Precursors of pyrimidine nucleotide biosynthesis for gravid Angiostrongylus cantonensis (Nematoda: Metastrongyloidea).

Gravid Angiostrongylus cantonensis can utilize radiolabelled bicarbonate, orotate, uracil, uridine and cytidine but not cytosine, thymine and thymidine for the synthesis of RNA and DNA. In cell-free extracts of the worm, a phosphoribosyltransferase was shown to convert orotate to OMP and uracil to UMP. A similar reaction was not observed with cytosine and thymine. Uridine was readily phosphorylated by a kinase but a similar reaction for thymidine and deoxyuridine was not found. Cytidine could be phosphorylated by a kinase or be deaminated by a deaminase to uridine. No deaminase for cytosine was detected. There was also no phosphotransferase activity for pyrimidine nucleosides in the cytosolic or membrane fractions. Pyrimidine nucleosides were, in general, converted to the bases by a phosphorylase reaction but only uracil and thymine could form nucleosides in the reverse reaction. The activity of thymidylate synthetase was also measured. These results indicate that the nematode synthesizes pyrimidine nucleotides by de novo synthesis and by utilization of uridine and uracil and that cytosine and thymine nucleotides are formed mainly through UMP. The thymidylate synthetase reaction appears to be vital for the growth of the parasite.     

Incorporation of deoxyribonucleosides into DNA of coryneform bacteria and the relevance of deoxyribonucleoside kinases

In order to obtain basic knowledge of the salvage pathways for DNA synthesis, the ability of Brevibacterium ammoniagenes ATCC 6872 and Micrococcus luteus ATCC 15932 for incorporation of nucleobases and nucleosides was investigated. Only adenine and uracil are incorporated by B. ammoniagenes, whereas M. luteus additionally can utilize deoxyadenosine and, less efficiently, thymidine. In M. luteus, the demonstration of deoxyadenosine kinase and thymidine kinase explains the incorporation data. Uptake of thymidine is of short duration because of rapid breakdown of exogenously supplied thymidine to thymine. At a 540-fold excess pyrimidine deoxyribonucleosides inhibit 14C incorporation from thymidine nearly totally and purine deoxyribonucleosides cut by half the uptake rate, probably by interfering with transport of thymidine. However, as no cessation of thymidine incorporation occurs at these concentrations of purine deoxyribonucleosides, incorporation is finally enhanced. During the initial period of this reduced uptake considerable protection of thymidine from breakdown to thymine is provided by deoxyguanosine, but not by deoxyadenosine. At a 108-fold excess there is actually no inhibition of thymidine uptake by deoxyguanosine and only an insignificant impairment by deoxyadenosine resulting in an ultimate enhancement of 14C incorporation up to 20% of the exogenously supplied thymidine. As there is no salvage pathway for thymidine in B. ammoniagenes due to the absence of thymidine kinase, labelling with adenine and hydrolyzing of the 'contaminated' RNA fraction with 1 M KOH is recommended for measurements of overall DNA synthesis in this strain.     

Metabolism of pyrimidine bases and nucleosides in Neisseria meningitidis.

In Neisseria meningitidis, uridine, deoxyuridine, cytosine, cytidine, or deoxycytidine could not be used by uracil-requiring mutants as pyrimidine sources. Consistent with these findings, only 5-fluorouracil of the different fluoropyrimidine bases and nucleosides showed any inhibitory effect on the growth of four prototrophic strains of N. meningitidis. Likewise, only radioactive uracil was readily incorporated into nucleic acids, whereas uptake of radioactive uridine, cytosine, or cytidine could not be demonstrated. Uracil was converted to uridine 5'-monophosphate by uracil phosphoribosyltransferase, whereas enzyme activities for conversion of cytosine or any of the nucleosides were not detectable in meningococcal extracts.     

超氧自由基与嘧啶碱基及其核苷反应的ESR研究

用自旋捕集技术和ＥＳＲ方法,以ＭＮＰ为捕集剂,研究了紫外线辐照核黄素产生超氧自由基等活性氧与嘧啶碱基及其核苷的反应,确定了尿嘧啶,胞嘧啶和胸腺嘧啶及其核苷所产生的自旋加合物自由基的类别,讨论了自由基的形成机制,揭示了超氧自由基与嘧啶碱基及其核苷的反应是不是直接进行的,而是通过羟基自由基来实现的。     

