Uridine monophosphate synthetase enables eukaryotic de novo NAD+ biosynthesis from quinolinic acid.

NAD+ biosynthesis is an attractive and promising therapeutic target for influencing health span and obesity-related phenotypes as well as tumor growth. Full and effective use of this target for therapeutic benefit requires a complete understanding of NAD+ biosynthetic pathways. Here, we report a previously unrecognized role for a conserved phosphoribosyltransferase in NAD+ biosynthesis. Because a required quinolinic acid phosphoribosyltransferase (QPRTase) is not encoded in its genome, Caenorhabditis elegans are reported to lack a de novo NAD+ biosynthetic pathway. However, all the genes of the kynurenine pathway required for quinolinic acid (QA) production from tryptophan are present. Thus, we investigated the presence of de novo NAD+ biosynthesis in this organism. By combining isotope-tracing and genetic experiments, we have demonstrated the presence of an intact de novo biosynthesis pathway for NAD+ from tryptophan via QA, highlighting the functional conservation of this important biosynthetic activity. Supplementation with kynurenine pathway intermediates also boosted NAD+ levels and partially reversed NAD+-dependent phenotypes caused by mutation of pnc-1, which encodes a nicotinamidase required for NAD+ salvage biosynthesis, demonstrating contribution of de novo synthesis to NAD+ homeostasis. By investigating candidate phosphoribosyltransferase genes in the genome, we determined that the conserved uridine monophosphate phosphoribosyltransferase (UMPS), which acts in pyrimidine biosynthesis, is required for NAD+ biosynthesis in place of the missing QPRTase. We suggest that similar underground metabolic activity of UMPS may function in other organisms. This mechanism for NAD+ biosynthesis creates novel possibilities for manipulating NAD+ biosynthetic pathways, which is key for the future of therapeutics.

Accumulation of Pyrimidine Intermediate Orotate Decreases Virulence Factor Production in Pseudomonas aeruginosa.

The impact of orotate accumulation in the medically important bacterium Pseudomonas aeruginosa was studied by deleting pyrE, the gene encoding orotate phosphoribosyltransferase and responsible for converting orotate into orotate monophosphate within the de novo pyrimidine synthesis pathway. The pyrE mutant accumulated orotate and exhibited decreased production of hemolysin, casein protease, and elastase. Feeding orotate at a concentration of 51.25 µM to the wild type, PAO1, likewise decreased production of these factors except for hemolysin, which was not affected. A significant increase in the pigments pyocyanin and pyoverdin was also observed. Pyocyanin increase in the pyrE mutant was heightened when the mutant was supplemented with orotate. Although pyoverdin production in the wild-type PAO1 was unaffected by orotate supplementation, a decrease in the mutant's production was observed when supplemented with orotate. These results indicate a significant reduction in virulence factor production in the pyrE mutant and reduction in some virulence factors in the wild type when supplemented with orotate.

Impact of 5-fluorouracil metabolizing enzymes on chemotherapy in patients with resectable colorectal cancer.

Although 5-fluorouracil (5-FU) is an important drug for colorectal cancer (CRC) treatment, no useful biomarker is currently available to predict treatment response. Since 5-FU is converted into active or inactive forms by orotate phosphoribosyltransferase (OPRT) or dihydropyrimidine dehydrogenase (DPD), a correlation between these enzymes and response to 5-FU has been suggested. However, such a correlation has not been investigated prospectively. Therefore, in the present study, we aimed to prospectively evaluate whether OPRT and DPD were predictive factors of the response to 5-FU treatment in patients with resectable CRC. The present investigation was designed as a multicenter prospective cohort study. OPRT and DPD activities were assessed in biopsy samples, obtained surgically from patients with resectable CRC. The OPRT/DPD ratio was calculated and the cut-off values for this ratio were determined for 5-year disease-free survival (DFS) and overall survival (OS). Patients were treated with 5-FU/leucovorin (LV) regimens and oral 5-FU. The endpoint of this study was the correlation between the OPRT/DPD ratio and 5-year DFS and OS. The cut-off value for the OPRT/DPD ratio was determined by using the maximum χ2 statistic method against 5-year DFS and OS. Sixty-eight patients were enrolled from July 2003 to May 2005. The median follow-up period was 1925 days. The OPRT/DPD ratio cut-off values for 5-year DFS and OS were 0.015 and 0.013, respectively. During the 5-year DFS and OS periods, patients with higher cut-off values had a better prognosis than those with lower ratios (P=0.03 and 0.02, respectively). In conclusion, our results suggest that the OPRT/DPD ratio could be a predictive factor for response to 5-FU/LV adjuvant chemotherapy.

