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PubMed2003

Influence of carbon source on pyrimidine synthesis in Pseudomonas mendocina.

The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in the bacterium Pseudomonas mendocina was studied. When glucose was the carbon source, orotic acid supplementation of P. mendocina cells produced the greatest depression of aspartate transcarbamoylase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities while P. mendocina cells grown in the presence of uracil caused the maximal decrease in dihydroorotase and OMP decarboxylase activities. After the pyrimidine starvation of an orotate phosphoribosyltransferase mutant strain of P. mendocina grown on glucose, the pyrimidine biosynthetic pathway enzyme activities were generally diminished. With respect to pyrimidine starvation studies, the carbon source glucose appeared to lessen regulation at the level of enzyme synthesis compared to what has been observed when succinate served as the carbon source. The regulation of the pyrimidine biosynthetic pathway by carbon source in P. mendocina appeared to differ from how carbon source influenced the control of pyrimidine biosynthesis in the closely-related species Pseudomonas stutzeri.

Aspartate Carbamoyltransferase↗
PubMed1982

Treatment of hyperargininaemia due to arginase deficiency with a chemically defined diet.

A brother and sister aged 11 and 17 years have been reported previously to have hyperargininaemia and arginase deficiency: they were treated with a semi-synthetic diet consisting of fat, carbohydrate, minerals, vitamins and essential amino acids in amounts equivalent to 0.55-0.65 g protein kg-1 day-1 for 2 years. Plasma arginine levels fell from 0.50-0.90 mumol/1 to 0.13-0.30 mumol/1 (normal range 0.02-0.15). Increased concentrations of arginine in the cerebrospinal fluid (CSF) fell from 0.069-0.098 mumol/l to 0.040-0.056 mumol/l (normal mean +/- SD = 0.020 +/- 0.006). Dibasic aminoaciduria returned to normal within 1 week. Substitution of the keto-acid analogues of five essential amino acids in the formula lowered arginine concentrations further, but proved to be unpalatable. Urinary concentrations of orotic acid, uridine and uracil fell toward normal but remained increased, even when the plasma ammonia concentration was measured as normal. Both patients showed a stable clinical improvement.

Adolescent↗
PubMed1986

[Adsorption on charcoal sorbents of the nucleic acids from the blood plasma of leukemia patients and from the ascitic fluid of mice with lymphoma NK/Ly].

Using 3H-thymidine, 14C-orotic acid and ethidium bromide identification of the nucleic acid double-stranded structures from biological fluids and the method of hemocarboperfusion the data are obtained on the active and irreversible adsorption of nucleic acids and nucleoproteins from plasma of leukaemic patients and ascitic fluid of mice with lymphoma NK/Ly.

Adsorption↗
PubMed1983

Synthesis of 3-N-ribosyluric acid 5'-monophosphate by red cells of the bovine fetus.

Although 3-N-ribosyluric acid (3-RUA) was not detected in the red cells of bovine fetuses, 3-N-ribosyluric acid 5'-monophosphate, the immediate precursor of 3-RUA, was synthesized from uric acid and 5-phosphoribosyl-1-pyrophosphate (PRPP) by hemolysates of fetal red cells. These preparations also synthesized 3-N-ribosylxanthine 5'-monophosphate from xanthine and PRPP. Partially purified extracts of fetal and adult erythrocytes of cattle had similar enzyme activity and the same specificity toward xanthine and orotic acid. Therefore, it was concluded that the absence of 3-RUA in fetal red cells was not a result of lack of the enzyme orotate phosphoribosyltransferase (EC 2.4.2.10).

Aging↗
PubMed1995

Antimalarial effects of C18 fatty acids on Plasmodium falciparum in culture and on Plasmodium vinckei petteri and Plasmodium yoelii nigeriensis in vivo.