Pyrimidine Salvage Pathways In Toxoplasma Gondii

ABSTRACT. Pyrimidine salvage enzyme activities in cell-free extracts of Toxoplasma gondii were assayed in order to determine which of these enzyme activities are present in these parasites. Enzyme activities that were detected included phosphoribosyltransferase activity towards uracil (but not cytosine or thymine), nucleoside phosphorylase activity towards uridine, deoxyuridine and thymidine (but not cytidine or deoxycytidine), deaminase activity towards cytidine and deoxycytidine (but not cytosine, cytidine 5'-monophosphate or deoxycytidine 5'-monophosphate), and nucleoside 5'-monophosphate phosphohydrolase activity towards all nucleotides tested. No nucleoside kinase or phosphotransferase activity was detected, indicating that T. gondii lack the ability to directly phosphorylate nucleosides. Toxoplasma gondii appear to have a single non-specific uridine phosphorylase enzyme which can catalyze the reversible phosphorolysis of uridine, deoxyuridine and thymidine, and a single cytidine deaminase activity which can deaminate both cytidine and deoxycytidine. These results indicate that pyrimidine salvage in T. gondii probably occurs via the following reactions: cytidine and deoxycytidine are deaminated by cytidine deaminase to uridine and deoxyuridine, respectively; uridine and deoxyuridine are cleaved to uracil by uridine phosphorylase; and uracil is metabolized to uridine 5'-monophosphate by uracil phosphoribosyltransferase. Thus, uridine 5'-monophosphate is the end-product of both de novo pyrimidine biosynthesis and pyrimidine salvage in T. gondii.     

Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells

The anabolism of pyrimidine ribo- and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo- and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine- and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidine-nucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.     

Pyrimidine base and ribonucleoside utilization by thePseudomonas alcaligenes group

Pyrimidine base and ribonucleoside utilization was investigated in the two type strains of thePseudomonas alcaligenes group. As sole sources of nitrogen, the pyrimidine bases uracil, thymine and cytosine as well as the dihydropyrimidine bases dihydrouracil and dihydrothymine supported the growth ofPseudomonas pseudoalcaligenes ATCC 17440 but neither these bases nor pyrimidine nucleosides supportedPseudomonas alcaligenes ATCC 14909 growth. Ribose, deoxyribose, pyrimidine and dihydropyrimidine bases as well as pyrimidine nucleosides failed to be utilized by eitherP. pseudoalcaligenes orP. alcaligenes as sole carbon sources. The activities of the pyrimidine salvage enzymes nucleoside hydrolase, cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase were detected in cell-free extracts ofP. pseudoalcaligenes andP. alcaligenes. InP. pseudoalcaligenes, the levels of cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase could be affected by the nitrogen source present in the culture medium.     

Pathways of Pyrimidine Salvage in Streptomyces

Using 5-fluoropyrimidine analogues, high-performance liquid chromatography (HPLC), and the feeding of pyrimidine compounds to pyrimidine auxotrophs, the pathways for salvage of exogenous pyrimidine nucleosides and bases in Streptomyces were established. Selection for resistance to the analogues resulted in the isolation of strains of S. griseus lacking the following enzyme activities: uracil phosphoribosyltransferase (upp) and cytidine deaminase (cdd). The conversion of substrates in the pathway was followed using reverse-phase HPLC. The strains deficient in salvage enzymes were also verified by this method. In addition, feeding of exogenous pyrimidines to strains lacking the biosynthetic pathway confirmed the salvage pathway. Data from the analogue, HPLC, and feeding experiments showed that Streptomyces recycles the pyrimidine base uracil, as well as the nucleosides uridine and cytidine. Cytosine is not recycled due to a lack of cytosine deaminase.     

Characterization of novel Na+-dependent nucleobase transport systems at the blood-testis barrier

In the testis, nucleosides and nucleobases are important substrates of the salvage pathway for nucleotide biosynthesis, and one of the roles of Sertoli cells is to provide nutrients and metabolic precursors to spermatogenic cells located within the blood-testis barrier (BTB). We have already shown that concentrative and equilibrative nucleoside transporters are expressed and are functional in primary-cultured rat Sertoli cells as a BTB model, but little is known about nucleobase transport at the BTB or about the genes encoding specific nucleobase transporters in mammalian cells. In the present study, we examined the uptake of purine ([3H]guanine) and pyrimidine ([3H]uracil) nucleobases by primary-cultured rat Sertoli cells. The uptake of both nucleobases was time and concentration dependent. Kinetic analysis showed the involvement of three different transport systems in guanine uptake. In contrast, uracil uptake was mediated by a single Na+-dependent high-affinity transport system. Guanine uptake was inhibited by other purine nucleobases but not by pyrimidine nucleobases, whereas uracil uptake was inhibited only by pyrimidine nucleobases. In conclusion, it was suggested that there might be purine- or pyrimidine-selective nucleobase transporters in rat Sertoli cells.     