De novo pyrimidine biosynthesis in the oomycete plant pathogen Phytophthora infestans.

The oomycete Phytophthora infestans, causal agent of the tomato and potato late blight, generates important economic and environmental losses worldwide. As current control strategies are becoming less effective, there is a need for studies on oomycete metabolism to help identify promising and more effective targets for chemical control. The pyrimidine pathways are attractive metabolic targets to combat tumors, virus and parasitic diseases but have not yet been studied in Phytophthora. Pyrimidines are involved in several critical cellular processes and play structural, metabolic and regulatory functions. Here, we used genomic and transcriptomic information to survey the pyrimidine metabolism during the P. infestans life cycle. After assessing the putative gene machinery for pyrimidine salvage and de novo synthesis, we inferred genealogies for each enzymatic domain in the latter pathway, which displayed a mosaic origin. The last two enzymes of the pathway, orotate phosphoribosyltransferase and orotidine-5-monophosphate decarboxylase, are fused in a multi-domain enzyme and are duplicated in some P. infestans strains. Two splice variants of the third gene (dihydroorotase) were identified, one of them encoding a premature stop codon generating a non-functional truncated protein. Relative expression profiles of pyrimidine biosynthesis genes were evaluated by qRT-PCR during infection in Solanum phureja. The third and fifth genes involved in this pathway showed high up-regulation during biotrophic stages and down-regulation during necrotrophy, whereas the uracil phosphoribosyl transferase gene involved in pyrimidine salvage showed the inverse behavior. These findings suggest the importance of de novo pyrimidine biosynthesis during the fast replicative early infection stages and highlight the dynamics of the metabolism associated with the hemibiotrophic life style of pathogen.

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy.

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.

Toll-like receptor 9-dependent activation of bone marrow-derived dendritic cells by URA5 DNA from Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis in immunocompromised patients. Recently, we reported that Toll-like receptor 9 (TLR9) is involved in host defense against C. neoformans: specifically, it detects the pathogen's DNA. In the present study, we aimed to elucidate the mechanisms underlying TLR9-mediated activation of innate immune responses by using the URA5 gene, which encodes a virulent component of this fungal pathogen. A PCR-amplified 345-bp URA5 gene fragment induced interleukin-12 p40 (IL-12p40) production by bone marrow-derived dendritic cells (BM-DCs) in a TLR9-dependent manner. Similar activity was detected in the 5' 129-bp DNA fragment of URA5 and in a synthesized oligodeoxynucleotide (ODN) with the same sequence. Shorter ODN fragments, which contained GTCGGT or GACGAT but had only 24 or 21 bases, induced IL-12p40 production and CD40 expression by BM-DCs, but this activity vanished when the CG sequence was replaced by GC or when a phosphorothioate modification was introduced. IL-12p40 production caused by active ODN was strikingly enhanced by treatment with DOTAP, a cationic lipid that increases the uptake of DNA by BM-DCs, though DOTAP failed to induce IL-12p40 production by inactive ODN and did not affect the activity of an ODN-containing canonical CpG motif. There was no apparent difference in intracellular trafficking between active and inactive ODNs. Finally, an extremely high dose of inactive ODN suppressed IL-12p40 production by BM-DCs that had been stimulated with active ODN. These results suggest that the C. neoformans URA5 gene activates BM-DCs through a TLR9-mediated signaling pathway, using a mechanism possibly independent of the canonical CpG motif.

Control of pyrimidine nucleotide formation in Pseudomonas fulva.

Control of pyrimidine formation was examined in Pseudomonas fulva ATCC 31418. Pyrimidine supplementation lowered pyrimidine biosynthetic pathway enzyme activities in cells grown on glucose or succinate as a carbon source indicating possible repression of enzyme synthesis. Pyrimidine limitation experiments were conducted using an orotidine 50-monophosphate decarboxylase mutant strain isolated in this study. Compared to uracil-supplemented, glucose-grown mutant cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities to increase about 6-, 13-, 3-, 15-fold, respectively, which confirmed regulation of enzyme synthesis by pyrimidines. At the level of enzyme activity, transcarbamoylase activity in Ps. fulva was strongly inhibited by pyrophosphate, CTP, GTP and GDP under saturating substrate concentrations.

