Pyrimidine Synthesis Pathway Research Articles

Anticancer Effects of a Chinese Herbal Medicine, Juzen-taiho-to, in Combination with or without 5-Fluorouracil Derivative on DNA-Synthesizing Enzymes in 1,2-Dimethylhydrazine Induced Colonic Cancer in Rats

Juzen-taiho-to (JTT; [Shi-quan-da-bu-tang], a Japanese modified Chinese herbal prescription) in combination with an anticancer drug UFT (5-fluorouracil derivative) prevented the body weight loss and the induction of the colonic cancer in rats treated with a chemical carcinogen 1,2-dimethylhydrazine (DMH), and suppressed markedly the activity of thymidylate synthetase (TS) involved in the de novo pathway of pyrimidine synthesis in colonic cancer induced by DMH.

The viral mechanism of Reye's Syndrome

In the initial stages of Reye's Syndrome, following an influenza infection, the viral RNA polymerase activates liver host cell ornithine decarboxylase by combining with this enzyme. Once the reaction has occured, ornithine decarboxylase is no longer available to combine with and to activate host cell RNA polymerase. The virally activated ornithine decarboxylase removes ornithine from participation in the urea cycle by metabolizing ornithine to putrescine which, in turn, is metabolized to spermidine. Once ornithine has been removed from participation in the urea cycle, mitochondrial carbamoyl phosphate levels increase until the carbamoyl phosphate passes from the mitochondria into the cytosol where it is metabolized by the de novo pyrimidine synthesis pathway. Through the implementation of this process, the virus has insured that: (a) host cell RNA polymerase in liver cells is inactivated, (b) viral RNA polymerase has complete access to newly synthesized pyrimidines, (c) production of pyrimidines for the synthesis of viral messenger RNA is initiated, (d) spermidine, a mRNA stabilizer is produced, (e) many of the components necessary for viral mRNA synthesis are provided by the host cell's RNA synthesizing mechanism.

Pyrimidine Pathway in Boron-deficient Cotton Fiber

Cotton ovules cultured in an insufficiency of boron (10 micromolar), showed inhibition of fiber growth by the ninth day in culture. Averaging data from eight to eleven days of culture under these conditions, total incorporation of [6-(14)C]orotic acid into fiber was inhibited by 59%. Inhibition was evident in all radioactively labeled pools, indicating that the effect may be at the membrane transport level or at an early stage of orotic acid metabolism. On a per cent basis, incorporation into RNA under boron deficiency was higher than under sufficiency. The effect is greater on the eighth day of culture, with a decreasing difference from controls up to the eleventh day. Conversely, the per cent incorporation into UDP-glucose was lower under boron deficiency than in controls, having a more or less constant value from 8 to 11 days of culture. Thus, a primary event of boron deficiency in cotton fiber culture is an alteration in the flow of metabolites through the pyrimidine synthesis pathway.

Insulin regulation of RNA synthesis

During periods of nitrogen exportation from the cell, mitochondrial carbamoyl phosphate is synthesized, thus initiating the urea cycle. During times of nitrogen conservation by the liver cell, carbamoyl phosphate is synthesized in the cytosol of the cell, whereupon the de novo pyrimidine synthesis pathway is initiated. The de novo pathway provides pyrimidines for increased ribonucleic acid synthesis. Formerly, it was believed that these two pathways functioned irrespective of one another. However, recent experimental evidence indicates that, when excess ammonia is present, mitochondrial carbamoyl phosphate passes from the mitochondria into the cell cytosol, where it is metabolized by the de novo pyrimidine synthesis pathway. When ornithine and excess ammonia are both present, mitochondrial carbamoyl phosphate no longer passes from the mitochondria into the cytosol to be metabolized by the de nova pathway. Thus the metabolic fate of mitochondrial carbamoyl phosphate, and that of excess nitrogen, is determined by the presence or absence of ornithine. In turn, this key molecule is the substrate for the cytoplasmic enzyme ornithine decarboxylase. When ornithine decarboxylase is stimulated by insulin, ornithine is metabolized to putrescine. The activated ornithine decarboxylase combines with ribonucleic acid polymerase, activating the later enzyme. When ornithine is acted upon by ornithine decarboxylase, it is no longer available for the perpetuation of the urea cycle and mitochondrial carbamoyl phosphate levels rise until the carbamoyl phosphate passes into the cytosol to be metabolized by the de novo pathway. Increased amounts of pyrimidines are available for the activated ribonucleic acid polymerase. Therefore insulin, through its stimulation of ornithine decarboxylase, achieves cellular nitrogen retention by regulating nitrogen incorporation into newly synthesized ribonucleic acid.

