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At least 1,153 records · Page 64Linked to original sources
PubMed1987

Modulation of transcription in rat liver by benzo[a]pyrene.

Single intraperitoneal administration of benzo[a]pyrene (B[a]P) at 20 mg/kg body weight to male Wistar rats caused an early (2 h) inhibition in liver of gross transcription as measured by incorporation of [14C] orotic acid into nuclear RNA. This inhibition was reversed gradually into a partial stimulation at 2-4 days after administration of B[a]P, followed by another period of inhibition at 7 days which persisted up to 14 days. The early reversible inhibition at 2 h was probed further by studies on in vitro transcription using isolated nuclei from treated rats. It was observed that total expression of RNA polymerase activity was only marginally inhibited because, while expression of RNA polymerases II and III showed remarkable inhibition, RNA polymerase I showed stimulation in activity.

Animals↗
PubMed1975

Effect of the exotoxin of Bacillus thuringiensis on the biosynthesis and maturation of mouse liver nuclear RNA.

The effect of the exotoxin of Bacillus thuringiensis on the in vivo incorporation of [14-C] orotic acid into mouse liver nuclear rRNA and low molecular weight RNA was studied. The following results were obtained. 1. The exotoxin does not inhibit the synthesis of 45 S pre-rRNA, but causes a breakdown of these molecules. 2. The exotoxin inhibits the conversion of 38 S pre-rRNA into 32 S and 21 S. 3. The exotoxin inhibits the labelling of nuclear 5 S RNA, whereas the labelling of 4.6 S pre-tRNA is not affected. It is suggested that 5 S RNA may control the processing of 45 S pre-rRNA.

Agar↗
PubMed1991

New shuttle vectors for direct cloning in Saccharomyces cerevisiae.

We have constructed new shuttle vectors to facilitate the screening of recombinant plasmids after direct transformation of yeast cells. The vectors are pBluescript-based shuttle vectors in which the lacZ marker has been replaced by an analogous system based on the Saccharomyces cerevisiae URA3 gene. DNA fragments are inserted in a polylinker located after the beginning of the URA3 coding sequence. Transformants are selected either by Trp or Leu prototrophy. Plasmids bearing an insert are selected by growth on 5-fluoro-orotic acid (5-FOA), a uracil analog toxic to cells containing a functional URA3+ gene (thus, this method requires the recipient strain to be ura3-); only cells containing a plasmid with an insert that disrupts the functional continuity of the URA3 gene can grow on medium containing 5-FOA. Using these plasmids, we were able to directly reclone the ACE1 gene from genomic DNA by directly transforming a strain deleted for ACE1. These vectors can be used for a variety of purposes including rapid cloning of genes by complementation or expression of fusion genes driven from the URA3 promoter.

Base Sequence↗
PubMed1978

Studies on the effects of chronic ethanol ingestion on the properties of rat brain ribosomes.

Previous observations have demonstrated decreased in vivo and in vitro protein synthesis by brain ribosomal systems following long-term ethanol ingestion. For further investigation of the properties of brain ribosomes, the 40S and 60S ribosomal subunits were successfully isolated from control and chronic 10% ethanol-drinking rats. For a successful dissociation of ribosomes into subunits NH4Cl, puromycin and a high-salt treatment at 10 degrees C were essential with a critical concentration of Mg2+ since ribosomes could not be resolved at less than 7 mM Mg2+. Analysis of the A260 profile of the subunits on the sucrose gradients showed no significant differences between the control and ethanol-ingesting groups. Studies on 3H-labeled ribosomes following in vivo RNA labeling showed correspondence of the radioactive profiles from the incorporation of [5(-3) H) orotic acid into RNA with the sucrose gradient absorbance profile of 60S and 40S ribosomal subunits. Furthermore, active reassociation of both subunits occurred at 37 degrees C as demonstrated by the increased [14 C]-phenylalanine incorporation in the presence of poly(U). Results further showed that the poly(U)-dependent [14C]phenylalanine incorporation was significantly reduced by the subunits from the ethanol-ingesting animals. These findings suggest that long-term ingestion of ethanol caused functional changes in the properties of brain ribosomes, specifically on the reassociation process of the two subunits.

Alcohol Drinking↗
PubMed2000

The mechanism of orotidine 5'-monophosphate decarboxylase: catalysis by destabilization of the substrate.

The mechanism of orotidine 5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) was studied using the decarboxylation of orotic acid analogues as a model system. The rate of decarboxylation of 1,3-dimethylorotic acid and its analogues as well as the stability of their corresponding carbanion intermediates was determined. The results have shown that the stability of the carbanion intermediate is not a critical factor in the rate of decarboxylation. On the other hand, the reaction rate is largely dependent on the equilibrium constant for the formation of a zwitterion. Based on these results, we have proposed a new mechanism in which ODCase catalyzes the decarboxylation of OMP by binding the substrate in a zwitterionic form and providing a destabilizing environment for the carboxylate group of OMP.

