Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “OROTIC ACID”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,315 records · Page 73Linked to original sources
PubMedDate not supplied

Age-dependence of nuclear RNA processing.

Following a 3 hour in vivo labelling of cytoplasmic RNA in rat liver with orotic acid-6-14C under conditions where ribosomal RNA synthesis was suppressed, the proportion of labelled messenger-like RNA released to the cytoplasm which contained polyadenylate (poly(A)) tracts was about 3.0 times higher in the livers of juvenile (50 day) as compared to adult (180 day) rats. This discrepancy was confirmed in a cell-free system which consisted of isolated prelabelled nuclei in fortified cytosol. Thus under conditions where approximately 80% of the released labelled RNA was messenger-like, the proportion of polyadenylated labelled RNA transported to the homologous cytosol was 3.4-fold greater in the systems derived from juvenile as compared to adult rat liver. Through comparisons of homologous and heterologous systems it was determined that the age-dependent change in the metabolism of polyadenylated messenger RNA resides in the nucleus and not in the cytoplasm. This change, furthermore does not involve the known ATP-dependence of nuclear RNA release. Rather it must involve other age-dependent changes in the processing or transport of polyadenylated messenger RNA.

Aged↗
PubMed1975

Messenger ribonucleoprotein complexes isolated with oligo(dT)-cellulose chromatography from kidney polysomes.

As an initial step towards understanding the role of mRNP complexes in translational regulation during compensatory renal hypertrophy, characteristics of polysome-associated mRNP isolated by affinity chromatography were studied. Renal mRNP contained 15-30 percent of the counts after a 1 hr pulse with -3H-orotic acid; it sedimented mainly between 10S and 100S and had a buoyant density of 1.42-1.44 g/cm-3. RNA derived from the mRNP sedimented between 5S and 40S on sucrose density gradients, with the greatest radioactivity in the region of 15S. After labeling with -3H-adenine for 1 hr, up to 17 percent of the radioactivity present in the mRNP-associated RNA was resistant to digestion by pancreatic and T1 ribonucleases. The mRNP protein moiety contained six polypeptides with molecular weights 69,000, 75,000, 80,000, 100,000, 109,000, and 118,000 daltons, which were undetected in the material not binding to oligo(dT)-cellulose.

Animals↗
PubMed1982

Effects of ethionine treatment of protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits.

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [14C]- or [3H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3.10(4) and 2.1.10(4) were decreased in ethionine-treated rate liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with N-[3H]ethylmaleimide or N-[14C]ethylmaleimide, and the 3H to 14C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.

Animals↗
PubMed1982

Evidence that free polysomes are not the precursors of membrane-bound polysomes in rat liver.

We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-14C) orotic acid and inorganic (32P] phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes.

Animals↗
PubMed1984

Production of the effector molecule thymidine by human lung alveolar macrophages.

Cultured human alveolar macrophages (HAM phi) were found to produce soluble factors which inhibit tritiated thymidine [( 3H]-TdR) incorporation into tumour cells in vitro. We present evidence that thymidine (TdR) can be detected in HAM phi culture supernatants by thin-layer chromatography. Moreover, TdR secretion by HAM phi is an active process. Using [6-14C]-orotic acid as an early precursor to TdR and [3H]-TMP as the phosphorylation product, we were able to show that cultured HAM phi transformed them into labelled TdR. The very efficient thin-layer chromatography of the labelled metabolites was backed up by high-pressure liquid chromatography of nucleotides. HAM phi produce TdR by de novo synthesis and dephosphorylation. This phenomenon is due to the lack of thymidine kinase in normal mature macrophages. Since TdR, in high concentrations, can inhibit DNA synthesis through the 'TdR blockade' phenomenon, it is suggested that TdR secretion by HAM phi is a mechanism of non-specific modulation of tumour cell growth but highly restricted to the immediate macrophage microenvironment in vivo. The effectiveness of the thymidine gradient may thus be quite narrow, but is worthy of interest.

Animals↗
PubMed1990

Effect of 3,7-bis-(4-trifluoromethylphenyl)-1,5,3,7-dioxadiazocane on pyrimidine and DNA synthesis in rat organs.