Orotic Acid and Pyrimidine Nucleotides of Ruminant Milks

Reports conflict on the apparent high content of orotic acid in ruminant milks compared to that (0 to 5μg/ml) in milks of other species and animal tissues. Amounts reported for mid-lactation ovine (sheep) milk and early lactation bovine (cow) milk range from none to over 320μg/ml based on chemical and microbiological assay procedures with the latter giving the higher results. To clarify this discrepancy, orotic acid in ovine and bovine milks was determined by a direct chemical and two microbiological assay procedures using mutant bacteria lacking different functional portions of the pyrimidine synthesis pathway before and after orotic acid. Nine mid-lactation ovine milks contained 26±9μg/ml of orotic acid and 331±51μg/ml (as uracil) of pyrimidines all of which were evaluated as orotic acid in the microbiological bioassay procedures showing a large amount. Bovine milks from early lactation were similar. Four bovine samples of first colostrum contained 21±7μg/ml of orotic acid and 279±96μg/ml (as uracil) of pyrimidines. Eight mid-lactation bovine milks contained 88±19μg/ml of orotic acid but only small amounts of pyrimidines. The orotic acid and pyrimidine contents of these milks interpreted from the chemical and microbiological assay procedures agree closely with those based on the isolation and separation of acid-soluble nucleotides. Pyrimidine nucleotides comprise 1 to 2% of the total nonfat solids of ovine milk with only relatively small amounts of orotic acid. In contrast, bovine milk contains only small.

Enzymes of Ammonia Detoxication after Portacaval Shunt in the Rat

At high systemic blood concentrations ammonia may be partially deviated into the pathway of pyrimidine synthesis, as has been observed in different genetic defects of the urea cycle. The portacaval shunt (PCS) rat presents an animal model to study ammonia detoxication without an underlying enzyme defect in the urea cycle. Since ammonia may induce a deviation into the pyrimidine pathway by influencing enzymatic reactions involved in this pathway, the activity of carbamylphosphate synthetase and aspartate transcarbamylase in liver as well as the excretion of orotic acid in the urine were measured in rats 10, 20 and 30 days after PCS. The results suggest that in this experimental model ammonia may be channeled into the pyrimidine pathway leading to a stimulation of the first enzymatic step and to an increased excretion of orotic acid.

Nucleotide Inhibition of Post-Orotate Pyrimidine Synthesis Pathway Enzymes of Bovine Mammary Tissue

Effects of various nucleotides are evaluated on enzymes of the post-orotate portion of the pyrimidine synthesis pathway from lactating bovine mammary tissue. Orotate phosphoribosyltransferase shows minimal activation by most nucleotides tested, but orotidine-5′-phosphate decarboxylase is highly inhibited by several nucleotides with xanthosine, cytidine, and uridine 5′-phosphates being the most effective. It is possible that product inhibition by uridine Diphosphate of orotidine 5′-phosphate decarboxylase is an important factor in the accumulation of orotate in bovine milk.

Carbamylphosphate synthetase of lactating bovine mammary tissue

Summary A carbamylphosphate synthetase has been partially purified from lactating bovine mammary tissue which uses ammonia and not L-glutamine as a primary substrate. It is stimulated by phosphoribosylpyrophosphate, does not require activation by N-acetyl-L-glutamate, and is inhibited by several nucleotides of which UTP is not the most effective. Although the enzyme resembles more closely the carbamylphosphate synthetase of the pyrimidine synthesis pathway than that of the urea cycle, its characteristics indicate that it is a different form than has been described.

Pyrimidine Synthesis Pathway Enzymes and Orotic Acid in Bovine Mammary Tissue

Studies were initiated to determine if the high orotic acid in bovine (ruminant) milk is indicative of a pathway of pyrimidine synthesis different from that in other bacterial and mammalian systems where this metabolic intermediate is normally low. Elevated orotic acid was not found in a variety of other body fluids or tissues of the bovine which included the apparent intracellular level in the tissue itself when corrected for its milk content. Analyses for three enzymes on the pathway of pyrimidine synthesis (dihydroorotic acid dehydrogenase, orotidine 5′-phosphate pyrophosphorylase, and orotidine 5′-phosphate decarboxylase) indicated adequate activity, thus, contrasting with other situations of orotic acid accumulation where activity of some of these enzymes is lacking. The results suggest that the normal pathway for pyrimidine synthesis is operational in bovine mammary tissue and that orotic acid accumulation must be explained by an unknown altered functioning of this pathway possibly related to the milk secretion process.

