinomycin D ([3H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [3H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [3H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [3H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [3H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [3H]AD to pure DNA in the absence of histones. The degree of [3H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD. 相似文献
6.
Con A, NaF, and eserine (lysosomotropic agents) induced marked translocation of acidic [3H] nonhistone proteins (NHP) from the cytoplasm to the nucleus in lymphocytes prelabeled with [3H]-2-mannose. The nuclear [3H] NHP contents were 38-120% higher in cells treated with these agents than in control cells. Tunicamycin, a strong inhibitor of N-glycosylation via the dolichol pathway, caused a concentration-dependent inhibition of [3H]-2-mannose incorporation into the nuclear [3H] NHP. Considerable amounts of nuclear [3H] NHP from lymphocytes labeled with either [3H]-2-mannose or [3H] leucine, bound specifically to Con A-Sepharose and could be eluted by alpha-methyl mannoside. Con A and NaF caused also nuclear translocation of acidic [3H] NHP in cells labeled with [3H] glucosamine, [3H] galactose, or [3H] fucose. Fractionation of the nuclear proteins by isoelectric focusing in a pH gradient of 2.5-6.5 showed that multiple species of acidic NHP were labeled with each of the four 3H-sugars. These results indicate that a fraction of the acidic nuclear NHP are N-glycosylated proteins and that gene activation and mitogenesis are associated with the translocation of these glycoproteins to the nucleus. Considering the known intracellular traffic of nascent glycoproteins our results suggest that at least some of the acidic NHP are synthesized and glycosylated in the endoplasmic reticulum and the Golgi (secretory pathway). It is likely that these proteins, after completion of synthesis and glycosylation, emerge from the trans-stack of the Golgi packaged in vesicles and accumulate in the cytoplasm. Induction of nuclear translocation of such NHP by various agents may be mediated by a vesicular transport mechanism. 相似文献
7.
The effects of lysosomotropic amines on protein degradation, migration of nonhistone proteins to the nucleus, and cathepsin D in lymphocytes 总被引:1,自引:0,他引:1
H Polet 《Journal of cellular physiology》1985,122(3):415-423
Various lysosomotropic amines have two parallel effects in human lymphocytes: they inhibit the degradation of cellular proteins and increase the migration of nonhistone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H] actinomycin D, indicating an altered structure of chromatin. The agents inhibit the degradation of short- and long-lived proteins equally. Fractionation of the [3H] NHP of the nucleus according to pH 2.5-6.5 shows that [3H] NHP with a high rate of degradation in untreated cells correspond to [3H] NHP with a high rate of migration in cells treated with the agents. Eserine, amantadine, nicotine, atropine, benzylamine, and propranolol inhibit cathepsin D in concentrations causing proteolytic inhibition in cell cultures or in concentrations believed to be attained in lysosomes. The agents strongly inhibit the cellular accumulation of [3H] chloroquine. The data support the proposal that the migration of NHP from the cytoplasm to the nucleus is the direct consequence of inhibited degradation of these proteins in lysosomes by the amines. 相似文献
8.
Herman Polet 《Experimental cell research》1986,166(2):455-464
[3H]Non-histone proteins ([3H]NHP), dissolved in the culture medium, are endocytosed by lymphocytes and equilibrate rapidly between the cytoplasm and the nucleus. During incubation, the proteins are gradually degraded in the lysosomes. The lysosomotropic agents conA, NaF, eserine and atropine have two parallel effects on resting lymphocytes, after they have endocytosed [3H]NHP: inhibition of degradation and increased translocation of [3H]NHP from the cytoplasm to the nucleus. This indicates that lysosomal degradation and translocation of [3H]NHP to the nucleus are linked and suggests that this translocation may be the result of inhibited lysosomal degradation of the [3H]NHP. The behaviour of endocytosed [3H]NHP appears similar to that of endogenous [3H]NHP in cells prelabeled with [3H]leucine, when subjected to the same lysosomotropic agents, reported previously (Polet, H, Exp cell res 148 (1983) 345). This observation may provide a model to study the mechanism(s) controlling nucleo-cytoplasmic traffic of NHP. 相似文献
9.
