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Role of Nonhistone Chomosomal Proteins in the Restriction of Mitotic Chromatin Template Activity
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Gary Stein John Farber 《Proceedings of the National Academy of Sciences of the United States of America》1972,69(10):2918-2921
RNA synthesis virtually ceases during mitosis in eukaryotic cells. This phenomenon is explainable, at least in part, by the reduced template activity for RNA synthesis of mitotic chromatin. The restriction in template activity can be accounted for by the presence in the chromatin of mitotic-specific nonhistone proteins. Chromatin reconstituted by gradient dialysis from the pooled histones of S-phase and mitotic chromatin and the nonhistone proteins of mitotic chromatin had a template activity similar to that of native mitotic chromatin, and lower than native S-phase chromatin or chromatin reconstituted with the pooled histones and the S-phase and nonhistone proteins. The template activity was identical for chromatin reconstituted from the pooled nonhistone proteins and either the histones from S-phase or mitotic chromatin. These results are supported with data showing that the procedure of reconstitution produces a chromatin indistinguishable in its protein composition both qualitatively and quantitatively from its native counterpart. 相似文献
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Cardiac hypertrophy was induced in adult female Wistar rats by daily subcutaneous injections of isoproterenol (0.3 mg/kg body weight). Heart weight increased 39% after eight days of treatment. Left ventricular pressure development (positive dP/dt) in hearts four days after hypertrophy induction was significantly increased, while negative dP/dt remained unchanged. RNA polymerase activity in isolated myocyte and nonmyocyte nuclei was stimulated 29 and 23%, respectively 24 h after a single isoproterenol injection. In the myocyte fraction, RNA polymerase activation progressively increased up to four days of treatment and then returned to control values after eight days. In the nonmyocyte nuclear subset, RNA polymerase activity showed no further stimulation and gradually returned to control values after eight days of treatment. Chromatin template function was substantially stimulated in the early stage (one to four days) of hypertrophy in both myocyte and nonmyocyte fractions. Titration of chromatin against a fixed amount of RNA polymerase (5 micrograms) in the presence of rifampicin and heparin showed that less chromatin from hypertrophied hearts was required to saturate the enzyme. These results indicate that both myocyte and nonmyocte chromatin from hypertrophied hearts can support greater enzyme binding than normal chromatin. The alkaline sucrose density centrifugation profile of DNA in myocyte and nonmyocyte chromatin from day 4 hypertrophied hearts was less fragmented. These observations suggest that during the early phase of isoproterenol-induced cardiac hypertrophy, enhanced RNA polymerase activity and chromatin template function play a coordinated role in RNA synthesis. The increased template activity could be due to alterations in chromatin composition which was indicated by the change in their enzyme binding capacity and DNA fragmentation profile. 相似文献
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John E. Mayfield James Bonner 《Proceedings of the National Academy of Sciences of the United States of America》1972,69(1):7-10
When two lobes of the liver of the adult rat are removed, the cells of the remaining lobe are aroused to renewed cell division. We have studied the early events in such regeneration. The first observable response to such partial hepatectomy is the production in the liver nuclei of rapidly-labeled high-molecular-weight RNA of sequences not produced by normal liver. This is followed, with a lag of about 1 hr, by the appearance of increased (above normal) amounts of chromosomal RNA, again of sequences not produced by normal liver. With a lag of another hour, the template activity in support of RNA synthesis of the liver chromatin increases substantially. These events occur before initiation of DNA synthesis in the cells of the regenerating liver. 相似文献
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Chromatin from the oviducts of estrogen-treated chicks was sheared and fractionated on sucrose gradients. This resulted in the production of several chromatin fractions which differ in their sedimentation properties, protein composition, the number of acceptor sites for the progesterone-receptor complex, and the ability to serve as a template for RNA synthesis. The pellet chromatin fraction shows an enhanced ability to bind the progesterone-receptor complex in vitro and in cell-free systems. Kinetic analysis indicates that the majority of the acceptor sites for the progesterone-receptor complex are located in the pellet chromatin fraction which is lowest in template activity. The sites may be important loci for initiating the changes in RNA synthesis following the exposure of target cells to steroid hormones. 相似文献