The administration of the lipophilic 3,7-bis-(4-trifluoromethylphenyl)- 1,5,3,7-dioxadiazocane (TFMPD) to rats induced the following effects on the biosynthesis of DNA in the liver, kidney, thymus and spleen: (a) The utilization of [3H]thymidine for the synthesis of liver DNA thymine was decreased after the administration of a single dose of the drug. The depression of the specific activities of DNA pyrimidines of liver DNA in experimental groups was observed also after an injection of [14C]orotic acid. (b) A decreased incorporation of labeled thymidine had occurred also in the spleen during the prereplicative period. Thereafter the specific activity of DNA thymine was higher than in the control group. (c) The observed mitogenic response in the spleen showed a protracted effect; after the administration of a single dose of the drug the specific activity of DNA thymine as well as the thymidine kinase activity of spleen cytosol have been rising up to the ninth day. The same holds true for DNA thymine of the thymus; the activity of thymidine kinase was not affected. (d) Both the single and repeated doses of TFMPD had no marked effect on the levels of microsomal cytochromes P-450 and b5 in the liver and kidney.

Administration, Oral↗
PubMed1988

The biosynthesis of highly branched N-glycans: studies on the sequential pathway and functional role of N-acetylglucosaminyltransferases I, II, III, IV, V and VI.

At least 6 N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V and VI) are involved in initiating the synthesis of the various branches found in complex asparagine-linked oligosaccharides (N-glycans), as indicated below: GlcNAc beta 1-6 GlcNAc-T V GlcNAc beta 1-4 GlcNAc-T VI GlcNAc beta 1-2Man alpha 1-6 GlcNAc-T II GlcNAc beta 1-4Man beta 1-4-R GlcNAc T III GlcNAc beta 1-4Man alpha 1-3 GlcNAc-T IV GlcNAc beta 1-2 GlcNAc-T I where R is GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAcAsn-X. HPLC was used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct (I. Brockhausen, J.P. Carver and H. Schachter (1988) Biochem. Cell Biol. 66, 1134-1151). The following sequential rules have been established: GlcNAc-T I must act before GlcNAc-T II, III and IV; GlcNAc-T II, IV and V cannot act after GlcNAc-T III, i.e., on bisected substrates; GlcNAc-T VI can act on both bisected and non-bisected substrates; both Glc-NAc-T I and II must act before GlcNAc-T V and VI; GlcNAc-T V cannot act after GlcNAc-T VI. GlcNAc-T V is the only enzyme among the 6 transferases cited above which can be essayed in the absence of Mn2+. In studies on the possible functional role of N-glycan branching, we have measured GlcNAc-T III in pre-neoplastic rat liver nodules (S. Narasimhan, H. Schachter and S. Rajalakshmi (1988) J. Biol. Chem. 263, 1273-1281). The nodules were initiated by administration of a single dose of carcinogen 1,2-dimethyl-hydrazine.2 HCl 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. The nodules had significant GlcNAc-T III activity (1.2-2.2 nmol/h/mg), whereas the surrounding liver, regenerating liver 24 h after partial hepatectomy and control liver from normal rats had negligible activity (0.02-0.03 nmol/h/mg). These results suggest that GlcNAc-T III is induced at the pre-neoplastic stage in liver carcinogenesis and are consistent with the reported presence of bisecting GlcNAc residues in N-glycans from rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (A. Kobata and K. Yamashita (1984) Pure Appl. Chem. 56, 821-832).

Animals↗
PubMed1976

Hypertrophy and hyperplasia of the neonatal rat exocrine pancreas induced by orally administered soybean trypsin inhibitor.

Measurement of RNA, DNA, protein and phospholipid synthesis in the neonatal rat pancreas following the oral administration of partially purified soybean trypsin inhibitor demonstrates an enhanced synthesis of all these constituents. Evidence of true hyperplasia in addition to this cellular hypertrophy is provided by an increased mitotic activity in the exocrine pancreas following a wave of enhanced [3H] thymidine incorporation into DNA. Complete inhibition of the stimulated RNA synthesis by low doses of actinomycin D indicates the importance of transcription as a regulatory step in the response of the exocrine pancreas to trophic stimulation by this means. Collateral observations of [3H] thymidine and [14C] orotic acid incorporation into liver DNA and RNA, respectively, fail to demonstrate comparable changes confirming the probable specificity of the trypsin inhibitor induced effect on the exocrine pancreas. It is suggested that the pronounced trophic effect of orally administered soybean trypsin inhibitor in the neonatal rat pancreas may serve as a useful model for the study of regulatory mechanisms of nucleic acid and protein synthesis in the mammalian pancreas.

Administration, Oral↗
PubMed1982

Effects of oestradiol-17 beta on the synthesis of RNA, proteins and lipids in the pyloric caeca of the female starfish Asterias rubens.

