Incorporation Of 14C-orotic Acid Research Articles

The effect of epinephrine and serotonin on hepatic poly (A) + RNA synthesis

In vivo administration of epinephrine or serotonin has been shown to stimulate the incorporation of 14C-orotic acid into Poly(A) + RNA. However, only epinephrine and not serotonin could stimulate DNA dependent RNA polymerase activity of isolated hepatic nuclei in in vitro experiments.

In vitro effects of aflatoxin B1 on the uptake of 14C-orotic acid into kidney, liver and muscle tissue of the Mongolian gerbil, Meriones unguiculatus.

Aflatoxins are a mixture of highly toxic metabolites produced by certain strains of the mold Aspergillu8 ~avu~ a contaminant of groundnuts and other grains. The chemical structures for these potent hepatotoxic and hepatocarcinogenic agents have been studied extensively and aflatoxin B I (AFB I) has been found to be the most abundant and toxic metabol~te (B~TLER 1966). A major area of study involving the aflatoxins has dealt with their mechanism of action. Often severe inhibition of protein synthesis by the cell can be measured by uptake and incorporation of various labeled precursors of protein and nucleic acids (SMITH 1963, CLIFFORD & REES 1966, 1967). One investigator reported inhibition Of nuclear and nucl~olar RNA synthesis (30% and 85%, respectively) as measured by 14C-orotic acid incorporation in Other work with rats showed rat livers (LEFARGE et al. 1966). i~ no inhibition of incorporation of C-orotic acid in in vivo studies of the nucleotide pool, but immediate inhibition in in vitro studies. However, the authors did find inhibition of nucleotide precursors into nuclear RNA and postulated inhibition of RNA polymerase as a possible explanation (CLIFFORD & REES 1967). Later work subsequently explored this hypothesis corroborating it for RNA polymerase and implicating metabolites of AFB I as the active intermediate forms responsible for the inhibitmon (AKINRIMIST et al. 1974). A metabolite of AFB I was also suggested as the actual inhibitor of RNA synthesis in another work where the incorporation of RNA precursors, 14C-orotic acid and 3H-orotic acid, was markedly inhibited by low levels of AFH I and AFGI, but not AFB 2 and AFG 2 (MCINTOSH et al. 1976). Most of the literature involving measurement of uptake and incorporation of precursors has dealt m~inly with the effects on rats and few recent studies have used 14C-orotic acid. It was the purpose of this study to determine the effects of AFB_ on the uptake of 14C-orotic acid into kidney, liver and muscle tissue of the Mongolian gerbil, Meriones unguicula~s. This animal appears to be less sensitive to the toxin than the rat (GODY & NEAL 1976, LLEWELLYN & THOMEN 1978).

Synergistic toxic effects of citrinin, ochratoxin A and penicillic acid in mice

G. A. Sansing, E. B. Lillehoj, R. W. Detroy and M. A. Miller. Synergistic toxic effects of citrinin, ochratoxin A and penicillic acid in mice. Toxicon 14, 213–220, 1976.—The ld 50's in mice of citrinin, ochratoxin A, and penicillic acid injected intraperitoneally were 89, 22, and 100 mg per kg of body weight, respectively. Paired combinations of the mycotoxins, citrinin: ochratoxin A (CI:OA), ochratoxin A:penicillic acid (OA:PA), and penicillic acid:citrinin (PA:CI) elicited synergistic lethal responses. After administration of citrinin, 14C-orotic acid incorporation into both liver and kidney increased significantly by 27 hr with a return to control levels at 51 hr. Treatment with penicillic acid also increased orotic acid incorporation into liver ribonucleic acid (RNA) at 27 hr and at 4 hr in the kidney. Ochratoxin A inhibited orotic acid incorporation into both liver and kidney RNA 6 hr after toxin injection with a subsequent return to control levels at 27 hr. Orotic acid incorporation was inhibited in both kidney and liver 6 hr after treatment with the toxin combination CI:OA, PA:CI stimulated precursor incorporation into kidney RNA at 27 hr and inhibited the function in liver. The OA:PA combination inhibited orotic acid incorporation in both organs 15–27 hr after toxin treatment.