Glucocorticoid inhibition of precursor incorporation into nuclear RNA of P1798 lymphosarcoma

1. Treatment of corticoid-sensitive P1798 lymphosarcoma-bearing BALB/c mice with 9α-fluoroprednisolone markedly depressed in vivo incorporation of uridine, uracil and guanosine into tumor nuclear RNA (about 50 %), while not affecting that of orotic acid. 2. No effect was observed on in vitro incorporation of uridine, uracil and guanosine following in vivo treatment with 9α-fluoroprednisolone, while the in vitro uptake and utilization of orotic acid were strongly inhibited. 3. In vivo uridine and in vitro orotic acid incorporation into nuclear RNA of corticoid-resistant P1798 lymphosarcoma were only slightly inhibited following administration of 9α-fluoroprednisolone; guanosine incorporation was depressed as much as or even more than in the corticoid-sensitive tumor. 4. These results suggest that glucocorticoid treatment of P1798 lymphosarcoma inhibits the salvage pathway of pyrimidine synthesis rather than the de novo synthesis of pyrimidine nucleotides from orotic acid, and that this effect is relevant to tumor regression.

TEMPERATURE SENSITIVITY OF MUTANTS OF BACILLUS ANTHRACIS CAUSED BY A BLOCK IN THYMINE-NUCLEOTIDE SYNTHESIS.

Mutants were isolated from the non-capsulated strain Vollum of Bacillus anthracis which were unable to grow above 34° in the absence of certain pyrimidines. At elevated temperatures, one of the mutants, YC–TdR–, was found to be dependent on thymidine while the other, VC–T–, required thymine. Both mutants, however, grew normally in the absence of pyrimidines at near to room temperature. A relatively high concentration of thymine was needed in order to overcome the thymidine requirement of mutant YC–TdR– at 37°, whereas a combination of a low concentration of thymine with different deoxyribosides (deoxyadenosine, deoxyguano-sine, deoxycytidine) gave good growth of the mutant. This observation is suggestive of the presence of a particular enzyme, trans-N-deoxyribosylase. in the mutant VC–TdR–, an enzyme which appears to be of limited distribution in nature. The second mutant. VC–T–, utilized added thymine readily at 37° and the base could not be substituted by its nucleoside, thymidine. In fact, thymidine and deoxyribonucleosides inhibited the growth of mutant VC–T– in the presence of thymine. Both mutants also grew well at 37° in the presence of thymidine-5-phosphate, which indicated that the de novo pathway of pyrimidine synthesis is blocked above 34° somewhere in the pathway between deoxycytidine-phosphate and thymidine-5-phosphate. This block in the pyrimidine synthesis occurring at elevated temperatures caused an unbalanced synthesis of macromolecules accompanied by an abnormal cell-wall formation. At 37°, germinated spores showed an abnormal elongation of the initial cell concomitant with a gradual loss of viability. At this temperature cell-wall formation was also abnormal at limiting concentrations of pyrimidines and minor deficiencies in cell-wall structure of the mutants were still apparent even in the presence of a large excess of pyrimidine. This, however, did not involve any change in virulence of mutant VC+TdR– in the homoiothermic mouse. It is assumed that the mutants produce an altered enzyme protein corresponding to a block in de novo synthesis of pyrimidine, or that an inhibitor is produced at high temperatures which diminishes and finally prevents the action of the normal enzyme.

Erratum: Neurospora Mutant Lacking an Arginione-Specific Carbamyl Phosphokinase

In the report "Neurospora mutant lacking an arginine-specific carbamyl phosphokinase" by R. H. Davis [ Science 142, 1652 (27 Dec. 1963)], the last sentence of paragraph 1 should read: "Early in the orotic-acid pathway of pyrimidine synthesis it [carbamy] phosphate] is used in the carbamylation of aspartate to form ureidosuccinate, a reaction catalyzed by aspartate transcarbamylase (ATC)." In the original, ornithine was substituted for aspartate .

Enzymes of uracil metabolism in tissues with different growth characteristics

Different tissues were investigated for their contents of uridine phosphorylase, uridine kinase, deoxyuridine phosphorylase and deoxyuridine kinase activities. These enzyme contents were considered to mirror the relative efficiency of an enzymic pathway using uracil for the biosynthesis of nucleic acid pyrimidines. Furthermore, the same tissues were investigated for their capacities to convert orotic acid to UMP, representing the de novo pathway of pyrimidine synthesis. In a survey of some normal tissues from rat, mouse and rabbit, a certain degree of correlation was found between the efficiency of this alternative pathway and the growth rate. During liver regeneration, marked increases in the enzymes studied were observed. Thus, uridine phosphorylase showed a maximal threefold increase between 36 and 72 h after hepactomy. Corresponding values were a fivefold increase for uridine kinase (24–36 h), a fourfold increase for deoxyuridine phosphorylase (about 48h), a tenfold increase for deoxyuridine kinase (36–48 h) and, for the enzymes transforming orotic acid to UMP a 5- tp 6-fold increase (about 48 h). 144 h after the operation, all enzymes had reverted to almost normal values. These rapid changes in the activities of enzymes involved in the synthesis of nucleotides, might form part of an homeostatic mechanism involved in pyrimidine biosynthesis.