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1.
Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid 总被引:1,自引:0,他引:1
J G Cory M M Mansell C B George D S Wilkinson 《Archives of biochemistry and biophysics》1974,160(2):495-503
Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [14C]cytidine nucleotides to [14C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [3H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [3H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo. 相似文献
2.
Several observations suggest an increased RNA synthesis in livers of protein-deprived rats, though the RNA/DNA ratio is decreased. A number of hormones may be involved in these changes. Therefore, we studied in RNA metabolism in isolated perfused livers taken from protein-fed and protein-deprived rats. (3H)-orotic acid was given to the rats 2 h before liver explantation, and (14C)-orotic acid was added to the perfusate. Other rats, called controls in vivo, whose livers were not transplanted were also given (3H)-orotic acid followed by (14C)-orotic acid. The livers of these rats, which were not hormone supplemented, were labelled for the same length of times as the livers in vitro. The ratio specific RNA radioactivity/specific nucleotide radioactivity x RNA/DNA was determined and taken as a measure of the RNA synthesis per liver cell. In the controls in vivo, this ratio was significantly higher for protein-deprived than for protein-fed rats. In livers from the protein-fed rats, labelling in vitro increased significantly when growth hormone, hydrocortisone, insulin and tri-iodothyronine were added to the perfusate. Labelling was also significantly higher in these livers than in the controls in vivo. In livers from protein-deprived rats, the ratio in question was the same whether the hormones were added to the perfusate or not, and was significantly lower than in the controls in vivo. Differences in RNA labelling are thus obtained in our in vitro system. Gel electrophoresis of RNA demonstrated normal RNA labelling, showing that the system is suitable for studying liver RNA synthesis. Further refinement can be made by studying the labelling of UTP and CTP. The results might suggest that the liver from a protein-fed rat, explanted in vitro, may increase its RNA synthesis under the influence of the four hormones in question, and that the RNA synthesis of the liver of a protein-deprived rat is high in-vivo and that it might decrease, when it is explanted to in vitro conditions. 相似文献
3.
《Insect Biochemistry》1981,11(4):487-489
Protein and RNA synthesis in the pea aphid, Acyrthosiphon pisum were studied at specific intervals during the entire life cycle. The pool size of [14C]-l-leucine in the aphid free amino acid pool and the incorporation of the same into the protein fraction increased with growth and reached a maximum between 12 and 16 days, corresponding to the period of active reproduction. The pool size of [3H]-orotic acid in the nucleotide pool and the incorporation of the same into the RNA fraction of the pea aphid also showed similar patterns. 相似文献
4.
The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after [3H]uridine injection or by the de novo pathway as shown after [3H]orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium--that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either [5-3H]uridine or [5-3H]orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloracetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after [3H]uridine and 72% after [3H]orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A+ RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after [3H] uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant. After [3H]-orotic acid injection, the number of silver grains over the nucleolus was negligible at all levels, whereas over the nucleoplasm the number was low in crypt cells, but high in villus cells with a peak in mid villus. The interpretation is that, except for a small amount of label incorporated into DNA from either precursor by crypt cells, the bulk of the label is incorporated into RNA as follows. In the crypts, cells make almost exclusive use of uridine, that is, of the salvage pathway, for the synthesis of ribosomal RNA in the nucleolus and of messenger and transfer RNA in the nucleoplasm. However, when cells pass from crypt to villus, they mainly utilize orotic acid--i.e., the de novo pathway--for the synthesis of messenger and transfer RNA within the nucleoplasm. 相似文献
5.
