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1.
Analogs of cyclic adenosine monophosphate (cAMP) (N6benzoyl cAMP and N6monobutyryl cAMP) as well as agents that increased the intracellular level of cAMP (glucagon and isobutylmethylxanthine) inhibited the EGF-stimulated DNA replication of adult rat hepatocytes in primary culture independently of cell density. This inhibition was strongly potentiated by the glucocorticoid dexamethasone. The effect of cAMP (and dexamethasone) was not due to toxicity, because the inhibition was reversible and the cell ultrastructure preserved. cAMP acted by decreasing the rate of transition from G1- to S-phase, the duration of G2- and S-phase of the hepatocyte cell cycle being unaffected. DNA replication started in the extranucleolar compartment of the nucleus and ended in the nucleolar compartment as described earlier for cells grown in the absence of cAMP (O.K. Vintermyr and S.O. Døskeland, J. Cell. Physiol., 1987, 132:12-21). The action of cAMP was very rapid: significant inhibition of the transition was noted 2 hr after the addition of glucagon/IBMX and half-maximal inhibition after 4 hours. The determination of extranucleolarly labelled nuclei in cells pulse-labelled with [3H]thymidine allowed precise analysis of rapid changes in the probability of transition from G1- to S-phase. The extranucleolar labelling index could also be determined in cells continuously exposed to [3H]thymidine.  相似文献   

2.
α-Amanitin acts in vitro as a selective inhibitor of the nucleoplasmic form B RNA polymerases. Treatment of Chinese hamster ovary (CHO) cells with this drug leads principally to a severe fragmentation of the nucleoli. While the ultrastructural lesions induced by α-amanitin in CHO cells and in rat or mouse liver are quite similar, the results diverge concerning the effect on RNA synthesis. It has been shown that in rat or mouse liver α-amanitin blocks both extranucleolar and nucleolar RNA synthesis. Our autoradiographic and biochemical evidence indicates that in CHO cells high molecular weight extranucleolar RNA synthesis (HnRNA) is blocked by the α-amanitin treatment, whereas nucleolar RNA (preribosomal RNA) synthesis remains unaffected even several hours after the inhibition of extranucleolar RNA synthesis. Furthermore, the processing of this RNA as well as its transport to the cytoplasm seem only slightly affected by the treatment. Finally, under these conditions, the synthesis of the low molecular RNA species (4–5S) still occurs, though less actively. The results are interpreted as evidence for a selective impairment of HnRNA synthesis by α-amanitin in CHO cells.  相似文献   

3.
RNA synthetic activity of human 2-16-cell embryos developing in vitro was studied by [3H]uridine light-microscope autoradiography. Parallelly cut thin sections were examined in the electron microscope. The first extranucleolar RNA synthesis was detected in 4-cell embryos, but nucleoli were never labelled until the 3rd cleavage (6-8-cell embryos). In 6-cell embryos the nucleolar labelling was mostly confined to a narrow peripheral zone. In later cleavage stages most of the blastomeres showed intensive labelling of nucleoli and extranucleolar chromatin. However, rather low levels of extranucleolar RNA synthesis and the absence of nucleolar activity were often seen even in blastomeres of fully compacted morulae. The activation of nucleolar RNA synthesis entailed a noticeable increase in the number of ribosomes (estimated by electron microscope morphometry) that followed a marked drop during the period between the 2-cell and 8-cell stages. The results indicate that the concentration of ribosomes in the preovulatory oocyte is a major factor of its developmental potential.  相似文献   

4.
The effect has been studied of Actinomycin D, Daunomycin (Da.), and Da. N acetyl derivative on mitotic activity and on the nucleic acid synthesis of in vitro HeLa cell cultures. The experiments were carried out by means of the radioautographic technique using stripping films. The relative uptake of thymidine-H3 and uridine-H3 was determined by means of the reduced silver grain count present in the nuclei of controls and treated cells. The mitotic activity and thymidine incorporation were noticeably reduced by Daunomycin and Actinomycin, whereas both processes appeared less affected by Da. N acetyl derivative. As regards nuclear RNA synthesis, all three antibiotics at low doses chiefly inhibit nucleolar RNA synthesis. On the other hand, whilst Actinomycin at higher doses causes an almost total inhibition of the synthesis of the whole nuclear RNA, in Daunomycin- and Da. N acetyl derivative-treated cells extranucleolar RNA synthesis is less susceptible to inhibition.  相似文献   