Stimulation of resting transformed cells (Chang liver cells), prelabeled with [3H] leucine, with fetal calf serum, caused increased nuclear translocation of [3H] nonhistone proteins ([3H] NHP) and DNA synthesis and a parallel inhibition of proteolysis of cellular proteins. [3H] NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5-6.5, showed that [3H] NHP fractions with high degradation rates in resting cells corresponded to the [3H] NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H] NHP are linked. Conditioned medium (COM) produced by Chang cells had similar effects as serum, suggesting that factors produced by these transformed cells, control cell growth by a mechanism that is similar to serum. The lysosomotropic amine eserine had similar effects as serum and COM. Based on the similarity of the effects, it would appear that serum and COM inhibit lysosomal proteolysis. It is proposed that serum and COM induce NHP migration to the nucleus by inhibiting lysosomal degradation of these proteins. Serum and COM caused also migration of [3H] histones to the nucleus, however the mechanism is not clear. 相似文献
10.
In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria. 相似文献
11.
Tryptophan, phenylalanine and leucine have two parallel effects in cultured lymphocytes, they inhibit cellular proteolysis and increase the translocation of non-histone proteins to the nucleus. The latter is associated with an increased cellular binding of [3H]actinomycin D, indicating an altered structure of chromatin. The amino acids also inhibit the cellular uptake of [3H]chloroquine, suggesting that inhibited protein degradation is lysosomal. Several amine catabolites of tryptophan and phenylalanine, some of which are known to play a role as biogenic amines, have similar actions, and can explain, at least in part, the effects of their parent amino acids. Fractionation of the nuclear 3H-labeled non-histone proteins according to pH 2.5-6.5 shows that such proteins with a high rate of degradation in untreated cells correspond to the 3H-labeled non-histone proteins with a high rate of translocation in tryptophan treated cells. These data suggest that the degradation and the translocation of the non-histone proteins are linked and that the increased translocation of the non-histone proteins to the nucleus may be the consequence of inhibited lysosomal degradation of these proteins by the amino acids. 相似文献
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13.
Germination of microcysts of Polysphondylium pallidum is characterized by an immediate rapid increase in incorporation of [3H]leucine into protein which is cycloheximide-sensitive but unaffected by actinomycin D. Significant RNA synthesis, as measured by [3H]uridine incorporation, does not begin until approx. 2 h after the onset of germination. The increase in [3H]uridine incorporation is prevented by actinomycin D. Germination and the increase in alkaline phosphatase and β-glucosidase enzyme activities are prevented by cycloheximide but unaffected by actinomycin D. The data strongly imply the presence of stable RNA in dormant microcysts and indicate a requirement for a discrete period of protein synthesis for germination of microcysts of P. pallidum. 相似文献
14.
We have previously shown that 3,5,3'-triiodo-L-thyronine (L-T3) stimulates cell growth and a 4- to 8-fold increase in growth hormone mRNA in GH1 cells. These effects appear to be mediated by a thyroid hormone nuclear receptor with an equilibrium dissociation constant for L-T3 of 0.2 nM and an abundance of about 10,000 receptors per cell nucleus. In this report, we show that L-T3 exerts a pleiotypic effect on GH1 cells to rapidly (within 2 h) stimulate [3H]uridine uptake to a maximal value of 2.5- to 3-fold after 24 h. This results from an increase in the number of functional uridine "transport sites" as shown by studies documenting an increase in the apparent Vmax with no change in the Km, 17 microM. Although the labeling of the cellular uridine pool and pools of all phosphorylated uridine derivatives was increased by L-T3, there was no change in the relative amounts of the individual pools in cells incubated with or without hormone. The intracellular concentration of [3H]uridine was estimated to be similar to that of the medium, suggesting that facilitated transport mediates [3H]uridine uptake. That this increase in [3H]uridine transport was nuclear receptor-mediated is supported by the excellent correspondence of the L-T3 dose-response curve for [3H]uridine uptake and that for L-T3 binding to receptor. Finally, inhibition of protein synthesis by cycloheximide and RNA synthesis by actinomycin D demonstrated that the L-T3 effect required continuing protein and RNA synthesis. These results are consistent with an effect of the L-T3-nuclear receptor complex to increase uridine uptake in GH1 cells by altering the expression of gene(s) essential for the transport process. 相似文献
15.
