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PubMed2003

Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris.

A pair of degenerate primers was used for amplification and cloning of a DNA fragment containing parts of the P. pastoris URA5 and SEC65 genes. Using additional information from a partial genomic sequence of P. pastoris, we cloned and sequenced a 1.9 kb chromosomal fragment containing the complete orotate-phosphoribosyltransferase-encoding URA5 gene. A disruption cassette was constructed by replacing a small part of the open reading frame with a kanamycin-resistance gene. The P. pastoris wild-type strain NRRL Y-11430 was transformed with the disruption cassette and an ura5 auxotrophic strain was identified. To generate marker constructs that can be reused in successive transformations of a single strain, we constructed two lacZ-PpURA3-lacZ and lacZ-PpURA5-lacZ cassettes and used them to disrupt PpOCH1. The PpURA3 and PpURA5 genes in the disruptants were then successfully recycled by selecting for resistance to 5'-fluoro-orotic acid. We also assembled a set of modular plasmids that can be used for the stable genetic modification of P. pastoris via a double cross-over event. The sequence presented here has been submitted to the EMBL data library under Accession No. AY303544.

Amino Acid Sequence↗
PubMed1981

Arginine requirement of mature protein-malnourished rats for maximal rate of repletion.

Male Sprague-Dawley rats (initial weight 400 g) were fed a 0.5% lactalbumin diet containing required amounts of all other known essential nutrients for 14 weeks. The body weights averaged 250 g after depletion when the animals were randomly assigned to four groups of four or five. Rats of one group were killed for baseline analyses on day 0 of repletion. The remaining three groups were repleted for 14 days with a complete L-amino acid diet containing 20% protein equivalent and 0, 0.75 or 1.5% Arg . HCl. Daily feed intakes averaged 11, 15 and 17 g and daily weight gains were 3.3, 7.4 and 8.3 g, respectively. Average values for corresponding groups were: nitrogen balance--116, 187 and 190 mg/day; urinary orotate--4.6, 0.8 and 0.15 mg/day; carcass lipid--27, 37 and 40 g/100 g dry matter. Liver weights per 100 g body weight for 0 arginine exceeded other groups by 40%. There were no differences in creatinine index, nor in plasma concentrations of insulin, glucagon and albumin. The maximal rate of recovery from protein-calorie malnutrition of mature rats required an exogenous source of arginine. Urinary orotic acid excretion provided a reliable measure for determining when arginine needs for maximal rate of repletion were not met.

Animals↗
PubMed1966

The effect of feeding with a tryptophan-free amino acid mixture on rat-liver polysomes and ribosomal ribonucleic acid.

1. Starving rats were given complete and tryptophan-deficient amino acid mixtures by stomach tube and were killed from 1 to 7hr. later. The polysome profile in the livers of rats fed with the tryptophan-deficient mixture showed a shift in distribution such that the large aggregates were decreased and the small aggregates were increased, particularly dimers. This polysome shift was reversed when the complete amino acid mixture was given by stomach tube 2hr. after administering the tryptophan-free amino acid mixture. 2. After removal of liver polysomes by centrifugation, some smaller ribosomal aggregates (oligosomes) remaining in suspension were harvested by prolonged centrifugation of the supernatant fluid. A large increase in the dimer population of this fraction was observed in the rats receiving the incomplete mixture. 3. When the polysome and oligosome fractions were incubated with cell sap, an energy-generating system and labelled amino acids dl-[1-(14)C]leucine and l-[Me-(14)C]tryptophan were incorporated into the cell fractions in the ratio 4.5:1. Preparations of polysomes and oligosomes from rats fed with the tryptophan-free amino acid mixture showed a decreased amino acid-incorporating activity compared with particulate preparations made from rats fed with the complete mixture. 4. The yield of free ribosomes prepared from the unfractionated liver microsomes by treatment with iso-octane was 40-50% greater in rats fed with the amino acid mixture deficient in tryptophan. 5. A post-microsomal fraction was prepared from cell sap and was shown to consist of ribosomal sub-units. When the animals were fed with the tryptophan-deficient mixture, there was an increase in content of this post-microsomal fraction and in the ratio 30s RNA/19s RNA. Rats were also given [5-(3)H]orotic acid at the time of feeding with the amino acids. Lack of tryptophan in the mixture caused a decrease in the specific activity of both RNA fractions which affected the 30s RNA more extensively than the 19s RNA. 6. These changes in the distribution and quantity of the cellular components engaged in protein synthesis are discussed in relation to RNA metabolism and amino acid-incorporating activity of the liver cell and their response to feeding with the tryptophan-free amino acid mixture.

