Mode of action of 5-fluorocytosine and 5-fluorouracil in dematiaceous fungi

The mode of action of 5-fluorocytosine (5FC) and 5-fluorouracil (5FU) in dematiaceous fungi was studied and compared with results of experiments in yeasts and Aspergillus species. In dematiaceous fungi 5FU is more potent than 5FC. The high activity of 5FU is related to a good and rapid uptake of this compound into the fungus cell. Both compounds exert fungistatic and fungicidal activity. A correlation exists between the amount of 5FU incorporated into RNA and its antifungal activity. The resistance frequency to 5FC varies from 2 x 10(-3) to 1 x 10(-7); resistance frequency to 5FU is generally lower. Addition of 5FC and 5FU to logarithmically multiplying cells inhibits increases of cell numbers and cell constituents after a delay period. The effects on the increase of protein and carbohydrate are more delayed than on the increase of DNA and RNA, indicating unbalanced growth. The concept of a dual biochemical mechanism, i.e. incorporation of 5FU into RNA and formation of 5-fluorodeoxy UMP leading to inhibition of DNA synthesis, previously proposed for the antifungal action of 5FC is also applicable to the action of 5FC and 5FU on the dematiaceous fungi.     

Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster

We report here the molecular cloning and characterization of the Drosophila neutral ceramidase (CDase). Using the BLAST program, a neutral CDase homologue (AE003774) was found in the Drosophila GenBank and cloned from a cDNA library of Drosophila imaginal discs. The open reading frame of 2,112 nucleotides encoded a polypeptide of 704 amino acids having five putative N-glycosylation sites and a putative signal sequence composed of 23 residues. When a His-tagged CDase was overexpressed in D. melanogaster Schneider's line 2 (S2) cells, the enzyme was continuously secreted into the medium through a vesicular transport system. Treatment of the secretory 86.3-kDa CDase with glycopeptidase F resulted in the generation of a 79.3-kDa protein, indicating that the enzyme is actually glycosylated with N-glycans. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, GM1a or sphingomyelin, and exhibited a peak of activity at pH 6.5-7.5, and thus was classified as a neutral CDase. RNAi for the enzyme remarkably decreased the CDase activity in a cell lysate as well as a culture supernatant of S2 cells mostly at neutral pH, indicating that both activities were derived from the same gene product.     

Yeast cytochrome c peroxidase: mutagenesis and expression in Escherichia coli show tryptophan-51 is not the radical site in compound I

Using oligonucleotide-directed site-specific mutagenesis, we have constructed a system for the mutation and expression of yeast cytochrome c peroxidase (CCP, EC 1.11.1.5) in Escherichia coli and applied it to test the hypothesis that Trp-51 is the locus of the free radical observed in compound I of CCP [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205]. The system was created by substituting a CCP gene modified by site-directed mutagenesis, CCP(MI), for the fol gene in a vector previously used for mutagenesis and overexpression of dihydrofolate reductase. E. coli transformed with the resulting plasmid produced the CCP(MI) enzyme in large quantities, more than 15 mg/L of cell culture, of which 10% is holo- and 90% is apo-CCP(MI). The apoenzyme was easily converted to holoenzyme by the addition of bovine hemin. Purified CCP(MI) has the same catalytic activity and spectra as bakers' yeast CCP. A mutation has been made in CCP(MI), Trp-51 to Phe. The Phe-51 mutant protein CCP(MI,F51) is fully active, and the electron paramagnetic resonance (EPR) spectrum, at 89 K, of its oxidized intermediate, compound I, displays a strong sharp resonance at g = 2.004, which is very similar to the signal observed for compound I of both bakers' yeast CCP and CCP(MI). However, UV-visible and EPR spectroscopy revealed that the half-life of CCP(MI,F51) compound I at 23 degrees C is only 1.4% of that observed for the compound I forms of CCP(MI) or bakers' yeast CCP. Thus, Trp-51 is not necessary for the formation of the free radical observed in compound I but appears to exert a significant influence on its stability.     

