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.     

Disappearance of radioactivity from the various ribonucleic acid pools and acid-soluble fractions of mouse liver and kidney after a single injection of labelled orotic acid. The effect of castration

1. An attempt was made to study the rate of synthesis as well as the distribution of RNA in the various cellular fractions in the livers and kidneys of normal and castrated mice. 2. The tissue was fractionated by the procedure of Blobel & Potter (1967). By using this method it was not possible to find any pronounced difference in the relative proportions of RNA in isolated subcellular fractions when kidneys from normal and castrated mice were compared. On the other hand there was an indication of a shift toward the bound ribosomes in livers from normal mice in comparison with livers from castrated mice. 3. Disappearance of the radioactivity followed the pattern of the first-order reaction. Comparing the half-lives of RNA in liver and kidneys it was found that in the latter in both groups of animals half-lives were shorter no matter which cellular fraction was studied. 4. The half-lives for total homogenate RNA, total ribosomal RNA and low-molecular-weight RNA from kidneys of castrated mice were approximately 20-25% longer than the half-lives for the corresponding fractions from normal mouse kidneys. 5. An explanation is put forward for the anomalous finding that RNA from the castrated-mouse kidneys has a higher specific radioactivity than that isolated from normal mice.     

The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats.

The response of muscle and liver protein metabolism to either a single or three successive daily injections of an endotoxin (Escherichia coli lipopolysaccharide, serotype 0127 B8; 1 mg/ml, 0.3 mg/100 g body wt.) was studied in vivo in the fed rat, and at 24 and 30 h after endotoxin treatment during fasting. In the fed rats there was a catabolic response in muscle, owing to a 60-100% increase in muscle protein degradation rate, and a 52% fall in the synthesis rate. Although there was a 20% decrease in food intake, the decrease in protein synthesis was to some extent independent of this, since rats treated with endotoxin and fasted also showed a lower rate of muscle protein synthesis, which was in excess of the decrease caused by fasting alone. The mechanism of this decreased protein synthesis involved decreased translational activity, since in both fed and fasted rats there was a decreased rate of synthesis per unit of RNA. This occurred despite the fact that insulin concentrations were either maintained or increased, in the fasted rats, to those observed in fed rats. In the liver total protein mass was increased in the fed rats by 16% at 24 h, and the fractional synthesis rate at that time was increased by 35%. In rats fasted after endotoxin treatment the liver protein mass was not decreased as it was in the control fasted rats, and the fractional synthesis rate was increased by 22%. In both cases the increased synthesis rate reflected an elevated hepatic RNA concentration. The extent of this increase in hepatic protein synthesis was sufficient at one point to compensate for the fall in estimated muscle protein synthesis, so that the sum total in the two tissues was maintained.     

Actions of dietary orotic acid on liver synthesis of phosphatidylcholine and phosphatidylethanolamine in rats

The in vivo rates of the reactions of the cytidine pathways of liver phosphatidylcholine and phosphatidylethanolamine synthesis were measured in rats after 1 day of feeding on a semisynthetic diet containing 1% orotic acid. The calculations were made from the specific and total radioactivity versus time curves of the precursors and products following intraportal injection of [1,2-14C]choline, [2-14C]ethanolamine, and [2-3H]glycerol. The liver CTP level was increased twofold and the rates of CDP-choline and phosphatidylcholine synthesis were stimulated 4.5-fold in the rats fed orotic acid. The rate of CDP-ethanolamine synthesis was increased but could not be accurately quantified because of its extreme rapidity. No change occurred in the rate of the ethanolaminephosphotransferase reaction and the overall rate of phosphatidylethanolamine synthesis was unchanged by orotic acid feeding. The catalytic activities of the enzymes of the cytidine pathways of phosphatidylcholine and phosphatidylethanolamine synthesis were not affected by feeding orotic acid for 1 day. Similar findings were obtained 3 h following intragastric administration of 100 mg of orotic acid. The results suggest the possibility that changes in the levels of liver CTP may play a role in regulation of the cytidine pathway of liver phosphatidylcholine synthesis but not of phosphatidylethanolamine synthesis, because the latter pathway appears to be tightly controlled at the ethanolaminephosphotransferase step.     