1. The effects of oestradiol-17 beta on processes of synthesis in the pyloric caeca of female Asterias rubens were studied. 2. In vitro treatment with 2.5 X 10(-7) M oestradiol-17 beta resulted in significantly higher RNA levels. 3. In vivo treatment with oestradiol-17 beta resulted in higher lipid levels in the pyloric caeca, but RNA levels, protein levels and the incorporation of 6-[14C]orotic acid, L-leucine-[14C] and sodium 1-[14C] acetate into RNA, proteins and lipids, respectively were not affected, neither were the indices of the gonads and pyloric caeca.

Animals↗
PubMed1996

Assembly and extension of yeast artificial chromosomes to build up a large locus.

For the assembly of a large human locus, overlapping regions on yeast artificial chromosomes (YACs) and cosmids were linked up using their regions of homology. By site-specific recombination a YAC of 410 kb was created accommodating the major part of the human lambda light chain locus in authentic configuration with 28 variable (V) genes, all joining (J) segments, all constant (C) genes and the downstream enhancer. A contiguous region was first constructed from three overlapping cosmids. Each of these was linearized at unique sites in the vectors and YAC arms were ligated to the 5' and 3' ends. After cells of Saccharomyces cerevisiae were transformed with the three cosmids, YACs of 120 kb were obtained which contained the reassembled 3' J-C region in authentic configuration. The assembled YAC was further extended by mitotic recombination with a YAC containing a 280-kb region of the C-proximal part of the V gene cluster with a 15-kb 3' overlap. For this, a simple three-way selection procedure was developed involving the integration of different selectable marker genes at specific sites in the left and right YAC arms. Rare recombination events between two overlapping YACs could be identified in yeast clones able to grow in lysine- and adenine-deficient medium in the presence of 5-fluoro-orotic acid which is toxic for yeast cells containing a YAC with a functional URA3 gene. This approach made it possible to assemble and extend large YACs from an unlimited number of smaller overlapping YACs by positive-negative selection.

Chromosomes, Artificial, Yeast↗
PubMed1996

Capillary electrophoresis for clinical problem solving: analysis of urinary diagnostic metabolites and serum proteins.

Many clinical laboratories employ gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) to detect abnormal compounds occurring in urine and serum due to disease. The methods, particularly GC-MS, often require laborious sample pre-treatment, and separation times may exceed an hour. We describe the use of capillary electrophoresis (CE) equipped a with a diode-array detector in an attempt to improve the efficiency of an analytical system routinely used for diagnosis of human metabolic disease. It was found that urine samples could be injected directly onto the CE instrument without any pre-treatment, and over 50 metabolites were separated in 15 min. Identification of abnormal metabolites was based on migration times and characteristic diode-array spectra. The method readily diagnosed adenolysuccinase deficiency, 5-oxoprolinuria, propionic acidemia and disorders have orotic acid as diagnostic metabolite (e.g. the HHH-syndrome). The results show that CE may become a useful additional tool for diagnosis of metabolic disease. In a different project CE was used to study sera from the Janus-bank. This large serum bank comprises samples collected at intervals from nearly 300,000 blood donors. As the sera are stored at -25 degrees C and not at a lower temperature, a major concern has been the stability of the specimens. GC-MS, 2D-protein electrophoresis, certain immunological assays and enzyme measurements have previously been used to evaluate the stability of the sera. We can now also show that the protein profile, as determined by CE, is remarkably stable even after 22 years of storage. The results moreover confirmed that the CE-method and traditional gel electrophoresis gave almost identical results, except for small amounts of fibrinogen which did not show up on the CE-pattern.

Blood Proteins↗
PubMed1993

Yeast artificial chromosome fragmentation vectors that utilize URA3 selection.

Two fragmentation vectors, pSE1 and pSE2, were developed for targeting yeast artificial chromosomes (YACs) containing human genomic DNA. Ura- yeast cells containing YACs were selected with 5-fluoro-orotic acid. Fragmented YACs were subsequently generated by transformation to a Ura+ phenotype. Over 80% of the transformants contained YACs of reduced molecular size. These fragmented YACs will prove to be useful in mapping the region of human chromosomes covered by the parental YAC. Fragmentation utilizing URA3 transformation provides a method for producing YAC deletion sets from YACs contained in AB1380 and other ura3- yeast stains. Linkage of a neomycin resistance gene to the URA3 gene facilitates functional analysis of these YACs in eukaryotic cells.

Acyltransferases↗
PubMed2003

Purine and pyrimidine metabolites in children's urine.

