Identification of three hydroxyproline O-arabinosyltransferases in Arabidopsis thaliana.

Hydroxyproline (Hyp) O-arabinosylation is a post-translational modification that is prominent in extracellular glycoproteins in plants. Hyp O-arabinosylation is generally found in these glycoproteins in the form of linear oligoarabinoside chains and has a key role in their function by contributing to conformational stability. However, Hyp O-arabinosyltransferase (HPAT), a key enzyme that catalyzes the transfer of the L-arabinose to the hydroxyl group of Hyp residues, has remained undiscovered. Here, we purified and identified Arabidopsis HPAT as a Golgi-localized transmembrane protein that is structurally similar to the glycosyltransferase GT8 family. Loss-of-function mutations in HPAT-encoding genes cause pleiotropic phenotypes that include enhanced hypocotyl elongation, defects in cell wall thickening, early flowering, early senescence and impaired pollen tube growth. Our results indicate essential roles of Hyp O-arabinosylation in both vegetative and reproductive growth in plants.

Subcellular localization of N-deoxyribosyltransferase in Lactobacillus fermentum: cell surface association of an intracellular nucleotide metabolic enzyme.

N-deoxyribosyltransferases are essential enzymes in the nucleotide salvage pathway of lactobacilli. They catalyze the exchange between the purine or pyrimidine bases of 2'-deoxyribonucleosides and free pyrimidine or purine bases. In general, N-deoxyribosyltransferases are referred to as cytoplasmic enzymes, although there is no experimental evidence for this subcellular localization. In this work, the subcellular localization of N-deoxyribosyltransferase II (NTD) from Lactobacillus fermentum was examined by subcellular fractionation, transmission electron microscopy, and fluorescence microscopy. Our results indicate that L. fermentum NTD are distributed not only in the cytoplasm but also on the cell wall surface, and further studies showed that surface-attached NTD can be released into the culture broth and conventional buffers.

Non-invasive molecular and functional imaging of cytosine deaminase and uracil phosphoribosyltransferase fused with red fluorescence protein.

INTRODUCTION: Increased expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) may improve the antitumoral effect of 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC), and thereby enhance the potential of gene-directed enzyme prodrug therapy. For the applicability of gene-directed enzyme prodrug therapy in a clinical setting, it is essential to be able to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using magnetic resonance spectroscopy and optical imaging to measure non-invasively CD and UPRT expression and function. MATERIALS AND METHODS: Expression vectors of CD or CD/UPRT fused to monomeric DsRed (mDsRed) were constructed and rat prostate carcinoma (R3327-AT) cell lines stably expressing either CD/mDsRed or CD/UPRT/mDsRed were generated. The expression of the fusion proteins was evaluated by flow cytometry, fluorescence microscopy, and Western blot analysis. The function of the fusion protein was confirmed in vitro by assessing 5-FC and 5-FU cytotoxicity. In vivo fluorine-19 magnetic resonance spectroscopy ((19)F MRS) was used to monitor the conversion of 5-FC to 5-FU in mice bearing the R3327-CD/mDsRed and R3327-CD/UPRT/mDsRed tumor xenografts. RESULTS: Sensitivity to 5-FC and 5-FU was higher in cells stably expressing the CD/UPRT/mDsRed fusion gene than in cells stably expressing CD/mDsRed alone or wild-type cells. Whole tumor (19)F MRS measurements showed rapid conversion of 5-FC to 5-FU within 20 min after 5-FC was administered intravenously in both CD/mDsRed and CD/UPRT/mDsRed tumors with subsequent anabolism to cytotoxic fluoronucleotides (FNucs). CD/UPRT/mDsRed tumor was more efficient in these processes. CONCLUSION: This study demonstrates the utility of these tumor models stably expressing CD or CD/UPRT to non-invasively evaluate the efficacy of the transgene expression/activity by monitoring drug metabolism in vivo using MRS, with potential applications in preclinical and clinical settings.