MECHANISM OF ACTION OF ALDOSTERONE IN ADRENALECTOMIZED MOUSE KIDNEY

Time-course of effect of aldosterone on the incorporation of 14C-orotic acid into kidney RNA from adrenalectomized and normal mice was examined with the following results.1. Stimulatory effect of aldosterone (0.55 μg/kg, s.c.) on 14C-orotic acid incorporation into RNA from kidney homogenate and its nuclear fraction was observed in early stages (1.53 hr) after aldosterone administration in adrenalectomized mice, however, the incorporation was not changed at 5 hr after aldosterone. RNA content in homogenate and subcellular fractions did not change in any cases.2. After the administration of larger dose of aldosterone (50μg/kg) to adrenalectomized mice, 14C-orotic acid incorporation into kidney RNA was not changed until 3 hr.3. Cortisol had no effect on RNA specific radioactivity and RNA amount in adrenalectomized mouse kidney. 4. 14C-orotic acid incorporation into kidney RNA was not changed until 5 hr between normal and adrenalectomized mice.5. 14C-orotic acid incorporation into kidney nuclear RNA from normal mice was increased until 3 hr after aldosterone (2 and 20μg/kg) administration and the maximum effect was observed at 1.5 hr later.6. The early effects of aldosterone on RNA synthesis in adrenalectomized mouse kidney are agreed with the general concept in “induction hypothesis” which has been offered by the study using adrenalectomized rat kidney and/or urinary bladder of toad.

Effects of insulin and glucose on subcellular fractions from rat adipose tissue.

Subcellular fractions of rat adipose tissue segments incubatedin vitro were obtained by differential centrifugation. In the presence of glucose, insulin produced the following effects: (a) increased incorporation of32P-orthophosphate and14C-glycine into the mitochondrial and microsomal fractions; (b) increased incorporation of32P-orthophosphate into nuclear RNA. In the absence of insulin, lack of glucose increased the incorporation of14C-orotic acid into both total RNA and nuclear RNA.

Incorporation of orally administered radioactive precursors into nucleic acids and ribosomes of housefly ovaries

After emergence female houseflies were fed for 4 days on a diet containing 14C-orotic acid and 3H-thymidine-5-triphosphate, or 3H-leucine. Nucleic acids and ribosomes were then isolated from the ovaries and studied by MAK column chromatography and sedimentation analysis respectively. The ultraviolet absorption and radioactivity of the fractions were also measured. After MAK column chromatography, the u.v. elution pattern showed that only tow distinct peaks, corresponding to tRNA and rRNA were present. A similar elution pattern was obtained by measuring the 14C from 14C-orotic acid incorporated into the RNA. Because of the small quantity present, DNA was not measurable by u.v. absorption, but by determining the incorporation of 3H from 3H-TTP, its presence was clearly evident. Sedimentation analysis of ovarian ribosomes revealed four polymeric forms besides subunits and monomers. The incorporation of 14C-orotic acid and 3H-leucine into the ribosomes was used to follow the synthesis of rRNA and rProtein respectively. Sucrose density gradient centrifugation of the rRNA indicated that the ovarian rRNA consisted primarily of 28 and 18 S particles.

Pentagastrin-stimulated incorporation of 14 C-orotic acid into RNA of gastric and duodenal mucosa.

SummaryFasted rats were injected 3 times over a 24 hr period with either pentagastrin, saline or histamine. 14C-Orotic acid was injected 1 hr prior to sacrifice. The incorporation of orotic acid into phenol extracted RNA was determined for gastric and duodenal mucosa and the liver. Pentagastrin injected animals incorporated 3 times as much orotic acid into RNA of the gastric and twice as much into RNA of the duodenal mucosa when compared to saline or histamine injected controls. Incorporation of orotic acid into liver RNA was not affected by pentagastrin.We conclude that pentagastrin stimulates the incorporation of orotic acid into mucosal RNA of the upper gastrointestinal tract and that is probably indicative of induction of RNA synthesis. These findings support the current evidence and hypothesis (3) that gastrin is a trophic hormone for gastric and duodenal mucosa.