Catalysis↗
PubMed2005

Urinary uracil in female patients with ornithine transcarbamylase deficiency.

BACKGROUND: Female patients with ornithine transcarbamylase deficiency (OTCD) show a wide range of clinical severity, from asymptomatic to lethal hyperammonemia. It is important to establish a simple method to distinguish symptomatic from asymptomatic patients. METHODS: Uracil and orotic acid concentrations were analyzed in three female patients with OTCD at both the hyperammonemia-attack and interval stages. These concentrations were compared with those in asymptomatic female patients reported previously. RESULTS: Uracil concentrations in symptomatic female patients were uniformly higher than those in asymptomatic female patients at both the hyperammonemia-attack and interval stages. CONCLUSION: Uracil may present a useful index for detecting OTCD female patients who are destined to suffer from hyperammonemia attack. Further data on uracil concentrations are necessary to establish the threshold for distinguishing symptomatic from asymptomatic subjects.

Child, Preschool↗
PubMedDate not supplied

The use of proline as a nitrogen source causes hypersensitivity to, and allows more economical use of 5FOA in Saccharomyces cerevisiae.

The use of proline as a nitrogen source causes hypersensitivity to 5-fluoro-orotic acid (5FOA) and allows up to 40-fold less of this drug to be used to select for the loss of URA3 function in Saccharomyces cerevisiae. 5FOA hypersensitivity is presumably due to the absence of nitrogen catabolite repression when proline is substituted for (NH4)2SO4 as a nitrogen source. There are two constraints to the use of the proline-5FOA combination: (1) S288c genetic background strains are hypersensitive to 5FOA when grown in proline as a nitrogen source but at least one other genetic background is resistant to low levels of 5FOA under these conditions. (2) The addition of some nutritional supplements confers phenotypic resistance to the 5FOA-proline combination.

Culture Media↗
PubMed2006

Control of ribosome turnover during growth of the haloalkaliphilic archaeon Natronococcus occultus.

The metabolism of ribosomes during growth of the haloalkaliphilic archaeon Natronococcus occultus was examined. The ribosome content was higher during exponential growth and diminished to 35% of the maximum in the stationary stage. The incorporation of H3-orotic acid and C14-uracil into rRNA was higher during exponential growth. After that, it decreased to 39% of the maximum in the stationary stage. The labeling of non-ribosomal RNA took place almost exclusively in the exponential stage. From loss of radioactivity, the half-life of rRNA was 11.43, 14.85, 5.28 and 7.14 h during the initial, exponential, late exponential and stationary growth stages, respectively. These results suggested that increased synthesis combined with diminished degradation were responsible for the high ribosome content displayed by Ncc. occultus during exponential growth. In contrast, diminished synthesis together with increased degradation provoked its posterior loss.

Carbon Radioisotopes↗
PubMed1971

Dibutyryl adenosine cyclic 3':5'-monophosphate effects on goldfish behavior and brain RNA metabolism.

Intraventricular administration of dibutyryl adenosine cyclic 3':5'-monophosphate into goldfish brains produced hyperactive animals. A study of the effects of the drug (25-50 mg/kg) on the incorporation of [5-(3)H] orotic acid, as a precursor of labeled uridine and cytidine, into newly synthesized RNA showed the formation of an RNA with a uridine to cytidine ratio 20-50% higher than that of the control. In double-labeling experiments with uridine as the labeled precursor, the synthesis of a nuclear RNA fraction (not produced in the absence of drug) was demonstrated. Some of this RNA was found to migrate into the cytoplasmic fraction and to become associated with polysomes. The results suggest that cyclic AMP might function as a "metabolic demand signal" for eliciting new RNA synthesis in goldfish brain.

Animals↗
PubMed1982

Adenosine- and deoxyadenosine-mediated altered pyrimidine metabolism in human adeosine deaminase-deficient lymphoblasts.