Following the demonstration of the antimalarial effect of the long chain saturated alcohol n-hentriacontanol ((CH2)29CH2OH), isolated from the Bolivian endemic solanaceous plant Cuatresia sp., we have tested the effect of the C18 fatty acids oleic, elaidic, linoleic, and linoleic on malaria parasites. These fatty acids inhibited the parasitemic development in mice infected with Plasmodium vinckei petteri or with Plasmodium yoelii nigeriensis in a 4-day suppressive test. To gain a deeper discernment of the antimalarial mode of action, the effects of these compounds were evaluated on Plasmodium falciparum growth in culture. Whereas n-hentriacontanol did not show any inhibition of this parasite, on the contrary, the C18 acids displayed a considerably inhibitory activity at < or = 200 micrograms/ml both in intact infected cells and in free parasites. In order to understand the mechanism of their antimalarial action, several tests were performed. No hemolysis of infected cells could be observed up to 500 microgram/ml. No effect on the lipid peroxidation, ATP levels, transport through the parasite-induced permeability pathways, or on the phagocytosis of the infected cells could be observed. The cytotoxic effect of the fatty acids was very rapid: full inhibition of nucleic acids and protein syntheses was observed in less than 30 min. This inhibition was not relieved by the addition of deferrioxamine or FeCl3, indicating that fatty acids (FA) do not act by facilitating the transport of iron. Inhibition was relieved in neither the presence of orotic acid or its methyl ester, indicating that FA do not act at the mitochondrial level of pyrimidine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Regulation of RNA degradation in cultured rat hepatocytes: effects of specific amino acids and insulin.

The regulation of RNA degradation by specific amino acids and insulin was investigated in cultured rat hepatocytes from fed rats previously injected in vivo with [6-(14)C]orotic acid. The effects of three groups of amino acids were compared to those of a complete amino acid mixture. The first one consisted of the eight amino acids (leucine, proline, glutamine, histidine, phenylalanine, tyrosine, methionine, tryptophan) previously found to be particularly effective in the control of proteolysis. The two other groups were defined from our study with single additions of amino acids, one consisting of proline, asparagine, glutamine, alanine, phenylalanine, and leucine and the other including the latter group with serine, histidine, and tyrosine. The results showed that these three groups were able to strongly inhibit deprivation-induced RNA breakdown at one and ten times normal plasma concentrations but to a lower extent than the complete amino acid mixture. Six amino acids (proline, asparagine, glutamine, alanine, phenylalanine, leucine) inhibited individually RNA degradation by more than 20%. However, the deletions of proline, asparagine, glutamine, or alanine from the group of these six amino acids were not followed by a loss of inhibitory effect. On the contrary, an important loss of inhibition was observed when leucine and phenylalanine were deleted. Furthermore, only these two amino acids exhibited an additive inhibitory effect. Thus leucine and phenylalanine could be considered as important inhibitors of RNA breakdown in cultured rat hepatocytes. Finally, insulin which had no significant effect on RNA degradation in the absence of amino acids, was able to potentiate the inhibitory effect of different amino acid groups.

Amino Acids↗
PubMed1987

Amino acid and hormonal control of macromolecular turnover in perfused rat liver. Evidence for selective autophagy.

The control of RNA degradation by amino acids, insulin, and glucagon was investigated in perfused livers of fed rats previously labeled in vivo with [6-14C] orotic acid; rates were determined from the release of [14C]cytidine in the presence of 0.5 mM cytidine to suppress reutilization. Studies with cyclically perfused livers showed that plasma amino acids at 10 times (10X) normal concentrations inhibited RNA breakdown by 85%. Similar inhibition was obtained with a known regulatory amino acid mixture (Leu, Met, Pro, Trp, and His), whereas leucine alone (0.8 mM) decreased degradation by 47%. Perfusions carried out in the single-pass mode with graded levels of plasma amino acids revealed that the acceleration of RNA degradation over the full range of amino acid deprivation (0 to 10X normal levels) was the same as that for protein breakdown (3.19 and 3.15% h-1, respectively), and both were equally suppressed by insulin (2.4 micrograms h-1). Glucagon (10 micrograms h-1), though, was far less effective in stimulating RNA than protein turnover. A direct comparison of the two dose responses revealed a strong dissociation at 1 and 2 times normal amino acid levels. These findings support the notion that RNA and protein are degraded within a single macroautophagic compartment during amino acid and insulin deprivation. Glucagon, however, appeared to induce a second pathway in which the proportion of sequestered RNA to protein was selectively reduced. Electron micrographs showed that the ratio of vacuoles containing rough as compared with smooth endoplasmic reticulum was decreased by nearly 80% under these conditions.

