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PubMed1966

Effects of pancreozymin, urecholine and actinomycin D on the metabolism of ribonucleic acid by canine pancreas.

1. Canine pancreas slices were incubated with [6-(14)C]orotic acid and the rate of its incorporation into RNA was measured. RNA was fractionated by shaking homogenates with phenol at 2 degrees , 50 degrees , 65 degrees and 80 degrees . Cytoplasmic RNA was extracted at the lowest temperature and nuclear RNA at the higher temperatures. The samples were centrifuged through sucrose gradients and the E(260) and (14)C-sedimentation patterns determined. Incorporation of orotic acid was very rapid into cytoplasmic 4s RNA. This probably represents end-group turnover. No incorporation into cytoplasmic ribosomal RNA was observed. 2. The nuclear 50 degrees -RNA exhibited two E(260) peaks, at 18s and 28s. This portion of the sample contained but moderate amounts of [(14)C]RNA. The highly labelled material had sedimentation coefficients in the range 35-50s. The nuclear 65 degrees -RNA showed an E(260) peak at 16s. The [(14)C]RNA peak occurred at 25-35s and this portion demonstrated the highest specific activity of any RNA fraction. 3. The 50 degrees -RNA, 65 degrees -RNA and 80 degrees -RNA were hydrolysed and their base compositions were determined. All three samples possess a ribosomal type of composition (G+C)/(A+U)=(1.4-1.7). For this reason they are considered to contain ribosomal precursor RNA as their major constituent. 4. Actinomycin D (0.5mug./ml.) in the incubation medium inhibited incorporation of orotic acid into both nuclear fractions but not into 4s RNA. 5. The cholinergic drug Urecholine inhibited incorporation into the heavy, high-specific-activity portions of the nuclear fractions but did not inhibit incorporation into the ribosomal precursor type of nuclear RNA. A similar result was also obtained with the hormone pancreozymin. Moderate inhibition of incorporation of orotic acid into 4s RNA likewise resulted from the presence of the drug and the hormone.

Animals↗
PubMed1980

Purine and pyrimidine metabolism in hereditary orotic aciduria: some unexpected effects of allopurinol.

Purine and pyrimidine metabolism have been investigated in the longest surviving case of hereditary orotic aciduria after 15 years of chronic uridine therapy. Several unusual features were recorded: 1. Although the uridine dosage (0.5 mmol/kg) was adequate to control an otherwise normal clinical status, orotic acid excretion was still excessive (in congruent 7 mmol/24 h). Urinary drug metabolites (uracil and uridine), however, accounted for less than 7% of the daily uridine dose, and no orotidine, or any abnormal pyrimidines or purines, were identified at any time. 2. Urinary uric acid excretion was high and plasma uric acid low, resulting in a clearance up to 4 times normal. This was attributed to the uricosuric effect of orotic acid. 3. In direct contrast to previous findings in gouty subjects and healthy male controls we noted: (i) no increase in plasma or urinary uric acid levels, or uric acid clearance, following the change from a low to a high nucleoprotein regime (normally up to two-fold); (ii) allopurinol reduced both urinary uric acid and total oxypurine levels by more than 50% on the low (normally unaffected) as well as the high (normally reduced 20-50%) nucleoprotein regime; (iii) a substantial (up to 70%) reduction in orotic acid excretion during allopurinol therapy (normally mild orotic aciduria), of similar magnitude and in parallel with the reduction in uric acid levels. Uric acid and orotic acid excretion were closely related throughout. These findings differ from those of a similar study of hereditary orotic aciduria and suggest there is competitive transport between exogenous (dietary) purines and pyrimidines, as well as an important interdependence between endogenous purine and pyrimidine metabolism, by mechanisms as yet undefined.

Adolescent↗
PubMed1985

Influence of ammonium ions on hepatic de novo pyrimidine biosynthesis.