To evaluate the importance of altered pyrimidine synthesis in the relationship between immune dysfunction and deficiency of ADA, this pathway has been evaluated in normal and ADA-deficient human lymphoblasts. Adenosine and deoxyadenosine effect comparable reductions of radiolabeled bicarbonate incorporation in uridine nucleosides of both normal and deficient cells, with this effect being more prolonged in deficient cells. In ADA-deficient lymphoblasts this altered pyrimidine metabolism is accompanied by reduced intracellular concentrations of PP-ribose-P with adenosine but not deoxyadenosine. The altered PP-ribose-P levels are accompanied by orotic acid accumulation, but this block does not fully account for the net reduction in uridine nucleotide synthesis. This suggests interference of the pyrimidine pathway at another locus, possibly by altered carbamyl phosphate synthetase activity. The mechanism for deoxyadenosine-mediated inhibition is obscure. Despite the demonstrable adenosine- and deoxyadenosine-mediated alterations of pyrimidine metabolism, these changes are not accompanied by marked interference of cell growth for both normal and ADA-deficient lymphoblasts. These data support the hypothesis that altered pyrimidine metabolism is not the basis for the immune disorder in patients with deficiency of ADA.

Adenosine↗
PubMed1968

Diversity and nature of ribosomal pools in hepatoma 7800 and host liver.

1. The ribosomal components in the postmitochondrial supernatant of a rat hepatoma (hepatoma 7800) and the corresponding host liver were examined for diversity and functional competence. 2. The ;free' and ;membrane-bound' polyribosomes of both tissues were equally active in vivo and had equilibrated with newly synthesized ribosomes 4hr. after administration of [6-(14)C]orotic acid. 3. The inactive monomer-dimer pool in hepatoma 7800 was unattached to membranes and a larger fraction of the polyribosomes was free in hepatoma than in liver. 4. By using sensitivity to puromycin as a criterion, evidence was obtained that most of the polyribosomes in hepatoma 7800 were active in vivo. 5. Actinomycin, azaguanine and carbon tetrachloride caused marked conversion of polyribosomes into inactive monomers and dimers in the host liver and moderate conversion in the hepatoma. 6. Significant accumulation of ferritin and shifts in the mean polyribosome size to the lighter species occurred in the host liver of rats bearing large hepatomas.

Animals↗
PubMed1980

[RNA-metabolism in aging (author's transl)].

After intraperitoneal application of 14C-orotic acid the tracer distribution and the labelling of total RNA in different organs of two groups of Wistar rats (5 and 22,5 months old) were represented. After 60 min 2/3 of tracer were found in liver and kidney. The uptake by liver of old animals is decreased significantly. The interactions between the tracer distribution in the organism, the time related uptake by the organs, the organ function (influenced by methimazol) and the specific activity of RNA were represented. From the results the conclusion is drawn that the comparison of the specific activities of total RNA alone--especially in comparison of young with old animals--is not sufficient for the examination of RNA synthesis.

Aging↗
PubMed1984

Ammonia: its effects on biological systems, metabolic hormones, and reproduction.

The physical, chemical properties of ammonia, its sources and detoxification, its effects in biological systems, its influence upon insulin action and glucose metabolism, and its possible effects on reproduction are discussed. Present chemical methods do not distinguish nonionic from ionic forms. At physiological pH, nonionic ammonia concentrations remain low but are primarily responsible for toxic effects. Thus, biologically significant changes of ammonia concentrations may not be revealed by determinations of ammonia in blood plasma. For these and other reasons the subacute toxicity of ammonia often is unrecognized, and its effects on intermediary metabolism and the hormonal milieu in normal and disease states remain poorly understood. Effects of ammonia may be stimulatory at low concentrations and inhibitory as concentrations rise or exposure is extended. Extensive experiments in eight ureotelic species, including man, show that urinary excretion of orotic acid becomes significantly elevated when the quantity of ammonia presented to the liver exceeds the capacity for normal detoxification. Present evidence with arginine and other intermediates of the urea cycle suggest that these substances influence glucose metabolism and insulin action. Recent studies of dairy cattle provide speculative evidence that high protein feeding or forms of protein that lead to elevated ammonia concentrations in tissue decrease conception rates and increase the calving to conception interval of dairy cows. Limited data concerning luteinizing hormone concentrations and steroid hormone metabolism are insufficient to establish whether differences in reproductive performance are due to changes of hormonal physiology, intrauterine environment, or metabolism.

Ammonia↗
PubMed1983

Interaction between the urea cycle and the orotate pathway: studies with isolated hepatocytes.