Amino Acids↗
PubMedDate not supplied

Hepatic UDP-glucose and UDP-glucuronic acid synthesis rates in rats during a reduced energy state.

Hepatic synthesis rates of UDP-glucose and UDP-glucuronic acid were determined in rats. Two high pressure liquid chromatographic methods were developed to quantitate and isolate UTP, UDP-glucose, and UDP-glucuronic acid from perchloric acid extracts of rat liver. The specific activities of UTP, UDP-glucose, and UDP-glucuronic acid were determined in liver samples obtained from rats killed by cervical dislocation at various times after [6-14C]orotic acid administration. Synthesis rates were calculated from the rate of change in specific activities of the compound of interest and its immediate precursor and the concentration of the compound of interest. Synthesis rates of UDP-glucose and UDP-glucuronic acid were 102 +/- 9 and 99 +/- 1 nmol X min-1 X g of liver-1, respectively. UDP-glucuronic acid synthesis apparently accounts for most of the UDP-glucose produced during a period (8 a.m.-10 a.m.) when glycogen synthesis is low. The effect of an ethionine-induced reduction of energy state on these basal synthesis rates was examined. UDP-Glucose and UDP-glucuronic acid synthesis rates were decreased by approximately 80%. In summary, the hepatic synthesis rates of UDP-glucose and UDP-glucuronic acid are approximately 100 nmol X min-1 X g of liver-1, and a reduced energy state can decrease these synthesis rates in vivo.

Animals↗
PubMed1985

Enhancement by uridine of the anabolism of 5-fluorouracil in mouse T-lymphoma (S-49) cells.

Uridine enhances the growth inhibition due to 5-fluorouracil (FUra) in a cultured mouse T-cell lymphoma (S-49). Using colony formation assays we found that cytotoxicity produced by 24-h continuous exposure to FUra (0.5 to 3.5 microM) was increased more than two-fold by simultaneous exposure to 10 microM uridine. Studies were undertaken to explain the mechanism by which uridine enhanced the cytotoxicity of FUra in S-49 cells. Uridine (10 microM) increased by about 50% both the anabolism of 1.0 microM [3H]FUra to acid-soluble metabolites and the incorporation of 1.0 microM [3H]-FUra into RNA. However, the incorporation of 1.0 microM [3H]FUra into these fractions was less than that seen with 2.4 microM [3H]-FUra, a dose which was equitoxic to 1.0 microM [3H]FUra plus 10 microM uridine. High-pressure liquid chromatography analysis of the acid-soluble metabolites of FUra did not show any selective change in specific FUra nucleotides, which could explain the increased cytotoxicity associated with 10 microM uridine. In addition, 5-fluoro-2'-deoxyuridine monophosphate levels and the amount of [3H]FUra which was incorporated into the alkali-stable, acid-insoluble fraction were not increased by uridine. Uridine (10 microM) inhibited de novo pyrimidine biosynthesis by 70%, while 5-phosphoribosyl-1-pyrophosphate levels were unchanged. Presumably, the inhibition of de novo pyrimidine biosynthesis decreased orotic acid levels and allowed more FUra to be anabolized to 5-fluorouridine monophosphate via orotate phosphoribosyl transferase. Furthermore, 2.4 microM FUra inhibited the incorporation of [3H]deoxyguanosine into DNA by 50% after 24 h of incubation. In contrast, 1.0 microM FUra plus 10 microM uridine did not inhibit the incorporation of [3H]deoxyguanosine into DNA. The data suggested that there was a qualitative difference in the mechanism by which 1.0 microM FUra plus 10 microM uridine killed S-49 cells as compared to 2.4 microM FUra alone, and that the enhancement by uridine of the cytotoxicity of FUra was due, in part, to the increased anabolism of FUra to ribonucleotides.