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Contraction modulates the capacity for protein synthesis during growth of neonatal heart cells in culture 总被引:4,自引:0,他引:4
Neonatal ventricular myocytes that were incubated in a well-defined serum-free medium containing 50 mM KCl did not contract and maintained stable cell size, as assessed by the protein/DNA ratio. The present study utilized KCl-arrested cells to examine the effect of constant rates of synchronous contraction in normal [K+]o (4 mM) as a physiological stimulus for myocyte growth. Cell growth increased following the onset of contraction when measured over 3 days. The rate of protein synthesis was accelerated in parallel by contraction, but the rate of protein degradation remained similar to rates in noncontracting cells. The capacity for protein synthesis was estimated by total RNA content and was increased in contracting as compared with KCl-arrested cells. This increase was accompanied by faster rates of RNA synthesis as determined from the incorporation of [3H]uridine into RNA and the specific activity of the cellular UTP pool. The rate of RNA degradation was accelerated during contraction but the difference between the rates of RNA synthesis and degradation resulted in net RNA accumulation of 49% after 3 days. These data demonstrated that 1) contractile activity stimulated myocyte growth through an increased capacity for protein synthesis and 2) the increased capacity for protein synthesis involved acceleration of the rate of RNA synthesis. Since enhancement of protein synthetic capacity is a common feature of myocyte hypertrophy in vivo and in vitro, this model can be used to examine the regulation of ribosome synthesis during hypertrophic growth. 相似文献
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We studied the in vitro effects of JH III and ecdysterone on RNA synthesis and secretory activities of the left colleterial gland (LCG) in mature females of Periplaneta americana during the reproductive cycle. We found that RNA synthesis is a cyclic and highly dynamic process. The major increases in poly(A)- RNA synthesis occurred 8 hr before the peak leucine incorporation into colleterial polypeptides, whereas high poly(A)+ RNA synthesis coincided with high leucine incorporation. Depending on the stage of the cycle JH III exhibited stimulatory or inhibitory effects, or caused no change in RNA synthesis. JH III stimulated RNA synthesis in glands from stages in the reproductive cycle with high endogenous RNA synthesis. The pattern of protein synthesis was not affected by JH III. Ecdysterone lowered RNA synthesis in the LCG and induced secretory activity in LCGs isolated at stages far removed from ovulation. JH III reduced the inhibitory effect of ecdysterone on RNA synthesis and inhibited the ecdysterone-induced secretory activity. The presence of ovarioles isolated at 62 hr of the reproductive cycle (onset of in vivo LCG secretory activity) in the incubation medium inhibited RNA synthesis in 32 hr LCG but induced secretory activity. We suggest that changes in synthetic and secretory activities are coordinated with ovulation and ootheca formation through the interactions between JH III and ovarioles, and ecdysterone may also play a role. 相似文献
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C K Osborne G Bolan M E Monaco M E Lippman 《Proceedings of the National Academy of Sciences of the United States of America》1976,73(12):4536-4540
The mechanisms of steroid and peptide hormone action in human breast cancer are poorly understood. We have previously characterized a cell line of human breast cancer in long-term tissue culture that possesses various steroid hormone receptors and responses, providing a model for the study of steroid hormone action. The present studies describe a human breast cancer in vitro that responds to physiologie concentrations of insulin with an increased rate of macromolecular synthesis and growth. Thymidine and uridine incorporation in cells in serum-free medium are stimulated by 10(-11) M insulin and are maximal with 10(-8) M. Leucine incorporation is stimulated by 5 X 10(-11) M insulin and is maximal with 10(-9) M. Significant stimulation of uridine and leucine incorporation is evident by 3 hr and maximal by 10 hr. A 10-hr lag period exists for insulin stimulation of thymidine incorporation, which is maximal form 14 to 24 hr. The effect of 10(-8) M insulin on macromolecular synthesis is accompanied by a 69% increase above controls in the number of cells after 24 hr. The effect on macromolecular synthesis is observed in glucose-free medium. Insulin's effect on protein synthesis is not blocked by inhibition of RNA synthesis with actinomycin D. Glucocorticoids partially inhibit the action of insulin in these cells. This system provides a model for studying insulin action, and suggests that some human breast cancer may show growth regulation by insulin. 相似文献
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D J Schneider B E Sobel 《Proceedings of the National Academy of Sciences of the United States of America》1991,88(22):9959-9963