3,7-Dichloroquinolinecarboxylic Acid Inhibits Cell-Wall Biosynthesis in Maize Roots 总被引:1,自引:0,他引:1
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The mode of action of the herbicide 3,7-dichloroquinolinecar-boxylic acid (quinclorac) was examined by measuring incorporation of [14C]glucose, [14C]acetate, [3H]thymidine, and [3H]uridine into maize (Zea mays) root cell walls, fatty acids, DNA, and RNA, respectively. Among the precursors examined, 10 [mu]M quinclorac inhibited [14C]glucose incorporation into the cell wall within 3 h. Fatty acid and DNA biosynthesis were subsequently inhibited, whereas RNA biosynthesis was unaffected. In contrast to the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile, quinclorac strongly inhibited cellulose and a hemicellulose fraction presumed to be glucuronoarabinoxylan. However, the synthesis of (1->3),(1->4)-[beta]-D-glucans was only slightly inhibited. The degree of inhibition was time- and dose-dependent. By 4 h after treatment, the concentration that inhibited [14C]glucose incorporation into the cell wall, cellulose, and the sensitive hemicellulose fraction by 50% was about 15, 5, and 20 [mu]M, respectively. Concomitant with an inhibition of [14C]glucose incorporation into the cell wall, quinclorac treatment led to a marked accumulation of radioactivity in the cytosol. The increased radioactivity was found mostly in glucose and fructose. However, total levels of glucose, fructose, and uridine diphosphate-glucose were not changed greatly by quinclorac. These data suggest that quinclorac acts primarily as a cell-wall biosynthesis inhibitor in a susceptible grass by a mechanism that is different from that of 2,6-dichlorobenzonitrile. 相似文献
6.
7.
The regulation of nucleic acid and protein synthesis in dormant, thermodormant, and after-ripened embryos of Vaccaria pyramidata (Caryophyllaceae) has been studied. Germination of after-ripened V. pyramidata seeds is prevented by inhibitors of protein, RNA, and DNA synthesis. The synthesis of both protein and RNA is activated at the beginning of imbibition, whereas [3H]thymidine incorporation does not start until the second period of the imbibition phase. [3H]Thymidine incorporation is greatly reduced in embryos treated with cycloheximide or 6-methylpurine. There is no correlation between the level of [3H]uracil and l-[14C]leucine incorporation into macromolecules and the physiological state of the seeds: tRNA, ribosomal RNA, and poly(A)-containing RNA (probably mRNA) as well as proteins are synthesized at the same rate in both dormant and thermodormant embryos as in after-ripened embryos. The protein patterns of dormant and after-ripened embryos are similar, as shown by electrophoresis and electrofocusing of double-labeled proteins. The level of DNA synthesis, measured as [3H]thymidine incorporation, may, on the other hand, indicate the physiological activity of the seeds: [3H]Thymidine is incorporated at a high rate in after-ripened embryos only and remains at a low level in dormant or thermodormant embryos. This correlation is, however, observed only in the axes. DNA synthesis in the cotyledons does not show any relation to the developmental stage of the seeds. These results are discussed in relation to the regulation of dormancy and after-ripening of seeds. 相似文献
8.
Summary Incorporation of three various amino acids ([3H]-tryptophan, [3H]-methionine or [3H]-leucine) into the non-histone chromatin proteins, synthesized in spleen cells of mice after immunization with IgG, is described.
Two new fractions of non-histone chromatin proteins (I-mol. wt. below 3 000 and B1-mol. wt. about 120 000), appearing during the immune reaction were labelled with [3H]-tryptophan and [3H]-methionine but not with [3H]-leucine. Synthesis of these fractions was observed only at the time of maximal RNA synthesis. A suggestion about the role
of tryptophan and methionine in non-histone chromatin proteins in the regulatory processes of gene activation is discussed
on the basis of their selective binding to DNA. 相似文献
9.
10.
P Daudel F Gachelin M C Delcey P Jacquignon N P Buu Hoi P Queval 《Chemico-biological interactions》1972,4(4):223-231
The effect of a series of bromomethylated polycyclic hydrocarbons on in vitro DNA and RNA synthesis has been studied by measurement of the incorporation of [3H]-dTMP or [14C]-AMP into new chains. The inhibition of RNA synthesis was less than 12% for 9-bromomethylanthracene, 9-methyl-10-bromomethylanthracene and 12-bromomethylbenzo(a)acridine, and more than 37% for 7-bromomethylbenzo(a)anthracene, 7,12-dibromomethylbenzo(a)anthracene and 7-bromomethylbenzo(c)acridine. Analogous results were found for the inhibition of DNA synthesis, except for 7-bromomethylbenzo(c)acridine which had little effect. Apart from this exception a good correlation was found between the inhibitory action of the bromo derivatives and the carcinogenicity of the non-halogenated parent hydrocarbons. 相似文献
11.
