A possible mechanism of inhibition of protein synthesis by dimethylnitrosamine

1. The incorporation of [¹⁴C]leucine into liver proteins of rats was measured in vivo at various times after treatment of the animals with dimethylnitrosamine and was correlated with the state of the liver ribosomal aggregates. Inhibition of incorporation ran parallel with breakdown of the aggregates. 2. Inhibition of leucine incorporation into protein and breakdown of ribosomal aggregates were not preceded by inhibition of incorporation of [¹⁴C]orotate into nuclear RNA of the liver. 3. Evidence was obtained of methylation of nuclear RNA in the livers of rats treated with [¹⁴C]dimethylnitrosamine. 4. Zonal centrifugation analysis of radioactive, nuclear, ribosomal and transfer RNA from livers of rats treated with [¹⁴C]dimethylnitrosamine revealed labelling of all centrifugal fractions to about the same extent. 5. It is suggested that methylation of messenger RNA might occur in the livers of dimethylnitrosamine-treated rats and the possible relation of this to inhibition of hepatic protein synthesis is discussed.     

Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions.

—The incorporation of [4,5-³H]lysine and [1-¹⁴C]leucine into the proteins of subcellular fractions of mouse brain was examined following a single electroconvulsive shock (ECS) or following cycloheximide injections. When the [³H]lysine was injected intraperitoneally immediately after the ECS the incorporation into total brain proteins was decreased by more than 50% as compared to sham controls. The proportion of lysine incorporated into the microsomal fraction was increased, but no changes were observed in the other subcellular fractions including the synaptosomal fraction. With extended pulses administered at various times after the ECS there was no change in total incorporation nor were selective effects seen in any subcellular fractions. With intracranial injections of both [³H]lysine and [¹⁴C]leucine the decreased incorporation caused by ECS was not observed, neither were there selective changes in any subcellular fraction. This lack of inhibition occurred because the intracranial injection itself severely inhibited [³H]lysine incorporation. Cycloheximide (30 mg/kg) which depressed [³H]lysine incorporation into brain proteins by 84% caused a selective depression of the incorporation into the cell-sap fraction and selective elevations into the microsomal and synaptosomal fractions. Similar changes were seen with a higher (amnestic) dose of cycloheximide (150 mg/kg) which inhibited incorporation by 94%. These data are interpreted in terms of the diverse mechanisms by which ECS and cycloheximide inhibit protein synthesis.     

Nuclear protein synthesis and phosphorylation in Friend erythroleukemia cells stimulated with DMSO

Patterns of nuclear protein synthesis and phosphorylation have been investigated in Friend erythroleukemia cells. The rate of incorporation of [³H]leucine and [³²P]phosphate remains relatively constant during the first 48 h of dimethylsulfoxide (DMSO) stimulation, when more than 90% of the cells commit to erythroid differentiation, but falls to 20% by 120 h. Histone H2A phosphorylation is greatly increased during DMSO treatment, but no significant changes were found in the non-histone phosphoprotein patterns as determined by gel electrophoresis. There is also a small, but reproducible, change in the relative amounts of the two sub-fractions of histone H2A. There are no striking changes in the electrophoretic patterns of [¹⁴C]leucine-labelled nuclear proteins during the first 48 h, but the amount and the synthesis of two proteins of 46 000 and 280 000 D are increased somewhat during this period. Another protein, of molecular weight 65 000, appears to be induced in low amounts.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

Pyrimidine biosynthesis and its regulation in the developing rat brain.

—Measurements of the incorporation of [¹⁴C]NaHCO₃ into orotic acid, uridine nucleotides and RNA in tissue minces establish the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in rat brain. Selective inhibition of the incorporation of various radiolabelled precursors into orotic acid by uridine demonstrates the operation of a feedback control mechanism in brain minces and indicates carbamoylphosphate synthetase to be the site of inhibition; purine nucleosides were similarly found to inhibit the de novo biosynthesis of pyrimidines. The activity of the orotate pathway, as assessed by the rate of incorporation of [¹⁴C]NaHCO₃ into orotic acid, was found to be very high in fetal brain and to decline rapidly with neurological development; the mature rat brain exhibits less than 1% of the activity of the fetal brain at 18 days of gestation. Comparative studies on the ability of minces of the brain and several extraneural tissues to utilize [¹⁴C]NaHCO₃ and [¹⁴C]aspartate as precursors of orotic acid lead us to speculate that variations in the ability of tissues to synthesize orotic acid de novo are determined by similar variations in their ability to synthesize carbamoylphosphate.     