Retinol-binding protein messenger RNA levels in the liver and in extrahepatic tissues of the rat

A retinol-binding protein (RBP) cDNA clone was used to examine the effect of retinol status on the level of RBP mRNA in the liver, and to explore whether extrahepatic tissues contain RBP mRNA. In the first series of experiments, poly(A+) RNA was isolated from the livers of normal, retinol-depleted, and retinol-repleted rats and the levels of RBP mRNA in these samples were determined by both Northern blot and RNA Dot blot analyses. The levels of RBP mRNA in liver were similar in all three groups of rats. These findings confirm and extend previous studies which showed that retinol did not alter the in vivo rate of RBP synthesis or the translatable levels of RBP mRNA. In a second series of experiments, the RBP cDNA clone was used to survey poly (A+) RNA isolated from 12 different rat tissues for RBP mRNA by Northern blot analysis. We found that, along with the liver, many extrahepatic tissues contained RBP mRNA. Kidney contained RBP mRNA at a level of 5-10% of that of the liver, and the lungs, spleen, brain, stomach, heart, and skeletal muscle contained 1-3% of that of the liver. Translation of kidney poly (A+) RNA in rabbit reticulocyte lysates and immunoprecipitation of the translation products with anti-RBP antiserum resulted in a protein band of the same size as liver preRBP. These data suggest that RBP is synthesized in many extrahepatic tissues.It is possible that this extra-hepatically synthesized RBP may function in the recycling of retinol from these tissues back to the liver or to other target organs.     

Ribonucleic acid labelling perfused livers of protein-fed and protein-deprived rats

Several observations suggest an increased RNA synthesis in livers of protein-deprived rats, though the RNA/DNA ratio is decreased. A number of hormones may be involved in these changes. Therefore, we studied in RNA metabolism in isolated perfused livers taken from protein-fed and protein-deprived rats. (3H)-orotic acid was given to the rats 2 h before liver explantation, and (14C)-orotic acid was added to the perfusate. Other rats, called controls in vivo, whose livers were not transplanted were also given (3H)-orotic acid followed by (14C)-orotic acid. The livers of these rats, which were not hormone supplemented, were labelled for the same length of times as the livers in vitro. The ratio specific RNA radioactivity/specific nucleotide radioactivity x RNA/DNA was determined and taken as a measure of the RNA synthesis per liver cell. In the controls in vivo, this ratio was significantly higher for protein-deprived than for protein-fed rats. In livers from the protein-fed rats, labelling in vitro increased significantly when growth hormone, hydrocortisone, insulin and tri-iodothyronine were added to the perfusate. Labelling was also significantly higher in these livers than in the controls in vivo. In livers from protein-deprived rats, the ratio in question was the same whether the hormones were added to the perfusate or not, and was significantly lower than in the controls in vivo. Differences in RNA labelling are thus obtained in our in vitro system. Gel electrophoresis of RNA demonstrated normal RNA labelling, showing that the system is suitable for studying liver RNA synthesis. Further refinement can be made by studying the labelling of UTP and CTP. The results might suggest that the liver from a protein-fed rat, explanted in vitro, may increase its RNA synthesis under the influence of the four hormones in question, and that the RNA synthesis of the liver of a protein-deprived rat is high in-vivo and that it might decrease, when it is explanted to in vitro conditions.     

Rates of sterol synthesis in the liver and extrahepatic tissues of the SHR/N-corpulent rat, an animal with hyperlipidemia and insulin-independent diabetes

The SHR/N-corpulent rat is a new genetically obese strain that exhibits both insulin-independent diabetes and hyperlipidemia. The present studies were undertaken to characterize various parameters of cholesterol metabolism in this model. At 11 weeks of age, the obese animals had markedly elevated plasma cholesterol, triglyceride, glucose, and insulin concentrations and elevated hepatic triglyceride concentrations compared to their lean littermates. The additional cholesterol in plasma was carried in the fractions of density less than 1.006, 1.020-1.055, 1.055-1.095, and 1.095-1.21 g/ml. In the obese rats the level of free cholesterol in the liver was decreased significantly while that of cholesteryl ester showed little change. Hepatic sterol synthesis was markedly suppressed in the obese animals. However, the rate of sterol synthesis in the small intestine and other extrahepatic tissues generally remained unchanged. Although hepatic synthesis was suppressed, whole animal sterol synthesis in the obese rats was similar to that in the lean controls. This resulted because, in the obese animals, not only was the reduced rate of hepatic synthesis partly balanced by a greater than 70% increase in liver mass, but the mass of the small intestine and adipose tissue was also increased more than 30% and 4-fold, respectively, thereby making these tissues quantitatively more important sites of sterol synthesis. When obese rats were pair-fed to the intake of their lean littermates for 10 weeks, there was only a modest reduction in body weight and plasma cholesterol concentration, and the rate of hepatic sterol synthesis remained very low. The suppression of synthesis in the liver also persisted when the obese rats were fed surfomer, a drug that specifically blocks cholesterol absorption. In contrast, feeding cholestyramine restored the rate of hepatic sterol synthesis to that found in lean animals. Bile acid pool size in the obese males and females was 2.5-fold greater than in their lean controls. The suppression of hepatic sterol synthesis in this model may be due to a change in the entero-hepatic circulation of bile acids arising from an expanded pool or, alternatively, it may represent a compensatory response to overproduction of sterol and its precursors in the intestinal and adipose compartments.     