Accelerated atherosclerosis accompanying diabetes mellitus, obesity, and some types of hypertension has been associated with hyperinsulinemia, augmented plasma plasminogen activator inhibitor type 1 (PAI-1), or both. We hypothesized that insulin and insulin-like growth factor type I (IGF-I) can influence synthesis of PAI-1, thereby potentially attenuating fibrinolysis. In HepG2 cells used as a model system, concentrations of insulin and IGF-I consistent with those seen in plasma independently stimulated PAI-1 synthesis. Accumulation of PAI-1 protein in conditioned medium over 24 hr was stimulated more with insulin alone than with the combination. Synergistic increases were evident, however, in the accumulation of PAI-1 protein over 48 hr with a concomitant increase in PAI-1 mRNA. A 10- to 20-fold increase in IGF binding protein I mRNA was seen 16-48 hr after exposure of the HepG2 cells to insulin and IGF-I, an increase abolished by cycloheximide. The results obtained are consistent with the hypothesis that hyperinsulinemia coupled with physiologic concentrations of IGF-I may attenuate fibrinolytic activity in vivo, thereby contributing to accelerated atherosclerosis. 相似文献
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Increased RNA Synthesis in Nuclear Monolayers of WI-38 Cells Stimulated to Proliferate 总被引:3,自引:3,他引:3
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Bernd M. Bombik Renato Baserga 《Proceedings of the National Academy of Sciences of the United States of America》1974,71(5):2038-2042
Nuclear monolayers of WI-38 cells prepared by the method of Tsai and Green were used to determine RNA synthesis in isolated nuclei in situ. In nuclear monolayers, incorporation of [(3)H]UTP into RNA is dependent on the presence of the other three nucleotide triphosphate and is abolished by actinomycin D. The extent of RNA synthesis under these conditions was measured in density-inhibited WI-38 human diploid fibroblasts at various intervals after cell proliferation was stimulated by a change of medium.RNA synthesis increases 15 min after the nutritional change and reaches a peak at 18 hr, which is also the peak of DNA synthesis. Thereafter RNA synthesis declines. Essentially similar results are obtained whether the endogenous RNA polymerase or a bacterial polymerase is used. Replacement of the stimulating medium by conditioned medium stops the increase in RNA synthesis that occurs in cultures subject to continuous stimulation. Finally, RNA synthesis in nuclear monolayers, using the endogenous RNA polymerase, occurs by chain elongation only, while re-initiation occurs with the bacterial RNA polymerase. 相似文献
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The in vitro template activity of chromatin isolated from submandibular gland increases threefold as rats age from two to twelve months. The increased capacity for DNA-primed RNA synthesis cannot be attributed to differences in nuclease or protease activity, and there are no variations in the ability of young and older submandibular gland DNA to transcribe RNA under these conditions. The histone content of young and older submandibular gland chromatin is similar. However, these proteins are less tenaciously bound to the DNA with age; consistent with the age-dependent increase in template activity. In contrast, there is an age-dependent decrease in the quantity of nonhistone chromosomal proteins associated with submandibular gland chromatin. 相似文献
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The rat zona glomerulosa has a renin-angiotensin system that appears to function as an autocrine or paracrine system in the regulation of aldosterone production. To further investigate dynamic changes of production of renin and aldosterone in vitro we developed a primary monolayer culture of rat adrenal glomerulosa cells in serum-free medium. Collagenase-dispersed glomerulosa cells were incubated in PFMR-4 medium containing 10% fetal calf serum for 48 hours; the medium was then replaced with serum-free PFMR-4 medium. The cell viability and the aldosterone secretion were stable over the additional 48 hours in the serum-free control medium. After incubation for 24 hours in the serum-free medium, the cells were exposed to high K+ or adrenocorticotropic hormone (ACTH) for another 24 hours. ACTH stimulated aldosterone secretion, and this increased secretion was associated with an increase in renin activity (cell active renin, from 15.56 +/- 0.71 to 45.75 +/- 5.69; cell inactive renin, from 0.67 +/- 0.54 to 8.75 +/- 3.40; medium inactive renin, from 5.58 +/- 1.16 to 106.20 +/- 14.01 pg angiotensin I (Ang I)/micrograms protein/3 hr). Aldosterone was also stimulated by high K+. This increase was also associated with an increase in active renin in the cells (from 15.08 +/- 1.80 to 23.26 +/- 2.15 pg Ang I/micrograms protein/3 hr) and an increase in inactive renin in the medium (from 10.87 +/- 1.62 to 21.37 +/- 3.20 pg Ang I/micrograms protein/3 hr). Addition of the angiotensin converting enzyme inhibitor lisinopril attenuated both ACTH- and high K(+)-stimulated aldosterone secretion significantly.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA. 总被引:18,自引:1,他引:18