Two enzymes catalyze the synthesis of carbamylphosphate (CP) in the liver. One is intramitochondrial and utilizes ammonia to make CP for ureagenesis; the second is cytoplasmic and utilizes glutamine to produce CP for pyrimidine biosynthesis. The extent to which the metabolic independence of the two pathways is abridged by the use of a common precursor was examined with measurements of the incorporation of [14C]NaHCO3 into orotic acid, uridine nucleotides, and urea in isolated hepatocytes. Pyrimidine synthesis was markedly stimulated by physiological concentrations of ammonia, and the stimulation was antagonized by ornithine. At intracellular concentrations of ornithine and levels of ammonia found in the portal circulation, some 90% of pyrimidine synthesis was ammonia-dependent. When the glutamine-dependent activity was released from feedback inhibition with galactosamine, the ammonia-dependent incorporation still accounted for 2/3 of pyrimidine synthesis. These results do not support the widely held view that the cytoplasmic enzyme is the sole source of CP for pyrimidine biosynthesis in the liver. They suggest instead that the bulk of the CP incorporated into hepatic pyrimidines is of mitochondrial origin. However, an experiment with intact animals failed to provide decisive evidence on this interpretation. Pyrimidine biosynthesis was sharply inhibited by the addition of uridine, but ureagenesis was unaffected. When physiological levels of ammonia were provided, the sensitivity of pyrimidine biosynthesis to uridine was lost. Although inhibition of the ammonia-dependent enzyme by pyrimidines has been observed with cell-free preparations, it was not evident in the intact cell. Thus, to the extent that the CP consumed in pyrimidine biosynthesis is of mitochondrial origin, feedback control of the orotate pathway appears to be thwarted.

Ammonia↗
PubMed1987

Differing effects of arginine deficiency on the urea cycle enzymes of rat liver, cultured hepatocytes and hepatoma cells.

We have confirmed that arginine-deficient diets increase the liver activities (units per 100 g) of the first four arginine biosynthetic enzymes of the urea cycle in Wistar rats, but not the activity of arginase. In contrast, rat liver cells cultured in monolayers for 48, 72 or 96 h in arginine-free L-15 or minimum essential medium showed no changes in carbamoyl-phosphate synthase (EC 6.3.4.16), ornithine transcarbamylase (EC 2.1.3.3), argininosuccinate synthase (EC 6.3.4.5), argininosuccinase (EC 4.3.2.1) or arginase (EC 3.5.3.1) activities. The arginine content of the cells grown on deficient medium was 36% of that of cells grown on 2.9 mM arginine-sufficient L-15, yet the urea excretion rate into the medium was reduced to 7% of the rate in control cells and the excretion of orotic acid was 400% of that in control cells. A Morris rat hepatoma cell line, 7800C1, which maintains activities of all five urea cycle enzymes, showed no consistent increases in the activities of the first four enzymes when the arginine in the medium was varied between 0 and 2 mM. Thus, in spite of severe arginine deficiency, cultured rat liver cells and hepatoma cells do not show the derepression-like response seen by other investigators when nonliver cells were cultured in arginine-deficient media. The difference between in vivo and in vitro effects of arginine deficiency on urea cycle activities remains unexplained.

Ammonium Chloride↗
PubMed1988

Estimation of rRNA synthesis and degradation rates in senescing wheat leaves.

Changes in cytoplasmic and chloroplast rRNA content and rates of rRNA synthesis and degradation of detached wheat leaves were determined. It was found that rRNA loss is proportionally higher in chloroplasts than in cytoplasm. Rates of synthesis were measured by incorporation of large amounts of [3H]orotic acid into rRNA. This approach overcame size differences between pyrimidine pools of cells under different physiological status. Furthermore, these pools reached nearly the same specific radioactivity as that of the administered solution. Rates of degradation were estimated either as the difference between synthesis and net variation of rRNA or by disappearance of radioactivity from 32P-labeled rRNA. Results indicated a decrease in the net rRNA synthesis capacity of leaves after 48 h of detachment. However, the fractional rates of rRNA synthesis were maintained in both cytoplasm and chloroplasts. Ribosomal RNA degradation rates were 2.5-fold higher in chloroplast than in cytoplasm. The observed chloroplast rRNA loss is due to an increased degradation rate which is 15-fold higher than the synthesis rate 48 h after detachment.

Chloroplasts↗
PubMed1999

Isolation and transformation of uracil auxotrophs of the edible basidiomycete Pleurotus ostreatus.

Uracil auxotrophs of Pleurotus ostreatus were isolated using the selectable marker, resistance to 5'-fluoro-orotic acid (5'-FOA). Two of the nine uracil auxotrophs obtained were transformed to prototrophy using plasmid pTRura 3-2 that contains the orotidine monophosphate decarboxylase (ura3) gene from Trichoderma reesei. Southern blot analyses of the transformants showed that the transforming DNA had integrated into the genome of the protoplasts. Using 2 x 10(7) protoplasts, this system gave a transformation efficiency of about 30 transformants per microg of DNA. Normal fruiting bodies were induced in the transformants by crossing them with wild-type monokaryons, and the basidiospores collected from these fruiting bodies showed a biased segregation rate to prototrophy. These results indicate the integrated DNA was stably inherited.

Aspartate Carbamoyltransferase↗