Methylation of newly synthesized ribonucleic acid by isolated rat liver nuclei. Characterization of the ribonucleic acid synthesized by nuclei from starved animals

1. Nuclei from rat liver incubated with S-adenosyl[methyl-(14)C]methionine incorporated radioactivity into RNA and into lipid and protein. 2. All of the labelled RNA was extracted from the nuclei with trichloroacetic acid at 90 degrees C. 3. The [(14)C]methyl-group incorporation into the hot-trichloroacetic acid extract was 30% inhibited by the addition of actinomycin D (100mug/mg of DNA) or by the omission of CTP, GTP and UTP. 4. Assuming that the main substrate for this triphosphate-dependent methylation was newly synthesized precursor rRNA containing one methyl group/30 uridylate residues, it was calculated that approx. 60% of the [(14)C]UMP incorporated under similar conditions represented precursor rRNA synthesis. 5. In agreement with this, low concentrations of actinomycin D (approx. 1mug/mg of DNA) sufficient to abolish the triphosphate-dependent incorporation of [(14)C]methyl group inhibited 68% of the [(14)C]UMP incorporation. 6. The incorporation of [(14)C]UMP by nuclei from starved animals decreased progressively with increasing periods of starvation, whereas the triphosphate-dependent [(14)C]methyl-group incorporation was not further decreased after 1 day of starvation. 7. This suggests that precursor rRNA synthesis decreased within 1 day whereas other species of RNA were affected only after longer periods of starvation.     

The effect of amino acid starvation on nucleoside uptake and RNA synthesis in Tetrahymena

The uptake of nucleosides and the synthesis of RNA in Tetrahymena thermophila were examined following amino acid starvation. Omission of leucine, phenylalanine, or arginine from the medium resulted in a rapid decrease in the incorporation of [3H]uridine into the acid-soluble pool and acid-insoluble material (RNA). Amino acid starvation inhibited the uptake of all ribo- and deoxyribonucleosides tested but did not affect the uptake of amino acids or glucose. In addition, under the conditions used, the omission of an amino acid did not result in a large decrease in amino acid incorporation into total protein. Treatment of cells with cycloheximide or emetine gave results similar to the effects of amino acid starvation, but in these experiments the inhibition of protein synthesis was essentially complete. Nucleotide pool sizes were also measured following amino acid starvation. ATP and UTP levels were essentially unchanged, but the dTTP pool size was decreased by 40%. The decrease in RNA synthesis in vivo in the absence of an essential amino acid was reflected in the endogenous RNA synthetic activity of isolated nuclei. However, when solubilized RNA polymerase activity was measured with calf thymus DNA as template, no significant difference was observed between control and amino acid-starved cells.     

Experimental conditions affecting ribonucleic acid polymerase in isolated rat liver nuclei. Effect of nucleoside triphosphate concentration, temperature, ammonium sulphate and heparin

1. The conditions affecting the activity of RNA polymerase in isolated rat liver nuclei were studied with Mg(2+) or Mn(2+) as activating ions. 2. The enzyme assayed with Mg(2+) and at low ionic strength is saturated by a lower concentration of nucleotide substrates than if assayed with Mn(2+) at low ionic strength or with either ion at high ionic strength. 3. At low and at high ionic strength the incorporation of AMP is affected in a similar way by variations in the temperature of incubation. Preincubation at 37 degrees impairs the AMP incorporation. 4. Heparin stimulates the RNA polymerase activity in the presence of Mn(2+). 5. Both ammonium sulphate and heparin ;restart' the reaction if added after 15min., the effect being more marked with ammonium sulphate than with heparin, and also more marked in the presence of Mn(2+) than of Mg(2+). 6. alpha-Amanitin abolishes the effect of ammonium sulphate and of heparin.     