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.     

Studies on ribonucleic acid and homopolyribonucleotide formation in neuronal, glial and liver nuclei

1. Various types of nuclear preparations, with different ratios of neuronal to glial nuclei, were isolated from guinea-pig cerebral grey matter and ox cerebral grey matter and white matter. Conditions appropriate for the separate assay of RNA and poly A formation were described. Comparative rates of RNA and poly A formation were studied in cerebral and liver nuclei. 2. RNA polymerase activity per nucleus is higher in neuronal nuclei than in glial nuclei. In liver nuclei, the activity is much lower than in cerebral nuclei. The physical relationship between RNA polymerase and deoxyribonucleoprotein seems to differ in neuronal, glial and liver nuclei. 3. Poly A polymerase activity in liver nuclei is selectively activated by Mn(2+) and inhibited by GTP, CTP and UTP. On a DNA basis, the activity in an aggregate enzyme is the same as in intact nuclei. Poly A polymerase activity per nucleus is much higher in liver nuclei than in neuronal nuclei. Glial nuclei show an intermediate activity. 4. It is suggested that, in neuronal nuclei, the synthesis of RNA is more prominent than that of poly A under conditions where both polymers are formed simultaneously. This contrasts with liver nuclei, where more poly A is made than RNA. 5. In neuronal nuclei, the rate of CTP incorporation is much higher than in glial and liver nuclei. This incorporation is most probably due to poly C synthesis.     

Developmental and regional variations in ribonucleic acid synthesis on cerebral chromatin

1. Chromatin was prepared from purified nuclei isolated from liver and cerebral regions of the rat. 2. The capacity of these preparations to promote RNA synthesis in the presence of bacterial RNA polymerase was determined. 3. The rate of RNA synthesis on chromatin was normally 12-21% of the rate observed with native DNA, but was markedly stimulated on addition of 200mm-ammonium sulphate. 4. At physiological concentrations (80mug./ml.), the brain-specific S-100 protein inhibited RNA synthesis on DNA and chromatin. 5. Cerebral chromatin from foetal and newborn animals was more active in RNA synthesis than were the analogous preparations from liver. 6. Cerebellar chromatin maintained a high rate of RNA synthesis during brain maturation. In contrast, RNA synthesis on chromatin from other brain regions and liver declined with age of the rat. 7. RNA synthesized on chromatin stimulated amino acid incorporation in an Escherichia coli ribosomal system and hybridized with homologous DNA. 8. RNA synthesized on chromatin from adult cortex or hindbrain hybridized with DNA to a greater extent than that synthesized on cerebellar chromatin. 9. The proportion of RNA formed on cerebral-cortical chromatin that hybridized with DNA increased with age of the rat. 10. The results indicate that the total amount and the types of RNA synthesized on cerebral chromatin vary regionally and during development.     

Comparison of glutathione S-transferase activity in the rat and birds: tissue distribution and rhythmicity in chicken (Gallus domesticus) liver.

1. Mature, male chickens, Bobwhite quail, and rats differed with respect to glutathione S-transferase (GST) activity in the kidney, duodenum and testis, but species differences were not observed in the liver. 2. GST activity was present in the heart, spleen, liver, duodenum, kidney, testis, cerebral cortex, cerebellum, optic tecta, and medulla oblongata of chickens with differences in tissues and breeds. 3. Renal GST activity was higher in female chickens, whereas enzyme activity in the brain was higher in males. 4. Hepatic GST activity fluctuated about a mean of 784 nmol min-1 mg protein-1 with a 12 hr periodicity which was not a feeding phenomenon. 5. The results demonstrate that GST activity occurs in diverse tissues of the chicken and Bobwhite quail with kidney greater than liver greater than duodenum greater than testis, compared to testis greater than liver greater than duodenum greater than kidney in the rat. Hepatic GST activity exhibits an ultradian periodicity.     