Mutants of yeast specifically resistant to petite induction by fluorinated pyrimidines

Induction of the cytoplasmic petite mutation in yeast by 5-fluorouracil (5FU) and 5-fluorocytosine (5FC) is known to depend on the incorporation of 5FU into some species of RNA; 5FC is active only following deamination to 5FU. Several mutants have now been isolated which are resistant to petite mutagenesis by 5FU but remain sensitive to growth inhibition by this analogue. They fall into two classes: those in class I are also resistant to mutagenesis by 5FC, while class II mutants retain partial sensitivity to the latter agent. The growth of both classes is sensitive to 5FC. The behavior of class II mutants requires that exogenous 5FU is specifically excluded from the site of synthesis of the target RNA involved in petite mutagenesis, while 5FC has access to it. The most likely explanation is that the RNA concerned is synthesized in the mitochondria, and that the mitochondrial membranes of class II mutants are impermeable to 5FU but not 5FC. This is supported by the finding that the membrane-active agent dimethylsulfoxide restored 5FU sensitivity to this class of mutants. No such effect was observed with class I mutants, and these are thought to have altered mitochondrial RNA-synthesizing systems which are unable to recognize fluorinated nucleotides.During the course of this work, S. G. O. was supported by a Medical Research Council Scholarship.     

19F nuclear magnetic resonance analysis of 5-fluorouracil metabolism in wild-type and 5-fluorouracil-resistant Nectria haematococca

A mutant (furA3) was isolated from the S1 wild-type strain of Nectria haematococca on the basis of its resistance to 5-fluorouracil (5FU). This mutant has greatly reduced activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme catalyzing the synthesis of UMP from uracil. The metabolism of 5FU was examined in both strains by using 19F nuclear magnetic resonance spectroscopy. In the S1 strain, 5FU appears to be metabolized by two pathways operating simultaneously: (i) conversion to fluoronucleotides and (ii) degradation into alpha-fluoro-beta-alanine. The furA3 mutant shows metabolic changes consistent with a uracil phosphoribosyltransferase lesion, since it takes up 5FU and forms a small amount of alpha-fluoro-beta-alanine but does not synthesize fluoronucleotides. Since pigment synthesis is strongly enhanced by 5FU in the S1 wild-type strain but not in the furA3 mutant, these results support the hypothesis that 5FU stimulation of secondary metabolism in N. haematococca is not mediated by the drug itself but involves a phosphorylated anabolite.     

Expression of cyclodextrinase gene from Paenibacillus sp. A11 in Escherichia coli and characterization of the purified cyclodextrinase

The expression of the Paenibacillus sp. A11 cyclodextrinase (CDase) gene using the pUC 18 vector in Escherichia coli JM 109 resulted in the formation of an insoluble CDase protein in the cell debris in addition to a soluble CDase protein in the cytoplasm. Unlike the expression in Paenibacillus sp. A11, CDase was primarily observed in cytoplasm. However, by adding 0.5 M sorbitol as an osmolyte, the formation of insoluble CDase was prevented while a three-fold increase in cytoplasmic CDase activity was achieved after a 24 h-induction. The recombinant CDase protein was purified to approximately 14-fold with a 31% recovery to a specific activity of 141 units/mg protein by 40-60% ammonium sulfate precipitation, DEAE-Toyopearl 650 M, and Phenyl Sepharose CL-4B chromatography. It was homogeneous by non-denaturing and SDS-PAGE. The enzyme was a single polypeptide with a molecular weight of 80 kDa, as determined by gel filtration and SDS-PAGE. It showed the highest activity at pH 7.0 and 40 degrees C. The catalytic efficiency (k(cat)/K(m)) values for alpha-, beta-, and gamma- CD were 3.0 x 10(5), 8.8 x 10(5), and 5.5 x 10(5) M(-1) min(-1), respectively. The enzyme hydrolyzed CDs and linear maltooligosaccharides to yield maltose and glucose with less amounts of maltotriose and maltotetraose. The rates of hydrolysis for polysaccharides, soluble starch, and pullulan were very low. The cloned CDase was strongly inactivated by N-bromosuccinimide and diethylpyrocarbonate, but activated by dithiothreitol. A comparison of the biochemical properties of the CDases from Paenibacillus sp. A11 and E. coli transformant (pJK 555) indicates that they were almost identical.     