CONTRIBUTION TO THE EXISTENCE OF REGULATORY MECHANISMS AT THE ADRENAL LEVEL

ABSTRACT Rat adrenal glands atrophied by the administration of cortisol acetate in vivo were used as a model for the study of early metabolic processes occurring in vitro. Atrophied adrenals incubated in the presence of 14C-leucine incorporated subnormal quantities of this amino acid per mg of protein for the first 120 min. When the incubation lasted for a total period of 180 or 240 min a supranormal rise in the 14C-leucine incorporation was observed. Similar changes occurred with some delay with regard to corticosterone production as expressed per 100 mg of tissue. No differences in 14C-leucine incorporation were observed between the control and atrophied adrenals in vivo. Homogenates from atrophied glands incorporated 14C-leucine to a greater extent than the control homogenates. The in vitro incorporation of 14C-orotic acid into the RNA was also higher in atrophied adrenals. The in vitro use of actinomycin D, cycloheximide and amphenone indicated that corticosterone production depended on the incorporation of 14C-leucine. The addition of cortisol to the incubation media markedly decreased the enhancement of 14C-lysine incorporation into the protein of atrophied adrenals. These, as well as additional results suggest rebound phenomena: once atrophic adrenals are transferred to cortisol-free media, reparative processes begin after a delay period. Such phenomena seem to be mediated by regulatory mechanisms at the adrenal level.

RNA synthesis during chloroplast development in Euglena gracilis

Changes in chloroplast and cytoplasmic RNA were measured during chloroplast development in Euglena gracilis over a 3-day period. The cells were grown in the dark and then illuminated by 1500 lx of white light at a cell density which was sufficiently high to prevent any cell division in illuminated cultures. Light stimulated incorporation of 14C-orotic acid into both chloroplast and cytoplasmic RNA. The most rapid rate of light-dependent incorporation occurred during the first 8 hr of illumination and before the main period of chlorophyll synthesis. The specific activity of chloroplast RNA was higher than that of cytoplasmic RNA, particularly during the first 24 hr of illumination. By 32 hr the rate of incorporation into total cell RNA had decreased to that of dark control cells. White there was no significant change in the amount of RNA per cell during chloroplast development, the amount of chloroplast RNA increased to a maximum of 7 per cent of the total cellular RNA by 16 hr. In growing autotrophic cultures, or in cultures in which chloroplast development was followed at low cell densities (3 × 10 6 cells per ml), chloroplast RNA accounted for about one-fifth of the total cellular RNA.

Effects of chlorpromazine on cell wall biosynthesis and incorporation of orotic acid into nucleic acids in Bacillus megaterium

The microorganism Bacillus megaterium was used as a model system for the study of the biochemical basis of action of chlorpromazine. At 33 μM, chlorpromazine (10 μg/ml) doubled the generation time of logarithmic phase cultures of B. megaterium. The effect of the drug on the incorporation of labeled precursors for macromolecules was compared at equivalent turbidities. Incorporation of 14C-orotic acid into RNA and DNA was immediately inhibited. In contrast, incorporation of 14C-adenine, 14C-formate and 14C-thymidine into nucleic acids was unaffected, indicating that nucleic acid synthesis was normal despite interference by the drug in the utilization of exogenous orotic acid. Incorporation of 14C- l-lysine was unaffected, but chlorpromazine immediately inhibited the incorporation of 14-C-diaminopimelic acid into cell wall. Uridine nucleotide precursors (colorimetric assay) for glycopeptide polymer of cell wall accumulated promptly upon addition of the drug. Accumulation was dose dependent and reached eight times that of control cells. Other phenothiazines including trifluoperazine (3 μM), promazine (58 μM), promethazine (156 μM) and chlorpromazine sulfoxide (2 mM) also caused significant accumulation of cell wall precursors. Although the mechanism of action is unknown, it appears that chlorpromazine specifically interferes with cell wall synthesis in B. megaterium.

