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PubMed1988

Altered erythrocyte nucleotide patterns are characteristic of inherited disorders of purine or pyrimidine metabolism.

This paper compares erythrocyte nucleotide levels in patients with eight different inherited purine or pyrimidine enzyme defects identified amongst a variety of patients referred predominantly for investigation of severe neurological abnormalities, or immunodeficiency syndromes. Characteristic nucleotide patterns were identified only in the six disorders (four involving purine and two pyrimidine metabolism) where there was clinical evidence of cellular toxicity. They were frequently related to the accumulation of abnormal metabolites in body fluids. These erythrocyte studies have demonstrated the following. 1. ATP depletion is not an invariable feature of adenosine deaminase (ADA) deficiency, but the accumulation of the deoxyribonucleotides dATP, or dGTP, is diagnostic of ADA, or purine nucleoside phosphorylase (PNP) deficiency, respectively. The early accumulation of dATP in foetal blood is a valuable aid to prenatal diagnosis of ADA deficiency. 2. GTP depletion appears to reflect the degree of CNS involvement in hypoxanthine-guanine phosphoribosyltransferase and PNP deficiency, as well as PP-ribose-P synthetase superactivity. Other diagnostic changes involving increased pyrimidine sugars and increased or decreased NAD levels, or ZTP in Lesch Nyhan erythrocytes, show no consistent correlation with the clinical manifestations. 3. These altered nucleotide levels afford a novel means for carrier detection of the X-linked defect associated with aberrant PP-ribose-P synthetase activity, where no other test is yet available. Measurement of erythrocyte nucleotide levels thus provides a simple and rapid aid to diagnosis and may sometimes be essential for determining prognosis, carrier detection, or monitoring therapy. These characteristic 'fingerprints' may give some insight into the mechanism by which the abnormal gene product produces disease. Such grossly altered nucleotide levels could also result in loss of erythrocyte flexibility, increased destruction and hence the anaemia, or other clinical manifestations, observed in some disorders.

Adenosine Diphosphate↗

PubMed1984

Tritrichomonas foetus: partly defined cultivation medium for study of the purine and pyrimidine metabolism.

A partly defined medium was successfully designed for the cultivation of Tritrichomonas foetus, an anaerobic protozoan parasite of cattle. The medium consists of hypoxanthine, uracil, and thymidine as the sole precursors of nucleotides in T. foetus. Elimination of any one of the three precursors from the medium led to cessation of T. foetus growth. The information provided by this medium verifies our previous observations that T. foetus is incapable of de novo purine and pyrimidine synthesis, that hypoxanthine can be converted to AMP and GMP, that uracil is incorporated into all pyrimidine ribonucleotides including UDP-glucose--the precursor of glycogen synthesis, and that thymidine is the only precursor of TMP. The omission of folate from the medium, without affecting growth of T. foetus, also supports our previous finding that the parasite does not have functioning dihydrofolate reductase or thymidylate synthetase. The successful plating of T. foetus on agar plates incorporating the partly defined medium with near 100% plating efficiency makes it possible to isolate T. foetus mutants for further studies of purine and pyrimidine metabolism in this parasite.

Agar↗

PubMed1984

Pyrazolo[3,4-d]pyrimidines as adenosine antagonists.

A large number of nitrogen heterocycles structurally related to caffeine and theophylline have been tested for activity as adenosine antagonists. Preliminary screening, utilizing displacement of [3H]N6-phenylisopropyladenosine (PIA) binding to rat brain membranes, identified several pyrazolo[3,4-d]pyrimidines with potential antagonist activity. These were then tested for their ability to antagonize adenosine-stimulated adenylate cyclase of guinea-pig slices and to block adenosine receptors which mediate presynaptic inhibition of transmitter release from cholinergic nerves in guinea-pig ileum. Of several compounds found to have antagonist activity, one of these, 4,6-bis-alpha- carbamoylethylthio -1-phenylpyrazolo[3,4-d]pyrimidine ( DJB -KK) was approximately an order of magnitude more potent than theophylline in both tests. GTP greatly reduces the potency of purine agonists, but not antagonists, as inhibitors of [3H] PIA binding; the potency of the pyrazolo[3,4-d]pyrimidine compounds was not altered by GTP. The compounds have no significant activity against [3H]adenosine uptake or on the binding of ligands to muscarinic cholinergic, beta-adrenergic, GABA or L-glutamate receptors.

Adenosine↗

PubMed1980

Studies on the influence of photoreactivation on the frequencies of UV-induced chromosomal aberrations, sister-chromatid exchanges and pyrimidine dimers in chicken embryonic fibroblasts.