The effects of starvation and refeeding on egg laying and the synthetic activity of the albumen gland in Bulinus truncatus, a snail vector of urinary schistosomiasis

1. The effects of starvation (for 1, 2, 3, 6, 9 and 12 days, respectively) and refeeding (12 days starvation and 1, 2 and 3 days refeeding, respectively) on egg laying and albumen gland activity in the freshwater snail, Bulinus truncatus were studied. 2. The egg laying of starving snails rapidly decreased and ceased by day 6 of starvation. Egg laying was restored 24 hr after refeeding. The recorded decrease in albumen gland wet weight was proportional to the starvation periods. The DNA contents of the glands of the different experimental groups was not statistically different from the controls. 3. Albumen gland synthetic activity expressed as 14C-glucose incorporation into galactogen/microgram DNA and 3H-amino acids into total protein was determined. The glands showed an abrupt decrease in synthetic activity after 1 day of starvation and gradually decreased further until days 9-12. The decrease in activity of the glands was more rapid than that of egg laying. Upon refeeding, the activity of the glands recovered rapidly, simultaneous with the increase in wet weight and egg laying. 4. In conclusion, there is a correlation between egg laying and the in vitro activity of albumen glands. The results show a short-term effect of starvation on the fecundity of the snails. Such studies could be useful in field studies as well as snail control by applying molluscicides under optimal conditions.     

短期饥饿和再投喂对岩原鲤仔鱼生存生长以及RNA/DNA和RNA/蛋白质比率的影响(英文)

研究通过对岩原鲤仔鱼在饥饿和再投喂条件下其生存、生长率、RNA/DNA和RNA/蛋白质比率的测定,评估了仔鱼对饥饿的耐受能力和恢复能力。在(19.5±0.5)℃水温下,将岀膜后第16天的岩原鲤仔鱼随机分成6个组:1个持续投饲对照组,实验组分别禁食1、2、3、4、5d后再投喂,实验共进行10d。每天分别从各组取9尾鱼测定体重、体长、RNA、DNA、蛋白质含量。实验结果显示,饥饿处理组仔鱼存活率和以上各项生长指标均随饥饿时间的增加而下降,在恢复投喂后均表现不同程度的补偿生长,其中饥饿1、2、3d的仔鱼在恢复投喂后显示出完全补偿生长,几乎弥补了饥饿所产生的影响,平均终体重与对照组比较无显著差异。饥饿4、5d的仔鱼显示部分补偿生长,恢复投喂只少量减轻了饥饿的影响,平均终体重与对照组相比存在显著差异。饥饿1、2、3d的仔鱼和4、5d的仔鱼在恢复投喂后分别需要1—2d和4d时间才能达到与对照组无显著差异水平。仔鱼生长率变动范围从0.59%到8.00%WW/day,仔鱼RNA/DNA比率、RNA/蛋白质比率与生长率的回归方程为:GR=3.63RNA/DNA 1.74(R2=0.80)和GR=120.14RNA/Protein 2.33(R2=0.31),两种比率均与生长率呈显著线性相关,RNA/DNA比率对生长变化的拟合度更好。结果表明,仔鱼阶段食物缺乏很可能是影响岩原鲤仔鱼存活、生长的主要因素。RNA/DNA更适合作为评定岩原鲤仔鱼营养条件和生长的指标。     

Studies on the pathogenesis of liver necrosis by α-amanitin. Effect of α-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei

1. Injection of alpha-amanitin to mice causes a decreased incorporation of [6-(14)C]-orotic acid into liver RNA in vivo. 2. The activity of RNA polymerase activated by Mn(2+) and ammonium sulphate is greatly impaired in liver nuclei isolated from mice poisoned with alpha-amanitin, and is inhibited by the addition of the same toxin in vitro. 3. The activity of the Mg(2+)-activated RNA polymerase is only slightly affected by alpha-amanitin either administered to mice or added in vitro.     

Isolation of chick intestinal nuclei. Effect of vitamin D3 on nuclear metabolism

1. Chick intestinal nuclei were isolated, with practically no contamination from other organelles and whole cells, by centrifugation through 2.4m-sucrose. 2. The proportions of RNA, DNA and protein of the isolated nuclei were unaffected by the vitamin D status of the birds. The RNA/DNA ratio was 0.15. 3. The incorporation of [5-(3)H]orotic acid into the rapidly labelled intestinal nuclear RNA, after a 10min. pulse of the orotic acid, was increased in vitamin D-deficient chicks only 10min. after a 125mug. dose of cholecalciferol. 4. There was no stimulation of the DNA-dependent RNA polymerase activity of the isolated nuclei from birds treated with cholecalciferol. 5. The results are discussed in relation to the changes occurring during the lag period, after administration of cholecalciferol and before Ca(2+) transport is detected, and the function of the vitamin.     