19F nuclear magnetic resonance analysis of 5-fluorouracil metabolism in wild-type and 5-fluorouracil-resistant Nectria haematococca.

A mutant (furA3) was isolated from the S1 wild-type strain of Nectria haematococca on the basis of its resistance to 5-fluorouracil (5FU). This mutant has greatly reduced activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme catalyzing the synthesis of UMP from uracil. The metabolism of 5FU was examined in both strains by using 19F nuclear magnetic resonance spectroscopy. In the S1 strain, 5FU appears to be metabolized by two pathways operating simultaneously: (i) conversion to fluoronucleotides and (ii) degradation into alpha-fluoro-beta-alanine. The furA3 mutant shows metabolic changes consistent with a uracil phosphoribosyltransferase lesion, since it takes up 5FU and forms a small amount of alpha-fluoro-beta-alanine but does not synthesize fluoronucleotides. Since pigment synthesis is strongly enhanced by 5FU in the S1 wild-type strain but not in the furA3 mutant, these results support the hypothesis that 5FU stimulation of secondary metabolism in N. haematococca is not mediated by the drug itself but involves a phosphorylated anabolite.     

Barley pathogenesis-related proteins with fungal cell wall lytic activity inhibit the growth of yeasts.

Proteins from intercellular fluid extracts of chemically stressed barley (Hordeum vulgare L.) leaves were separated by native polyacrylamide gel electrophoresis at alkaline or acid pH. Polyacrylamide gels contained Saccharomyces cerevisiae (bakers' yeast) or Schizosaccharomyces pombe (fission yeast) crude cell walls for assaying yeast wall lysis. In parallel, gels were overlaid with a suspension of yeasts for assaying growth inhibition by pathogenesis-related proteins. The same assays were also performed with proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. In alkaline native polyacrylamide gels, only one band corresponding to yeast cell wall lytic activity was found to be inhibitory to bakers' yeast growth, whereas in acidic native polyacrylamide gels one band inhibited the growth of both yeasts. Under denaturing nonreducing conditions, one band of 19 kD inhibited the growth of both fungi. The 19-kD band corresponded to a basic protein after two-dimensional gel analysis. The 19-kD protein with yeast cell wall lytic activity and inhibitory to both yeasts was found to be different from previously reported barley chitosanases that were lytic to fungal spores. It could be different from other previously reported lytic antifungal activities related to pathogenesis-related proteins.     

Synthesis of F16 conjugated with 5‐fluorouracil and biophysical investigation of its interaction with bovine serum albumin by a spectroscopic and molecular modeling approach

5‐Fluorouracil (5‐FU) has been widely used as a chemotherapy agent in the treatment of many types of solid tumors. Investigation of its antimetabolites led to the development of an entire class of fluorinated pyrimidines. However, the toxicity profile associated with 5‐FU is significant and includes diarrhea, mucositis, hand–foot syndrome and myelosuppression. In aiming at reducing of the side effects of 5‐FU, we have designed and synthesized delocalized lipophilic cations (DLCs) as a vehicle for the delivery of 5‐FU. DLCs accumulate selectively in the mitochondria of cancer cells because of the high mitochondrial transmembrane potential (ΔΨ_m). Many DLCs exhibited anti‐cancer efficacy and were explored as potential anti‐cancer drugs based on their selective accumulation in the mitochondria of cancer cells. F16, the DLC we used as a vehicle, is a small molecule that selectively inhibits tumor cell growth and dissipates mitochondrial membrane potential. The binding of the conjugate F16–5‐FU to bovine serum albumin (BSA) was investigated using spectroscopic and molecular modeling approaches. Fluorescence quenching constants were determined using the Stern–Volmer equation to provide a measure of the binding affinity between F16–5‐FU and BSA. The activation energy of the interaction between F16–5‐FU and BSA was calculated and the unusually high value was discussed in terms of the special structural block indicated by the molecular modeling approach. Molecular modeling showed that F16–5‐FU binds to human serum albumin in site II, which is consistent with the results of site‐competitive replacement experiments. It is suggested that hydrophobic and polar forces played important roles in the binding reaction, in accordance with the results of thermodynamic experiments. Copyright © 2012 John Wiley & Sons, Ltd.     

