Pyrimidine biosynthesis in rat brain

—Studies on the incorporation of [¹⁴C]NaHCO₃ into both orotic acid and RNA in tissue slices reveal the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in the rat brain. A comparison of the rates of incorporation of bicarbonate into orotic acid and RNA in tissue slices of brain and liver indicate the brain to be one-fourth to one-half as active as the liver in the de novo biosynthesis of pyrimidines. The results of this study favor the proposal that the adult rat brain can meet its needs for pyrimidines through de novo synthesis and is not dependent upon salvage activity and an extraneural supply of pyrimidines.     

Effect of Endotoxin and Cortisone on Synthesis of Ribonucleic Acid and Protein in Livers of Mice

The effect of cortisone and endotoxin, singly and in combination, on ribonucleic acid (RNA) synthesis in livers of adrenalectomized mice was determined. This was accomplished by measuring the incorporation either of inorganic (32)P or of (14)C-orotic acid into the RNA. Under similar conditions, the effect of these agents on the rate of protein synthesis was examined with the use of (14)C-leucine. Bacterial endotoxin was found to augment the uptake of isotope in the RNA and in the protein of the liver. These reactions did not appear to be mediated via the pancreatic hormone insulin, which was found to depress the incorporation of the radioactive compounds into RNA. Cortisone increased the uptake of isotope in liver RNA but depressed the incorporation of leucine into hepatic protein. These results indicate that the previously observed ability of endotoxin to prevent the hormone induction of hepatic enzymes, such as tryptophan oxygenase, is not associated with impaired synthesis of liver RNA or protein.     

Incorporation of orotic acid into nucleotides and RNA in mouse organs during 60 minutes.

Mouse kidney and liver were found to increase their levels of radioactivity above that of serum from 2 to 60 min after administration of [6-14C]orotic acid. In spleen, thymus and brain, the radioactivity level reached a maximum soon after the injection and then decreased, as did that in serum. Sixty minutes after the injection, 44% of the administered isotope dose was found in the kidneys, 22% in the liver and 0.75% in the spleen. The 14C activity in liver UTP increased rapidly and then remained constant for 60 min. The ratio between the activities in uridine phosphates and UDP-sugars was 3:4 from 10- 60 min after injection. In the liver and kidneys, the RNA 14C activities at 60 min after injection were 15% of the activity in their acid-soluble fractions. Intraperitoneal administration was found to be preferable to intravenous administration for studies on nucleotides and RNA in mouse liver, due to the delayed incorporation of the [14C]orotic acid activity into the nucleotide pool.     

14C] Alanine incorporation into proteins and their contents in the tissue of intact and adrenalectomized rabbits after hydrocortisone and cortitropin administration

A single administration of hydrocortisone to intact rabbits increases the incorporation of [14C] alanine into proteins of the brain and liver tissue homogenates and soluble fractions as well as in blood plasma proteins and reduces the label incorporation into the brain tissue proteins and reduces its incorporation into the blood plasma proteins. Adrenalcetomy is followed by an increase in the incorporation of [14C] alanine into proteins of the brain and muscle tissue homogenates and soluble fraction and into proteins of blood plasma and liver tissue homogenates as well as by reducing the label incorporation into the spleen soluble fraction proteins. ACTH administered to adrenalectomized rabbits reduces incorporation of [14C] alanine into the brain and muscle tissue proteins, total proteins of liver tissue homogenate and increases it into the proteins of the spleen tissue soluble fraction. Multiple administration of the soluble fraction hormones both to intact and adrenalectomized rabbits inhibits the label incorporation into the studied tissue proteins. Parallel with the change in [14C] alanine incorporation into proteins under the hormones effect certain shifts in their contents were also established.     

Age related changes in total DNA and RNA and incorporation of uridine and thymidine in rat brain

1. Total brain DNA and total brain RNA and the incorporation of thymidine[14C] and uridine[3H] were measured in young and aged rats. 2. From 20 days to the time of sexual maturation, both DNA and RNA levels increase. Total RNA exceeds total DNA at all ages. Comparatively, the ratio of total DNA/RNA is higher in young than in aged animals. 3. The incorporation of thymidine[14C]/g of DNA and of uridine[3H]/g of RNA decreases with age. This decrease is rapid in young animals. After 350 days of age, the incorporation becomes very low. The significance of data is discussed.     