F L Yu I H Geronimo W Bender R J Dowe 《Journal of biochemical and biophysical methods》1986,13(6):333-342
The [3H]XTPs are used widely to monitor RNA synthesis in vitro. Recently, we discovered that they reflected only 40-45% of the true rate of nuclear RNA synthesis. Thus, when [8-14C]GTP was used, 1466 pmol [8-14C]GMP was incorporated per mg DNA/10 min. On the other hand, when [8-3H]GTP was used, only 564 pmol [8-3H]GMP was incorporated per mg DNA/10 min. There are three obvious factors that could have contributed to this greater than 2-fold difference in the apparent incorporation rate: commercial [8-3H]GTP sample was contaminated with substances causing the assay medium to be less efficient in RNA synthesis; 3H exchange occurred during acid washing of the [3H]RNA; and there was a greater quenching effect on [3H]RNA. Experiments were designed to test each of these alternatives. We are able to conclude that none of the above three are contributing factors. Our data also show that the 3H label was removed after it was incorporated into RNA. Similar differences were observed when 3H and 14C labeled pairs of ATP, UTP and CTP were compared. Furthermore, when nuclei were fractionated into nucleolar and nucleoplasmic fractions and carried out RNA synthesis, the loss of 3H label was observed mainly from the nucleoplasmic fraction. 相似文献
12.
Raymond D. Mosteller Shing F. Kwan 《Biochemical and biophysical research communications》1976,69(2):325-332
Mutants of K-12 which are sensitive to glucose starvation were isolated by an enrichment procedure using thymine starvation to select for nongrowing cells. Eleven independent isolates were obtained by this method. The mutants are also sensitive to glycerol starvation and to a lesser extent to nitrogen or amino acid starvation. The mutants are more sensitive than the parental strain to inhibitors of protein synthesis but not inhibitors of RNA or DNA synthesis. [3H]-leucine incorporation experiments indicate that protein synthesis is blocked in the mutants during recovery from glucose starvation or chloramphenicol inhibition. Incorporation of [3H]uridine in amino acid-starved cells demonstrates that the mutants are partially relaxed for control of RNA synthesis. Physiological and genetic experiments indicate that these mutants are different from previously isolated relaxed-control mutants. 相似文献
13.
Effects of monensin on posttranslational processing of myelin proteins 总被引:13,自引:11,他引:2
Rat brain slices were incubated with [3H]palmitic acid and [14C]glycine to label the lipid and protein moieties, respectively, of myelin proteolipid protein (PLP). The effects of monensin on posttranslational processing of proteins were examined by measuring the appearance of [14C]glycine- and [3H]palmitate-labeled proteins in myelin and myelin-like fractions. At 0.01 and 0.10 microM, monensin did not appreciably affect total lipid or protein synthesis; higher concentrations caused increased inhibition. Monensin at 0.10 microM markedly decreased the appearance of [14C]glycine-labeled PLP in myelin, but had little effect on the 14C basic proteins or the incorporation of [3H]palmitic acid into total or myelin PLP. The same relative effect was apparent at higher monensin concentrations. In the myelin-like fraction, monensin at 0.10 microM also depressed entry of [14C]glycine into protein comigrating with PLP, and again had no effect on incorporation of [3H]palmitic acid. In addition, monensin increased the [3H]palmitate label associated with two high-molecular-weight proteins in the myelin-like fraction with no concomitant increase in [14C]glycine label. 相似文献
14.
Problems associated with the labelling of RNA with radioactive precursors in vivo (author's transl)]
H G Dahnke 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1975,356(10):1555-1563
The radioactivity of RNA, DNA and proteins in the liver, muscles and cerebrum of 30-day-old rats after labelling with [3H]uridine, [14C]uridine, [3H]cytidine or [3H]orotic acid was measured. It was found that after administration of [3H]uridine, the proteins were 5 - 10 times more radioactive than the RNA. After administration of [14C]uridine, the proteins were 1 - 2 times more heavily labelled than the RNA. Hydrolysis of the proteins followed by chromatography of the amino acids revealed that the protein labelling was mostly due to [3H]glutamate. In the liver, [3H]orotic acid produced very specific labelling of the RNA. The radioactivity of the proteins is very slight. However, the specific labelling of the RNA in the muscles and cerebrum is not so pronounced with this precursor. [3H]Cytidine is an ideal precursor for RNA. The labelling of protein in all three organs examined is very slight, and furthermore, the specific activity of the RNA is 10 - 20 times higher than after labelling with uridine. We were also able to show that after labelling with radioactive uridine, the method of isolation of RNA by alkaline hydrolysis gives incorrect results, because [3H]amino acids interfere with the measurement of the specific activity of the RNA. The heavy labelling of proteins by [3H]-uridine must also be taken into account in histoautoradiography, because our experiments showed that in liver, the proteins in the cell nucleus are 3 times as radioactive as the nucleic acids. The particulate components of the cytoplasm are even 20 times more radioactive than the nucleic acids. 相似文献
15.