ATP and polyphosphate-dependent bacterial NAD+-kinases

Measurable levels of activity of NAD+ kinases of actinomycetes Micrococcus luteus and Corynebacterium ammoniagenes were observed after substituting inorganic tripolyphosphate for ATP, whereas the enzyme from the eubacterium Escherichia coli was not active with this substrate. Gradient PAGE found two molecular isoforms of NAD+ kinase in C. ammoniagenes and E. coli; four forms were found in M. luteus. All isoforms of this enzyme found in C. ammoniagenes and M. luteus displayed a NADP-synthesizing activity in the presence of either ATP or tripolyphosphate. Because of its capability of utilizing inorganic tripolyphosphate, M. luteus is the most promising NADP producer organism.     

Preparation of 2,3′-Anhydropyrimidine Nucleosides using N,N-Diethylaminosulfur Trifluoride (DAST)

Abstract

2,3′-Anhydro-2′-deoxy-5′-0-(triphenyl methyl) and 5′-0-(monomethoxytriphenylmethyl) pyrimidine nucleosides of uracil, thymine, and cytosine were synthesized in a single step from their 2′-deoxy-5′-0-(triphenylmethyl) or 5′-0-(monomethoxytriphenylmethyl) precursors using N,N-diethylaminosulfur trifluoride (DAST). The anhydronucleosides were either isolated or directly converted to their respective 2-deoxy-β-D-threo-pentofuranosyl nucleosides using sodium hydroxide in ethanol.     

γ射线辐照嘧啶核苷水溶液的电子自旋共振（ESR）研究

亚硝基特丁烷（ＭＮＲ）为捕集剂,利用自旋捕集技术,对γ射线辐照嘧啶苷水溶液进行了ＥＳＲ研究,确定了胸苷、尿苷和胞苷可产生的自肇捕集自由基的类别,并讨论了自由基形成的机制。     

Giardia lamblia: uptake of pyrimidine nucleosides

The aerotolerant, anaerobic parasite Giardia lamblia, which depends solely upon salvage pathways for its pyrimidine requirements, was found to transport uridine, cytidine, and thymidine by a carrier mediated mechanism. Support for this conclusion comes from the facts that uptake of radiolabeled uridine, cytidine, and thymidine exhibited saturation kinetics, and uptake of these same radiolabeled nucleosides was inhibited by unlabeled homologs, certain pyrimidine analogs, iodoacetate, and N-ethylmaleimide. Uridine and cytidine (perhaps uracil and cytosine also) are postulated to be transported at a common site which is distinct from the site for thymidine transport. Thymidine does appear to bind nonproductively to the uridine/cytidine transport site, but the reverse of this does not appear to occur.     

Pyrimidine scavenging by Mycobacterium leprae

Mycobacterium leprae incorporated exogenously supplied pyrimidines as bases or nucleosides, but not as a nucleotide, into its nucleic acids. Notably, thymine was incorporated approximately 5 times more rapidly than thymidine by both suspensions of, or intracellular M. leprae. Thymine incorporation was significantly inhibited by clofazamine and dapsone at near-pharmacological levels. Therefore, incorporation of thymine is preferable as an activity for assessing viability of M. leprae. Nucleosides were converted to nucleotides through kinases, bases through phosphoribosyltransferases. Alternatively, thymine and uracil could first be converted to nucleosides. Cytosine and uracil bases were interconvertible, and uracil alone could supply all the pyrimidine requirements of M. leprae, though conversion to the thymine base was extremely slow. Overall, pyrimidine scavenging occurs at a slower rate than, and appears not to be so important as purine scavenging in M. leprae.     

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.     

Nucleotide,nucleoside and base nutritional requirements of the mosquito Culex pipiens

Nucleic acid subcomponents needed to satisfy the dietary nucleic acid requirement of Culex pipiens were studied in growth experiments using synthetic media in which nucleosides, bases and alternative nucleotides were variously substituted in mixtures of 3 nucleotides (adenylic acid, thymidylic acid, and either cytidylic or uridylic acid) previously shown to be adequate replacements for whole nucleic acid. Any or all 3 nucleotides could be replaced by corresponding nucleosides without adverse effect, except that adenosine substitution moderately delayed pupation. All base substitutions were unsatisfactory: substitution of thymine for thymidylic acid allowed development to the adult stage but at a greatly reduced rate; single substitution of adenine, cytosine or uracil for the corresponding nucleotides allowed scarcely more development than in the total absence of nucleic acid derivatives. Inosinic acid or inosine were adequate substitutes for adenylic acid, but orotic acid or orotidine were ineffective in place of the pyrimidine ribonucleotides, cytidylic or uridylic acids. Deoxyadenylic acid could take the place of adenylic acid, though inefficiently, but deoxycytidylic and deoxyuridylic acids were very poor replacements for the corresponding ribonucleotides. The minimal required nucleic acid derivatives thus appear to be a purine ribonucleotide (adenylic or inosinic acids), a pyrimidine ribonucleoside (either uridine or cytidine), and the pyrimidine deoxyribonucleoside, thymidine.     