Chicken embryonic fibroblasts, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the induction of pyrimidine dimers, sister-chromatid exchanges (SCEs) and chromosomal aberrations by 254 nm UV. While photoreactivation (PR) efficiently removed most of the induced dimers (75-95%), the frequencies of SCEs and chromosomal aberrations were reduced only by about 30-65%, in parallel experiments. Since pyrimidine dimers are the only photoreactivable photolesions known, the reduction in the frequencies of SCEs and chromosomal aberrations on PR has been interpreted as due to disappearance of pyrimidine dimers, implying that these lesions are the primary events responsible for the induction of the biological end points studied. The possible reasons for the lack of quantitative relationship between the frequencies of dimers and the frequencies of SCEs and chromosomal aberrations are discussed.

Animals↗

PubMed1981

Strand cleavage at psoralen adducts and pyrimidine dimers in DNA caused by interaction between semi-purified uvr+ gene products from Escherichia coli.

Partially purified extracts of Escherichia coli containing either uvrA+ or a mixture of uvrB+ and uvrC+ gene products were tested for an endonuclease activity on DNA treated with 8-methoxypsoralen plus 360-nm light. Neither of these fractions was active alone. The combined fractions, however, caused extensive strand cleavage of the psoralen-treated DNA. The endonuclease activity was dependent upon addition of ATP and Mg2+ to the reaction mixtures, and hence appeared similar to the UV-endonuclease activity previously shown to be reconstituted from the same fractions. It is concluded that the uvr+ gene products in these fractions interact to cause breakage of both psoralen-treated and UV-irradiated DNA. An examination of the dose-dependence relationship of the break formation in psoralen-treated DNA revealed that the enzyme acts upon psoralen mono-adducts. By varying the experimental conditions to increase the ratio of interstrand cross-links to mono-adducts it was found that the enzyme also acts upon cross-links, but with lower efficiency than for mono-adducts. Further studies of break formation in UV-irradiated DNA showed that elimination of pyrimidine dimers by treatment with photoreactivating enzyme in the light resulted in a loss of endonuclease-sensitive sites. This shows directly that pyrimidine dimers are the lesions recognized by the complemented uvr+ gene products in UV-irradiated DNA. For comparison, another endonuclease acting at pyrimidine dimers in DNA, the Micrococcus luteus UV-endonuclease, was also tested with psoralen-treated DNA, but no activity was observed. This and other data indicate that the repair endonuclease encoded by the uvr+ genes in E. coli is basically different from the other dimer-specific endonucleases previously characterized.

Bacterial Proteins↗

PubMed1981

Sensitive determination of pyrimidine dimers in DNA of UV-irradiated mammalian cells. Introduction of T4 endonuclease V into frozen and thawed cells.

Endonuclease V from E. coli infected with phage T4 was used to evaluate the frequency and the removal of pyrimidine dimers from DNA in cultured mammalian cells. Cellular membranes were made permeable to the enzyme by two cycles of rapid freezing and thawing. The number of endonuclease-sensitive sites in DNA was assayed by sedimentation in alkaline sucrose gradients upon which the cells were lysed directly. Comparison of the frequency of endonuclease-sensitive sites with the frequency of pyrimidine dimers determined by chromatographic analysis of hydrolysed DNA indicated that about 50% of the dimers in the permeabilized cells were substrates for T4 endonuclease V. This was confirmed by observation that when DNA treated with the enzyme in situ was purified, it contained the expected additional number of endonuclease-sensitive sites if again treated with the enzyme. The percentage of pyrimidine dimers recognized by T4 endonuclease V was enhanced to nearly 100% by exposing the permeabilized cells to 2 M NaCl before the enzyme was introduced. This method allowed the measurement of frequencies of endonuclease-sensitive sites after doses of UV irradiation at low as 0.5 J/m2. Loss of endonuclease sites from cellular DNA was observed during post-irradiation incubation of V79 Chinese hamster cells and several human cell strains. A comparison of the results obtained in human cells with or without the high-salt exposure before endonuclease treatment suggested that the dimers recognized under low-salt conditions may be removed slightly faster than those recognized only after high-salt exposure.

Animals↗

PubMed1988

Ultraviolet radiation-induced lethality and repair of pyrimidine dimers in fish embryos.