BIOCHEMICAL EFFECTS OF THYROID DEFICIENCY ON THE DEVELOPING BRAIN

Abstract— The effects of neonatal thyroidectomy on some constituents of the cerebrum, cerebellum and liver of the rat have been studied during the first 7 weeks of life. In the normal rat between the 6th and 14th post-natal days the RNA content per unit of DNA in the brain increased by 70 per cent. Although the brain continued to grow from the 14th to the 35th day, the amount of RNA relative to DNA decreased by about 20 per cent. The ratio of protein to DNA increased during the whole period studied and in the cerebral cortex it was more than trebled between the age of 6 and 35 days. The growth of the cerebellum extended over a longer period than that of the cerebrum, its weight increasing by 88 per cent between the ages of 14 and 35 days as compared with a cerebral increase of 34 per cent. The DNA content showed a 50 per cent increase during this period. Qualitatively these maturational changes were not affected by neonatal thyroidectomy. Quantitative changes, which applied equally to the cerebral cortex and brain as a whole, were observed. At the age of 35 days, the weights of the cerebral hemispheres and cerebellum were reduced by thyroidectomy by 20 per cent; the overall DNA content per organ did not change, but the amounts of protein and RNA relative to DNA decreased significantly. It is therefore inferred that thyroid deficiency affects the size of the cells in brain and cerebellum rather than their total number. Conversely, the cell population of the liver was only a quarter of that in the control. There was a small but significant decrease in the hepatic protein and RNA content in the hypothyroid animal. The activities of the following enzymes which served as markers for subcellular fractions in homogenates of cerebral cortex were determined: lactate dehydrogenase for the supernatant, glutamate dehydrogenase for the mitochondrial and glutamate decarboxylase for the synaptosomal fractions. When the activities were expressed on a fresh weight basis a significant decrease by comparison with the control values was observed only in the case of glutamate decarboxylase (—15 per cent at the age of 17–32 days); when the activities were based on DNA content all values were reduced, probably as a result of the general decrease in cell size. Pyrimidine metabolism of brain and liver, studied after the administration of [6-¹⁴C]-orotic acid, was not affected in either tissue by neonatal thyroidectomy. A small but significant reduction in the incorporation of labelled pyrimidine nucleotides in liver RNA was observed, but no significant decrease in the incorporation in cerebral RNA was found in the hypothyroid rats.     

Effect of adrenocorticotropic hormone on nucleic acid and protein synthesis and content in the brain of rat embryos in the early stages of neurogenesis]

The injection of adrenocorticotropic hormone (ACTH) of prolonged effect at the doses of 4 and 10 M. U. to the intact rats from the 11th till the 15th day of pregnancy resulted in the twofold increase of protein content in the brain and its decrease in the liver of 15 days old embryos, as compared with the control ones. The content of DNA, RNA and proteins in the placenta of experimental animals increased as well. The rate of incorporation of 3H-thymidine in the liver DNA and 14C-leucine in the liver and brain acid-soluble protein decreased within small intervals of time following the treatment. The total radioactivity of proteins in the liver, brain and placenta calculated per DNA unit was similar to the control one whereas the specific radioactivity of total protein in the liver of experimental embryos was higher than in the control.     

Biosynthesis of the Brain-specific 14-3-2 Protein in a Cell-free System from Wheat Germ Extract Directed with Poly(A)-containing RNA from Rat Brain

Abstract: 14-3-2 Protein is a neuron-specific protein with a molecular weight of 46,000. Poly(A)-containing RNA was prepared from free polysomes of rat whole brains by means of phenol-chloroform extraction and oligo (dT)-cellulose chromatography. This RNA directed the synthesis of 14-3-2 protein in a cell-free, protein-synthesizing system derived from wheat germ. 14-3-2 Protein was not detected in the products of endogenous incorporation and the products directed with liver poly(A)-containing RNA. These results indicate that mRNA for 14-3-2 protein contains the poly(A) sequence and resides only in the brain.     

