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PubMed1992

Immunochemical detection of unrepaired cyclobutane-type pyrimidine dimers of DNAs extracted from human skin tumours.

Unrepaired cyclobutane-type pyrimidine dimers of DNA extracted from human skin tumours were examined by an immunoblotting method using polyclonal antibodies raised against UV-irradiated calf thymus DNA. A total of 40 DNA samples extracted from seven SCC lesions, two AK lesions, two lymphomas, one basal cell epithelioma, one eccrine poroma, one neurofibroma of Recklinghausen's disease, on verruca vulgaris, four femoral normal skins and white blood cells of 21 humans were studied by immunoblotting using this antibody. Two of the 40 DNAs examined, one from facial actinic keratosis (AK) and one from a squamous cell carcinoma (SCC) which developed form facial AK formed immunoprecipitates. It was found, using photoreactivation enzyme plus visible light, that both immunoprecipitates were cyclobutane-type pyrimidine dimers. In addition, immunofluorescent studies on AK tissue were positive in an immunoblotting assay and revealed that the unremoved photodamage in DNA remained in the nucleus of AK cells. These findings indicate that these tumour cells may be deficient in the enzyme function for repairing photoproduct damage. The unrepaired cyclobutane-type pyrimidine dimer in AK cells might reflect the genetic process in multistage carcinogenesis as well as in xeroderma pigmentosum.

Adult↗

PubMed1983

Determination of ribonucleosides, deoxyribonucleosides, and purine and pyrimidine bases in adult rabbit cerebrospinal fluid and plasma.

Purine and pyrimidine base and nucleoside levels were measured in adult rabbit cisternal CSF and plasma by reversed-phase high-performance liquid chromatography. The concentrations of bases, nucleosides, and nucleoside phosphates were similar in plasma and CSF except for the adenosine phosphates and uracil which were higher in the plasma. In plasma and CSF, adenosine levels were low (0.12 microM) and guanosine, deoxyadenosine, deoxyguanosine, and deoxyinosine were not detectable (less than 0.1 microM); inosine and xanthine concentrations were 1-2 microM and hypoxanthine concentrations were approximately 5 microM; uridine (approximately 8 microM), cytidine (2-3 microM), and thymidine, deoxyuridine, and deoxycytidine (0.5-1.4 microM) were easily detectable. In both plasma and CSF, guanine, and thymine were undetectable (less than 0.1 microM), adenine and cytosine were less than 0.2 microM, but uracil was present (greater than 1 microM). Adenosine, inosine, and guanosine phosphates were also detectable at low concentrations in CSF and plasma. These results are consistent with the hypothesis that purine deoxyribonucleosides are synthesized in situ in the adult rabbit brain. In contrast, pyrimidine deoxyribonucleosides and ribonucleosides, and purine and pyrimidine bases are available in the CSF for use by the brain.

Adenine↗

PubMed1983

Purine and pyrimidine base and nucleoside concentrations in human cerebrospinal fluid and plasma.

Purine and pyrimidine base and nucleoside levels were determined in adult human lumbar (CSF) and plasma by reversed-phase high performance liquid chromatography (HPLC). Guanine, thymine, cytosine and uracil were not detectable (less than 0.1 microM) in human CSF or plasma. Adenine was detectable in plasma (0.3 microM) but was not found in CSF (less than 0.2 microM). Hypoxanthine and xanthine levels in CSF were each approximately 2.5 microM. Plasma levels of hypoxanthine and xanthine were considerably lower (0.4-0.6 microM). Purine and pyrimidine ribonucleosides in human CSF were less than or equal to 0.2 microM with the exception of uridine which was present at concentrations of 2-3 microM. Although low concentrations of thymidine and deoxyuridine (0.2 microM) were present in human plasma, purine and pyrimidine deoxyribonucleosides were less than 0.1 microM in human lumbar CSF.

Chromatography, High Pressure Liquid↗

PubMed1993

Immunohistochemical examination of tumor-suppressor gene p53 product and pyrimidine dimer in solar keratosis.

In order to find biomarkers to measure the effects of UV irradiation, we examined the accumulation of p53 protein and pyrimidine dimers in 18 solar keratosis specimens. Frozen or AMeX-fixed solar keratosis specimens were immunohistochemically stained by anti-p53 mouse monoclonal antibody, pAb1801 and polyclonal anti-(pyrimidine dimer) antibody. Nuclear accumulation of p53 protein was found in 5/18 (28%) solar keratosis lesions. The percentage of cases showing nuclear p53 protein varied according to the histological type; in the bowenoid type it was 4/7 (57%); in the atrophic type it was 1/7 (14%). Nuclear pyrimidine dimers were not stained in solar keratosis, although the skin of UV-irradiated nude mice was positive. Accumulation of p53 protein is a good marker for early precancerous change caused by UV exposure.