Comparison of overall rates of RNA synthesis in implanting and delayed implanting mouse blastocysts in vitro

Implanting and delayed implanting mouse embryos were incubatedin vitro with [³H]uridine for 2–24 hr. The size and specific activity of the [³H]UTP pools were determined by means of a double isotope technique using copolymer synthesis with the [³H]UTP in the embryos, exogenous [¹⁴C]ATP, andE. coli RNA polymerase. Using the rate of incorporation of [³H]uridine into acid-insoluble material and the specific activity of the [³H]UTP pools, it was possible to calculate the overall rate of incorporation of uridine into RNA by the embryos. In implanting embryos it was constant for 24 hr. In contrast, the initial rate of uridine incorporation by the delayed implanting embryos was only 31% of that in implanting embryos (i.e., per cell); this increased steadily during the incubation period, reaching 81% of the rate in implanting embryos after 24 hr. This activation of RNA synthesis by delayed implanting embryosin vitro occurred in the absence of any uterine stimulatory factors. Further, it was shown that although 10% mouse serum would support trophoblastic outgrowthin vitro, it did not influence uptake, distribution of label into nucleotides, or rate of uridine incorporation into RNA in either implanting or delayed implanting embryos. Therefore, it is suggested that if depression and activation of metabolic activity in blastocysts are part of the mechanims of delayed implantation, and if trophoblast outgrowthin vitro is analogous to the process of implantationin vivo, then these two aspects of embryo activation are under different controls.     

Influence of Synchronized Sleep on the Biosynthesis of RNA in Neuronal and Mixed Fractions Isolated from Rabbit Cerebral Cortex

Abstract: Adult male rabbits implanted with dural electrodes were injected intraventricularly with [³H]orotate and killed 1 h later. During the period of incorporation they were left undisturbed while their EEG activity was continuously monitored. In the fraction of neuronal perikarya prepared from cerebral cortex by a method developed by Satake and Abe in 1966, the relative content of radioactive RNA of the nuclear particulate showed a twofold increase in the transition from 0 to 100% synchronization. On the other hand, a slight but significant decline was observed in the corresponding cytoplasmic compartment. A marked increase in the relative content of radioactive RNA was similarly observed in the nuclear particulate prepared from the mixed cellular fraction. The corresponding cytoplasmic compartment showed a nonsignificant increase. These results indicate that during sleep neuronal nuclei accumulate newly synthesized RNA (presumably hnRNA) at a faster rate. Under the same conditions the process of RNA transfer to the cytoplasm (presumably rRNA) may be reduced. These effects may be only partly shared by other cerebral cells.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [¹⁴C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [¹⁴C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [³H]choline or [¹⁴C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [¹⁴C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [¹⁴C] ethanolamine into [¹⁴C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

Incorporation of Label from Acetate and Laurate into the Mannan of Leishmania donovani via the Glyoxylate Cycle

Leishmania donovani promastigotes in late-stationary phase incorporated label from [2-¹⁴C]acetate and [1-¹⁴C]laurate into the mannose residues of mannan, thus confirming the presence of a functional glyoxylate bypass in these parasitic protozoa. Isolated, washed calls also incorporated label from [2-¹⁴C]acetate and [1-¹⁴C]laurate into mannan during a 1-hr incubation in buffer. Glucose had no effect on label incorporation into mannan, but glutamate caused over a four-fold increase in incorporation from [2-¹⁴C]acetate and a 2.4-fold increase from [1-¹⁴C]laurate. Staurosporine, a protein kinase inhibitor that inhibits glutamate and alanine oxidation, did not inhibit label incorporation from [2-¹⁴C]acetate into mannan. Hyperosmolality caused about a 33% inhibition of label incorporation into mannan. These results show the glyoxylate cycle and/or the subsequent biosynthetic pathway from fructose-6-phosphate to mannan are subject to regulation.     

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Compartmentation with respect to the synthesis of heterogeneous nuclear RNA and precursors to ribosomal RNA.