Effect of acetylcholine on incorporation of 14C-precursors into uridylic and cytidylic nucleotides of the RNA of digestive glands

The incorporation rate of [2-14C]orotic acid and [2-14C]uridine into the cytidylic RNA nucleotides is significantly lower than into the uridylic ones. In the liver it was twice as low as in the stomach mucosa or in pancreas of albino rats. The administration of acetylcholine in combination with proserine has no influence on the RNA content and its nucleotide composition in the tissues. The administered drugs however caused changes in the relation of the incorporation rates of both labels into uridylic and cytidylic RNA nucleotides, which evidences for the uridylic nucleotide synthesis. In the liver such changes are not detected, but utilization of the labeled uridine is shown to be more intensive for the cytidylic RNA nucleotides synthesis.     

Two pathways of pyrimidine nucleotide biosynthesis in birds

Dillerent chicken tissues are shown to display a clearly pronounced specificity relative to [2-14C] orotic acid and [5-3H]uridine as precursors of synthesis of the pool and RNA pyrimidine nucleotides. The fraction of pyrimidine nucleotides synthetized relative to the reserve pathway (uridine utilization) decreases in the series: kidneys greater than duodenum mucosa greater than lungs greater than liver greater than pancreas greater than bone marrow greater than brain greater than spleen. The results of [2-14C]orotic acid and [53H]uridine incorporation into UMP and CMP of the liver and spleen tissues RNA are interpreted in terms of the concept on existence of separate pools of pyrimidine phosphates--RNA precursors.     

Incorporation of 14C-thymidine into liver and brain DNA of protein-deficient rats.

The authors studied the effect of a protein-free diet on 14C-thymidine incorporation into rat liver DNA in vivo and found, after 2-3 weeks, a marked decrease in uptake of the radioactive base into the liver DNA, followed by a decrease in the proportion of DNA in liver cell homogenates. The total nuclear count/mg liver tissue displayed an increase during protein depletion, except for the 5th week, when a decrease was recorded. The incorporation of 14C thymidine into the brain DNA likewise displayed no great differences, although a significant drop was observed during the 2nd to 4th week of depletion. In the 5th week we recorded an increase in uptake of the radioactive base by brain DNA, exceeding the incorporation values in the controls.     

Effect of amines on fibrinogen synthesis

L-Epinephrine, serotonin, and isoproterenol stimulate the incorporation of [14C]leucine into thrombin-induced clottable protein; this stimulation was abolished by actinomycin D. The incorporation of 32P into total RNA of rat liver, the site of fibrinogen synthesis, was stimulated by epinephrine and was highest at 2 h after 32P administration. [14C]Orotic acid incorporation into polysomal RNA of liver was also increased significantly by epinephrine and serotonin. The immunoprecipitation of newly synthesized protein by monospecific antibody raised against pure rat fibrinogen clearly demonstrates that L-epinephrine increased fibrinogen formation in vivo under the experimental condition. Translation of poly (A)-containing RNA from total polysomal RNA clearly indicates that L-epinephrine increased mRNA specific for fibrinogen.     

Incorporation of [2-14C orotic] acid into chromatin RNA of tissues of animals of various ages

The structure and biological activity (the level of the labelled precursor incorporation into RNA) of active and repressed chromatin of the liver and small intestine mucosa were studied in adult (6-8 months) and old (24-26 months) rats. The content of repressed chromatin fraction in both tissues is found to increase with age. In the liver of old rats the level of [14C[ orotic acid incorporation into RNA of chromatin fractions decreases, radioactivity of the acid-soluble fraction being unchanged. In the small intestine mucosa a high leve of [14C] orotic acid incorporation into chromatin RNA with ageing is due to an increase in permeability of the mucosa cells.     

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.     

Liver growth, biosynthesis of cytidine nucleotides and level of cytochrome P-450 in rat liver after administration of alpha-hexachlorocyclohexane.