Inhibition of cell division in amoebae: the incorporation of tritiated precursors into Amoeba proteus after the injection of non-homologous cytoplasm.

The injection of non-homologous cytoplasm into any strain of large free-living amoebae leads to a 60% inhibition of division amongst recipient cells. When the post-microsomal supernatant fraction of Amoeba discoides was injected into A. proteus, this inhibition of division was as high as 95%. The incorporation of tritiated precursors, either [3H]uridine or 3H-amino acids, into these inhibited amoebae was studied at various times after the injection of the inhibitory material using autoradiography. When cells were grown in [3H]uridine, autoradiographs indicated that RNA synthesis had ceased 2 days after the injection of non-homologous material. However, if [3H]uridine was injected into the inhibited cells, some synthesis of RNA could be detected up to 4 days after the injection of inhibitor. These results suggested that uptake of [3H]uridine was impaired and that one site of action of the inhibitory molecules was RNA synthesis for membrane components. Experiments with a variety of 3H-amino acids suggested that protein synthesis continued for at least 9 days after the injection of non-homologous cytoplasm, and that in these cells some informational RNA molecules were long-lived. There seemed to be accumulation of material containing [3H]lysine in the nuclei of control cells taken at random from cultures, and this was seen in the nuclei of inhibited cells 1 day after injection. However, 2 days after the injection of inhibitor, no accumulation of [3H]lysine-containing material was found in the nuclei.     

Fractionation of nuclei from brain by zonal centrifugation and a study of the ribonucleic acid polymerase activity in the various classes of nuclei

1. The nuclei of the cells of the whole rat brain have been fractionated in a B-XIV zonal rotor with a discontinuous gradient of sucrose. Five fractions were obtained. Zone (I) contained neuronal nuclei (70%) and astrocytic nuclei (23%). Zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (15%). Zone (III) contained astrocytic nuclei (84%) and oligodendrocytic nuclei (15%). Zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained only oligodendrocytic nuclei. 2. The content of DNA, RNA and protein per nucleus was determined for each zone. Although the amount of DNA per nucleus is constant (7pg) the RNA varies from 4.5 to 2.5pg/nucleus and the protein from 38 to 17.6pg/nucleus. The neuronal nuclei have the greatest amounts of protein. The oligodendrocytic nuclei have the least content of RNA and protein. 3. The effects of pH, ionic strength, and Mg(2+) and Mn(2+) concentration on the activity of the nuclear system for synthesis in vitro of RNA have been investigated for unfractionated nuclei. From these studies a standard set of conditions for the assay of nuclear RNA polymerase has been established. 4. The activity of the RNA polymerase in each of the zonal fractions has been determined in the presence and in the absence of alpha-amanitin. Zone (II) is the most active, followed by zone (I). The nuclei of zones (IV) and (V) have comparable activity, which is 40% of that of zone (II). 5. The extent of incorporation of each of the four labelled nucleoside triphosphates by the nuclei from each zone has been measured. These values have been used to calculate the base composition of the RNA synthesized in vitro in each class of nucleus. 6. The effect of changes in the condition of assay of RNA polymerase in the different classes of nuclei has been investigated. Significant differences in the response to concentrations of metal ions and ammonium sulphate have been observed. 7. Homopolymer formation in each zone of brain nuclei has been determined. The extent of formation of the four homopolymers roughly parallels the RNA polymerase activity.     