The incorporation of [14C]orotate and [14C]uridine into UMP residues of hnRNA (heterogeneous nuclear RNA) and pre-rRNA (precursors to rRNA) of Eharlich ascites-tumour cells was compared: orotate was incorporated at a markedly higher rate into hnRNA. On the other hand, the rate of incorporation of uridine into pre-rRTNA was even somewhat higher than into hnRNA. The ratio of specific radioactivities of CMP to UMP residues in pre-rRNA and hnRNA was studied. At all times of labelling this ratio was similar for both RNA species independently of the precursor used. On addition of excess unlabelled uridine, the CMP/UMP labelling ratio in both pre-rRNA and hnRNA rose. However, this increase was much more pronounced with hnRNA. It is concluded that nuclear pyrimidine nucleotide pool for RNA synthesis is compartmentalized. The synthesis of hnRNa is supplied preferentially by the large and the small compartment, respectively. A detailed model for the cellular compartmentation of uridine nucleotide precursors to RNA is proposed.U     

Effect of a single dose of dimethylnitrosamine on biosynthesis of nucleic acid and protein in rat liver and kidney

1. Administration of a single dose of dimethylnitrosamine to rats temporarily fed on a protein-deficient diet causes a high incidence of kidney tumours. The effect of such a dose of dimethylnitrosamine (40mg/kg body wt.) on metabolism of nucleic acids and protein in rat liver and kidneys was examined during the week immediately after administration. 2. Incorporation of [(14)C]leucine and [(14)C]orotate into hepatic macromolecules was inhibited within 5h of injection of dimethylnitrosamine, and did not recover for at least 5 days. Interpretation of these results is complicated by the concomitant extensive hepatic necrosis. 3. Renal RNA synthesis was assayed by incorporation of [(14)C]orotate in vivo and measurement of DNA-dependent RNA polymerase activity in vitro. Both systems indicate biphasic inhibition; minimal activity was recorded 9h and 3 days after treatment. Changes in incorporation of [(14)C]leucine into renal protein were similar but less marked. 4. Sucrose-density-gradient analysis of renal cytoplasmic RNA indicated increased synthesis of rRNA 24h after injection of the nitrosamine. The rate of loss of radioactivity from kidney ribosomes pre-labelled with [(14)C]orotate was not modified by dimethylnitrosamine. 5. Dimethylnitrosamine increased incorporation of [(3)H]-thymidine into renal DNA. The three distinct periods of stimulated synthesis observed are discussed, with particular reference to recently published morphological studies of the sequential development of kidney tumours induced by dimethylnitrosamine in protein-depleted rats.     

Errors from using 3H-labeled ribonucleoside triphosphates to monitor nuclear RNA synthesis in vitro

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.     

Collagen and elastin synthesis in the developing chick aorta

Thoracic aortas from 8- to 18-day embryonic chicks were incubated in vitro for 30 min with [³H]glycine and the newly synthesized, labeled proteins were subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The gels were fractionated and the incorporation of label into procollagen (125,000 M_r) and tropoelastin (70,000 M_r) was estimated by summation of the radioactivity found in the appropriate regions of the gel. The analyses showed that at Day 8 approximately 14% of the incorporated [³H]glycine was found in procollagen and 22% in tropoelastin. In the following 6 days of development, there was a significant decline in the relative incorporation into procollagen and an increase into tropoelastin so that at Days 14–18 less than 10% of the label was found in collagen and 40% was now found in tropoelastin. Since glucocorticoids have been shown to alter the rate of synthesis of other proteins in the developing chick, 150 μg of hydrocortisone was injected into 8-day eggs and 24 h later the aortas were incubated and treated as described above. The pattern of protein synthesis exhibited by the hormone-treated aortas resembled that of 14- to 18-day embryos. Furthermore, incubation of 8-day aortas with 10^?8m hydrocortisone for 24 h produced a significant increase in the rate of elastin synthesis relative to that of other proteins. These results demonstrate that collagen and elastin synthesis vary during development of the chick aorta and they suggest that glucocorticoids may be involved in the control of their synthesis.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

REVERSIBLE, THERMOTROPIC ALTERATION OF NUCLEAR MEMBRANE STRUCTURE AND NUCLEOCYTOPLASMIC RNA TRANSPORT IN TETRAHYMENA

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [¹⁴C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28° to 18°C (or above) decreases [¹⁴C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18°C but above 10°C causes the reverse. [¹⁴C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20° and 16°C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.     

Regulation of Pyrimidine Biosynthesis in Intact Cells of Cucurbita pepo

The occurrence of the complete orotic acid pathway for the biosynthesis de novo of pyrimidine nucleotides was demonstrated in the intact cells of roots excised from summer squash (Cucurbita pepo L. cv. Early Prolific Straightneck). Evidence that the biosynthesis of pyrimidine nucleotides proceeds via the orotate pathway in C. pepo included: (a) demonstration of the incorporation of [¹⁴C]NaHCO₃, [¹⁴C]carbamylaspartate, and [¹⁴C]orotic acid into uridine nucleotides; (b) the isolation of [¹⁴C]orotic acid when [¹⁴C]NaHCO₃ and [¹⁴C]carbamylaspartate were used as precursors; (c) the observation that 6-azauridine, a known inhibitor of the pathway, blocked the incorporation of early precursors into uridine nucleotides while causing a concomitant accumulation of orotic acid; and (d) demonstration of the activities of the component enzymes of the orotate pathway in assays employing cell-free extracts.     