Animals↗
PubMed1996

Clinical and biochemical heterogeneity in females of a large pedigree with ornithine transcarbamylase deficiency due to the R141Q mutation.

A large family with ornithine transcarbamylase deficiency due to mutation R141Q was ascertained through a propositus who presented with acute neonatal hyperammonemic coma. Of 13 females at risk, 11 were evaluated clinically and had laboratory studies performed. Seven were found to be heterozygous for the mutation. Of these seven, five had chronic clinical symptoms and two were asymptomatic. None of the heterozygotes had elevated plasma ammonia on random testing. Of the five symptomatic females, three had markedly elevated plasma glutamine levels on random testing, while two had levels in the upper range of normal. Plasma citrulline and arginine levels were somewhat lower in the symptomatic individuals but still within the normal range. Five heterozygotes who were tested had either spontaneous orotic aciduria or elevated orotic acid following ingestion of allopurinol, whereas one unaffected female and one unaffected male had normal allopurinol tests. A higher than expected proportion of female heterozygous for the R141Q mutation were clinically and biochemically symptomatic but remained undiagnosed for many years. Plasma glutamine determination and allopurinol testing should be performed in females who present with a combination of relatively non-specific symptoms detailed in this report.

Allopurinol↗
PubMed1970

The methylation of transfer ribonucleic acid during regeneration of the liver.

Transfer ribonucleic acid(1) is methylated after the molecule is synthesized; at least eight enzymes are involved in the transfer of methyl groups (derived from methionine). The time courses of methylation and synthesis of tRNA during rat liver regeneration have been compared in an in vivo radioisotopic study, using 6-orotic acid-(14)C and (3)H-methyl-L-methionine as precursors in double label pulses. Liver regeneration is a synchronized system in which biochemical events of the cell cycle are separable. Transfer RNA methylation increase precedes by several hours tRNA synthesis during regeneration, although the curves overlap. A ratio of the relative rate of methylation to the relative rate of synthesis has been made; that curve positively correlates with the rise and fall of protein synthesis during regeneration. It is clear that methylation and synthesis of tRNA are only weakly coupled; changing methyl content of the tRNA "pool" resulting from differential tRNA methylase and polymerase activities may regulate the rate of protein synthesis in the cell cycle at the translational level. The "pool sizes" of uridine monophosphate (UMP) and S-adenosylmethionine (SAM) were measured indirectly; UMP and SAM were isolated from perchloric acid supernatants and their specific activities were computed. Differential changes in radioactivity available to tRNA methylases and polymerases are not a source of artifact. That is, the control of both the synthesis and methylation of tRNA is at the enzyme level in vivo, rather than at some enzymatic step prior to those enzymatic reactions.

Acyltransferases↗
PubMed1978

[The influence of allopurinol on purine- and pyrimidinesynthesis (author's transl)].

Allopurinol inhibits xanthinoxydase. This results in a decrease of the serum and urinary uric acid. Simultaneously the renal excretion of hypoxanthine and xanthine increases. In most patients, however, the decrease of urinary uric acid is not completely replaced by the increase of oxypurines. This is attributed to an additional inhibition of de novo purine biosynthesis. The different effect of allopurinol on the endogenous and exogenous urates offers an alternative approach to explain the "purine deficit". The effect of allopurinol on the pyrimidine metabolism is due to an inhibition of orotidylic decarboxylase by the ribonucleotides of allopurinol, xanthine and oxipurinol. This inhibition is followed by an increase in the urinary excretion of orotidine and orotic acid. The additional administration of ribonucleic acid leads to a striking decrease of the allopurinol induced orotaciduria. The continuous administration of allopurinol also produces an increase in the activity of orotate phosphoribosyltransferase and orotidylic decarboxylase. At this time the mechanism of this increase in activity is not completely understood. Allopurinol is metabolized rapidly. Only 3-10% of an administered dose are excreted unchanged in the urine. Most of the allopurinol is oxidized to oxipurinol. A small portion is converted to the ribonucleosides and ribonucleotides1 of allopurinol and oxipurinol.

Allopurinol↗
PubMed1974

Some physiological and biochemical effects of a Coxiella burneti lipopolysaccharide preparation on guinea pigs.