Pimephales promelas (fathead minnow) embryos were used to show a correlation between induction of pyrimidine dimers in DNA and embryo death. Embryo killing was measured by a lack of heart-beat and blood circulation at 48 h post-ultraviolet radiation (UVR). When the embryos were exposed to various doses of UVR from a FS-40 sunlamp followed by exposure to photoreactivating light (PRL) (320-400 nm), the number of pyrimidine dimers decreased significantly. The photorepair of dimers was accompanied by a substantial increase in embryo survival. When embryo killing was examined as a function of the number of dimers present, dimers were identified as a major lesion involved in UVR-induced killing in these fish embryos. This in vivo study on photoreactivation treatment of fish embryos shows a direct association between UVR-induced pyrimidine dimers and embryo killing. In addition, when embryos were held in the dark for 9 h after UVR, 50% of the dimers were removed by excision repair.

Animals↗

PubMed1996

Inhibition of RNA and DNA synthesis in UV-irradiated normal human fibroblasts is correlated with pyrimidine (6-4) pyrimidone photoproduct formation.

UV-irradiation of living cells results in an inhibition of RNA and DNA synthesis. The purpose of this study was to determine whether specific photoproducts or the total combined yield of lesions were responsible for these effects. Asynchronously dividing human fibroblasts from normal donors were irradiated with UVC (254 nm), broad spectrum UVB (290-320 + nm, Westinghouse FS20 lamp) or narrow spectrum UVB (310-315 nm, Philips TL01 lamp) at fluences which induce known yields of cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts or Dewar isomers. DNA synthesis was approximately 3-4 times more sensitive to both UVC and UVB irradiation than RNA synthesis. The immediate inhibition of RNA and DNA synthesis was correlated with (6-4) rather than overall photoproduct formation suggesting that the (6-4) photoproduct is the mediator of these inhibitory effects. In support of this suggestion we found that photoreactivation of cells cultured from the marsupial, mouse Sminthopsis crassicaudata, resulted in removal of 70% of pyrimidine dimers from the overall genome, but had only a slight effect on the recovery of RNA synthesis.

Animals↗

PubMed1989

The removal of UV-induced pyrimidine dimers from DNA of rat skin cells in vitro and in vivo in relation to aging.

Young and old rats were compared with respect to the capacity of their skin fibroblasts and epidermal keratinocytes to remove low levels of ultraviolet light (UV) induced UV-endonuclease sensitive sites (pyrimidime dimers) from their DNA, in vitro and in vivo, respectively. In vitro, over a 24-h time period, fibroblasts from both young and old rats were found to remove about 20% of the pyrimidine dimers originally induced by 4.6 J/m2 of UV-C. In vivo, after 2.6 kJ/m2 of UV-B hardly any UV lesions were found to be present in fibroblasts, as demonstrated by immunohistochemistry using an anti-thymine dimer antibody. As reported earlier (Mullaart et al., J. Invest. Dermatol., 90 (1988) 346-349) cultured epidermal keratinocytes do not differ from cultured fibroblasts in UV repair kinetics, whereas in vivo they remove at least 50% of the pyrimidine dimers induced by 4 kJ/m2 of UV-B within 3 h. We now show that epidermal keratinocytes from old rats are not deficient in their in vivo repair characteristics upon this low UV-B dose. However, since a considerable fraction of the pyrimidine dimers appeared to be persistent in fibroblasts and keratinocytes, demonstrated by both enzymatic and immunochemical assays, the possibility is discussed that long-term exposure of skin cells to UV may lead to an accumulation of DNA damage with age.

Aging↗

PubMed1982

Effects of acivicin and PALA, singly and in combination, on de novo pyrimidine biosynthesis.

The inhibitory activities of two oncolytic amino acid analogs, acivicin and N-(phosphonacetyl)-L-aspartate, on pyrimidine biosynthesis have been examined in a murine tumor line, the Lewis lung carcinoma. Acivicin, an antimetabolite elaborated by Streptomyces sviceus, inhibits a spectrum of L-glutamine utilizing enzymes including carbamoyl phosphate synthetase II, the inaugurating enzyme of de novo pyrimidine biosynthesis. Profound inhibition of carbamoyl phosphate synthetase II activity by acivicin is demonstrated in vitro as well as in vivo. N-(Phosphonacetyl)-L-aspartate, a rationally-designed transition-state analog of the reaction catalyzed by L-aspartate transcarbamylase, the second enzyme of the pathway, is a potent and specific inhibitor of L-aspartate transcarbamylase. Both agents, at therapeutic doses, exert marked inhibitions of their respective target enzymes and impede flux through the pathway as monitored by inhibition of pyrazofurin-provoked accumulation of orotate and orotidine. Additionally, synergistic effects are observed when acivicin and N-(phosphonacetyl)-L-aspartate are used in combination, both in terms of biochemical and therapeutic endpoints. The salient features of the actions of these drugs on pyrimidine biosynthesis in the Lewis lung carcinoma are summarized in Table 6. Comparison of the effects of acivicin with those of N-(phosphonacetyl)-L-aspartate suggest divergent actions on nucleotide biosynthesis. In spite of its pronounced sensitivity to acivicin, carbamoyl phosphate synthetase II appears not to be a critical target for the antineoplastic activity of this drug.