Carbamyl phosphate (CP) is synthesized in the liver by two separate enzymes, CPS I and CPS II. CPS I, an intramitochondrial enzyme involved in ureogenesis, has a relative activity of 500- to 1000-fold greater than CPS II, a cytoplasmic enzyme which initiates the sequence of reactions for pyrimidine biosynthesis. The contributions of NH4Cl (substrate for CPS I) ang glutamine (substrate for CPS II) as precursors for pyrimidine biosynthesis in isolated hepatocytes were compared by measuring their effect on uracil nucleotide pool size, the incorporation of NaH14CO3 into these pools, and the accumulation of orotic acid. Physiological concentrations of NH4Cl caused a marked stimulation of incorporation of radioactivity into uracil nucleotides (6-fold increase at 0.5 mM NH4Cl), and radioactive orotate appeared in both the cells and the medium. In contrast, glutamine (at concentrations up to 10 mM) had no effect on the incorporation of radioactivity into uracil nucleotides, and no orotic acid was detected. Uracil nucleotide pools were expanded up to 50% by low levels of NH4Cl, but there was no expansion of this pool in the presence of added glutamine. NH4Cl-driven pyrimidine de novo biosynthesis was insensitive to feedback inhibition by an expanded uracil nucleotide pool, to galactosamine treatment, and to acivicin treatment, indicating that NH+4 stimulated pyrimidine biosynthesis as a result of CP synthesis by mitochondrial CPS I. The consequence of intramitochondrially produced CP being available for pyrimidine biosynthesis is that the controlling step of this pathway (CPS II) is bypassed. The appearance of orotic acid following NH4Cl stimulation indicated that the rate-controlling step of hepatic de novo pyrimidine synthesis under these conditions was orotate phosphoribosyl transferase. These data indicate that, at physiological concentrations of NH+4, the majority of uracil nucleotides synthesized in isolated rat hepatocytes was derived from intramitochondrially generated CP. The effect of NH4Cl on the output of uridine by the isolated perfused rat liver was examined. In the presence of a single addition of 20 mM NH4Cl, the excretion of uridine was increased from 100-200 to 375 nmol h-1 g-1 liver and orotic acid was released into the circulating perfusate reaching a maximum of 2 microM (in 220 ml of perfusate) after 2 h. With 40 mM NH4Cl, uridine export was increased to 450 nmol h-1 g-1 and a maximum of 5 microM orotic acid was released into the perfusate after 2 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonia↗
PubMed1975

Increased synthesis of low-molecular-weight nuclear ribonucleic acids of rat liver after gamma irradiation and hepatectomy.

Incorporation of [3H]orotic acid into low-molecular-weight nRNA of rat liver, fractionated on polyacrylamide gels, increased 6-12h after partial hepatectomy and 6h after gamma-irridation at 2000 R. The incorporation of orotic acid was particularly increased into the 4.5S, 5S and approx. 10S nRNA fractions. If the irradiation was given after 6h of regeneration and RNA was isolated from the nucleus 12h after hepatectomy then the incorporation of orotic acid into these low-molecular-weight nRNA components was greater than after hepatectomy or irradiation alone.

Animals↗
PubMed1997

[Morphological aspects of the effect of magnesium orotate therapy on changes in the vascular walls induced by cholesterol-rich diet].

Heart and blood vessel disease are one of the leading causes of death, so their prevention and therapy are very important. In the present study the effects of magnesium chloride, magnesium orotate and orotic acid were tested. New Zealand rabbits were fed with enriched (2%) cholesterol diet during 112 days: starting with day 56 all rabbits were treated with MgCl2, Mg-orotate or orotic acid (orally). Aortas, coronaries, renal and femoral arteries were removed and evaluated by morphological and morphometric methods. Atherosclerotic alterations in each vessel could be influenced moderately by Mg-chloride, quite well by orotic acid and excellently by Mg-orotate. From these results one can conclude that orotic acid and Mg-orotate have a beneficial effect in the prevention and therapy of heart and vessels diseases.

Animals↗
PubMed1987

Inhibition of pyrimidine de novo synthesis by DUP-785 (NSC 368390).

The mechanism of action of NSC 368390 (DUP-785, 6-fluoro-2-(2'-fluoro-1, 1'-biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid sodium salt) was studied using three different approaches. First, we studied growth inhibition by DUP-785 in L1210 leukemia cells and M5 melanoma cells. The concentrations causing 50% growth inhibition after 48 hr of culture were 5.8 and 0.6 microM, respectively. DUP-785 had to be present continuously throughout culture. Growth inhibition by 25 microM DUP-785 could be prevented by addition of 1 mM uridine or orotic acid to cultures of these cell lines; in M5 cells cytidine was also able to prevent growth inhibition. Dihydro-orotic acid (DHO) and carbamyl-aspartate were not able to prevent growth inhibition by DUP-785. Second, we studied accumulation of orotic acid and of orotidine induced by incubation with 1 microM pyrazofurin, an inhibitor of the orotate phosphoribosyl-transferase-orotidine-monophosphate decarboxylase complex. Addition of DUP-785 to the culture medium prevented the orotic acid accumulation. Furthermore, DUP-785 prevented accumulation of H14CO3- into orotic acid of pyrazofurin-treated L1210 cells. Third, we measured the effect of DUP-785 on DHO-dehydrogenase (DHO-DH), since the results indicated that this enzyme was affected by DUP-785. DHO-DH was assayed in isolated rat liver mitochondria. The Km for L-DHO was about 12 microM. DUP-785 appeared to be a potent inhibitor of DHO-DH with an apparent Ki of about 0.1 microM and an apparent Ki' of about 0.8 microM. The mode of inhibition appeared to be linear mixed type. After exposure of L1210 cells to 25 microM DUP-785 for 2 hr DHO-DH was almost completely inhibited. After suspension in fresh medium without drug, DHO-DH activity was recovered to about 60% after 24 hr. In conclusion, DUP-785 is a potent inhibitor of pyrimidine de novo biosynthesis, by inhibition of the mitochondrial enzyme DHO-DH.