Aged↗

PubMed1994

The antimitotic drug 4,6-dimethyl-2-amino-3,4,5- trimethoxyphenyl-pyrimidine inhibits the nucleoside transport system of cells from various animal species.

4,6-dimethyl-2-amino-3,4,5-trimethoxyphenyl-pyrimidine (B-31) is a pyrimidine derivative previously reported to arrest the mitotic cycle of mammalian cells in metaphase. In the present study it is shown that B-31 also acts as a specific inhibitor of the cellular uptake of nucleosides. The uptake of purinic and pyrimidinic nucleosides is inhibited by 80-90% at concentrations in the range 5-20 micrograms/ml, whereas those of nucleobases, leucine, choline and glucose are unaltered at the maximum nontoxic dose of B-31 (25 micrograms/ml). Various mammalian (human, monkey and murine) and avian cell are equally sensitive to the inhibition of nucleoside transport. The antimitotic effect of B-31, by contrast, is species-specific: human cells are the most sensitive whereas monkey and chicken fibroblasts appear resistant to this effect. Both effects can be reversed by removal of B-31 from the medium; inhibition of nucleoside transport can also be reversed by high doses of the nucleosides themselves.

Animals↗

PubMed1993

Synthesis and fungitoxicity of some pyrimidine derivatives.

A series of 12 pyrimidine derivatives were prepared and tested in vitro against growth, sporulation and nucleic acid content of Fusarium oxysporum f. sp. lycopersici and Helminthosporium oryzae. Introduction of a thiazole ring together with two aryl groups into 2-aminopyrimidine brought about drastic toxicity for both fungi. Pyrimidine derivatives with aryl groups alone were less toxic. Nitro groups were found to enhance the toxicity of the pyrimidine derivatives especially when substituted in the ortho-position of the aryl groups. Inhibition of nucleic acid synthesis of both fungi was attributed mainly to the presence of the thiazole ring.

Antifungal Agents↗

PubMedDate not supplied

PhrA, the major photoreactivating factor in the cyanobacterium Synechocystis sp. strain PCC 6803 codes for a cyclobutane-pyrimidine-dimer-specific DNA photolyase.

A new broad-host-range plasmid, pSL1211, was constructed for the over-expression of genes in Synechocystis sp. strain PCC 6803. The plasmid was derived from RSF1010 and an Escherichia coli over-expression plasmid, pTrcHisC. Over-expressed protein is made with a removable N-terminal histidine tag. The plasmid was used to over-express the phrA gene and purify the gene product from Synechocystis sp. strain PCC 6803. PhrA is the major ultraviolet-light-resistant factor in the cyanobacterium. The purified PhrA protein exhibited an optical absorption spectrum similar to that of the cyclobutane pyrimidine dimer (CPD) DNA photolyase from Synechocuccus sp. strain PCC 6301 (Anacystis nidulans). Mass spectrometry analysis of PhrA indicated that the protein contains 8-hydroxy-5-deazariboflavin and flavin adenine dinucleotide (FADH2) as cofactors. PhrA repairs only cyclobutane pyrimidine dimer but not pyrimidine (6-4) pyrimidinone photoproducts. On the basis of these results, the PhrA protein is classified as a class I, HDF-type, CPD DNA photolyase.

ATP-Binding Cassette Transporters↗

PubMed2007

Looking for organization patterns of highly expressed genes: purine-pyrimidine composition of precursor mRNAs.

