Control of pyrimidine nucleotide formation in Pseudomonas aurantiaca.

The control of pyrimidine nucleotide formation in the bacterium Pseudomonas aurantiaca ATCC 33663 by pyrimidines was studied. The activities of the pyrimidine biosynthetic pathway enzymes were investigated in P. aurantiaca ATCC 33663 cells and from cells of an auxotroph lacking orotate phosphoribosyltransferase activity under selected culture conditions. All activities of the pyrimidine biosynthetic pathway enzymes in ATCC 33663 cells were depressed by uracil addition to the minimal medium when succinate served as the carbon source. In contrast, all pyrimidine biosynthetic pathway enzyme activities in ATCC 33663 cells were depressed by orotic acid supplementation to the minimal medium when glucose served as the carbon source. The orotidine 5'-monophosphate decarboxylase activity in the phosphoribosyltransferase mutant strain increased by more than sixfold in succinate-grown cells and by more than 16-fold in glucose-grown cells after pyrimidine limitation showing possible repression of the decarboxylase by a pyrimidine-related compound. Inhibition by ATP, GTP, UTP and pyrophosphate of the in vitro activity of aspartate transcarbamoylase in ATCC 33663 was observed. The findings demonstrated control at the level of pyrimidine biosynthetic enzyme synthesis and activity for the P. aurantiaca transcarbamoylase. The control of pyrimidine synthesis in P. aurantiaca seemed to differ from what has been observed previously for the regulation of pyrimidine biosynthesis in related Pseudomonas species. This investigation could prove helpful to future work studying pseudomonad taxonomic analysis as well as to those exploring antifungal and antimicrobial agents produced by P. aurantiaca.

Synthesis and evaluation of two series of 4'-aza-carbocyclic nucleosides as adenosine A2A receptor agonists.

The synthesis of two series of 4'-aza-carbocyclic nucleosides are described in which the 4'-substituent is either a reversed amide, relative to the carboxamide of NECA, or an N-bonded heterocycle. Using established purine substitution patterns, potent and selective examples of agonists of the human adenosine A(2A) receptor have been identified from both series. The propionamides 14-18 and the 4-hydroxymethylpyrazole 32 were determined to be the most potent and selective examples from the 4'-reversed amide and 4'-N-bonded heterocyclic series, respectively.

Inhibiting the teratogenicity of the immunosuppressant leflunomide in mice by supplementation of exogenous uridine.

Leflunomide is an immunosuppressant drug displaying teratogenicity in mice, rats, and rabbits. Its immunosuppressive effect occurs via inhibition of dihydroorotate dehydrogenase (DHODH) and tyrosine kinases. In this study, we coadministered Leflunomide and uridine, a precursor substance of pyrimidine nucleotides, to pregnant CD-1 mice, and examined whether or not a decreased level of intracellular pyrimidine nucleotides with inhibition of DHODH is related to the teratogenicity of Leflunomide. Then we examined the alteration of the nucleotide level in fetal tissue by Leflunomide and the effect of coadministered uridine. We administered Leflunomide with or without uridine to pregnant mice on gestation day 10, and used the vehicle of Leflunomide as a control. Leflunomide caused multiple malformations in all fetuses, but coadministration with uridine inhibited most of its teratogenicity. Leflunomide decreased the concentration of pyrimidine nucleotides, not purine nucleotides, whereas uridine coadministered with Leflunomide partially restored the level of pyrimidine nucleotides. These results indicate that the inhibitory effect of DHODH activity is related to the teratogenicity of Leflunomide.

Kinetics of N-(phosphonacetyl)-L-aspartate and pyrazofurin depletion of pyrimidine ribonucleotide and deoxyribonucleotide pools and their relationship to nucleic acid synthesis in intact and permeabilized cells.

Pools of uridine triphosphate and cytidine triphosphate are greatly (90%) reduced in cultured L1210 cells exposed to N-(phosphonacetyl)-L-aspartate (PALA) or pyrazofurin; the concentration of the deoxynucleotides deoxycytidine triphosphate, deoxythymidine triphosphate, and deoxyguanosine triphosphate also decreases, but deoxyadenosine triphosphate pools are enlarged. Associated with these pool depletions is a pronounced inhibition of DNA synthesis even when pools are only moderately reduced; RNA synthesis is only slightly inhibited under these same conditions. DNA synthesis in permeabilized preparations of L1210 cells was also more sensitive than was RNA synthesis when the concentrations of ribonucleotide and deoxyribonucleotide triphosphates presented were equivalent to those found in PALA- or pyrazofurin-treated cells. The specific sensitivity to depletion of DNA precursors was also seen in protection of both DNA synthesis and growth of L1210 cells by deoxycytidine and thymidine. This supplement restored deoxycytidine triphosphate, deoxythymidine triphosphate, and deoxyguanosine triphosphate pools to normal but of course did not affect the marked depletions of uridine triphosphate and cytidine triphosphate or the less marked effect of PALA on RNA synthesis. The relative ability of PALA to reduce uridine triphosphate and cytidine triphosphate pool size in L1210 ascites and Lewis lung carcinoma in vivo correlates with the intrinsic sensitivity to this agent.

Purine and pyrimidine metabolism.

The pathways of purine biosynthesis and degradation have been elucidated during the last 30 years; the regulation of the mechanisms involved is not yet fully understood, particularly with respect to quantitative aspects. Research into inborn errors of purine metabolism has provided valuable insights into purine synthesis and salvage pathways. Nutrition experiments using purine-free formula diets and supplements with defined purine sources permit precise descriptions of the influence of various dietary purines on uric acid formation. Supplements of dietary purines produce dose-proportional increases in plasma uric acid concentrations, uric acid pool size and renal uric acid excretion. The magnitude of these increases depends on the type of purine compound administered, which may limit the value of food tables for human dietetics. Purine content of food must be related not only to weight but also to energy and to protein, particularly if new foodstuffs or a vegetarian diet are ingested. Dietary purines appear to influence the biosynthesis of pyrimidines. In contrast to dietary purines, pyrimidines in the diet, if administered as nucleosides or nucleotides, are utilized in animals for the synthesis of nucleic acids. Much further work is necessary for a better understanding of the inter-relationships of purine and pyrimidine metabolism.

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Characteristics related to quantitative studies of RNA metabolism.

Ehrlich ascites-tumour cells incubated in a mineral medium supplemented with glucose and glutamine intensely incorporated labelled uridine into free nucleotides and RNA. Detailed kinetic studies of the labelling of total acid-soluble pools of uridine and cytidine nucleotides and of RNA under standard conditions and after chase with non-radioactive uridine were carried out. The relative distribution of the radioactivity between the individual uridine phosphates as well as their total cellular contents were also determined under standard and chase conditions. The labelling kinetics of the mononucleotides and RNA by radioactive uridine and orotate were compared, and some turnover parameters were estimated by mathematical analysis of a steady-state model. The following conclusions were drawn. (1) The observed chase effect of addition of non-radioactive uridine is due to a rapid expansion of the acid-soluble uridine nucleotide pool and consequently a severalfold lowering of its specific radioactivity. (2) The free uridine nucleotides are partly separated into a large and a small compartment, labelled preferentially by exogeneous orotate and uridine respectively. (3) The half-life of the mononucleotides, at least those located in the smaller compartment, is a few minutes only, owing to a rapid exchange with the uridylic acid residues of unstable RNA species. (4) This process of reutilization of RNA-degradation products accounts for 85% (a minimum estimate) of the overall turnover of the nucleotide pool in resting cells.