The biosynthesis of cytidine nucleotides and the level of microsomal cytochrome P-450 in intact and regenerating rat liver after repeated administration of alpha-hexachlorocyclohexane (alpha-HCH) were compared. In alpha-HCH treated animals the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides is suppressed. In 24-h regenerating liver the incorporation of labelled orotic acid into cytidine nucleotides is markedly activated; the degree of activation is lower in regenerating livers of alpha-HCH treated animals. The changes in the level of cytochrome P-450 vary inversely with the changes in the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides. The activity of cytidine triphosphate synthetase of liver cytosol increases shortly after the administration of alpha-HCH; uridine-cytidine kinase is enhanced in the later stages of the drug action. Within 15-45 min after the administration of alpha-HCH the uptake of [U-14 C] cytidine into the liver and its incorporation into RNA cytosine are increased. After the administration of the drug the uptake of [2-14 C] uridine and its incorporation into RNA uracil is also enhanced whereas its utilization for the synthesis of cytidine nucleotides of the acid-soluble extract as well as for the RNA cytosine are suppressed.     

Effect of chloramphenicol on ribonucleic acid synthesis in liver cells in suspension

1. Chloramphenicol has a stimulatory effect on the incorporation of radioactive phosphate into the RNA of perfused rat-liver slices, whole liver homogenates or the liver-cell suspensions, and no effect on the incorporation of [(14)C]adenine and [(14)C]uracil into the RNA of the tissue slices. 2. Chloramphenicol completely inhibits the incorporation of labelled adenine and uracil into the RNA of the cell suspensions, or into the RNA of homogenates derived from the whole liver tissues. 3. Chloramphenicol has at most a slight inhibitory effect on the transport of labelled adenine or uracil in the hepatic cells in suspension; in the slices, the transport of these bases is not inhibited at all. 4. The above observations indicate that: (a) unlike the tissue slices, hepatic cells in suspension are permeable to chloramphenicol; (b) in the presence of chloramphenicol, for reasons that are not clear, the conversion of the base into the appropriate nucleotide does not proceed.     

Effect of rearing conditions on nucleic acid synthesis in the brain of rats of different genotypes

The incorporation of [3H]thymidine into DNA and of [14C]uridine into RNA has been investigated in different areas of brain and in liver of inbred August rats, non-inbred Wistar rats and hybrids between male wild-type and female Wistar rats which were kept either under normal conditions or under intensive sensory stimulation at different age. DNA metabolism has been found to display tissue, regional, age-dependent and interlinear differences. Sensory stimulation influences considerably the synthesis of nucleic acids which is maximal at early ontogenesis stages and in the brain of hybrid animals.     

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.     

Mechanism of action of a novel antitumor preparation of chlofiden. Effect on RNA synthesis

Study of the influence of the antitumor preparation chlofiden on RNA synthesis by sarcoma 45 and liver cells was carried out using 14C-orotic acid. Chlofiden was shown to activate RNA transport from the nucleus to the cytoplasm, and to reduce label incorporation into the nuclear and cytoplasmic RNA. Chlofiden increased the rate of RNA processes, which promoted some increase of label incorporation into low molecular RNA fraction of the nucleus and the cytoplasm. High and stable antitumor activity of chlofiden on sarcoma 45 was established.     

In vivo protein synthesis from 14C-substrates in porcine tissues during the transition to postnatal development

[2-14C] leucine, [1-14C] alanine, [1-14C] glucose, [1-14C] lactate and [1-14C] pyruvate utilization in the protein synthesis has been studied in vivo at early stages of postnatal development of piglets. It has been established, that during the first 24 hours after birth the protein synthesis intensity, judging by [2-14C] leucine incorporation, in liver, skeletal muscle, duodenal wall and subcutaneous tissue of piglets increases 5, 7, 6.5 and 2.1 times respectively. At the age of 1-2 h the radioactive carbon incorporation from [1-14C] glucose into the brain proteins is more pronounced than into the proteins of liver and skeletal muscle. During the first days of life the intensity of the label incorporation from [1-14C] glucose into liver and skeletal muscle proteins of piglets is enhanced, whereas in brain it remains at the same level. The degree of 14C carbon incorporation from [1-14C]-alanine, [1-14C] pyruvate and [1-14C] lactate into the liver and skeletal muscle proteins of 5-days-old piglets is approximately the same, 14C substrates of protein synthesis in brain and subcutaneous adipose tissue having some peculiarities.