Profiles of pyrimidine biosynthesis,salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.     

Biochemistry and metabolism of Giardia

Giardia lamblia, an aerotolerant anaerobe, respires in the presence of oxygen by a flavin, iron-sulfur protein-mediated electron transport system. Glucose appears to be the only sugar catabolized by the Embden-Meyerhof-Parnas and hexose monophosphate pathways, and energy is produced by substrate level phosphorylation. Substrates are incompletely oxidized to CO2, ethanol and acetate by nonsedimentable enzymes. The lack of incorporation of inosine, hypoxanthine, xanthine, formate or glycine into nucleotides indicates an absence of de novo purine synthesis. Only adenine, adenosine, guanine and guanosine are salvaged, and no interconversion of these purines was detected. Salvage of these purines and their nucleosides is accomplished by adenine phosphoribosyltransferase, adenosine hydrolase, guanosine phosphoribosyltransferase and guanine hydrolase. The absence of de novo pyrimidine synthesis was confirmed by the lack of incorporation of bicarbonate, orotate and aspartate into nucleotides, and by the lack of detectable levels of the enzymes of de novo pyrimidine synthesis. Salvage appears to be accomplished by the action of uracil phosphoribosyltransferase, uridine hydrolase, uridine phosphotransferase, cytidine deaminase, cytidine hydrolase, cytosine phosphoribosyltransferase and thymidine phosphotransferase. Nucleotides of uracil may be converted to nucleotides of cytosine by cytidine triphosphate synthetase, but thymidylate synthetase and dihydrofolate reductase activities were not detected. Uptake of pyrmidine nucleosides, and perhaps pyrimidines, appears to be accomplished by carrier-mediated transport, and the common site for uptake of uridine and cytidine is distinct from the site for thymidine. Thymine does not appear to be incorporated into nucleotide pools. Giardia trophozoites appear to rely on preformed lipids rather than synthesizing them de novo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pathways of Pyrimidine Salvage in <Emphasis Type="Italic">Pseudomonas</Emphasis> and Former <Emphasis Type="Italic">Pseudomonas</Emphasis>: Detection of Recycling Enzymes Using High-Performance Liquid Chromatography

Pyrimidine salvage pathways are vital for all bacteria in that they share in the synthesis of RNA with the biosynthetic pathway in pyrimidine prototrophs, while supplying all pyrimidine requirements in pyrimidine auxotrophs. Salvage enzymes that constitute the pyrimidine salvage pathways were studied in 13 members of Pseudomonas and former pseudomonads. Because it has been established that all Pseudomonas lack the enzyme uridine/cytidine kinase (Udk) and all contain uracil phosphoribosyl transferase (Upp), these two enzymes were not included in this experimental work. The enzymes assayed were: cytosine deaminase [Cod: cytosine + H₂O → uracil + NH₃], cytidine deaminase [Cdd: cytidine + H₂O → uridine + NH₃], uridine phosphorylase [Udp: uridine + P_i ↔ uracil + ribose – 1 - P], nucleoside hydrolase [Nuh: purine/pyrimidine nucleoside + H₂O → purine/pyrimidine base + ribose], uridine hydrolase [Udh: uridine/cytidine + H₂O → uracil/cytosine + ribose]. The assay work generated five different Pyrimidine Salvage Groups (PSG) designated PSG1 – PSG5 based on the presence or absence of the five enzymes. These enzymes were assayed using reverse phase high-performance liquid chromatography techniques routinely carried out in our laboratory. Escherichia coli was included as a standard, which contains all seven of the above enzymes.     

Chemostat selection of Escherichia coli mutants secreting thymidine,cytosine, uracil,guanine, and thymine

Escherichia coli mutants which secreted thymidine, thymine, uracil, cytosine, and guanine into the culture medium were isolated. The isolation strategy was based on the combination of a sensitive screening method and a mutant-generating system. The screening method made use of a thyA mutant of E. coli. These cells, when spread on the agar surface with the 3-galactosidase indicator X-gal, will grow into bule colonies if a minute amount of thymidine is supplied to them from a nearby secretor colony. A chemostat was used as a mutant-generating system to select for E. coli mutants that were resistant to inhibitors of the pyrimidine biosynthetic pathway. Although many mutants were selected based on their secretion of thymidine, other kinds of nucleosides and nucleobases, such as cytosine, uracil, guanine, and thymine, were also present in larger quantities. This rational selection strategy should be applicable to other species of micro-organisms for the isolation of better producers of nucleosides. The production of nucleosides and nucleobases by fermentation could then become a possibility.