5.
Hepatocytes, isolated from adult (250-350 g) rats, attached and survived well in primary culture on highly diluted (less than 1 microgram/cm2) collagen gel in a synthetic medium without serum or hormones. About 20% of the cells "spontaneously" entered S phase during the first 4 days of culturing, and mitoses were easily demonstrated at the near physiological concentration (1.25 mM) of Ca++ prevailing in the medium. Cultures given 9 nM epidermal growth factor (EGF) and 20 nM insulin 20 h after inoculation showed vigorous DNA synthesis and mitotic activity. Autoradiography of such cells exposed to [3H]thymidine allowed the determination of the following cell cycle parameters: Lag period from EGF/insulin stimulation till onset of increased DNA synthesis, 17 h; rate of entry into S phase (kG1/S), 0.028/h; duration of S phase, 8.4 h; duration of G2 phase, 2.7 h. The peak DNA synthesis (pulse labelling index, 24%) and peak mitotic activity (mitotic index, 1.7%) occurred 35 and 43 h, respectively, after the stimulation with EGF/insulin. These values are comparable to those reported during the in vivo compensatory hyperplasia following partial hepatectomy of adult rats. A marked variation of the intranuclear [3H]thymidine pulse labelling pattern was noted: During the first 1.5 h of the S phase, the labelling was extranucleolar and during the last 1.5 h chiefly nucleolar. The cells survived well in the absence of glucocorticoid, whose effect on cell cycle parameters therefore could be studied. Dexamethasone (25-250 nM) did not appreciably affect the durations of S phase and G2 phase or the pattern of preferential extranucleolar and nucleolar DNA synthesis within the S phase.  相似文献   

6.
The degree of inhibition of semiconservative DNA replication induced by nickel chloride (NiCl2) was analyzed by radiolabeled-thymidine incorporation alone or with cesium chloride (CsCl) density gradient centrifugation. The onset and duration of this Ni2+-induced inhibition was time- and concentration- dependent, but the degree of inhibition was not. A maximal reduction in the rate of DNA synthesis was observed within the first hour of treatment with 2.5 mM NiCl2, which was the highest noncytotoxic concentration utilized. After six hours, 500 μM and 1 mM as well as 2.5 mM NiCl2 all produced the same 50% to 60% reduction in [3H]-thymidine incorporation into DNA. The inhibitory effect of nickel ions on DNA synthesis was reversible. The rate of DNA synthesis following a 500 μM or 1 mM NiCl2 treatment began to increase after washout of nickel, but a six-hour exposure of cells to 2.5 mM NiCl2 produced a sustained 50% to 60% suppression of DNA synthetic activity for at least 36 hours. At all concentrations of NiCl2 used in this study, some inhibition of DNA synthesis persisted for at least 48 hours, but by 72 hours after treatment, the rate of [3H]-thymidine incorporation was actually 10% above the control. Examination of autoradiographic slides of cells treated with 2.5 mM NiCl2 for six hours demonstrated a 60% reduction of silver grains, but there was no preferential reduction in the quantity of grains in the nucleolus or any other region. Cesium chloride density gradient analysis of the replication of nucleolar DNA in cells treated with 2.5 mM nickel supported the autoradiographic findings. The inhibitory effect of NiCl2 on DNA replication was prevented by the addition of magnesium chloride (MgCl2) to cells maintained in a simple salts/glucose medium (SGM). This effect did not appear to be due to an antagonism of the cellular uptake of nickel by Mg2+, since the maximally effective dose of Mg2+ reduced 63Ni2+ uptake by no more than 25% while the inhibition of replication was completely reversed.  相似文献   

7.
A HEAT-SENSITIVE CELLULAR FUNCTION LOCATED IN THE NUCLEOLUS   总被引:10,自引:3,他引:7       下载免费PDF全文
Striking nucleolar lesions occur in cultured cells after exposure to supranormal temperatures. These lesions appear at 42°C and consist of a loss of the granular ribonucleoprotein (RNP) component and intranucleolar chromatin, and a disappearance of the nucleolar reticulum. The material remaining in the morphologically homogeneous nucleolus is a large amount of closely packed fibrillar RNP. The lesions remain identical as temperature increases to 45°C. These alterations are reversible when the cells are returned to 37°C and are associated with the reappearance of an exaggerated amount of intranucleolar chromatin and granular RNP. High-resolution radioautography indicates that after thermic shock nucleolar RNA synthesis is inhibited whereas extranucleolar sites are preserved: it also suggests that the granular RNP is reconverted to fibrillar RNP probably by simple unraveling. The results prove the existence of heat-sensitive cellular functions in the nucleolus which deal with the DNA-dependent RNA synthesis. The precise site of action is assumed to involve hydrogen bonds, resulting in configurational changes in nucleolar RNP and affecting the stability of the DNA molecule. The subsequent events in nucleolar RNA synthesis are discussed in light of the morphologic and biochemical effects of actinomycin D on the nucleolus.  相似文献   