Enhanced RNA polymerase activity in skeletal muscle undergoing hypertrophy

Increased microsomal RNA is characteristic of skeletal muscle undergoing hypertrophy and appears to contribute to enhanced protein synthesis in the tissue. Accordingly, RNA synthesis was studied in rat solei undergoing compensatory hypertrophy by measurement of the incorporation of 14C-orotic acid into RNA in vivo and by assay of the activity of RNA polymerase in isolated nuclei at several time intervals after tenotomy of the synergistic gastrocnemius muscles. Solei from sham-operated contralateral limbs served as controls. Increased synthesis of RNA in vivo was apparent within 24 hr after the stimulus to hypertrophy. However, augmented activity of RNA polymerase in isolated nuclei in vitro was not demonstable until 48 hr later, at a time when microsomal RNA content was already elevated. The results are compatible with the view that an early alteration in the intracellular environment leads to increased activity of RNA polymerase in the cell resulting in the increased RNA synthesis characteristic of early hypertrophy. On the other hand, the increased activity of RNA polymerase in isolated nuclei in vitro, observed considerably later, appears to represent a secondary adaptation to physiologic stress in which the actual quantity of RNA polymerase is increased.

Nickel carbonyl inhibition of 14C-orotic acid incorporation into rat liver RNA.

Nickel carbonyl, a metallic carcinogen inhibited 14C-orotic acid incorporation into liver RNA. Forty min after an ip injection of 14C-orotic acid, the mean specific activity of RNA from livers of control rats was 318 (SD, ± 133) μmoles of 14C/mole of ribose. In rats which also received an intravenous injection of nickel carbonyl (2.2 mg of Ni/100 g) 24 hr before sacrifice, the mean specific activity of liver RNA was 81 (SD, ± 23) μmoles of 14C/mole of ribose (p vs controls = <0.01). The incorporation of 14C-orotic acid into liver RNA was not significantly affected by injection of phenobarbital (10 mg/100 g, ip) 20 hr before sacrifice. This work supports the hypothesis that nickel carbonyl inhibits the synthesis of hepatic RNA.

Incorporation of 14C-orotic acid into RNA of different-sized nucleoli of rat livers following partial hepatectomy or after force-feeding a threonine-devoid diet

Incorporation of 14C-orotic acid into RNA of different-sized nucleoli of rat livers following partial hepatectomy or after force-feeding a threonine-devoid diet

Effect of Dietary Composition on the Incorporation of14C-Orotic Acid into Ribonucleic Acid of the Liver

Growing rats were fed a protein-free diet or a diet containing 25% casein for seven days. The rate of RNA synthesis in the liver was examined by determining the incorporation of 14C-orotic acid into CMP and UMP of liver acid-soluble fraction and RNA hydrolysate on the basis of relative specific radioactivity.The rate of RNA synthesis was raised in the rats fed protein-free diet up to 170% of that with 25% casein diet. The rate of RNA synthesis was also measured when dietary components, such as casein, butter, and starch were individually given for three hours to rats which had been adapted, by training, to spaced feeding for three hours every day. The rate of RNA synthesis was raised in rats given casein, butter, and starch, up to 117%, 144%, and 146%, respectively, of that in rats fasted. From these results, it is presumed that the enhanced RNA synthesis in the livers of rats fed the protein-free diet may be due to the increased uptake of starch which was contained at 90% in the diet.

Effect of growth hormone and hydrocortisone on the synthesis of rapidly labeled, high molecular weight, nuclear RNA in rat liver.

The effects of growth hormone and hydrocortisone on the synthesis of rat liver RNA have been studied. Administration of growth hormone stimulated the incorporation of 14C-orotic acid into 45 S preribosomal nucleolar RNA, and had no effect on the 6–50 S polydisperse nuclear RNA of extra-nucleolar origin. On the other hand, hydrocortisone stimulated the synthesis of all types of nuclear RNA, but mainly that of the 18 S class of 6–50 S polydisperse nuclear RNA.