Partial purification of d-glucose-6-phosphate dehydrogenase from bakers' yeast by affinity partitioning using polymer-bound triazine dyes

d-Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate:NADP⁺ 1-oxidoreductase EC 1.1.1.49) has been purified from bakers' yeast by liquid-liquid extraction using phase-restricted triazine dyes (Procion Yellow HE-3G, Procion Olive MX-3G, Procion Navy MX-RB and Cibacron Blue F3G-A). This method was combined with fractional precipitation with poly(ethylene) glycol) and batchwise treatment with DEAE-cellulose. This rapid procedure gave an enzyme preparation with a specific activity of 0.92 kat per kg protein within 5 h. The affinity extraction step can easily be scaled up and the good recovery of ligand-poly(ethylene glycol) should make the process useful for larger amounts of enzyme. The technical possibilities are discussed.     

Product release is rate-limiting in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase

Yeast cytosine deaminase (yCD), a zinc metalloenzyme, catalyzes the hydrolytic deamination of cytosine to uracil. The enzyme is of great biomedical interest because it also catalyzes the deamination of the prodrug 5-fluorocytosine (5FC) to form the anticancer drug 5-fluorouracil (5FU). yCD/5FC is one of the most widely used enzyme/prodrug combinations for gene-directed enzyme prodrug therapy for the treatment of cancers. A pH indicator assay has been developed for the measurement of the steady-state kinetic parameters for the deamination reaction. Transient kinetic studies have shown that the product release is a rate-limiting step in the activation of the prodrug 5FC by yCD. The rate constant of the chemical step for the forward reaction (250 s(-)(1)) is approximately 8 times that of the product release (31 s(-)(1)) and approximately 15 times k(cat) (17 s(-)(1)). The transient kinetic results are consistent with those of the steady-state kinetic analysis in the sense that the k(cat) and K(m) values calculated from the rate constants determined by the transient kinetic analysis are in close agreement with those measured by the steady-state kinetic analysis. NMR experiments have demonstrated that free 5FU is in slow exchange with its complex with yCD but has a low affinity for yCD. The transient kinetic and NMR results together suggest that the release of 5FU is rate-limiting in the activation of the prodrug 5FC by yCD and may involve multiple steps.     

Kinetics and inhibition of cyclomaltodextrinase from alkalophilic Bacillus sp. I-5

The cyclomaltodextrinase from alkalophilic Bacillus sp. I-5 (CDase I-5) was expressed in Escherichia coli and the purified enzyme was used for characterization of the enzyme action. The hydrolysis products were monitored by both HPLC and high-performance ion chromatography analysis that enable the kinetic analysis of the cyclomaltodextrin (CD)-degrading reaction. Analysis of the kinetics of cyclomaltodextrin hydrolysis by CDase I-5 indicated that ring-opening of the cyclomaltodextrin was the major limiting step and that CDase I-5 preferentially degraded the linear maltodextrin chain by removing the maltose unit. The substrate binding affinity of the enzyme was almost same for those of cyclomaltodextrins while the rate of ring-opening was the fastest for cyclomaltoheptaose. Acarbose and methyl 6-amino-6-deoxy-alpha-d-glucopyranoside were relatively strong competitive inhibitors with K(i) values of 1.24 x 10(-3) and 8.44 x 10(-1) mM, respectively. Both inhibitors are likely to inhibit the ring-opening step of the CD degradation reaction.     

Determination of 5-fluorouracil in plasma with HPLC-tandem mass spectrometry

In this article, we describe a fast and specific method to measure 5FU with HPLC tandem-mass spectrometry. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled 5FU (1,3-15N2-5FU) was used as an internal standard. 5FU was measured within a single analytical run of 16 min with a lower limit of detection of 0.05 microM. The intra-assay variation and inter-assay variation of plasma with added 5FU (1 microM, 10 microM, 100 microM) was less then 6%. Recoveries of the added 5FU in plasma were > 97%. Analysis of the 5FU levels in plasma samples from patients with the HPLC tandem mass spectrometry method and a HPLC-UV method yielded comparable results (r2 = 0.98). Thus, HPLC with electrospray ionization tandem mass spectrometry allows the rapid analysis of 5FU levels in plasma and could, therefore, be used for therapeutic drug monitoring.     