Amides↗
PubMed2005

Regulation of pyrimidine biosynthesis in Acidovorax delafieldii.

The regulation of de novo pyrimidine biosynthesis in the nutritionally versatile bacterium Acidovorax delafieldii ATCC 17505T was influenced by carbon source and pyrimidine supplementation. Uracil supplementation of succinate-grown A. delafieldii ATCC 17505T cells produced a greater decrease in the de novo pyrimidine biosynthetic pathway enzyme activities than did orotic acid supplementation. The presence of orotic acid or uracil in the medium of the glucose-grown wild type cells generally increased the pyrimidine biosynthetic enzyme activities. After the pyrimidine limitation of an A. delafieldii orotate phosphoribosyltransferase mutant strain, the pyrimidine biosynthetic pathway enzyme activities in the glucose-grown mutant cells were more highly derepressed than in the succinate-grown mutant cells.

Comamonadaceae↗
PubMed2003

Quantification of non-protein nitrogen components of infant formulae and follow-up milks: comparison with cows' and human milk.

The composition of fourteen infant formulae and six follow-up milks with regard to their free amino acids (including taurine), free nucleotides, orotic acid, and free and total l-carnitine content was studied. The levels found were compared with the limits established in European legislation and with the composition of human and cows' milk samples. HPLC methodologies, optimized and validated for the matrices under study, were used, except for free and total l-carnitine contents that were quantified using a flow-injection manifold, also optimized and validated for the matrices under study. Global statistical treatment of the results by cluster analysis indicated similarities between the contents of the N compounds under study of infant formulae, follow-up milks and cows' milk and differences with regard to human milk composition. The principal component analysis showed that 60.2 % of the variation in data was due to the first principal component, and the second component represented 23.8 % of the total information. Nucleotide profiles, orotic acid, and free and total l-carnitine contents explain the main differences observed between human milk and the other milks studied (cows' milk, infant formulae and follow-up milks). Cows' milk is distinguished from infant formulae and follow-up milks mainly owing to the different uric acid contents and free amino acids profiles.

Amino Acids↗
PubMed1988

Ultrasonic propagation properties (@ 100 MHz) in excessively fatty rat liver.

The effects on the ultrasonic propagation properties of livers of the addition of 1% orotic acid to rat diets were examined. In rats, dietary orotic acid exerts several effects on lipid metabolism; its overall consequence is that excessively high hepatic fat concentrations are built up over a short period of time, thus making this an ideal model to study the ultrasonic propagation properties as a function of sequential development of fatty liver. Over a 16-day period on the orotic acid diet, the supplemented rat liver lipid concentrations increased from a normal range of 2%-4% to the lower 20's%; hepatic water concentration decreased from a normal value of approximately 70% to approximately 50%; total protein concentration decreased slightly from a normal range of 17%-20% to 11%-16%; and rat liver weight increased from approximately 11 g to around 20 g. Ultrasonic attenuation coefficient and speed were assessed in liver tissue with the scanning laser acoustic microscope at 100 MHz. As hepatic lipid increased, ultrasonic attenuation at 100 MHz increased temporally from a normal range of 12-14 dB/mm to a maximum of 54 dB/mm and ultrasonic speed decreased from a normal range of 1553-1584 m/s to a minimum of 1507 m/s. Multivariant linear regression was used in the analysis of covariance to fit the least-squares estimates to the linear regression model. Strong correlates of ultrasonic speed with both water concentration and fat concentration in the liver were observed.

Animals↗
PubMed1980

Non-specific incorporation of nucleic acid precursors in Blastomyces dermatitidis and Histoplasma capsulatum.