8.
Removal of serum from BHK-21/C13 cells in culture results in a decline in thymidine incorporation extending over five days. Additional removal of any of several amino acids results in a rapid decrease in incorporation of thymidine to negligible levels by 24 hours. Replacement by complete medium then provokes a synchronous wave of DNA synthesis after only ten hours with DNA synthesis first increased at six hours. Starvation for glutamine results in a rapid decline in protein synthesis over the 24 hour period when DNA synthesis is falling. However, there is considerable degradation of total protein during this period, and RNA degradation is also greatly increased. Concurrently, synthesis of RNA falls to less than 10% of that in control cells.  相似文献   

9.
Cultures of human diploid fibroblasts (HDFs) exhibiting density dependent inhibition of replication (DDIR) resumed their progression through the cell cycle following medium replacement and, after a lag period of two hours, showed a dramatic increase in the incidence of isonucleolinar 4 cells and in the levels of uptake of 3H-uridine into the nucleoli. Between five and ten hours after refeeding these nucleolar changes were maximal, leveling off at the highest values, in periods corresponding to late G1 and early S. Concomitantly, a parallel increase in the number of nucleolini per cell occurred. As cells progressed through S and G2 phases the nucleolini decreased in number and reverted to the aniso-nucleolinar type. The intensity of nucleolar labeling by 3H-uridine and its correlate, the frequency of cells with labeled nucleoli, also decreased during these cell cycle stages. Both pre- and postreplicative periods of mitotic quiescence were characterized by high levels of anisonucleolinosis (60–80% of the cells) and by very low levels of nucleolar 3H-uridine incorporation. The magnitude of these nucleolar changes occurring during G1 stage was found to be strongly dependent on: (1) the length of time of contact between the cells and the fresh medium, at least eight hours of contact being necessary for a maximal response; (2) the amount of serum in the medium, the optimal serum concentration being between 10 and 50%, and (3) the pH of the medium. The nucleolar response was completely abolished at pH values below 7.0. These nucleolar changes were very sensitive to the presence of cycloheximide (10 μg/ml) and actinomycin D (0.003 μg/ml). The behavior of the nucleoli in response to these parameters was similar to the activation response of the cells to initiate DNA synthesis. During the time period of maximal nucleolar (activation) the onset of DNA synthesis as well as the morphological and autoradiographic manifestations of the nucleolar activation were completely inhibited by very low levels of actinomycin D (Ellem and Mironescu, '72), a selective inhibitor of nucleolar RNA synthesis (Perry, '65). This suggested a possible role of nucleolar metabolism, in normal diploid cells, in the initiation of DNA synthesis. Our results, however, seem to indicate that the nucleolar changes are necessary but not sufficient for the subsequent initiation of DNA synthesis, since with graded serum concentrations or medium volumes, smaller levels of a stimulus were needed to produce maximal isonucleolinosis than to effect a maximum replicative response in the cells.  相似文献   

10.
The nucleolar and extranucleolar RNA synthesis was studied in the mink blastocysts at different stages of embryonic diapause and during the periimplantation period using cytoradioautography. The data obtained suggest a differential and stage specific activity of the embryonic mRNA and rRNA synthesis during the period of delayed implantation.  相似文献   

11.
12.
The nucleolar and extranucleolar RNA synthesis was studied in the mink blastocysts at different stages of embryonic diapause and during the periimplantation period using cytoradioautography. The data obtained suggest a differential and stage specific activity of the embryonic mRNA and rRNA synthesis during the period of delayed implantation.  相似文献   

13.
The effects of zinc on the ribonucleoprotein (RNP) constituents of HEp-2 cells have been analyzed. Pulse-chase autoradiographic experiments show a preferential inhibition of nucleolar RNA synthesis and a block in the transport of nucleolar and extranucleolar RNA in zinc-treated cells. Concomitantly with the disturbance in RNA metabolism and in protein synthesis, nucleolar condensation, accumulation of perichromatin granules and fibrils, condensation of interchromatin fibrils, and appearance of dense granular bodies occur. Accumulation of perichromatin fibrils and condensation of interchromatin fibrils appear to be related to the block in the transport of heterogeneous nuclear RNA. Depletion of certain proteins required for the assembly of RNP particles could share in the abnormal behavior of RNA and lead to the accumulation of perichromatin granules and the appearance of dense granular bodies.  相似文献   

14.
Summary Parenchyma cells of dormant tubers ofHelianthus tuberosus L. cv. OB1 (Jerusalem artichoke) contain a very low amount of hormones, therefore they respond to 2,4-D or IAA treatment by dividing and synthesizing RNA, DNA, and polyamines.In particular the activation of the dormant tissues induces an early synthesis of DNA, which reaches the maximum at 3 hours, much before the beginning of the S phase (12 hours). By supplying [6-3H] thymidine and carrying out electron microscopic autoradiography, we were able to determine that plastids and mitochondria were the organelles responsible for this early synthesis while the DNA in the nucleus first appeared labeled at 15 hours.In addition, ultrastructural observations carried out to compare the dormant cells with activated ones, showed an increase in the nucleolar volume, a different organization of the tubular complex of the plastids and several other ultrastructural changes which indicate that at 3 hours some fundamental metabolic processes are already active; they become even more evident later on.The implications of these results in the physiology of the tuber cells during activation are discussed.  相似文献   