A lipopolysaccharide obtained in a dialyzed phenol extract from the rickettsia Coxiella burneti produced the following effects in guinea pigs after intraperitoneal injection: hyperthermia, loss of body weight, increased liver weight and concomitant lipid infiltration, elevated levels of hepatic and plasma cortisol, increased incorporation of [(3)H]orotic acid into hepatic 28S and 18S ribosomal ribonucleic acid, increased incorporation of (14)C-labeled amino acids into liver and plasma protein, and leukocytosis. Most of these events also occur during infection of guinea pigs with C. burneti, and a causal relationship between the rickettsial lipopolysaccharide and the biochemical changes that occur during Q fever is suggested.

Amino Acids↗
PubMed1970

The effect of feeding with a tryptophan-free amino acid mixture on rat liver magnesium ion-activated deoxyribonucleic acid-dependent ribonucleic acid polymerase.

1. The Widnell & Tata (1966) assay method for Mg(2+)-activated DNA-dependent RNA polymerase was used for initial-velocity determinations of rat liver nuclear RNA polymerase. One unit (U) of RNA polymerase was defined as that amount of enzyme required for 1 mmol of [(3)H]GMP incorporation/min at 37 degrees C. 2. Colony fed rats were found to have a mean RNA polymerase activity of 65.9muU/mg of DNA and 18h-starved rats had a mean activity of 53.2muU/mg of DNA. Longer periods of starvation did not significantly decrease RNA polymerase activity further. 3. Rats that had been starved for 18h were used for all feeding experiments. Complete and tryptophan-deficient amino acid mixtures were given by stomach tube and the animals were killed 15-120min later. The response of RNA polymerase to the feeding with the complete amino acid mixture was rapid and almost linear over the first hour of feeding, resulting in a doubling of activity. The activity was still elevated above the starvation value at 120min after feeding. The tryptophan-deficient amino acid mixture produced a much less vigorous response about 45min after the feeding, and the activity had returned to the starvation value by 120min after the feeding. 4. The response of RNA polymerase to the feeding with the complete amino acid mixture was shown to occur within a period of less than 5min to about 10min after the feeding. 5. Pretreatment of the animals with puromycin or cycloheximide was found to abolish the 15min RNA polymerase response to the feeding with the complete amino acid mixture, but the activity of the controls was unaffected. 6. The characteristics of the RNA polymerase from 18h-starved animals and animals fed with the complete or incomplete amino acid mixtures for 1h were examined. The effects of Mg(2+) ions, pH, actinomycin D and nucleoside triphosphate omissions were determined. The [Mg(2+)]- and pH-activity profiles of the RNA polymerase from the animal fed with the complete mixture appeared to differ from those of the enzyme from the other groups, but this difference is probably not significant. 7. [5-(3)H]Orotic acid incorporation by rat liver nuclei in vivo was shown to be affected by the amino acid mixtures in a similar manner to the RNA polymerase. 8. The tryptophan concentrations of plasma and liver were determined up to 120 min after feeding with the amino acid mixtures. Feeding with the complete mixture produced a rapid increase in free tryptophan concentrations in both plasma and liver, but feeding with the incomplete mixture did not alter the plasma concentration. The liver tryptophan concentration increased at about 45min after feeding with the tryptophan-deficient diet. 9. There was a good correlation between the liver tryptophan concentration and RNA polymerase activity in all groups of animals. 10. It was concluded that the rat liver nucleus responded to an increase in amino acid supply by increased synthesis of RNA as a result of synthesis of RNA polymerase de novo. The correlation of tryptophan concentration and RNA polymerase activity appears to reflect the general amino acid concentration required to support hepatic protein synthesis and to produce new RNA polymerase. This new polymerase appears to differ from the basal RNA polymerase by its rapid synthesis and destruction, which may be a means of regulating RNA synthesis by the amino acid concentration in the liver.

Amino Acids↗
PubMed1983

Effect of proline on arginine uptake and nitrogen metabolism of lactating goats.

The hypothesis that addition of proline may save dietary protein for milk production was tested by administration of proline to two goats through a duodenal cannula. Because proline is synthesized by the mammary gland in vitro from arginine, the effect of proline supplementation on arginine uptake by the gland was tested in vivo. Arginine uptake, calculated from arterio-venous difference, dropped significantly in both animals, especially in the morning 1 h after milking, when a low-protein diet was fed. Milk production and total nitrogen in milk were not affected significantly by proline supplementation. A trend was toward decrease of milk orotic acid and an increase of milk fat due to proline supplementation.

Animals↗
PubMed1978

Aspects of the role of lecithin:cholesterol acyltransferase in metabolism of triglycerides.