Cerivastatin enhances the cytotoxicity of 5-fluorouracil on chemosensitive and resistant colorectal cancer cell lines

Cerivastatin is one of the synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors used for the treatment and prevention of hypercholesterolaemia. The observation that patients receiving this drug had a lower incidence at cancer led to our interest in using it as a putative anticancer agent. In this study, we tested the cytotoxicity of cerivastatin on a panel of 5-fluorouracil (5FU) sensitive and resistant cell lines in vitro. Cerivastatin was cytotoxic to both 5FU sensitive and resistant cells. Cerivastatin significantly augmented the cytotoxic effect of 5FU on drug sensitive (6-22-fold) and resistant (229-310-fold) cell lines. Cerivastatin and 5FU acted synergistically. Cerivastatin inhibited nuclear factor kappaB DNA binding activity. The enhancing effect of cerivastatin on 5FU was partially mevalonate pathway independent. Cerivastatin may allow successful 5FU therapy in chemoresistant patients.     

The concentration of 5-phosphoribosyl 1-pyrophosphate in monolayer tumor cells and the effect of various pyrimidine antimetabolites

5-Phosphoribosyl 1-pyrophosphate (PRPP) was determined in several murine and human cancer cell lines grown in monolayer, and harvested by trypsinization. For all cell lines a large variation in the PRPP concentration (5-1300 pmol/ 10(6) cells was found. A 1-hr incubation in Dullbecco's medium reduced the variation in PRPP concentration. After this incubation the highest concentration was found in the murine B16 melanoma cell line (about 200 pmol/10(6) cells). The human melanoma cell lines IGR3 and M5 and the human colon carcinoma cell line WiDr contained about 100 pmol/10(6) cells. After this preincubation of 1-hr these cell suspensions were used to study the effect of several antimetabolites on PRPP concentration. A 2-hr incubation with 1mM N-(phosphonacetyl)-L-aspartate (PALA) increased the PRPP concentration only in M5 cells, whereas methotrexate caused an increase in all cell lines. When 5-fluorouracil (5FU) was added, no significant decrease was found in any cell line. Addition of 5FU after a 2-hr preincubation with PALA resulted in a lower concentration in B16, M5 and WiDr cells. The prodrug, 5-fluoro-5' deoxyuridine altered the PRPP concentration only in in WiDr cells when it was added after PALA. The activity of the 5FU metabolizing enzyme orotate phosphoribosyl transferase was comparable in B16, M5 and WiDr cells, but much lower in IGR3 cells.     

Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA.

To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNA(Val), resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNA(Phe) mutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNA(Val) mutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12. Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNA(Val), it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the 19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAVal indicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil. However, the signal from FU59, in the T-loop of tRNA(Val), was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7.A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. An unanticipated finding was that the 19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.     

Enhanced DNA-directed effects of FdUMP[10] compared to 5FU

FdUMP[N] molecules and conjugates are much more effective at inhibiting the proliferation of human tumor cells than is the widely used anticancer drug 5-fluorouracil (5FU). We have evaluated the inhibition of thymidylate synthase (TS), the extent of DNA damage, cell cycle arrest, and the induction of apoptosis by FdUMP[10] and 5FU in the human colorectal cancer cell line HT29. The magnitude and duration of TS inhibition following exposure of HT29 cells to FdUMP[10] at 1 x 10(-8) M was greater than that which occurred following exposure of these cells to 5FU at 1 x 10(-6) M. FdUMP[10] exposure also resulted in much more extensive DNA damage to HT29 cells than occurred following exposure to 100-fold higher concentrations of 5FU. Although exposure of HT29 cells to both drugs resulted in S-phase arrest, more complete accumulation of cells in S-phase was achieved following FdUMP[10] exposure at much lower drug concentrations. FdUMP[10] was also much more effective at inducing apoptosis in HT29 cells than was 5FU. The results are consistent with FdUMP[10] being much more efficient that 5FU at inducing DNA damage that results in apoptotic cell death in colon cancer cells.