Animals↗

PubMed1987

Effects of N,N-dimethylformamide and sodium butyrate on enzymes of pyrimidine metabolism in cultured human tumor cells.

Effects of a 7-day treatment with the maturational agents DMF and sodium butyrate on enzymes of pyrimidine metabolism, growth rate and cell maturation were assessed in 5 human tumor cell lines, ARH-77 (myeloma), K-562 (chronic myeloid leukemia), KG-1 (myeloid leukemia), HL-60 (promyelocytic leukemia) and RWLy-1 (non-Hodgkin's lymphoma). DMF lengthened the doubling times of all five cell lines while sodium butyrate lengthened only those of K-562, HL-60 and RWLy-1. Full maturation was induced only in HL-60 by either agent and in K-562 by butyrate. Exposure resulted in a decreased activity of the anabolic enzyme orotate phosphoribosyltransferase (EC 2.4.2.10) and increased activities of the catabolic enzymes thymidine phosphorylase (EC 2.4.2.4) and dihydrouracil dehydrogenase (EC 1.3.1.2). Changes in the amphibolic enzyme, uridine phosphorylase (EC 2.4.2.3) did not follow any apparent pattern. This study indicates that the pattern of pyrimidine metabolism differs between the differentiated and slowly growing, and undifferentiated rapidly growing counterpart of several human tumors, suggesting that enzymes of pyrimidine metabolism can be used as markers for cellular growth and/or maturity.

Butyrates↗

PubMed1988

Purine and pyrimidine metabolism in human muscle and cultured muscle cells.

Using radiochemical methods, we determined the activities of various enzymes of purine and pyrimidine metabolism in homogenates of human skeletal muscle and of cultured human muscle cells. Results show a large discrepancy between the enzyme activities in muscle and cultured cells. With regard to purine metabolism, adenylate (AMP) deaminase activity was only 1-3% in cultured cells compared to that in muscle, whereas the activity of adenosine deaminase, purine-nucleoside phosphorylase, adenosine kinase, adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase was 7-15-fold higher in the cultured cells. The enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase and uridine kinase showed activity of 100-200-fold higher in cultured cells than in adult muscle. The differences in enzyme activity are probably related to the low differentiation stage and the absence of contractile activity in the cultured muscle cells. Care must be taken when using these cells as a model for studying purine and pyrimidine metabolism of adult myofibers.

5'-Nucleotidase↗

PubMed1995

Effect of long-term zidovudine exposure on salvage and de novo purine and pyrimidine nucleotide syntheses.

Salvage and de novo purine and pyrimidine nucleotide syntheses were studied in H9 (a human lymphoid cell line) and H9-AZT cells (chronically zidovudine-exposed H9 cells). H9-AZT cells incorporated 18% and 27% more hypoxanthine and uridine, respectively, than H9 cells. The incorporation of the formate and bicarbonate was similar in both cell lines. Purine and pyrimidine de novo synthesis was inhibited by hypoxanthine and uridine, respectively. Hypoxanthine and uridine salvage pathways, however, were not affected by formate or bicarbonate. Short-term AZT exposure of cells had no effect on nucleotide synthesis. Some of the problems encountered in the studies of purine and pyrimidine synthesis are also discussed.

Bicarbonates↗

PubMed1988

Excision repair of pyrimidine dimers in human peripheral blood lymphocytes: comparison between mitogen stimulated and unstimulated cells.

Excision repair kinetics of UV-induced pyrimidine dimers in DNA of phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes were compared to unstimulated lymphocytes using a dimer-specific endonuclease from Micrococcus luteus in conjunction with agarose gel electrophoresis. Removal of pyrimidine dimers could be detected within 6 h after irradiation only in PHA-stimulated lymphocytes. However, incorporation of [3H]thymidine as UV-induced unscheduled DNA synthesis was detected in the unstimulated lymphocytes in the 6-h period. The number of pyrimidine dimers remaining in unstimulated lymphocytes was approximately 85% after 24 h as compared to less than 25% in stimulated cells.