Ornithine decarboxylase (ODC) and the polyamines are thought to play a role in maturation of mammalian tissues. Daily postnatal administration of alpha-difluoromethylornithine (DFMO, a specific inhibitor of ODC) to newborn rats caused organ-specific deficits in tissue weight gain, with brain and kidney as the major targets. Subnormal organ weights were associated with deficits in the levels of nucleic acids and proteins in the affected tissues, and examination of the synthetic rates of DNA ([3H]thymidine incorporation), RNA ([3H]uridine incorporation) and protein ([14C]leucine incorporation) confirmed that macromolecule synthesis was inhibited in DFMO-treated pups. The time of onset of effect of DFMO on the synthesis of nucleic acids and proteins was the same as that reported for depletion of polyamines by this treatment. Potential adverse effects of DFMO on cell survival were also assessed by labeling DNA with [3H]thymidine on day 3 and examining retention of label 12 days later; DFMO did not cause an increase in cell death. In contrast to the sensitivity of brain and kidney to postnatally administered DFMO, development of cardiac tissue was relatively resistant to growth inhibition despite polyamine depletion. The organ specificity of effect of DFMO results, in part, from the different timetables for cellular events in tissue development displayed by each organ type; administration of DFMO earlier in development (during days 15 to 17 of gestation) did produce deficiencies in cardiac growth and nucleic acid levels similar to those which had been seen for brain and kidney. These data support the view that polyamines play a key role in cell replication, differentiation and growth during critical periods of mammalian organ development through their regulation of DNA, RNA, and protein synthesis. 相似文献
16.
Renaud Guy Soler-Argilaga Carlos Infante Recaredo 《Biochemical and biophysical research communications》1980,95(1):220-227
After a single injection of Cerium chloride to female rats, liver triglycerides concentration increases sharply and plasma lipids decrease to about one half the initial level. In these conditions, the respiratory quotient of treated rats decreases by 20% indicating a lowered fatty acid oxidation. Incorporation of [3H]-oleate into liver lipids shows an important accumulation of newly synthetized triglycerides without any significant effect on phospholipids. Lipoproteins production estimated by [3H]-oleate and [14C]-leucine incorporation into plasma very low-, low- and high-density lipoproteins shows a 50% inhibition synthesis of lipoprotein. 相似文献
17.
A Bilecka 《Folia histochemica et cytochemica》1975,13(3-4):139-149
An over 9 per cent increase was found in the protein content of callus and tumorous tissues of Nicotiana tabacum cultured for 39 days in a medium with an addition of hydroxyurea at a concentration of 100 mg/l. The inhibitory effect of this compound on incorporation of [14C]leucine was demonstrated, differences in the intensity of labelling between the nucleus and cytoplasm being present only in the tumorous tissue. These differences may indicate either disturbances in the migration of proteins from cytoplasm to nuclei or a specific blockade of histone synthesis. Hydroxyurea inhibits incorporation of [3H]arginine in callus tissues, whereas in tumorous tissues, apart from inhibition (at 75 and 100 mg HU/1), the stimulation of incorporation of this amino acid was observed (at 10 and 50 mg HU/1). The inhibitory effect of hydroxyurea on incorporation of [3H] lysine was demonstrated in both tissues examined, but it was stronger in the callus tissue. On the basis of the results concerning the influence of hydroxyurea on the content and synthesis of nucleic acids [4, 5] and the results of the present study it may be supposed that this compound induces unbalanced growth of cells of both tissues (inhibition of DNA and RNA synthesis and simultaneous accumulation of proteins), thus leading to their death. 相似文献
18.
Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells 总被引:13,自引:0,他引:13
We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [3H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [3H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [3H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis.Using the assay of [3H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [14C]leucine or [14C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis.We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [3H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons. 相似文献
19.
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
20.
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