The effect of X-radiation on the incorporation of 14C-orotic acid into rapidly labeled nuclear and cytoplasmic RNA

The effects of X-radiation on 14C-orotic acid incorporation into nuclear and cytoplasmic rat liver RNA were studied in animals killed 24 hr after partial hepatectomy. The animals received total-body doses of 1500 R and were injected with orotic acid 10, 20, 40 and 60 min prior to sacrifice. X-radiation inhibits the incorporation of orotic acid into nuclear and cytoplasmic RNA in the livers of animals injected with the precursor 10 or 20 min before they are killed. Sixty minutes after orotic acid injection no inhibition of the labeling of nuclear and cytoplasmic RNA was observed. The results suggest that X-radiation retards the incorporation of 14C-orotic acid into the rapidly labeled nuclear and cytoplasmic RNA and that in the cytoplasm X-radiation preferentially interferes with the labeling of RNA with rapid turnover. The retardation of the incorporation of 14C-orotic acid into nuclear RNA can be detected 1 hr after irradiation and reaches a maximum at 6 hr. It is possible that the primary injury produced by X-radiation involves the DNA molecules which at the time of the irradiation function as template for the synthesis of rapidly labeled RNA.

Effect of Dietary Composition on the Incorporation of 14C-Orotic Acid into Ribonucleic Acid of the Liver

Growing rats were fed a protein-free diet or a diet containing 25% casein for seven days. The rate of RNA synthesis in the liver was examined by determining the incorporation of 14C-orotic acid into CMP and UMP of liver acid-soluble fraction and RNA hydrolysate on the basis of relative specific radioactivity. The rate of RNA synthesis was raised in the rats fed protein-free diet up to 170% of that with 25% casein diet. The rate of RNA synthesis was also measured when dietary components, such as casein, butter, and starch were individually given for three hours to rats which had been adapted, by training, to spaced feeding for three hours every day. The rate of RNA synthesis was raised in rats given casein, butter, and starch, up to 117%, 144%, and 146%, respectively, of that in rats fasted. From these results, it is presumed that the enhanced RNA synthesis in the livers of rats fed the protein-free diet may be due to the increased uptake of starch which was contained at 90% in the diet.

Further studies on the steroid inhibition of adrenal protein synthesis.

Steroids have been reported to inhibit radioactive amino acid incorporation into rat adrenal protein in vitro. To study the possibility that this effect might be due to an inhibition of amino acid transport, testosterone was incubated with rat adrenal quarters and amino acid transport was measured. There was a 15% inhibition of 14C-alpha amino isobutyric acid transport but no inhibition of 14C-cycloleucine transport. When testosterone was incubated with adrenal homogenates, there was a 50% inhibition of radioactive amino acid incorporation into protein, which suggested that this steroid effect was not primarily due to an inhibition of amino acid transport. In a further study of the mechanism by which these steroids inhibit adrenal protein synthesis, it was found that the incorporation of 14C-orotic acid into RNA was inhibited by testosterone and corticosterone but not by aldosterone. (Endocrinology 83: 18, 1968)

Regulation of Ferritin Synthesis in Rat Liver

Abstract Ferritin synthesis in rat liver is greatly increased by prior treatment of intact animals with iron salts. Liver slices from treated rats incorporate increased amounts of 14C-leucine into ferritin in vitro, with net synthesis of the protein. Slices of spleen, testis, intestine, and kidney also show increased incorporation following iron. The response of the liver to iron has been studied to determine whether increased transcription of genes and the formation of messenger RNA are involved, or whether control is at a later stage in the biosynthetic pathway. Three lines of evidence support the existence of transcriptional control. (a) Actinomycin D inhibits the response to iron. (b) Iron treatment stimulates incorporation of 14C-orotic acid into a rapidly labeled fraction of liver nuclear RNA. (c) Direct addition of iron to liver slices in vitro has failed to stimulate ferritin synthesis, although 59Fe penetrates liver cells.

Biochemical effects of prednisolone on human liver cells in tissue culture

The growth-inhibiting effect of prednisolone on human liver cells (Chang) grown in monolayer cultures, was most pronounced during the first hours of incubation. Prednisolone in concentrations (125 μM) which inhibited completely cell multiplication during the first 3 hr, had no effect on the concurrent incorporation of 14C labeled thymidine into DNA and of 14C-methionine into protein. The incorporation of 14C-orotic acid into RNA was slightly reduced by higher concentrations (250 μM) of prednisolone. The glycogen content of the cells was markedly increased after incubation with prednisoline for 20–30 hr. Experiments with labeled glucose indicated that the extra glycogen was not derived from added glucose. The incorporation of labeled pyruvate into glucose and glycogen was not affected by prednisolone, while the steroid increased the incorporation of radioactivity from labeled alanine.