Effect of fasting and refeeding on in vitro muscle cell proliferation in rainbow trout (Oncorhynchus mykiss)

The effects of short-term fasting and refeeding were studied on satellite cells extracted from white epaxial muscle of juvenile rainbow trout (1-3 g body weight). In vitro changes in the proliferation of satellite cells were analyzed using bromodeoxyuridine (BrdU) incorporation over a 24-h period. Proliferation in fed control fish was characterized by an initial basal proliferation rate of 5-10% BrdU-labeled nuclei x day(-1), followed by an exponential increase at a rate of +18-20% x day(-1), up to a maximum of 60-70% BrdU-labeled nuclei x day(-1). Characteristics of satellite cells extracted from starved fish, namely extraction yield, morphology, and proliferation, were different from those of fed fish. Fasting (8-10 days) completely suppressed initial proliferation of satellite cells in vitro over a period of 4 days. After this delay, proliferation resumed and changes in proliferation rates over time were similar to those of the control group. In fish fed for 4 days after an 8-day fast, the initial proliferation rate and the changes in proliferation rates over time were completely restored. These findings demonstrate that satellite cells express different behavior depending on feeding status, which could be due to the presence of different satellite cell populations.     

The effect of aflatoxin B1 on normal and cortisol-stimulated rat liver ribonucleic acid synthesis

1. Aflatoxin B(1), administered in vivo, inhibits the incorporation of [(14)C]orotic acid in vivo into rat liver nuclei, and also inhibits both Mg(2+)- and Mn(2+)-dependent RNA polymerase activities in nuclei assayed in vitro. 2. Aflatoxin B(1) inhibits the cortisol-induced increase in incorporation of [(14)C]leucine in vivo, but does not affect the control value of this activity. 3. Aflatoxin B(1) administered in vivo inhibits the increase in nuclear Mg(2+)-dependent RNA polymerase activity, assayed in vitro, which results from the treatment with cortisol. 4. Adrenalectomy causes a decrease in Mg(2+)-dependent RNA polymerase activity. The effect on this enzymic activity of adrenalectomy plus treatment with aflatoxin B(1) is no greater than that of treatment with aflatoxin B(1) alone. 5. These results suggest that the inhibition of cortisol-stimulated biochemical pathways by aflatoxin B(1) is due to an inhibition of cortisol-stimulated RNA synthesis. 6. The cytoplasmic action of aflatoxin is thought to be due to a competition for receptor sites on the endoplasmic reticulum between steroid hormones and aflatoxin B(1). No evidence was obtained for a similar competition for nuclear receptor sites between [(3)H]cortisol and aflatoxin B(1). 7. No differences were observed between the activities of RNA polymerase preparations solubilized from control or aflatoxin-inhibited nuclei. 8. No differences in ;melting' profiles were observed between DNA and chromatin preparations isolated from control nuclei or from aflatoxin-inhibited nuclei. 9. It is suggested that aflatoxin B(1) exerts its effect on RNA polymerase by decreasing the template capacity of the chromatin and that the aflatoxin ;target' area of the chromatin includes that region which is stimulated by cortisol. This process, however, does not involve inhibiting the movement of cortisol from the outside of the hepatic cell to the nuclear chromatin.     

Catecholamines inhibit Ca(2+)-dependent proteolysis in rat skeletal muscle through beta(2)-adrenoceptors and cAMP

Overall proteolysis and the activity of skeletal muscle proteolytic systems were investigated in rats 1, 2, or 4 days after adrenodemedullation. Adrenodemedullation reduced plasma epinephrine by 95% and norepinephrine by 35% but did not affect muscle norepinephrine content. In soleus and extensor digitorum longus (EDL) muscles, rates of overall proteolysis increased by 15-20% by 2 days after surgery but returned to normal levels after 4 days. The rise in rates of protein degradation was accompanied by an increased activity of Ca(2+)-dependent proteolysis in both muscles, with no significant change in the activity of lysosomal and ATP-dependent proteolytic systems. In vitro rates of Ca(2+)-dependent proteolysis in soleus and EDL from normal rats decreased by ~35% in the presence of either 10(-5) M clenbuterol, a beta(2)-adrenergic agonist, or epinephrine or norepinephrine. In the presence of dibutyryl cAMP, proteolysis was reduced by 62% in soleus and 34% in EDL. The data suggest that catecholamines secreted by the adrenal medulla exert an inhibitory control of Ca(2+)-dependent proteolysis in rat skeletal muscle, mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.     