Significance of 5-fluorouracil-related enzyme activities in predicting sensitivity to 5-fluorouracil in bladder carcinoma.

BACKGROUND: The association between 5-fluorouracil (5-FU)-related enzyme activity and the sensitivity of bladder urothelial carcinoma (BUC) to 5-FU were investigated, and methods to improve 5-FU sensitivity were analyzed. MATERIALS AND METHODS: Tumor specimens were obtained from 127 patients. Orotate phosphoribosyl transferase (OPRT) activity was analyzed by the paper disc method and thymidine phosphorylase and dihydropyrimidine dehydrogenase (DPD) activities were measured by ELISA. 5-FU sensitivity was assessed in 99 cases by an in vitro chemosensitivity test. RESULTS: A significant positive correlation between OPRT activity level and the sensitivity of BUC to 5-FU was identified. Moreover, the combination of 5-FU and 5-chloro-2,4-dihydroxypyrimidine significantly enhanced 5-FU sensitivity in BUC, particularly in cases showing higher DPD activity. CONCLUSION: OPRT was the most important enzyme in predicting sensitivity to 5-FU in BUC. These results may have implications for tailor-made medication using 5-FU-related compounds as postoperative adjuvant chemotherapy in BUC patients.

Clinical implications of thymidylate synthetase, dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase activity levels in colorectal carcinoma following radical resection and administration of adjuvant 5-FU chemotherapy.

BACKGROUND: A number of studies have investigated whether the activity levels of enzymes involved in 5-fluorouracil (5-FU) metabolism are prognostic factors for survival in patients with colorectal carcinoma. Most reports have examined thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in unresectable or metastatic cases, therefore it is unclear whether the activity of these enzymes is of prognostic value in colorectal cancer patients treated with radical resection and adjuvant chemotherapy with 5-FU. METHODS: This study examined fresh frozen specimens of colorectal carcinoma from 40 patients who had undergone curative operation and were orally administered adjuvant tegafur/uracil (UFT) chemotherapy. TS, DPD and orotate phosphoribosyl transferase (OPRT) activities were assayed in cancer tissue and adjacent normal tissue and their association with clinicopathological variables was investigated. In addition, the relationships between TS, DPD and OPRT activities and patient survival were examined to determine whether any of these enzymes could be useful prognostic factors. RESULTS: While there was no clear relationship between pathological findings and TS or DPD activity, OPRT activity was significantly lower in tumors with lymph node metastasis than in tumors lacking lymph node metastasis. Postoperative survival was significantly better in the groups with low TS activity and/or high OPRT activity. CONCLUSION: TS and OPRT activity levels in tumor tissue may be important prognostic factors for survival in Dukes' B and C colorectal carcinoma with radical resection and adjuvant chemotherapy with UFT.

Prognostic value of 5-fluorouracil metabolic enzyme genes in Dukes' stage B and C colorectal cancer patients treated with oral 5-fluorouracil-based adjuvant chemotherapy.

5-Fluorouracil (5-FU) is the most commonly used anticancer drug for colorectal cancer (CRC). In this study, we aimed to clarify the prognostic value of the expression of the 5-FU metabolic enzyme genes, including orptate phosphoribosyl transferase (OPRT), dihydropyrimidine dehydrogenase (DPD), thymidylate synthetase (TS) and thymidylate phosphorylate (TP) genes in CRC patients treated with oral 5-FU-based adjuvant chemotherapy. We examined 103 CRC patients with Dukes' stage B and C who underwent oral 5-FU-based adjuvant chemotherapy. Formalin-fixed, paraffin-embedded tumor specimens from primary CRC tissues were dissected by laser-captured microdissection and quantification of mRNA levels of OPRT, DPD, TS and TP were measured by real-time reverse transcription (RT) PCR. The relationship between these 5-FU metabolic enzyme gene levels and disease-free and overall survival rates were examined. The disease-free and overall survival curves of the OPRT mRNA high-expression group were significantly longer than that of the OPRT mRNA low-expression group. The disease-free and overall survival curves of the DPD mRNA high-expression group were significantly shorter than that of the DPD mRNA low-expression group. In contrast, there were no significant differences between the TS or TP mRNA high- expression and low-expression groups in the disease-free and overall survival curves. In a multivariate Cox regression analysis, it was demonstrated that the OPRT mRNA level is an independent prognostic variable for disease-free and overall survival. These results suggest that the OPRT mRNA is a useful indicator in the prediction of disease-free and overall survival in Dukes' B and C stage CRC patients treated with oral 5-FU-based adjuvant chemotherapy.