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J G Levin M J Rosenak 《Proceedings of the National Academy of Sciences of the United States of America》1976,73(4):1154-1158
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Effects of estrogen on gene expression in chick oviduct: nuclear receptor levels and initiation of transcription. 总被引:5,自引:0,他引:5
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S Y Tsai M J Tsai R Schwartz M Kalimi J H Clark B W O''''Malley 《Proceedings of the National Academy of Sciences of the United States of America》1975,72(11):4228-4232
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A factor that initiates myocardial hypertrophy in hypertension 总被引:2,自引:0,他引:2
A lack of correlation between blood pressure and myocardial hypertrophy was established in spontaneously hypertensive rats, suggesting that factors other than blood pressure control might be responsible for the modulation of myocardial hypertrophy. An in vitro system that is independent of blood pressure and hemodynamic effects was developed by use of isolated myocytes to study myocardial protein synthesis. The validity of this system was determined by means of morphology, by receptor integrity, and by studying the incorporation of tritiated leucine into myocyte protein (dpm/mg/hr). Addition of a supernatant of spontaneously hypertensive rat myocardial homogenate (centrifuged at 1500 g) to the myocyte system resulted in a significant increase in tritiated leucine incorporation into myocyte protein when compared with the addition of homogenates from normal controls. The protein from the homogenate was partially purified by high performance liquid chromatography. The resultant purified protein also stimulated protein synthesis by 70%. Furthermore, a significant increase in the specific activity of the transfer RNA and the rate of protein synthesis was observed after addition of homogenate from hypertrophied heart (4.02 +/- 0.3 vs 7.0 +/- 0.2 pmol leucine/microgram protein/hr; p less than 0.05). These data demonstrate the existence of a soluble factor in the hypertrophied myocardium that stimulated protein synthesis. This factor may play a key role in modulation of myocardial structure during development or regression of myocardial hypertrophy in hypertension. 相似文献
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In vitro transcription of heat-shock-specific RNA from chromatin of Drosophila melanogaster cells. 总被引:6,自引:0,他引:6
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H Biessmann B Levy B J McCarthy 《Proceedings of the National Academy of Sciences of the United States of America》1978,75(2):759-763
Polyadenylylated RNA synthesized after heat shock was isolated from polysomes of cultured cells of Drosophila melanogaster and used as template to prepare cDNA. An excess of poly(A)-RNA from heat-shocked cells hybridized to 80% of the cDNA, whereas cytoplasmic poly(A)-RNA from cells grown at 25 degrees could drive only half of the cDNA probe into hybrid. These sequences were removed from the cDNA population by annealing to poly(A)-RNA from cells grown at 25 degrees. The unreacted material represented only heat-shock-induced mRNA sequences, as shown by a second cycle of hybridization. Isolated chromatin was transcribed in vitro at 25 degrees with Escherichia coli RNA polymerase, with mercurated UTP as precursor. RNA transcribed from chromatin that was prepared from cells 1 hr after the temperature was shifted to 37 degrees hybridized with 100-fold faster kinetics to the heat-shock-specific cDNA probe than did RNA transcribed from chromatin of cells grown at 25 degrees. Therefore, heat shock results in a change in chromatin structure recognizable by E. coli RNA polymerase. 相似文献
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Early Stimulation of RNA Synthesis by Erythropoietin in Cultures of Erythroid Precursor Cells 总被引:5,自引:2,他引:5
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George M. Maniatis Richard A. Rifkind Arthur Bank Paul A. Marks 《Proceedings of the National Academy of Sciences of the United States of America》1973,70(11):3189-3194
The effect of erythropoietin on cultured erythroid precursor cells from 13-day mouse-fetal livers was examined. Within 1 hr, erythropoietin causes a 2- to 3-fold stimulation of uridine incorporation into RNA by these cells. The types of RNA preferentially stimulated by erythropoietin during the first hour of exposure of the cells to the hormone include ribosomal RNAs and their precursors, as well as 4-5S RNA. No unique RNA species, not present in control cells, could be detected by sucrose gradient sedimentation or gel electrophoresis. Inhibition of protein synthesis for up to 1 hr does not abolish the stimulatory effect of erythropoietin on RNA synthesis, suggesting that the effect of the hormone on RNA synthesis is not mediated by a newly synthesized protein. 相似文献