The effect of feeding with a tryptophan-free amino acid mixture on rat liver magnesium ion-activated deoxyribonucleic acid-dependent ribonucleic acid polymerase

1. The Widnell & Tata (1966) assay method for Mg(2+)-activated DNA-dependent RNA polymerase was used for initial-velocity determinations of rat liver nuclear RNA polymerase. One unit (U) of RNA polymerase was defined as that amount of enzyme required for 1 mmol of [(3)H]GMP incorporation/min at 37 degrees C. 2. Colony fed rats were found to have a mean RNA polymerase activity of 65.9muU/mg of DNA and 18h-starved rats had a mean activity of 53.2muU/mg of DNA. Longer periods of starvation did not significantly decrease RNA polymerase activity further. 3. Rats that had been starved for 18h were used for all feeding experiments. Complete and tryptophan-deficient amino acid mixtures were given by stomach tube and the animals were killed 15-120min later. The response of RNA polymerase to the feeding with the complete amino acid mixture was rapid and almost linear over the first hour of feeding, resulting in a doubling of activity. The activity was still elevated above the starvation value at 120min after feeding. The tryptophan-deficient amino acid mixture produced a much less vigorous response about 45min after the feeding, and the activity had returned to the starvation value by 120min after the feeding. 4. The response of RNA polymerase to the feeding with the complete amino acid mixture was shown to occur within a period of less than 5min to about 10min after the feeding. 5. Pretreatment of the animals with puromycin or cycloheximide was found to abolish the 15min RNA polymerase response to the feeding with the complete amino acid mixture, but the activity of the controls was unaffected. 6. The characteristics of the RNA polymerase from 18h-starved animals and animals fed with the complete or incomplete amino acid mixtures for 1h were examined. The effects of Mg(2+) ions, pH, actinomycin D and nucleoside triphosphate omissions were determined. The [Mg(2+)]- and pH-activity profiles of the RNA polymerase from the animal fed with the complete mixture appeared to differ from those of the enzyme from the other groups, but this difference is probably not significant. 7. [5-(3)H]Orotic acid incorporation by rat liver nuclei in vivo was shown to be affected by the amino acid mixtures in a similar manner to the RNA polymerase. 8. The tryptophan concentrations of plasma and liver were determined up to 120 min after feeding with the amino acid mixtures. Feeding with the complete mixture produced a rapid increase in free tryptophan concentrations in both plasma and liver, but feeding with the incomplete mixture did not alter the plasma concentration. The liver tryptophan concentration increased at about 45min after feeding with the tryptophan-deficient diet. 9. There was a good correlation between the liver tryptophan concentration and RNA polymerase activity in all groups of animals. 10. It was concluded that the rat liver nucleus responded to an increase in amino acid supply by increased synthesis of RNA as a result of synthesis of RNA polymerase de novo. The correlation of tryptophan concentration and RNA polymerase activity appears to reflect the general amino acid concentration required to support hepatic protein synthesis and to produce new RNA polymerase. This new polymerase appears to differ from the basal RNA polymerase by its rapid synthesis and destruction, which may be a means of regulating RNA synthesis by the amino acid concentration in the liver.     

Intracellular Substrates for Endogenous Metabolism During Long-Term Starvation of Rod and Spherical Cells of Arthrobacter crystallopoietes

Cells of Arthrobacter crystallopoietes, harvested during growth as spheres and as rods, were starved by shaking at 30 C in phosphate buffer for 30 days, during which time they maintained 100% viability. Changes in cellular components and the activity of specific enzyme pathways were monitored. A glycogen-like polysaccharide comprised 40% of the dry weight of growing spherical cells and 10% of the dry weight of rod cells. This material was utilized at approximately the same rate, on a percentage basis, during starvation of both cell forms. The rods degraded intracellular protein at approximately twice the rate of the spheres. At the end of 30 days, the rods had degraded 40% and the spheres 20% of their initial content of protein. Ribonucleic acid (RNA) was degraded significantly more rapidly in the rods. After 30 days starvation, 85 and 32% of the initial RNA of rods and spheres, respectively, had been depleted. Magnesium ion followed this same general pattern; the rods lost 65% and the spheres 45% of their initial content during 28 days of starvation. Deoxyribonucleic acid increased by 20% during the first few hours of starvation of both cell forms and then remained constant. The ability of glucose-, succinate-, and 2-hydroxypyridine (2-HP)-grown cells to oxidize glucose remained constant during 14 days of starvation. The ability of succinate-grown cells to oxidize succinate decreased rapidly during the first few hours of starvation to a rate which remained constant for 14 days. Cells adapted to growth on 2-HP completely lost their ability to oxidize this substrate after 3 days starvation.     