Regulation of pyrimidine formation in Pseudomonas oryzihabitans.

The regulation of pyrimidine formation in the opportunistic human pathogen Pseudomonas oryzihabitans was investigated at the level of enzyme synthesis and at the level of activity for the pyrimidine biosynthetic pathway enzyme aspartate transcarbamoylase. Although pyrimidine supplementation of succinate-grown P. oryzihabitans cells produced little effect on the de novo pyrimidine biosynthetic pathway enzyme activities, pyrimidine limitation experiments undertaken using an orotidine 5'-monophosphate decarboxylase mutant strain isolated from P. oryzihabitans ATCC 43272 indicated that repression of enzyme synthesis by pyrimidines was occurring. Following pyrimidine limitation of the succinate-grown decarboxylase mutant strain cells, aspartate transcarbamoylase and dihydroorotase activities were found to increase by about 3-fold while dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities were also observed to increase relative to their activities in the mutant strain cells grown on excess uracil. At the level of enzyme activity, aspartate transcarbamoylase in P. oryzihabitans was strongly inhibited by pyrophosphate, ADP, ATP and GTP in the presence of saturating substrate concentrations.

Expression of orotate phosphoribosyl transferase in human pancreatic cancer: implication for the efficacy of uracil and tegafur-based adjuvant chemotherapy.

The enzyme orotate phosphoribosyl transferase (OPRT) is involved in the metabolism of the anticancer drug 5-fluorouracil (5-FU), and is a key enzyme for conversion of 5-FU to its active form in tumor tissue. Little is known regarding the significance of OPRT in human pancreatic cancer. The present study was designed to assess the association between the activity of OPRT in the tumor, and the clinicopathological status and prognosis of human resectable pancreatic cancer, especially regarding its relevance to the efficacy of adjuvant chemotherapy with uracil and tegafur (UFT), cyclophosphamide (CPA) and/or gemcitabine (GEM). The present study included 99 resectable pancreatic cancers, which were all invasive ductal tubular carcinomas. OPRT was immunostained with a rabbit anti-human OPRT polyclonal antibody. OPRT was positively stained in 54 (54.5%) of 99 pancreatic cancers. The post-surgical survival rate of the OPRT (+) pancreatic cancers was significantly higher than that of the OPRT (-) ones. In the OPRT (+) group, the survival rate of the patients, who received adjuvant chemotherapy (ACT) with UFT, CPA or GEM, was significantly higher than that of the patients without ACT; however, in the OPRT (-) group, there was no difference in the survival between the ACT (+) and (-) groups. Multivariate analyses demonstrated that for all patients, primary tumor, status of nodal involvement (pN), residual tumor, level of dissection and CPA were significant variables for the prognosis: in OPRT (+) groups, primary tumor, nodal involvement, GEM and CPA were significant variables. In contrast, in the OPRT (-) group, pN was the only significant variable. The present study is the first report on the significance of OPRT in human pancreatic cancer, and the results indicate that the expression of OPRT may be useful to predict the response to adjuvant chemotherapy in human pancreatic cancer.

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha.

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha ATCC 9890 was investigated and the pyrimidine biosynthetic pathway enzyme activities were affected by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. In pyrimidine-grown ATCC 9890 cells, the activities of four de novo enzymes could be depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the succinate-grown mutant strain cells, pyrimidine limitation of this strain caused dihydroorotase activity to increase about 3-fold while dihydroorotate dehydrogenase and orotidine 5'-monophosphate decarboxylase activities rose about 2-fold. Regulation of de novo pathway enzyme synthesis by pyrimidines appeared to be occurring. At the level of enzyme activity, aspartate transcarbamoylase activity in P. synxantha ATCC 9890 was strongly inhibited in vitro by pyrophosphate, UTP, ADP, ATP, CTP and GTP under saturating substrate concentrations.

[Clinical and prognostic significance of protein and gene expression of orotate phosphoribosyltransferase in gastric carcinoma].

