UPTAKE OF THE NUCLEOSIDE URIDINE BY THE LIVER FLUKE, FASCIOLA-HEPATICA

1. Uptake of the nucleoside uridine by adult Fasciola hepatica during a 2 min period is a linear function of concentration over the range 0.01-2.5 mM.

Preparation of Potential Cell-Permeant Nucleoside-2',3'-Cyclic Phosphate Precursors

Uridine-3'-phosphorothiolate triesters bearing lipophilic moieties were prepared via Michaelis-Arbuzov chemistry. Subsequent deprotection of the S-cholesteryl phosphorothiolate triester afforded the corresponding diester which underwent spontaneous Cyclization to cleanly afford uridine 2',3'-cyclic phosphate. This transesterification reaction could be expedited by treatment with iodine under mild, neutral conditions.

Analysis of UDP-galactose 4'-epimerase mutations associated with the intermediate form of type III galactosaemia

Type III galactosaemia is a hereditary disease caused by reduced activity in the Leloir pathway enzyme, UDP-galactose 4'-epimerase (GALE). Traditionally, the condition has been divided into two forms-a mild, or peripheral, form and a severe, or generalized, form. Recently it has become apparent that there are disease states which are intermediate between these two extremes. Three mutations associated with this intermediate form (S81R, T150M and P293L) were analysed for their kinetic and structural properties in vitro and their effects on galactose-sensitivity of Saccharomyces cerevisiae cells that were deleted for the yeast GALE homologue Gal10p. All three mutations result in impairment of the kinetic parameters (principally the turnover number, k(cat)) compared with the wild-type enzyme. However, the degree of impairment was mild compared with that seen with the mutation (V94M) associated with the generalized form of epimerase deficiency galactosaemia. None of the three mutations tested affected the ability of the protein to dimerize in solution or its susceptibility to limited proteolysis in vitro. Finally, in the yeast model, each of the mutated patient alleles was able to complement the galactose-sensitivity of gal10 Delta cells as fully as was the wild-type human allele. Furthermore, there was no difference from control in metabolite profile following galactose exposure for any of these strains. Thus we conclude that the subtle biochemical and metabolic abnormalities detected in patients expressing these GALE alleles likely reflect, at least in part, the reduced enzymatic activity of the encoded GALE proteins.

Low cytosine triphosphate synthase 2 expression renders resistance to 5-fluorouracil in colorectal cancer

Understanding the determinants of resistance of 5-fluorouracil (5FU) is of significant value to optimizing administration of the drug, and introducing novel agents and treatment strategies. Here, the expression of 92 genes involved in 5FU transport, metabolism, co-factor (folate) metabolism and downstream effects was measured by real-time PCR low density arrays in 14 patient-derived colorectal cancer xenografts characterized for 5FU resistance. Candidate gene function was tested by siRNA and uridine modulation, and immunoblotting, apoptosis and cell cycle analysis. Predictive significance was tested by immunohistochemistry of tumors from 125 stage III colorectal cancer patients treated with and without 5FU. Of 8 genes significantly differentially expressed between 5FU sensitive and resistant xenograft tumors, CTPS2 was the gene with the highest probability of differential expression (p = 0.008). Reduction of CTPS2 expression by siRNA increased the resistance of colorectal cancer cell lines DLD1 and LS174T to 5FU and its analog, FUDR. CTPS2 siRNA significantly reduced cell S-phase accumulation and apoptosis following 5FU treatment. Exposure of cells to uridine, a precursor to the CTPS2 substrate uridine triphosphate, also increased 5FU resistance. Patients with low CTPS2 did not gain a survival benefit from 5FU treatment (p = 0.072), while those with high expression did (p = 0.003). Low CTPS2 expression may be a rationally-based determinant of 5FU resistance.

THE EFFECT OF DIAMPHENETHIDE ON PROTEIN-SYNTHESIS BY THE LIVER FLUKE, FASCIOLA-HEPATICA

The effect of the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 mug ml-1) on the uptake and incorporation by adult Fasciola hepatica of radioactively labelled precursors of DNA, RNA and protein synthesis ([H-3]thymidine, [H-3]uridine and [H-3]leucine, respectively) was measured by liquid scintillation counting. Comparison was made between the effects of DAMD and those of specific inhibitors of DNA, RNA and protein synthesis, namely, 5-fluorouracil, cordycepin and cycloheximide, respectively. DAMD caused a significant decrease in the overall uptake and incorporation of [H-3]uridine by F. hepatica, decreased the incorporation of [H-3]leucine and also caused a significant decrease in the overall protein content of the flukes. The effect of DAMD was similar to that of cycloheximide (I x 10(-3) M), a potent inhibitor of protein synthesis, which also caused a significant decrease in the incorporation of [H-3]leucine by the fluke and a decrease in the overall protein content of the fluke. Cordycepin(100 mug ml-1) caused a significant decrease in the protein content of the fluke, but had no effect on the uptake or incorporation of [H-3]uridine. 5-Fluorouracil (I x 10(-4) m) did not affect the uptake or incorporation of VH]thymidine, nor did it decrease the protein content of the fluke. The results indicate that DAMD inhibits protein synthesis by F. hepatica, possibly by inhibiting RNA synthesis. The results are also consistent with previous morphological investigations involving DAMD.

The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate

Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaP(CT)) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaP(CT) allowing us to obtain highly purified protein. We demonstrate that WbaP(CT) is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaP(CT) domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.

Phosphoramidates of 2'-beta-D-arabinouridine (AraU) as phosphate prodrugs; design, synthesis, in vitro activity and metabolism

2'-Beta-D-arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.

Substrates modified by advanced glycation end-products cause dysfunction and death in retinal pericytes by reducing survival signals mediated by platelet-derived growth factor

AIMS/HYPOTHESIS: Premature death of retinal pericytes is a pathophysiological hallmark of diabetic retinopathy. Among the mechanisms proposed for pericyte death is exposure to AGE, which accumulate during diabetes. The current study used an in vitro model, whereby retinal pericytes were exposed to AGE-modified substrate and the mechanisms underlying pericyte death explored. METHODS: Pericytes were isolated from bovine retinal capillaries and propagated on AGE-modified basement membrane (BM) extract or non-modified native BM. The extent of AGE modification was analysed. Proliferative responses of retinal pericytes propagated on AGE-modified BM were investigated using a 5-bromo-2-deoxy-uridine-based assay. The effect of extrinsically added platelet-derived growth factor (PDGF) isoforms on these proliferative responses was also analysed alongside mRNA expression of the PDGF receptors. Apoptotic death of retinal pericytes grown on AGE-modified BM was investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling labelling, mitochondrial membrane depolarisation and by morphological assessment. We also measured both the ability of PDGF to reverse Akt dephosphorylation that was mediated by AGE-modified BM, and increased pericyte apoptosis. RESULTS: Retinal pericytes exposed to AGE-modified BM showed reduced proliferative responses in comparison to controls (p

UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors

Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 μM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.