Cells, Cultured↗

PubMed1984

Prebiotic syntheses of purines and pyrimidines.

The work done in many laboratories during the last two decades has confirmed that hydrogen cyanide and cyanoacetylene are the two major precursors for the prebiotic synthesis of purines and pyrimidines, respectively. Although several different pathways for the synthesis of purines have been described, they are all variations of the initial mechanism proposed by Oró and Kimball, where hydrogen cyanide leads first to the formation of a 4,5-di-substituted imidazole derivative, and then to the closing of the purine ring with a C1 compound. A number of experiments have shown that purines and pyrimidines can also be obtained from methane, ammonia (nitrogen), and water mixtures, provided an activating source of energy (radiation, electric discharges, etc.) is available. However, in this case the yields are lower by about two orders of magnitude because of the intermediate formation of hydrogen cyanide and cyanoacetylene. The latter two compounds have been found in interstellar space, Titan and other bodies of the solar system. They were probably present in the primordial parent bodies from the solar nebula in concentrations of 10(-2) to 10(-3) M as inferred from recent calculations by Miller and coworkers obtained for the Murchison meteorite. These concentrations should have been sufficient to generate relatively large amounts of purine and pyrimidine bases on the primitive Earth.

Adenine↗

PubMed1981

In vitro screening of potential anti-cancer chemicals: effect of purine pyrimidine analogues on seed germination.

Germinating Cicer arietinum seeds were used to simulate embryonic tissue for evaluating toxicity of purine and pyrimidine analogues. 6-Mercaptopurine stimulated germination, whereas 8-azaadenine, 8-azaguanine, 2,6-diaminopurine, 6-amino, 2-hydroxypurine and 5-fluorouracil inhibited germination. 6-Methyluracil, 5-aminouracil and methylxanthines, viz. caffeine, theophylline, theobromine and uric acid exhibited little inhibitory effects. RNA synthesis ensued immediately after water imbibition and reached maximum at 12 h of germination. 2,6-Diaminopurine, 8-azaadenine, 8-azaguanine and 5-fluorouracil inhibited RNA synthesis. Cyclic-AMP, adenosine, indole-3-acetic acid (IAA) and folic acid partially reversed the inhibitory effects produced by purine pyrimidine analogues. Several hitherto untested pyrimidine analogues inhibited germination as well as RNA synthesis. Inhibition of Escherichia coli dihydrofolate reductase was used for purposes of comparison.

Antineoplastic Agents↗

PubMed1977

Chemical evolution XXIX. Pyrimidines from hydrogen cyanide.

Dilute (0.1 M) solutions of HCN condense to oligomers at pH 8-9. Hydrolysis of these oligomers at pH 8.5 or with 6 N HCl yields 4,5-dihydroxypyrimidine, as the most abundant pyrimidine product along with orotic acid and 5-hydroxyuracil. These results, together with the earlier data, demonstrate that the three major nitrogen-containing classes of biomolecules could have originated from HCN on the primitive earth. The observation of the formation of orotic acid and 4-aminoimidazole-5-carboxamide by the hydrolysis of the HCN oligomers suggests that once the initially formed pyrimidines and purines were consumed, those life forms persisted which evolved enzymes for conversion of these intermediates to the pyrimidines and purines present in contemporary RNA.

Chemical Phenomena↗

PubMed1996

Salvage pathway of pyrimidine synthesis: divergence of substrate specificity in two related species of teleostean fish.

For nucleotide synthesis, cells use purine and pyrimidine nucleosides generated either through de novo synthesis or through utilization of salvage pathways. In the pyrimidine salvage pathway, thymidine is taken up by transport proteins and phosphorylated by the enzyme thymidine kinase to thymidine monophosphate. So far, all vertebrates analyzed are able to use radioactively labeled thymidine for the biosynthesis of nucleotides in brain tissue. However, when standard autoradiographic, immunohistochemical and biochemical procedures were applied for the detection of the incorporation of tritiated thymidine and the thymidine analogue 5-bromo-2'-deoxyuridine into DNA to two species of gymnotiform fish, a divergence in substrate specificity has been revealed. Although brain cells of the two species, Apteronotus leptorhynchus and Eigenmannia sp., can utilize 5-bromo-2'-deoxyuridine for pyrimidine synthesis, only Eigenmannia sp. is able to incorporate tritiated thymidine into DNA during the S phase of the cell cycle. We hypothesize that this inability to use thymidine for nucleotide synthesis is caused either by a defect in the transport system mediating the uptake of thymidine or by a deficiency in the thymidine kinase of A. leptorhynchus.

Animals↗