Although orotate phosphoribosyltransferase (OPRT EC 2.4.2.10) is a key enzyme related to the first-step activation process of 5-fluorouracil, and therefore it has been shown to be an important enzyme that enables to predict sensitivity to 5-fluorouracil, the clinical and prognostic significance of protein and/or gene expression of OPRT has not been well established in gastric carcinoma. We examined the protein level, and mRNA expression of OPRT in gastric carcinoma tissues and relationships with clinicopathologic factors and prognosis were evaluated. A total of 75 surgically-resected gastric carcinoma tissues were subjected to the study. An enzymelinked immunosorbent assay (ELISA) was used to accurately assess intratumoral OPRT, and gene expressions of OPRT were examined using a real-time PCR method. Survival of patients with gastric carcinoma in relation to OPRT protein levels was analyzed using Kaplan-Meier methods along with log-rank test. The mean value of OPRT was 5.4+/-3.6 ng/mg protein, and it was significantly higher in patients with differentiated-type and invasive-type gastric carcinoma. The prognosis of patients in the high OPRT group was better than for those with low OPRT (p<0.05). There was a significant correlation between OPRT levels measured by ELISA and OPRT mRNA expression (p<0.05). Determination of OPRT levels is a useful tool to predict the biological characteristics of gastric carcinoma and possibly predict sensitivity to fluoropyrimidine-based anticancer chemotherapy,particularly dihydropyrimidine dehydrogenase-inhibitory fluoropyrimidine, in patients with gastric carcinoma.

Orotate phosphoribosyltransferase gene polymorphism predicts toxicity in patients treated with bolus 5-fluorouracil regimen.

PURPOSE: We investigated whether the determination of orotate phosphoribosyltransferase (OPRT) and thymidylate synthase (TYMS) polymorphisms could predict the toxicity of 5-fluorouracil (5-FU) in colorectal cancer patients. EXPERIMENTAL DESIGN: The determination of OPRT and TYMS genotypes were done in genomic DNA extracted from blood by PCR amplification in 69 patients treated with bolus 5-FU as adjuvant chemotherapy. Associations between these polymorphisms and toxicity were evaluated retrospectively. RESULTS: The Ala allele in OPRT Gly213Ala polymorphism and the two tandem repeats (2R) in TYMS promoter polymorphism were associated with grade 3 to 4 neutropenia and diarrhea. The multivariate logistic regression models revealed that only TYMS promoter polymorphism had an independent value to predict grade 3 to 4 neutropenia [odds ratio, 19.2 for patients with the 2R allele compared with patients with homozygous with the three repeat (3R) alleles], whereas both OPRT and TYMS promoter polymorphisms were independent predictive factors for grade 3 to 4 diarrhea (odds ratio, 13.3 for patients with the Ala allele compared with patients in the Gly/Gly genotype and 11.1 for patients with the 2R allele compared with patients in the 3R/3R genotype). A significant difference was observed in the time to onset of severe toxicity, defined as grade 4 neutropenia and/or grade 3 to 4 gastrointestinal toxicities according to OPRT and TYMS promoter polymorphisms. CONCLUSION: OPRT Gly213Ala polymorphism seems to be a useful marker for predicting toxicity to bolus 5-FU chemotherapy. Prospective translational treatment trials including larger number of patients are needed to confirm our results.

Prognostic impact of orotate phosphoribosyl transferase activity in resectable colorectal cancers treated by 5-fluorouracil-based adjuvant chemotherapy.

BACKGROUND AND OBJECTIVES: Phosphoribosylation of 5-fluorouracil (5-FU) is an essential step which leads to tumor growth inhibition and orotate phosphoribosyl transferase (OPRT) is the main enzyme that involves in this conversion of 5-FU to 5-fluorouridine monophosphate. This retrospective study was aimed to evaluate the correlation between tumor OPRT activity and the clinical outcome in colorectal cancer (CRC) patients treated by oral 5-FU-based adjuvant chemotherapy. METHODS: Surgical specimen was obtained from resectable 124 CRC patients who were subsequently treated by oral 5-FU-based adjuvant chemotherapy. OPRT activity in the extract of tumor tissue was enzymatically determined. The cut-off value of intratumor OPRT activity against disease free survival was determined by maximal chi2 method. The disease free survival and overall survival in each group were calculated using the Kaplan-Meier method. RESULTS: Patients were divided into two groups by determined cut-off value of intratumor OPRT (0.147 nmol/min/mg protein) (high group: n = 102, low group: n = 22). Five-year DFS (P = 0.035) and OS (P = 0.020) were significantly better for high OPRT group. CONCLUSIONS: This study demonstrated that an assay of tumor OPRT contributes to the determination of 5-FU-based adjuvant chemotherapy outcome and application in clinical practice should be included in tumor analysis prior to 5-FU-based adjuvant chemotherapy.