Growth hormone regulates the abundance of insulin-like growth factor I RNA in adult rat liver

Insulin-like growth factor I (IGF-I) is a mitogenic polypeptide present in the plasma of man and rat that is thought to mediate the actions of pituitary growth hormone on cartilage to promote skeletal elongation. In the rat, plasma levels of IGF-I show both developmental and hormonal regulation: levels are low at birth, increase with age, and are decreased in growth hormone-deficient adult animals. The present study demonstrates that these changes in plasma IGF-I reflect the abundance of IGF-I RNA in rat liver. A human IGF-I cDNA probe hybridized to multiple RNA species in adult rat liver with sizes 8.6, 4.6, 3.2, 2.1, and 1.0-1.4 kilobases. These RNA species were decreased by greater than 80% in neonatal (2- and 12-day-old) rat liver and by greater than 90% in liver from adult rats made growth hormone-deficient by hypophysectomy. Treatment of hypophysectomized rats with growth hormone increased the abundance of all species of IGF-I RNA. These results suggest that growth hormone regulates the expression of its physiological mediator by altering the synthesis, stability, or both of IGF-I RNA in rat liver.     

Effect of post-ischaemic recovery on albumin synthesis and relative amount of translatable albumin messenger RNA in rat liver.

In liver cells recovering from reversible ischaemia, total protein synthesis by postmitochondrial supernatant and membrane-bound and free polyribosomes is not different from that in sham-operated controls. However, the relative proportion of specific proteins is changed, since the incorporation of [3H]leucine in vivo into liver albumin, relative to incorporation into total protein, as determined by precipitation of labelled albumin with the specific antibody, decreases by 40-50% in post-ischaemic livers. Cell-free synthesis by membrane-bound polyribosomes and poly(A)-enriched RNA isolated from unfractionated liver homogenate shows that the decrease in albumin synthesis in liver of rats recovering from ischaemia is due to the relative decrease in translatable albumin mRNA.     

Effect of growth hormone on ribonucleic acid metabolism. The template activity of the chromatin and molecular species of ribonucleic acid synthesized after treatment with the hormone

Growth hormone stimulates the synthesis of RNA in hypophysectomized rat liver. The question whether the hormonal stimulation of RNA synthesis is due to the activation of repressed cistrons or to other factors was studied. Nuclear RNA from the livers of adult female hypophysectomized and growth-hormone-treated rats was examined for molecular homology by hybridization techniques: no new species of RNA were detected after hormone treatment. The template activity of the chromatin for RNA synthesis is also not increased by the action of growth hormone. Short- and long-pulse-labelling experiments demonstrate that the hormonal stimulation of RNA synthesis is most marked in experiments where the period of incorporation of radioactive precursors is limited to 1-2hr. It is concluded that the hormone influences essentially the rate of RNA synthesis in these tissues.     

Effect of fasting on the metabolism of cytidine nucleotides in the liver of intact and alpha-hexachlorocyclohexane-treated rats

The utilization of (2-14C)orotic acid for the synthesis of cytidine components of the acid-soluble extract and for the RNA cytosine is decreased in the liver of rats which fasted for 24 or 72 h. The depression of the specific activity of the cytidine components is greater in animals which received alpha-HCH during the 24-hour interval after removal of food than in the control group; by contrast, the specific activity of the cytidine components again increases in rats fasting for 72 h. Analogous changes also occurred in the specific activity of RNA cytosine. Both the (U-14C)cytidine uptake and its utilization for the synthesis of RNA cytosine are enhanced in fasting rats; the administration of alpha-HCH has a potentiating effect. The total content of cytidine components of the acidsoluble extract of 1 g of liver tissue is enhanced 24 h after the animals of the control and experimental group were deprived of food. There are no marked differences in the concentration of the uridine components. Fasting has an additive effect on the increase of cytochrome P-450 level in the alpha-HCH treated rats. Alpha-HCH = alpha-1,2,3,4,5,6-hexachlorocyclohexane.