Changes of beta-endorphin and somatostatin concentrations in different hypothalamic areas of female rats after chronic starvation

To study the effect of starvation on hypothalamic beta-endorphin and somatostatin (SRIF) concentrations in relation to starvation induced anestrus, groups of 8 rats were fed 50% of their normal daily chow consumption. Rats were sacrificed after 4, 8, 12, and 16 days during diestrus or anestrus. beta-endorphin concentrations decreased in the preoptic suprachiasmatic area (0.52 +/- 0.13 vs 0.21 +/- 0.05 ng/mg tissue wet weight) and increased in the posterior hypothalamus (0.31 +/- 0.06 vs 0.57 +/- 0.11 ng/mg) after 4 days of starvation. No significant change occurred in the arcuate nucleus or in the median eminence. On day 8 and 12 of starvation, beta-endorphin was unaltered in all areas compared to controls. Vaginal smears showed constant diestrus in a significant number of rats (5 out of 8) after 12 days. beta-endorphin concentrations in the arcuate nuclei of these rats were significantly reduced on day 16 (1.00 +/- 0.33 vs 0.30 +/- 0.11 ng/mg). The SRIF levels changed only in the median eminence with increased concentrations on day 12 (45.2 +/- 8.4 vs 79.5 +/- 14.8 ng/mg). At this time serum levels of luteinizing hormone (LH), prolactin (PRL), and growth hormone (GH) were significantly reduced. The results indicate that changes in hypothalamic beta-endorphin accompany the events leading to starvation induced anestrus.     

Biochemical and Molecular Responses to Loss of Renal Mass: Membranes and Protein Synthesis: Metabolic Response to Renal Compensatory Growth

Forty-eight hours after unilateral nephrectomy in young male Sprague-Dawley rats the concentrations of free methionine, alanine and tyrosine in renal cortical tissue were increased by 15-65 percent while the corresponding plasma concentrations decreased by 23-35 percent. The renal cortical concentrations of valine and leucine increased by 41 percent and 26 percent while plasma concentrations remained unchanged. The cortical concentrations of ornithine, serine and threonine remained unchanged while the plasma concentration decreased by approximately one-third. The total free amino acid contained in the cortex was not changed, while total free amino acids in plasma decreased by 7 percent. These data are thought to reflect an increased uptake of methionine and tyrosine into renal cells during compensatory hypertrophy, and an increased incorporation into renal protein of serine, threonine and ornithine. All these changes as well as all other biochemical changes accompanying compensatory hypertrophy with the exception of an increase of the RNA/DNA ratio were prevented by starvation for 48 hours after unilateral nephrectomy.In young male Sprague-Dawley rats and adult male Charles River mice, the incorporation of ¹⁴C-choline into acid-insoluble phospholipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin) was already accelerated 5 minutes after contralateral nephrectomy and further rose to +68 ± 7 percent within 20 minutes to 3 hours. Incorporation of ¹⁴C-choline into phospholipids remained accelerated for two to three days and reflected increased rates of phospholipid synthesis rather than increased choline uptake. Three hours after unilateral nephrectomy in mice, incorporation of i.p. injected ¹⁴C-choline into phospholipids was accelerated 25 percent. The rate of turnover of free labelled renal phospholipids was not accelerated during compensatory renal growth. The very early increase of choline incorporation into phospholipids after contralateral nephrectomy, therefore, appears to reflect an increased rate of synthesis of membrane material.