When [3H] Actinomycin D (Act. D) is used to interact with nuclei and nucleoli in vitro, it binds preferentially to nucleolar chromatin. The preferential binding is no longer detectable, when purified nuclear and nucleolar DNAs are used. In parallel, Act. D preferentially inhibits nucleolar over nuclear RNA synthesis when chromatin templates are used, and the preferential inhibition is lost when purified nuclear and nucleolar DNAs are used. It is concluded: 1) the preferential inhibition of nucleolar over nuclear RNA synthesis by Act. D is a direct reflection of the preferential binding of Act. D to the nucleolar chromatin; and 2) the nucleolar chromosomal proteins, not the nucleolar DNA, confer the preferential binding of Act. D. 相似文献
16.
Prostaglandin production by methylcholanthrene-transformed mouse BALB/3T3. Requirement for protein synthesis 总被引:3,自引:0,他引:3
Stimulation of prostaglandin synthesis in transformed mouse fibroblasts by serum, thrombin, and bradykinin was blocked by actinomycin D and cycloheximide. These RNA and protein synthesis inhibitors did not affect prostaglandin synthetase in vitro or in vivo; nor did they affect the acylation of arachidonic acid into phospholipids. Serum-stimulated release of arachidonic acid and prostaglandins from [3H]arachidonic acid-labeled cells also was inhibited by actinomycin D and cycloheximide. RNA and protein synthesis appear to be required for expression of phospholipase activity; a prerequisite for prostaglandin synthesis by these cells. 相似文献
17.
Hiroshi Yamasaki Taira Enomoto Nicole Martel Yoshiki Shiba Yoshinobu Kanno 《Experimental cell research》1983,146(2)
A tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), reversibly inhibits the onset and maintenance of cell-cell communication measured by electrophysiological method. We have now studied the mechanism by which TPA inhibits communication of human cells (FL) in culture. Using [3H]phorbol-12,13-dibutyrate ([3H]PDBu), we found a class of specific, high-affinity, saturable binding sites in intact FL cells; they have a dissociation constant of 15.4 nM, and at saturation about 3 × 105 PDBu molecules were bound to each cell. The binding of [3H]PDBu to FL cells was inhibited by TPA, phorbol-12-13-didecanoate and mezerein, whereas phorbol and 4α-phorbol-12-13-didecanoate had no effect. There is a close correlation between the ability of the former compounds to inhibit [3H]PDBu binding and their capacity to inhibit cell-cell communication. When FL cells are dispersed with EDTA and plated onto a culture dish, they start to couple electrically within 2 h; such cell coupling was not affected by the presence of cycloheximide or actinomycin D. TPA inhibits the formation of electrical cell coupling as well as its maintenance, even in the presence of cycloheximide; the recovery of cell-cell communication after the removal of TPA was not significantly affected by the addition of cycloheximide or actinomycin D. Taken together, these results suggest that TPA-mediated reversible inhibition of intercellular communication is mediated by specific binding of TPA to cellular receptors and that macromolecular synthesis is not necessary. 相似文献
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Nuclear ribonucleoprotein particles of 75S were obtained from rat liver nuclei after mild sonication and isotonic salt extraction only when the preparation was carried out in the presence of a cytosolic ribonuclease inhibitor. Particles of 38S were isolated in the absence of inhibitor. The 38S nuclear ribonucleoprotein (nRNP) particles showed a protein/RNA ratio of 8, and a buoyant density of 1.39 g/ml in cesium chloride solution. They were further characterized by the pattern of their proteins on sodium dodecylsulfate (SDS)-acrylamide gel electrophoresis. Incorporation of [3H]cytidine into nuclear RNA was reduced to approx. 20% of controls 3 and 6 h after administration of galactosamine or actinomycin D. However, when [3H]cytidine was administered 30 min prior to the drugs a decrease of radioactivity in 38S nRNP particles to 43 and 81% of controls was found after 3 h. The yield of 38S particles 3 h after galactosamine or actinomycin D dropped to 41% and 78% of controls, and after 6 h to 43 and 70%, respectively. Six hours after galactosamine or actinomycin D treatment, the protein to RNA ratio increased to 13.3 and 9.1. No significant changes in protein patterns 3 h after treatment with galactosamine or actinomycin D were observed. Possible mechanisms, such as impaired transport of 38S nRNP particles after actinomycin D treatment or increased loss of particles due to a defective nuclear membrane after galactosamine administration are discussed. 相似文献