Various enzyme defects in the metabolic pathways of purines and pyrimidines are known, which result in different diseases occurring in children. They mainly affect kidney function, central nervous system, immunological and blood system. For example, complete deficiency of HPRT (hypoxanthine-guanine-phosphoribosyl-transferase) causes the Lesch Nyhan syndrome, which is characterized by hyperuricemia, mental retardation, choreoathetosis and compulsive self-mutilation. XDH deficiency (xanthine-dehydrogenase) causes in arthropathia and myopathia. For screening for these and other enzyme defects, urinary purine and pyrimidine excretion is considered a simple diagnostic tool. The purpose of the present study was to establish a reverse phase HPLC screening method for urinary purines and pyrimidines and to establish age related reference ranges in children for the urinary excretion of orotic acid, uracile, pseudouridine, uric acid, hypoxanthine, xanthine, thymine, 7-methylguanine, inosine, guanosine and adenosine.

Adolescent↗
PubMed1979

Measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography.

A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in combination with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 10(3) concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed.

Chromatography, High Pressure Liquid↗
PubMed1997

Diagnostic applications of chromatography and capillary electrophoresis.

Capillary electrophoresis (CE) equipped with a diode-array detector, and GC-MS have been used to determine diagnostic metabolites occurring in urine of patients with various metabolic disorders. The urine samples were injected directly onto the CE instrument without any pretreatment. GC-MS required extraction and derivatisation before separation. Identification of abnormal metabolites was based on migration times and characteristic diode-array spectra, or mass spectral library search when GC-MS was used. The CE method has previously been shown capable of diagnosing several metabolic diseases, and was now used on more difficult cases. CE readily diagnosed glyceric aciduria and the secondary metabolite in lysinuric protein intolerance, orotic acid. Methylmalonic aciduria required pressure elution in addition to high voltage to accomplish diagnosis. In mevalonic aciduria the characteristic metabolite had weak light absorption and the mevalonate peak also co-eluted with endogenous aromatic acids making diagnosis difficult. Both in the latter case and with the disorders glutaric aciduria I and glyceroluria, GC-MS was the method of choice. A possible role of CE in the routine system for diagnosing metabolic disorders, might be to use this method for pre-testing all urine samples. Samples with abnormal CE-profiles would subsequently be given high priority for more elaborate analysis with GC-MS and amino acid analyzer. In a different project a CE instrument designed for serum protein analysis was used to study sera from patients with myelomatosis. The method also allowed identification of the various immunoglobulins using immunosubtraction. Samples collected after diagnosis as well as many years prior to disease were available through the Janus-bank. This large serum bank comprises samples collected since 1973 at intervals from nearly 300000 blood donors. It was found that the monoclonal immunoglobulins characteristic of the disease started to appear in serum up to 15 years before clinical diagnosis.

Acidosis↗
PubMed1975

Methylated messenger RNA in mouse kidney.

Polyadenylated messenger RNA from mouse kidney labeled in vivo exhibited a pattern of methylation distinct from that of rRNA and tRNA. After mice were given L-[methyl-3H]methionine, 4% of the polyribosomal RNA label was bound to oligo (dT)-cellulose; 20-24% of orotate- or adenine-labeled polyribosomal RNA eluted in the poly(A)+ RNA fraction under similar conditions. [3H]Methyl radioactivity was not incorporated into low molecular weight (5-5.8 S) rRNA, indicating the extent of nonmethylpurine ring labeling was negligible. [3H]Methyl-labeled poly(A)+ RNA sedimented heterogeneously in sodium dodecyl sulfate containing gradients similarly to poly(A)+ mRNA labeled with [3H]orotic acid. Based on an average molecular length of 2970 nucleotides, renal mRNA was estimated to contain 8.6 methyl moieties per molecule. Analysis of alkaline-hydrolyzed RNA sampled by DEAE-Sephadex-urea chromatography provided estimates of the relative amounts of base and ribose methylation. Although 83% of the [3H]methyl radioactivity in rRNA was in the 2'-0-methylnucleotide fraction, no methylated dinucleotides were found in mRNA. In poly(A)+ mRNA 60% of the [3H]methyl label was in the mononucleotide fraction; the remainder eluted between the trinucleotide and tetranucleotide markers and had a net negative charge between -4 and -5. The larger structure, not yet charcterized, could result from two or three consecutive 2'-0-ribose methylations and is estimated to contain 2.6 methyl residues. Alternatively, the oligonucleotide could be a 5'-terminal methylated nucleotide species containing 5'-phosphate(s) in addition to the 3'-phosphate moiety resulting from alkaline hydrolysis. Either structure could have a role in the processing or translation of mRNA in mammalian cells.

Animals↗