β-Hydroxybutyrate as a Precursor to the Acetyl Moiety of Acetylcholine

Abstract— Rat brain cortex slices were incubated with 10 mm -glucose and trace amounts of [6-³H]glucose and [3-¹⁴C]β-hydroxybutyrate. The effects of (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase; methylmalonate, an inhibitor of β-hydroxybutyrate dehydrogenase; and increasing concentrations of unlabeled acetoacetate were examined. The incorporation of label into lactate, citrate, malate, and acetylcholine (ACh) was measured and ³H:¹⁴C ratios calculated. Incorporation of [¹⁴C]β-hydroxybutyrate into lactate was limited because of the low activity of gluconeogenic enzymes in brain, whereas incorporation of ¹⁴C label into Krebs cycle intermediates and ACh was higher than in previous experiments with [³H-,¹⁴C]-glucose. (–)-Hydroxycitrate (5.0 m^M) reduced incorporation of [³H]glucose and [¹⁴C]β-hydroxybutyrate into ACh. In contrast, slices incubated with methylmalonate (1 mm ) showed a decrease in ¹⁴C incorporation without appreciably affecting glucose metabolism. The effects of high concentrations of methylmalonate were nonselective and yielded a generalized decrease in metabolism. Acetoacetate (1 mm ) also produced a decreased ¹⁴C incorporation into ACh and its precursors. At 10 mm , acetoacetate reduced ³H and ¹⁴C incorporation into ACh without substantially affecting total ACh content. From the results, it is suggested that in adult rats β-hydroxybutyrate can contribute to the acetyl moiety of ACh, possibly via the citrate cleavage pathway, though it is quantitatively less important than glucose and pyruvate. This contribution of ketone bodies could become significant should their concentration become abnormally high or glucose metabolism be reduced.     

Inhibitory effects of orotate on precursor incorporation into nucleic acids.

The influence of orotic acid on the incorporation of precursors into nucleic acids was studied in mice and rats and in isolated cells. In vivo, orotate levels were modified by two diets which are known to increase the rate of pyrimidine nucleotide synthesis in rat liver. Of these diets, a 1% orotate diet had greater inhibitory effects than an arginine-deficient diet on the incorporation of [3H]orotate into RNA of mouse kidney than mouse liver. This contrasted with the situation in the rat where there was a greater effect in the liver than the kidney. The situation in the rat was more readily interpreted than in the mouse in terms of previously established effects of these diets on ribonucleotide pool sizes. However, studies using [3H]adenosine as a precursor for incorporation into RNA suggested that even in the mouse the effects of orotate were on pool sizes rather than an inhibitory effect on RNA synthesis. The incorporation of [3H]thymidine into DNA was inhibited by orotate to a similar degree in cultured HTC hepatoma cells and a line of rat liver epithelial cells. An effect on DNA synthesis rather than solely on pool sizes was suggested by the observation that the pool size of dTTP was not increased by 5 mM orotate under conditions in which there was a four-fold increase in the level of UTP in HTC cells. An inhibitory effect of orotate on DNA synthesis was further supported by an observation of decreased incorporation of [3H]deoxyadenosine into DNA and a lower rate of cellular proliferation.     

Relation between DNA synthesis and germination of vaccaria Pyramidata seeds

Dormant seeds of Vaccaria pyramidata are characterized by a high level of [¹⁴C]Ieucine incorporation into proteins, a high level of [³H]uracil incorporation into RNA and an undiminished synthesis of poly(A)⁺-RNA; however, DNA synthesis is very much reduced. Germination of dormant Vaccaria seeds can be induced either partially by cytokinins or more completely by cytokinins and ethylene. Cytokinins cause a drastically increased [³H]thymidine incorporation in cotyledons of imbibing seeds. This effect is enhanced by ethylene. The incorporation of [³H]thymidine in radicles is also stimulated, but to a lesser extent. Cytokinin-dependent stimulation of [³H]thymidine incorporation can be referred to as an activation of nuclear DNA synthesis.