Animals↗
PubMed1995

Inhibition of UDP-glucuronyltransferase activity in rat liver microsomes by pyrimidine derivatives.

Thirty-one differently substituted pyrimidine bases were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol and phenolphthalein by rat liver microsomes. 5-Nitrouracil (compound 1) and its isomer 4,6-dihydroxy-5-nitropyrimidine (compound 2) were the most potent and selective inhibitors of 4-nitrophenol glucuronidation, without any effect on the phenolphthalein conjugating activity of UDP-glucuronyltransferase (UGT). Kinetic studies with compound 1 revealed a mixed type of inhibition toward the acceptor substrate 4-nitrophenol and an atypical competitive type of inhibition toward UDP-glucuronic acid, with apparent Ki values of 0.11 and 0.2 mM, respectively. Two benzylamino-substituted pyrimidines (compounds 10 and 12) and an orotic acid derivative (compound 25) inhibited both 4-nitrophenol and phenolphthalein UGT activities.

Animals↗
PubMedDate not supplied

[Comparative study of the effects of essentiale and the novel Russian hepatoprotective agent "phospholiv" in a model of acute hepatitis in rats].

Curative effect of new preparation "Phospholiv", elaborated in Institute of Biomedical Chemistry, in acute CCl4 induced rat hepatit model was studied. The preparation consists of polyunsaturated phosphatidylcholine and glycyrrhizinic acid salt. Recovery of damaged biosynthesis of albumin and total cell liver RNA--by incorporation of C14-leucine and C14-orotic acid--were observed after 3 days Phospholiv administration, that showed on reparation of damaged protein-synthesis system. Label incorporation into liver fraction > 80S--that was decreased under CCl4 influence--was also restored after Phospholiv treatment, that may testify on its regenerative effect on wholeness of subcellular hepatocytes structures. Substantial decrease of morphologic damages of liver tissue was demonstrated as well. Other phospholipid preparation--known hepatoprotector Essentiale--gave some positive effects too, but Phospholiv influence on biochemical and morphological liver features were 1.5-2 fold as compared with that of Essentiale. Results show on efficiency of polyunsaturated phospholipids in the treatment of acute hepatit in rats--as a result of influence on hepatocytes cell membrane--and on preferential effect of new hepatoprotector Phospholiv.

Acute Disease↗
PubMed1999

Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi.

Uracil auxotrophic mutants of the hyperthermophilic archaeon Pyrococcus abyssi were isolated by screening for resistance to 5-fluoro-orotic acid (5-FOA). Wild-type strains were unable to grow on medium containing 5-FOA, whereas mutants grew normally. Enzymatic assays of extracts from wild-type P. abyssi and from pyrimidine auxotrophs demonstrated that the mutants are deficient in orotate phosphoribosyltransferase (PyrE) and/or orotidine-5'-monophosphate decarboxylase (PyrF) activity. The pyrE gene of wild-type P. abyssi and one of its mutant derivatives were cloned and sequenced. This pyrE gene could serve as selectable marker for the development of gene manipulation systems in archaeal hyperthermophiles.

Amino Acid Sequence↗
PubMed1999

The URA5 gene encoding orotate-phosphoribosyl transferase of the yeast Kluyveromyces lactis: cloning, sequencing and use as a selectable marker.

A pair of degenerate primers was used for amplification and cloning of an internal fragment of the K. lactis URA5 gene. Primers were designed on the basis of highly conserved motifs within protein sequences predicted for URA5 genes from several microorganisms. Using the amplified fragment as a probe, we finally cloned and sequenced a 1.9 kb chromosomal fragment containing the orotate-phosphoribosyltransferase-encoding URA5 gene and an incomplete open reading frame strikingly similar to SEC65 of Saccharomyces cerevisiae and other yeasts, in which the gene encodes a subunit of the signal recognition particle. Uracil-requiring mutants of K. lactis CBS 683 were selected on media containing 5-fluoro-orotic acid and used as recipients in transformation experiments using K. lactis URA5 as the selectable marker, thereby proving functionality of the cloned gene.