We analyzed precursor messenger RNAs (pre-mRNAs) of 12 eukaryotic species. In each species, three groups of highly expressed genes, ribosomal proteins, heat shock proteins, and amino-acyl tRNA synthetases, were compared with a control group (randomly selected genes). The purine-pyrimidine (R-Y) composition of pre-mRNAs of the three targeted gene groups proved to differ significantly from the control. The exons of the three groups tested have higher purine contents and R-tract abundance and lower abundance of Y-tracts compared to the control (R-tract-tract of sequential purines with Rn>or=5; Y-tract-tract of sequential pyrimidines with Yn>or=5). In species widely employing "intron definition" in the splicing process, the Y content of introns of the three targeted groups appeared to be higher compared to the control group. Furthermore, in all examined species, the introns of the targeted genes have a lower abundance of R-tracts compared to the control. We hypothesized that the R-Y composition of the targeted gene groups contributes to high rate and efficiency of both splicing and translation, in addition to the mRNA coding role. This is presumably achieved by (1) reducing the possibility of the formation of secondary structures in the mRNA, (2) using the R-tracts and R-biased sequences as exonic splicing enhancers, (3) lowering the amount of targets for pyrimidine tract binding protein in the exons, and (4) reducing the amount of target sequences for binding of serine/arginine-rich (SR) proteins in the introns, thereby allowing SR proteins to bind to proper (exonic) targets.

Animals↗

PubMed2005

Comprehensive analysis of pyrimidine metabolism in 450 children with unspecific neurological symptoms using high-pressure liquid chromatography-electrospray ionization tandem mass spectrometry.

To evaluate the significance of inborn metabolic disorders of the pyrimidine degradation pathway, 450 children with unspecific neurological symptoms were comprehensively studied; 200 healthy children were recruited as controls. Uracil and thymine as well as their degradation products in urine were determined with an improved method based on reversed-phase HPLC coupled with electrospray ionization tandem mass spectrometry and detection by multiple-reaction monitoring using stable-isotope-labelled reference compounds as internal standards. From the results of the control group we established age-related reference ranges of all pyrimidine degradation products. In the patient group, two children with dihydropyrimidine dehydrogenase (DPYD) deficiency were identified; one of these was homozygous for the exon 14-skipping mutation of the DPYD gene. In addition, two patients with high uracil, dihydrouracil and beta-ureidopropionate were found to have ornithine transcarbamylase deficiency. In the urine of 9 patients, beta-alanine was markedly elevated owing to treatment with vigabatrin, an irreversible inhibitor of GABA transaminase, which interferes with beta-alanine breakdown. Four patients had exclusively high levels of beta-aminoisobutyrate (beta-AIB) due to a low activity of the D-beta-AIB-pyruvate aminotransferase, probably without clinical significance. In conclusion, quantitative investigation of pyrimidine metabolites in children with unexplained neurological symptoms, particularly epileptic seizures with or without psychomotor retardation, can be recommended as a helpful tool for diagnosis in clinical practice. Sensitive methods and age-related reference ranges enable the detection of partial enzyme deficiencies.

Adolescent↗

PubMed1983

A 13C tracer method for quantitating de novo pyrimidine biosynthesis in vitro and in vivo.

Gas chromatographic/mass spectrometric methods for the measurement of the flux through the de novo pyrimidine biosynthetic pathway by quantitating the incorporation of [13C]bicarbonate and 13CO2 into the uracil nucleotide pool in L1210 tumors are reported. Simultaneous measurements of the incorporation of [13C]bicarbonate and the more commonly used [14C]bicarbonate into uridine of L1210 cells in vitro showed that the two methods were comparable. A modification of the method was applied to in vivo studies where the incorporation of 13CO2 into the uracil nucleotide pool of L1210 tumors in mice was quantitated. The measurements were used to determine changes in the flux through the de novo pyrimidine pathway in animals pretreated with known inhibitors of the pathway. A comparison of control animals with those pretreated with 6-azauridine, acivicin, and pyrazofurin resulted in mean percentage inhibitions of 87, 95, and 94%, respectively. This technique should allow investigation of the respective contributions of salvage and de novo synthesis in the formation of pyrimidines in vivo and the effects of agents designed as enzyme inhibitors of the de novo pathway.

Animals↗

PubMed1989

Determination of pyrimidine dimers in DNA by high-performance liquid chromatography/gas chromatography and electron capture detection.

Exposure of DNA to uv radiation results in the formation of a number of photoproducts including the cyclobutyl pyrimidine dimers. At low uv fluences the concentrations of these dimeric compounds are only a small fraction of the corresponding DNA pyrimidine concentration (e.g., as low as 0.02% or less of the total thymine content). Sensitive methods of analysis are therefore required for accurate determinations. Analytical methodology based upon HPLC fractionation and electrophore labeling followed by GC/electron capture detection (ECD) has been developed to quantitate these species. Separation of thymine-thymine, thymine-uracil, and uracil-uracil from the monomeric bases and from other constituents present in acid-hydrolyzed DNA is achieved by reversed-phase HPLC. Isolation of the dimeric fractions is followed by off-line derivatization to form pentafluorobenzyl products for analysis by GC/ECD. All active hydrogens are alkylated, yielding products with high response factors and detection limits in the low femtomole range. The overall analytical scheme for the determination of pyrimidine dimers in DNA is presented.