15.
The induction of ornithine decarboxylase and the stimulation of nucleolar RNA synthesis following dilution of stationary phase Friend Leukemia Cells into fresh medium were studied. Ornithine decarboxylase activity and the rate of nucleolar RNA synthesis reached maximum values within 4 hours after dilution, with ornithine decarboxylase levels increasing 10–20 fold and nucleolar RNA synthesis increasing by about 60% during this period. 0.5 mM putrescine effectively inhibited the rise in ornithine decarboxylase following cell transfer, but did not prevent increases in the rate of nucleolar RNA synthesis.  相似文献   

16.
The rates of DNA, RNA and protein synthesis were investigated by incorporation of radioactive precursors into the excised root tips of V. faba. 2-h exposure to 0.1% caffeine resulted in inhibition of protein synthesis to about 60% of the control rate. RNA synthesis was reduced in the range of 20–30%. The same concentration of caffeine did not affect the rate of DNA synthesis even during 12-h incubation, but concentrations higher than 1% caused a significant decrease in [3H]thymidine incorporation.  相似文献   

17.
The effects of 5-hydroxyuracil (5-HU) (isobarbituric acid) upon cell elongation, mitosis, and DNA synthesis were studied in Vicia faba roots. 5-HU had no consistent effect upon root elongation. It blocked DNA synthesis (analyzed by photometric measurements of Feulgen dye in nuclei) during the first 6 hours of treatment; the block spontaneously disappeared by the 12th hour of treatment. Uracil and thymine had no effect upon this block of synthesis. Both thymidine and uridine reversed the block in 6 and 9 hours respectively. In all cases blockage of DNA synthesis was followed by inhibition of mitosis (determined by changes in the percentage of cells in mitosis) and resumption of DNA synthesis was followed by resumption of mitosis. Inhibition indices calculated from the mitotic data indicated a competitive relationship between 5-HU and thymidine and 5-HU and uridine. 5-HU is considered to block DNA synthesis by competing with thymidine for sites on enzymes involved in the synthesis. It is suggested that uridine reverses the block in synthesis by undergoing a conversion to thymidine.  相似文献   

18.
The effects of 5-hydroxyuracil (5-HU) (isobarbituric acid) upon cell elongation, mitosis, and DNA synthesis were studied in Vicia faba roots. 5-HU had no consistent effect upon root elongation. It blocked DNA synthesis (analyzed by photometric measurements of Feulgen dye in nuclei) during the first 6 hours of treatment; the block spontaneously disappeared by the 12th hour of treatment. Uracil and thymine had no effect upon this block of synthesis. Both thymidine and uridine reversed the block in 6 and 9 hours respectively. In all cases blockage of DNA synthesis was followed by inhibition of mitosis (determined by changes in the percentage of cells in mitosis) and resumption of DNA synthesis was followed by resumption of mitosis. Inhibition indices calculated from the mitotic data indicated a competitive relationship between 5-HU and thymidine and 5-HU and uridine. 5-HU is considered to block DNA synthesis by competing with thymidine for sites on enzymes involved in the synthesis. It is suggested that uridine reverses the block in synthesis by undergoing a conversion to thymidine.  相似文献   

19.
The effect of hydroxyurea and 5-fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L?1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached. FdUrd (25 mg·L?1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L?1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2-h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.  相似文献   

20.
1. By using ultrasonic treatment in media of high ionic strength, the RNA polymerase activities associated with prostatic nuclei and nucleoli can be completely solubilized. Such enzyme preparations are entirely dependent on the provision of added DNA for full activity. 2. The solubilized enzymes from the nucleolar and extranucleolar regions can be separated by ion-exchange chromatography. 3. Based on differences in the optimum DNA templates, pH optima and the effects of ammonium sulphate on the activities in vitro, Mn(2+)- and Mg(2+)-specific enzymes are associated with both the nucleolar and extranucleolar regions of prostatic nuclei. 4. Androgenic hormones administered in vivo have a particularly pronounced effect on the activity of Mg(2+)-dependent enzyme associated with the isolated prostatic nucleolus. 5. Time-course experiments in vivo show that androgens induce a rapid stimulation of the solubilized Mg(2+)-dependent nucleolar enzyme before a pronounced activation of nucleolar chromatin can be measured. 6. The implications of these findings to the mechanism of action of androgenic steroids are discussed.  相似文献   

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