Amino Acid Sequence↗
PubMed1990

Rates of RNA degradation in isolated rat hepatocytes. Effects of amino acids and inhibitors of lysosomal function.

1. RNA degradation in isolated rat hepatocytes was measured as the release of radioactive cytidine from fed rats previously labeled in vivo for 60 h with [6-14C]orotic acid. Rates were determined from the linear accumulation of [14C]cytidine between 30 and 120 min of incubation in the presence of 0.5 mM unlabeled cytidine to suppress reutilization. 2. In the absence of amino acids, rates of RNA degradation in isolated hepatocytes averaged 3.97%/h. A complete mixture of amino acids added at 10-20 times normal plasma concentration inhibited RNA degradation by 65-70%. However, at physiological concentrations of amino acids, RNA degradation in isolated rat hepatocytes was less responsive as compared to perfused rat livers. 3. Numerous and large autophagic vacuoles at various stages of digestion were identified throughout the cytoplasm of isolated hepatocytes after 2 h of incubation in the absence of amino acids. The addition of amino acids at 20 times normal plasma concentration abolished almost completely the appearance of autophagic vacuoles. Furthermore, prophylamine, which accumulates in lysosomes, suppressed RNA degradation by 65% and the inhibitor of autophagic vacuole formation, 3-methyladenine, inhibited 70-80% of the degradation. Taken together, these results strongly suggest a contribution of the lysosomal system in the increase of RNA degradation rates in isolated rat hepatocytes.

Amino Acids↗
PubMed1976

Effect of whole-body irradiation on the synethesis of ribonucleic acid associated with nuclear ribonucleoprotein particles of rat liver.

Synthesis of the RNA of rat liver nuclear RNP particles ("informofers") was studied within 12 hrs after 1930 rad whole-body gamma-irradiation. 14C-orotic acid was administratered intravenously and nuclear RNP particles were extracted by 0.1 M and by 0.3 M salt solutions at pH 8.0. Radioactivity of the RNP particles 1.5--2.0 times higher than that of the unirradiated controls up to 6 hrs after irradiation, exhibiting a maximum at the first hour. The labelling of the 0.3 M RNP particles was higher and chased less rapidly than that of the 0.1 M RNP particles. The RNA to protein ratio and the protein composition of the RNP the RNP particles did not change after irradiation as examined by CsCl density gradient centrifugation and by acrylamide gel electrophoresis, respectively. In the cytoplasm enhanced synthesis of rapidly labelled RNA sedimenting with 40 S cytoplasmic RNP paricles could also be demonstrated after irradiation. It is concluded that the synthesis of messenger-type RNA associated with liver nuclear RNP particles increases and more RNA is transferred from the nucleus to the cytoplasm after whole-body irradiation.

Animals↗
PubMed1975

A high molecular weight dihydro-orotate dehydrogenase of Neurospora crassa. Purification and properties of the enzyme.