Incorporation of thymidine, thymidine monophosphate (TMP), thymidine triphosphate (TTP), uridine and orotic acid into DNA, RNA and protein in Blastomyces dermatitidis and Histoplasma capsulatum was studied utilizing a specific acid hydrolysis technique developed for these fungi. Thymidine was incorporated to the greatest extent (approximately 0.5% of added label) followed by uridine, orotic acid, TMP and TTP. In Blastomyces, uridine and orotic acid labeled primarily RNA. TMP and TTP labeled RNA, DNA and protein at nearly the same level. In Histoplasma RNA was labeled poorly by any of these precursors. TMP and TTP labeled DNA predominately and protein to a slightly lower level. Deoxyadenosine or uridine media supplements of 250 micrograms/ml did not enhance incorporation. All precursors tested were found to be nonspecific in that RNA, DNA and protein were labeled. All data indicate that neither RNA nor DNA synthesis can be specifically measured in whole cells or acid precipitates by any of these precursors. Specific radiometric monitoring with these isotopes therefore requires the separation of these macromolecules.

Animals↗
PubMed1976

The effect of cycloheximide on hepatic RNA synthesis and nucleolar size in rats force-fed a threonine-devoid diet.

Young rats were force-fed a complete or threonine-devoid diet for 3 days. On the fourth morning, rats of each group were injected intraperitoneally with cycloheximide (150 mug/100 g body weight) or saline with [6-14C]orotic acid 30 minutes later, 2 hours before killing. Incorporation of [6-14C]orotic acid into hepatic RNA fractions (whole homogenate, postmitochondrial supernatant, microsomes, ribosomes, nuclei and soluble) revealed elevated levels (cpm/mg RNA) in rats force-fed the threoninedevoid diet in comparison to those of rats force-fed the complete diet. However, treatment with cycloheximide decreased the incroporation of [6-14C]orotic acid into the hepatic RNA fractions of the rats force-fed the threonine-devoid diet to levels that were similar to those in rats force-fed the complete diet with or without cycloheximide treatment. Studies dealing with nucleoli isolated by sucrose gradients from livers of control and experimental rats revealed heavier nucleoli and more radioactive labeled RNA in nucleoli ([6-14C]orotic acid administered 30 minutes before killing) of rats force-fed the threonine-devoid diet than in those force-fed the complete diet for 3 days. Treatment with cycloheximide decreased the elevated incorporation in the experimental rats. Also, electron microscopic studies revealed that after cycloheximide treatment, the enlarged hepatic nucleoli of the experimental rats became smaller and returned to a more normal pattern, as found in the control rats. The studies suggest that active hepatic protein synthesis is involved in the increased hepatic RNA synthesis in rats force-fed the threonine-devoid diet and that following inhibition of protein synthesis, as induced by cycloheximide, there is a rapid inhibition of the accelerated hepatic RNA synthesis observed in rats force-fed the threonine-devoid diet with a rapid reversal toward a normal level, i.e. toward that found in control rats.

Animals↗
PubMed1977

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.

Animals↗
PubMed2002

Control of pyrimidine formation in Pseudomonas putida ATCC 17536.

The regulation of de novo pyrimidine biosynthesis in Pseudomonas putida ATCC 17536 by pyrimidines was explored. The pathway enzyme activities were higher in glucose-grown cells than in succinate-grown cells, indicating catabolite repression by succinate. In P. putida cells grown on succinate as a carbon source, only aspartate transcarbamoylase activity was greatly diminished by uracil supplementation. When glucose was the carbon source, orotic acid supplementation significantly decreased orotate phosphoribosyltransferase and orotidine 5'-monophosphate (OMP) decarboxylase activities. Uracil auxotrophs, deficient for dihydroorotase activity or with reduced phosphoribosyltransferase activity, were isolated. After pyrimidine limitation of both auxotrophs, the greatest derepression of enzyme activity was observed for OMP decarboxylase independent of carbon source. Orotic acid induced both phosphoribosyltransferase and decarboxylase activities in glucose-grown cells of the dihydroorotase-deficient strain. Regulation at the transcriptional level of de novo pyrimidine biosynthetic enzyme synthesis in P. putida ATCC 17536 was observed, which contrasts with previous observations.

Aspartate Carbamoyltransferase↗
PubMed1994

Inborn errors of urea synthesis.

Inborn errors of urea synthesis can present in the newborn period as a catastrophic illness or later in childhood or adulthood with an indolent course punctuated by hyperammonemic episodes. Because symptoms mimic other neuropsychiatric disorders, it is common for there to be a delay in diagnosis, often with dire consequences. Diagnosis relies on the combination of clinical suspicion and the measurement of ammonium, lactate, and amino acids in plasma and organic acids and orotic acid in urine. Treatment involves nitrogen restriction combined with the stimulation of alternate pathways of waste nitrogen excretion. More recently liver transplantation has been performed as enzyme replacement therapy. The outcome is poor in children who survive prolonged neonatal hyperammonemic coma, with most manifesting developmental disabilities. The etiology of neuronal injury in this disorder is unclear but may involve some combination of ammonia/amino acid accumulation, neurotransmitter alterations, and excitotoxic injury. Gene therapy holds the promise of improved treatment in the future.