An increased TG secretion from the liver may lead to an increased secretion of LCAT. Studies on isolated rat hepatocytes have shown: 1. Oleic acid added to the medium stimulates TG and LCAT secretion in a similar way. 2. Cycloheximide, colchicine, 4-amino-pyrazolopyrimidine, and D-galactosamine depress the secretion of both TG and LCAT. 3. There is a positive linear correlation between TG and LCAT secretion in cell preparations from rats operated on with ventromedial hypothalamic lesions. Treatment of rats with orotic acid resulted in maintained plasma LCAT activity despite very low TG concentration. This indicates that LCAT secretion is not dependent upon an intact TG secretion.

Animals↗
PubMed1975

Different binding of poly(A)-containing and poly(A)-free fractions of nuclear ribonucleic acid to ribosomes from rat liver.

Total nuclear RNA extracted from nuclei of rat liver cells by phenol/chloroform in the presence of sodium dodecyl sulphate was separated by combined gel filtration on Sepharose 4 B and affinity chromatography on poly(U) Sepharose into fractions differing in their molecular weights and contents of poly(A) sequences. The poly(A)-containing 45-S RNA became labelled most rapidly if rats were administered [3H] orotic acid. This fraction showed a high template activity when added to postmitochondrial supernatants of the Krebs ascites tumour. Fractions of nRNA, free of poly(A) sequences, had no stimulating effect on protein synthesis in this system. The 45-S RNA-containing poly(A) was readily bound to crude polyribosomes from rat liver at 0 degrees C and both ATP and GTP were necessary for this reaction. Sucrose gradient analyses provided evidence that this RNA species is bound predominantly to 80-S ribosomes. No binding was obtained with polyribosomes washed with 0.5 M KCl. The binding ability of washed polyribosomes was restored by the addition of the ribosomal wash fraction or rat liver cytosol. Crude polyribosomes bound significantly lower quantities of nRNA species free of poly(A) when compared with poly(A)-45-S RNA. The label was scattered through the whole ribosomal sedimentation pattern with no predominant peaks and the binding reaction required neither soluble factors nor nucleotide cofactors. The labelling kinetics and high template activity of poly(A)-45-S nRNA indicate that this fraction contains precursors of cytoplasmic mRNA. Requirements for soluble factors and nucleotide cofactors in the binding of this RNA species to 80-S ribosomes suggest that this binding, unlike that of other nRNA species, has a specific mechanism resembling that of mRNA binding during peptide initiation.

Animals↗
PubMedDate not supplied

[Quantitative evaluation of the anxiolytic and nootropic effects of potassium orotate in a wide range of doses].

Chronic administration of potassium orotate to rats per os in doses of 5, 10, 20, 50 and 100 mg/kg was followed by different correlations of the training under stress with anxiolytic activity. The doses of the drug were defined, at which the maximally pronounced effects under consideration were in optimal correlations, as were doses which entailed inhibition of the training in the presence of a relatively low tranquilizing activity, low emotional responsiveness and aggressiveness. It is suggested that natural analogs of pyrimidine, whose precursor is orotic acid, are universal endogenous regulators of mnemonic and antianxiety functions.

Animals↗
PubMedDate not supplied

6-(14C)-orotate uptake and rna synthesis by liver from thyroidectomized rats treated with 3,5,3'-triiodo-1-thyronine (t3).

It has been shown that T3 administration to thyroidectomized rats induces a marked increase of the in vivo labelling of liver nuclear RNA by 6-[14C]-orotic acid and, although to a lesser extent, of the radioactivity of the acid-soluble fraction. Therefore, in evaluating the real increase in RNA synthesis induced by T3 administration it has been taken into account that the hormone stimulates the uptake of the labelled precursor by the liver and enhances the specific activity of the nucleoside and nucleotide pool. The greater penetration of orotate into the liver does not depend on the increased RNA synthesis since it is not prevented by the administration of actinomycin D which markedly inhibits the transcriptional process. These findings indicate that the increased uptake of orotate and the stimulation of RNA synthesis represent distinct effects of the thyroid hormone on its target tissue.

Animals↗
PubMed2000

Urinary pyrimidine analysis in healthy newborns, infants, children, adults and patients with congenital metabolic diseases.