Some of the unusual molecular and catalytic properties of a high molecular weight dihydro-orotate dehydrogenase (DHOD) from Neurospora crassa have been determined. Comparison of the properties of this enzyme with the properties of the soluble biosynthetic enzyme of prokaryotes has revealed several important differences. The fungal enzyme is located in a mitochondrial membrane in a position consistent with linkage with the respiratory chain through ubiquinone (Miller, R. W.: Arch. Biochem, Biophys. 146, 256-270 (1971)). Release of the enzyme from the membrane results in a solubilized protein complex containing bound lipids and inactive hydrophobic proteins. Non-specific protein aggregation is minimized during purification by Triton-X-100 and phospholipase treatments. The catalytically active enzyme has an apparent molecular weight of 210 000. In contrast to soluble DHOD preparations the high molecular weight enzyme has no endogenous dihydro-orotate oxidase (EC 1.3.3.1) activity and is relatively insensitive to inactivation by sulfhydryl-reactive reagents in the presence of dihydro-orotate (DHO). The enzyme activity is highly sensitive to conditions causing oxidation of flavin mononucleotide (FMN). The activity cannot be restored by cysteine or other means. FMN is present in all purified preparations in a bound, non-fluorescent (reduced) form until dihydro-orotic acid is removed or oxidized. Catalytic efficiency of the purified enzyme was 12 000 mol DHO oxidized per minute per mole FMN. This high turnover rate is due in part to the small flavin content of the purified enzyme, equivalent to 1 mol FMN per 120 000 g of catalytically active protein. Iron was detected in the purified enzyme by atomic absorption spectroscopy but labile sulfide was absent. Thenoyltrifluoroacetone, an iron chelator, only partially inhibited DHO oxidation regardless of electron acceptor. Fatty acids interact with a hydrophobic site of the enzyme in non-competitive fashion but under certain conditions appear to significantly alter the Km for ubiquinone. Orotate, by comparison, is a purely competitive inhibitor. Both types of inhibitor may function to regulate the biosynthesis of orotate in vivo. Superoxide anion is not produced in significant quantities by the DHO-reduced enzyme unless both ubiquinone and a suitable single electron carrier such as phenazine methosulfate are present. DHOD has been proposed as a source of superoxide anion in mammalian mitochondria (Forman, H. J. & Kennedy, J. A.: J. Biol. Chem. 250, 4322-4326 (1975)).

Binding Sites↗
PubMed2002

Pyrimidine nucleotide and nucleic acid synthesis in embryos and megagametophytes of white spruce (Picea glauca) during germination.

Pyrimidine nucleotide synthesis was investigated in isolated germinating zygotic embryos and separated megagametophytes of white spruce by following the metabolic fate of 14C-labelled orotic acid, uridine, and uracil, as well as by measuring the activities of the major enzymes participating in nucleotide synthesis. The rate of nucleic acid synthesis in these tissues was also examined by tracer experiments and autoradiographic studies conducted with labelled thymidine, and by conventional light microscopy. From our results, it emerges that changes in the contribution of the de novo and salvage pathways of pyrimidines play an important role during the initial stages of zygotic embryo germination. Preferential utilization of uridine for nucleic acid synthesis, via the salvage pathway, was observed at the onset of germination, before the restoration of a fully functional de novo pathway. Similar metabolic changes, not observed in the gametophytic tissue, were also documented in somatic embryos previously. These alterations of the overall pyrimidine metabolism may represent a strategy for ensuring the germinating embryos with a large nucleotide pool. Utilization of 14C-thymidine for nucleic acid synthesis increased in both dissected embryos and megagametophytes during germination. Autoradiographic and light microscopic studies indicated that soon after imbibition, DNA synthesis was preferentially initiated along the embryonic axis, especially in the cortical cells. Apical meristem reactivation was a later event, and the root meristem became activated before the shoot meristem. Taken together, these results indicate that precise changes in nucleotide and nucleic acid metabolism occur during the early phases of embryo germination.

Journal Article↗
PubMed1975

Ethanol and brain ribosomes.

The effects of chronic ethanol intake by mice and rats have been determined on brain ribosomes. Under conditions of ethanol administration, when physical dependence on ethanol either does or does not develop in rats, significant inhibition of polypeptide synthesis was observed in comparison with animals not receiving ethanol. In both rats and mice drinking a 10% ethanol solution, in vitro protein synthesis was found to be diminished more in the free than membrane-bound polyribosomes (polysomes) when compared to similar fractions obtained from control animals. Addition of exogenous amino acids stimulated protein synthesis of free polysomes to a greater extent than that of bound polysomes obtained from both groups of micemin vivo incorporation of [5-3H]-orotic acid into RNA was more inhibited in polysomes than ribosomes of ethanol-drinking mice, suggesting that ethanol affects messenger RNA. In addition, chronic ethanol ingestion produced a decreased in vivo incorporation in ribosomal RNA of a mixed population of ribosomes and polysomes. Chronic ethanol ingestion by mice led also to a differential effect on the polysomal population of the brain. It decreased the amount and the incorporation of precursor label into RNA of free polysomes while exerting an opposite effect on bound polysomes. The significance of these findings relative to brain metabolism is discussed.

Animals↗