Amino Acid Metabolism, Inborn Errors↗
PubMed1998

Partition analysis of an enzyme acting concurrently upon two substrates in a continuous multiwavelength assay.

We describe a multiwavelength method for measuring an enzyme's discrimination of one substrate over another by continuously monitoring the reactions of the two substrates simultaneously. This method is generally applicable to ultraviolet-visible diode array or rapid-scanning spectrophotometers and the measurement requires a single incubation of enzyme with two substrates. Rates at each of the wavelengths may be fit globally by using a nonlinear least-squares fitting procedure which provides adequate statistical analysis. The specificity of trypsin for N-alpha-benzoyl-L-arginine p-nitroanilide (BRpNA) over N-t-butyloxycarbonyl-L-alanine-p-nitrophenylester (BocApNP) was 2.1 as measured by the multiwavelength partition method and 2.3 by comparing the individual kcat/K(m)'s for the two substrates. Multiwavelength analysis was applied to two enzymes in the biosynthetic pathway for fungal melanin: scytalone dehydratase and trihydroxynaphthalene reductase from Magnaporthea grisea. The specificity of trihydroxynaphthalene reductase for 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one compared to scytalone, a natural substrate for the enzyme, was 95. Scytalone dehydratase was eight-fold more specific for 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one than it was for scytalone. Multiwavelength analysis was also used to measure an equilibrium constant of 0.040 for the reaction ¿dihydroorotate + oxonic acid<-->orotate + dihydrooxonic acid¿ catalyzed by dihydroorotate dehydrogenase. Advantages, limitations, and further applications of this steady-state method, which directly measures relative substrate specificities, are delineated. All studies described in this paper were at pH 7.0 and 25 degrees C.

Ascomycota↗
PubMed1997

Pyrazolylborate-Zinc Complexes of RNA Precursors and Analogues Thereof.

The Tp ligand tris(3-cumenyl-5-methylpyrazolyl)borate was found to stabilize zinc complexes of nucleobases, of their natural precursors, and of nucleoside and nucleotide derivatives. Dihydroorotic acid and orotic acid are bound as monodentate carboxylate ligands. Uracil is coordinated via its deprotonated N1; 6-methylthiouracil acts as a bidentate ligand via N1 and S. Analogously, xanthine is a monodentate ligand bound by its deprotonated N7, while 6-mercaptopurine seems to bind in a bidentate fashion via N7 and S. Spectroscopic evidence indicates coordination of uridine and 2',3'-O-isopropylideneuridine via their deprotonated N3, as well as of xanthosine via N7. The hydrolytic cleavage of 2',3'-O-isopropylideneuridine 5'-(bis(p-nitrophenyl) phosphate) by TpZn-OH is preceded by an attachment of one TpZn unit to the deprotonated uracil base, presumably via N3.

Journal Article↗
PubMed1981

Photochemistry of pyrimidine bases as studied by e.s.r. and spin-trapping.

The direct photoexcitation of pyrimidine bases in D2O solutions yields free radicals which could be conveniently identified by spin-trapping with 2-methyl-2-nitrosopropane. Most of the radicals formed were attributed to D-addition to one end of the 5,6 double bond. However, orotic acid and iso-orotic acid yielded N(3) centred free radicals, formed by homolytic cleavage of the N-H bond. No indication could be found for a free radical involvement in the photocleavage of cyclobutane-type pyrimidine dimers.

Electron Spin Resonance Spectroscopy↗
PubMed1985

Detection of dihydroorotate dehydrogenase activity as a marker of de novo nucleic acid synthesis in germinal centers of spleens following antigen administration.

A histochemical technique, developed for the localization of DHO-D activity in tissue, has been utilized to study the distribution of this activity in rabbit spleen. This enzyme is involved in the de novo synthesis of pyrimidines of nucleic acids via orotic acid as an intermediate. The enzyme was found to be most active at the periphery of follicles in the resting spleen. Following antigenic challenge, enzyme activity became prominent for several days in macrophages within germinal centers, but not in the intensely pyroninophilic cells which are the dominant cell type of these structures. Antigenicity of the injected compound appeared to be necessary for the production of this effect. The pattern of localization of DHO-D is similar to the pattern of distribution of antigen within splenic tissue.

Animals↗