BACKGROUND: In previous reports, the reference range for urinary pyrimidine was determined on the basis of a small number of samples, with data for only a few patients being reported. In the present study, we measured urinary pyrimidine compounds in 25 healthy newborns, 33 healthy infants, 130 healthy children and 166 healthy adults. In addition, we also analyzed urinary pyrimidine compounds in various patients with abnormal pyrimidine metabolism, such as congenital pyrimidine metabolism disorders and urea cycle disorders. METHODS: We analyzed urines by high-performance liquid chromatography with column switching. Analyses were performed with both a reverse-phase column and an anion-exchange column. The columns were connected by a column switch, with all systems being controlled automatically by a computerized system controller. RESULTS: The excretion of pyrimidine compounds in patients with abnormal pyrimidine metabolism (containing heterozygotes) was out of our reference ranges. CONCLUSIONS: These results suggest that urinary pyrimidine analysis is a useful index for the diagnosis of pyrimidine metabolism disorders, urea cycle disorders and these heterozygotes. Based on this large-group analysis of healthy individuals, we were able to determine the reference ranges of urinary orotic acid, dihydrouracil and uracil for each age group.

Adolescent↗
PubMed1975

Hereditary orotic aciduria: results of a screening survey.

Urine from 1,358 mentally retarded subjects was screened for the presence of elevated concentrations of orotic acid and orotidine. This survey was conducted in search of occult variants of hereditary orotic aciduria which might be associated with mental retardation. Although no homozygous variants were detected, 9 subjects with persistently abnormal urinary screening tests were discovered. Assays of erythrocyte orotidylate decarboxylase and phosphoribosyltransferase enzymes showed deficient activities for 2 of these subjects typically found in red cells of persons heterozygous for hereditary orotic aciduria. The same studies were conducted on urine and blood samples from the families of the affected subjects, and additional family members were also found to be affected. Detection of two unrelated heterozygotes among so small a screened population suggests, as previously noted, a higher frequency of the abnormal gene than that indicated by the extreme rarity of the homozygous condition. This study demonstrates the usefulness of the urinary screening test mass surveys and indicates the need for further study of the prevalence of the mutant gene.

Adult↗
PubMed2004

A suspected case of ornithine transcarbamylase deficiency in a cat.

An 18 month-old, intact female American Shorthair cat was presented for evaluation of stunted growth and postprandial depression. Fasting serum ammonia and serum bile acid concentrations were above reference ranges at 396 microg/dl and 6.5 micromol/ l and their postprandial concentrations were 785 microg/dl and 9.5 micromol/l, respectively. The initial tentative diagnosis of a portosystemic shunt was excluded by mesenteric portography and histopathology of the liver. The cat was then suspected of a urea cycle enzyme deficiency and its urine was analyzed by gas chromatography-mass spectrometry. A presumptive diagnosis of ornithine transcarbamylase deficiency was made on the basis of the detection of orotic acid and uracil.

Alanine Transaminase↗
PubMed2000

Influence of dose and age on the response of the allopurinol test for ornithine carbamoyltransferase deficiency in control infants.

Measurement of urinary orotidine and orotic acid after an oral allopurinol challenge is an important diagnostic test for ornithine carbamoyltransferase deficiency that is sometimes used in infants (< 1 year of age), although there is little information on normal test results in this age group. We found higher orotidine excretion in normal infants than in older children given the test, whereas orotate excretion was similar in both groups. The increased orotidine excretion appears to be due to the use in the infants of higher allopurinol doses per kilogram of body weight than in the children. The normalized-dose dependency of the orotidine response extends even to adult age. Thus, dose-normalized responses should be used in the test and there is no need for careful age-matching of the controls.

Adolescent↗
PubMed1996

Prolonged metabolic correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors.

A murine model of ornithine transcarbamylase (OTC) deficiency was used in this study to evaluate the efficacy of recombinant adenoviruses for correcting the metabolic defect in liver. Recombinant adenoviruses deleted in E1 and containing a human OTC cDNA expressed little functional OTC enzyme in vivo and had no observable impact on the underlying metabolic abnormalities of the OTC-deficient mouse (i.e. elevated urinary orotate and serum glutamine). E1-deleted vectors were improved through the use of the strong constitutive promoter from cytomegalovirus driving the normal murine homolog of OTC cDNA and the ablation of E2a with a temperature-sensitive mutation. Infusion of this improved vector into the mouse model was associated with a complete normalization of liver OTC enzyme activity that persisted for at least 2 months with complete but transient correction in serum glutamine and urine orotic acid. These studies illustrate the utility of improved adenoviral vectors in the treatment of liver metabolic disease.

Adenoviridae↗