Overexpression of the orotate phosphoribosyl-transferase gene enhances the effect of 5-fluorouracil on gastric cancer cell lines.

OBJECTIVE: Orotate phosphoribosyl-transferase (OPRT) is the initial enzyme of the 5-fluorouracil (5-FU) metabolic pathway, converting 5-FU into 5-fluorouridinemonophosphate, which is the most important mechanism of 5-FU activation. We therefore investigated whether overexpression of the OPRT gene enhances sensitivity to 5-FU. METHODS: An expression vector of the OPRT gene (pTARGET-OPRT) was transfected into two gastric cancer cell lines, TMK-1 and MKN-45, with low baseline expression levels of OPRT. The sensitivity to and anti-tumor activity of 5-FU were then investigated in vitro and in vivo in these two transfected clones (TMK-OPRT and MKN-OPRT). RESULTS: Although cell growth was unaltered compared to parent cells, overexpression of the OPRT gene was confirmed by Western blotting in both the TMK-OPRT and MKN-OPRT cells. OPRT enzyme activity increased 38-fold in TMK-OPRT cells and 8.0 fold in MKN-OPRT cells compared to their parent cells. Interestingly, although the sensitivity to Adriamycin, cis-platinum, mitomycin C and paclitaxel was unaltered in the transfected clones, the sensitivity to 5-FU was increased 14.2- and 6.0-fold in TMK-OPRT and MKN-OPRT cells, respectively, compared to their parent cells. Moreover, enhanced sensitivity was also confirmed in the in vivo study. CONCLUSION: The results indicate that overexpression of the OPRT gene plays an important role in the antiproliferative effect of 5-FU and might therefore be a predictive factor of response to 5-FU in gastric cancer patients.

Effect of carbon source on pyrimidine formation in Pseudomonas fluorescens ATCC 13525.

The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in the type strain of Pseudomonas fluorescens was investigated. The de novo pyrimidine biosynthetic enzyme activities were measured in P. fluorescens ATCC 13525 cells and in cells from an auxotroph deficient for orotate phosphoribosyltransferase activity where glucose or succinate served as the carbon source. Pyrimidine supplementation to the culture medium affected the biosynthetic enzyme activities in ATCC 13525 cells. Pyrimidine Limitation of the mutant strain indicated transcriptional regulation of the biosynthetic pathway by pyrimidines that was influenced by carbon source. Transcriptional regulation of pyrimidine synthesis in P. fluorescens appeared to be more highly regulated than was observed for the taxonomically related species Pseudomonas aeruginosa or Pseudomonas putida.

Functional analysis of the pyrimidine de novo synthesis pathway in solanaceous species.

Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. In recent years, the molecular organization of nucleotide biosynthesis in plants has been analyzed. Here, we present a functional analysis of the pyrimidine de novo synthesis pathway. Each step in the pathway was investigated using transgenic plants with reduced expression of the corresponding gene to identify controlling steps and gain insights into the phenotypic and metabolic consequences. Inhibition of expression of 80% based on steady-state mRNA level did not lead to visible phenotypes. Stepwise reduction of protein abundance of Asp transcarbamoylase or dihydro orotase resulted in a corresponding inhibition of growth. This was not accompanied by pleiotropic effects or by changes in the developmental program. A more detailed metabolite analysis revealed slightly different responses in roots and shoots of plants with decreased abundance of proteins involved in pyrimidine de novo synthesis. Whereas in leaves the nucleotide and amino acid levels were changed only in the very strong inhibited plants, the roots show a transient increase of these metabolites in intermediate plants followed by a decrease in the strong inhibited plants. Growth analysis revealed that elongation rates and number of organs per plant were reduced, without large changes in the average cell size. It is concluded that reduced pyrimidine de novo synthesis is compensated for by reduction in growth rates, and the remaining nucleotide pools are sufficient for running basic metabolic processes.