Chromatography, Gas↗

PubMed1982

Purine and pyrimidine salvage pathways in Leishmania donovani.

Leishmania donovani, grown in culture, salvaged radiolabeled purine bases which were distributed into adenine and guanine ribonucleotides and into the RNA of these cells. De novo synthesis of purines in L. donovani does not occur [J. J. Marr, R. L. Berens and D. J. Nelson, Biochim. biophys. Acta 544, 360 (1978)]. [8-14C]Adenine was rapidly deaminated to hypoxanthine via the action of an adenine aminohydrolase (EC 3.5.4.2). [8-14C]Guanine was also rapidly deaminated by guanase (EC 3.5.4.3) to form zanthine in these cells. Therefore, the formation of nucleotides of hypoxanthine and xanthine are the first committed steps of purine salvage in L. donovani. While purines are efficiently conserved by this parasite, the salvage of pyrimidines is not so dramatic. [2-14C]Orotic acid was converted to OMP and then incorporated into the pyrimidine nucleotides and into RNA, indicating the existence of the later steps of de novo pyrimidine synthesis. [6-14C]Thymidine was salvaged by L. donovani, being incorporated into the thymine deoxyribonucleotides and into DNA. The major pathway of thymidine metabolism in this parasite, however, was cleavage of the deoxyriboside linkage to form thymine, probably via the action of a thymidine phosphorylase (EC 2.4.2.4).

Animals↗

PubMed1983

Combined action of acivicin and D-galactosamine on pyrimidine nucleotide metabolism in hepatoma cells.

The glutamine antagonist acivicin, L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, strongly reduced CTP and GTP contents in AS-30D rat hepatoma cells in suspension. UTP only dropped to 63% of the respective control after 4 hr; however, by combining acivicin with the uridylate-trapping sugar analogue D-galactosamine, a synergistic decrease in UTP contents to 7% of control was induced. Incorporation of 14CO2 into purine and pyrimidine nucleotides followed by radio-high performance liquid chromatography showed marked inhibition of purine and pyrimidine biosynthesis de novo; the latter was reduced to 35% of control. The inhibitory potency of acivicin on glutamine-dependent carbamoyl-phosphate synthetase and consequently on de novo uracil nucleotide formation was also reflected by the complete suppression of the D-galactosamine-induced rise in total uridylate. Induction of UTP deficiency by interference with the first and rate-limiting step in pyrimidine biosynthesis de novo together with a trapping of uridylate by D-galactosamine may provide a promising approach to the chemotherapy of hepatocellular carcinoma.

Animals↗

PubMed1984

Regulation of pyrimidine biosynthesis in cultured L1210 cells by 3-deazauridine.

The effect of 3-deazauridine on the synthesis of uracil nucleotides by de novo and salvage pathways was investigated in intact cultured L1210 cells. De novo pyrimidine biosynthesis, as measured by sodium [14C]bicarbonate incorporation into uracil nucleotides, was inhibited 40-85% at intracellular 3-deazauracil nucleotide concentrations of 1-6 nmoles/10(6) cells. The inhibition was not due to an increase in the size of the uracil nucleotide pool since this pool was only 97-66% of control level at 3-deazauracil nucleotide concentrations of 1-6 nmoles/10(6) cells. Furthermore, intracellular 3-deazauracil nucleotide concentrations of 0.5 to 5.2 nmoles/10(6) cells inhibited the salvage of [14C]uridine by 25-75%. The data indicate that 3-deazauridine may potentiate its own inhibition of CTP synthetase by reducing the concentration of competing uracil nucleotides by inhibiting de novo pyrimidine biosynthesis and pyrimidine salvage. It is postulated that the biochemical mechanism by which 3-deazauridine inhibits uracil nucleotide synthesis is by acting as a fraudulent allosteric regulator of carbamyl phosphate synthetase II and uridine/cytidine kinase.

3-Deazauridine↗

PubMed1989

Inhibition of phosphoribosylpyrophosphate synthetase by 4-methoxy-(MRPP) and 4-amino-8-(D-ribofuranosylamino) pyrimido[5,4-d]pyrimidine (ARPP).

The basis for the antitumor activities of the exocyclic amino nucleosides 4-amino-(ARPP) and 4-methoxy-8-(D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP) was investigated. The primary target of these nucleosides appeared to be 5-phospho-alpha-D-ribofuranose-1-pyrophosphate (PRPP) synthetase. MRPP-5'-monophosphate was a competitive inhibitor (Ki = 40 microM) of the activation of this enzyme by the cofactor inorganic phosphate (K alpha = 2.2 mM). Consequently, ARPP and MRPP treatment of WI-L2 cultures rapidly inhibited both de novo pyrimidine and purine synthesis as well as the nucleotide salvage reactions dependent on PRPP, ARPP or MRPP treatment completely prevented [14C]bicarbonate incorporation into acid-soluble pyrimidine and purine nucleotides. The rate of salvage of [8-14C]hypoxanthine to form IMP was decreased by 85%. Treatment of cells with these agents caused a 50% reduction in the steady-state level of PRPP. When the capacity of the treated cells for sustained synthesis of PRPP was examined by adenine incorporation, the rate of adenine uptake was inhibited by greater than 50%. In vivo treatment of BDF1 mice with a single dose of ARPP (173 mg/kg) or MRPP (62 mg/kg) extended the mean life span of the mice, which had been inoculated intraperitoneally 1 day earlier with 1 x 10(6) L1210 murine leukemia cells, by 62 and 82% respectively. These studies indicate that MRPP and ARPP inhibit PRPP synthetase, and that PRPP synthetase may be a viable target in the development of certain antitumor agents.

Adenosine Deaminase↗

PubMed1980

Reverse phase partition HPLC for determination of plasma purines and pyrimidines in subjects with gout and renal failure.

A reverse phase partition mode of high pressure liquid chromatography was developed for the estimation of purine, pyrimidine and pyrazolopyrimidine nucleosides and bases. This method has been applied to the investigation of purine and pyrimidine metabolism in subjects with gout and/or renal failure during allopurinol therapy. Plasma levels of hypoxanthine and xanthine were quantitated in all subjects. No significant differences were observed between healthy males and gouty subjects with or without allopurinol medication. There was a significant increase in xanthine levels in patients with gout and renal failure, or renal failure on allopurinol, and plasma levels of oxipurinol were increased, but no correlation with glomerular filtration rate (GFR) was observed. The separation method was also used to examine urines from patients with known abnormalities of purine and pyrimidine metabolism in an attempt to evaluate its usefulness as a urine screening technique.

Acute Kidney Injury↗

PubMed1983

Erythrocyte nucleotide pattern in two children in a Norwegian family with pyrimidine 5'-nucleotidase deficiency.

In the erythrocytes from two Norwegian children, a brother and a sister, with a hemolytic anemia due to pyrimidine 5'-nucleotidase deficiency, the pyrimidine and purine nucleotides have been investigated using HPLC with a strong anionic exchanger. The standard procedure was complemented with some additional elution systems which made it feasible to separate in the extract and to analyse, in addition to the conventional mono-, di- and triphosphates, UDP-glucose, UDP-N-acetylglucosamine, CDP-choline and CDP-ethanolamine. The two different purine nucleotides (A, G) and the two different pyrimidine nucleotides (U, C) exhibited normal ratios (energy charge ratios) between the conventional nucleotides. This would indicate that the erythrocytes have a sufficient energy production. It is suggested that the partly intravascular hemolysis might be due to disturbed synthesis of phospholipids.

5'-Nucleotidase↗

PubMed1985

The degradation of 5'-deoxy-5-fluorouridine by pyrimidine nucleoside phosphorylase in normal and cancer tissues.

We describe an assay procedure for the quantification of pyrimidine nucleoside phosphorylase, the enzyme thought to degrade the pro-drug 5'-deoxy-5-fluorouridine to 5-fluorouracil. The method is based on the known differences in the ultraviolet absorption spectra in alkaline medium between pyrimidines and their nucleosides. Analogous to the cleavage of uridine by uridine phosphorylase, the enzymatic degradation of 5'-deoxy-5-fluorouridine in the presence of inorganic phosphate yields 5-fluorouracil and ribose-1-phosphate. We have shown that the rate of phosphorolysis of this pyrimidine nucleoside could be readily measured by spectrophotometry. The method described is simple, specific for the 'pro-drug' as well as reproducible. We have also applied this method to define tissue enzyme activities in extracts of human tumours, normal tissues of the same organ and tissues of mice.

Animals↗