Proteomic analysis reveals metabolic changes during yeast to hypha transition in Yarrowia lipolytica.

Fungal dimorphism is important for survival in different environments and has been related to virulence. The ascomycete Yarrowia lipolytica can grow as yeast, pseudomycelial or mycelial forms. We have used a Y. lipolytica parental strain and a Deltahoy1 mutant, which is unable to form hypha, to set up a model for dimorphism and to characterize in more depth the yeast to hypha transition by proteomic techniques. A two-dimensional gel electrophoresis (2-DE) based differential expression analysis of Y. lipolytica yeast and hyphal cells was performed, and 45 differentially expressed proteins were detected; nine with decreased expression in hyphal cells were identified. They corresponded to the S. cerevisiae homologues of Imd4p, Pdx3p, Cdc19, Sse1p, Sol3p, Sod2p, Xpt1p, Mdh1p and to the unknown protein YALIOB00924g. Remarkably, most of these proteins are involved in metabolic pathways, with four showing oxidoreductase activity. Furthermore, taking into account that this is the first report of 2-DE analysis of Y. lipolytica protein extracts, 35 more proteins from the 2D map of soluble yeast proteins, which were involved in metabolism, cell rescue, energy and protein synthesis, were identified.

Pyrimidine nucleoside phosphorylase and dihydropyrimidine dihydrogenase activities as predictive factors for the efficacy of doxifluridine together with mitomycin C as adjuvant chemotherapy in primary colorectal cancer.

BACKGROUND/AIMS: Pyrimidine Nucleoside Phosphorylase (PyNPase) converts 5'-deoxy-5-fluorouridine (5'-DFUR, doxifluridine) to 5-fluorouracil (5-FU). While this reaction is taking place Dihydropyrimidine Dihydrogenase (DPD) catalyzes 5-FU to inactive molecules. Mitomycin C (MMC) elevates the PyNPase level in tumor cells. METHODOLOGY: We investigated 17 colorectal cancer patients' PyNPase and DPD activities in tumor and normal tissues using an enzyme-linked immunosorbent assay (ELISA) to assess their clinical significance as indicators for selecting colorectal cancer patients for 5'-DFUR together with MMC as adjuvant chemotherapy. RESULTS: Six of 17 patients developed experienced a recurrence. Tumor DPD activity of the 6 patients who had a recurrence were higher than those of the 11 patients with no recurrence (p = 0.047). On the other hand, there were no significant differences in both the PyNPase and the PyNPase/DPD (P/D) ratio between the group with recurrence and the group without recurrence. For survival analyses, we designed the cut-off value of tumor PyNPase, DPD and P/D ratio as their median value and classified patients into a higher group and a lower group, but there were no significant differences between the groups. CONCLUSIONS: The DPD activity in the tumor may be a useful indicator for selecting patients likely respond to 5'-DFUR together with MMC as adjuvant chemotherapy. If tumor DPD is high, we had better select a different anticancer drug.

Measurement of fibrosis marker xylosyltransferase I activity by HPLC electrospray ionization tandem mass spectrometry.

BACKGROUND: Xylosyltransferase I (XT-I), the key enzyme in the biosynthesis of glycosaminoglycan chains in proteoglycans, has increased activity in the blood serum of patients with connective tissue diseases. Therefore, the measurement of serum XT-I activity is useful to monitor disease activity in these patients. METHODS: We developed an HPLC electrospray ionization tandem mass spectrometry method to assay XT-I activity in serum by use of a synthetic peptide (Bio-BIK-F) as the XT-I substrate. On the basis of XT-I-mediated transfer of D-xylose from UDP-D-xylose to the synthetic peptide to form Bio-BIK-F-Xyl, we determined XT-I activity in human serum samples. RESULTS: Multiple calibration curves for the analysis of Bio-BIK-F-Xyl exhibited consistent linearity and reproducibility in the range of 0.20-20 mg/L, corresponding to XT-I activity of 1.14-114 mU/L under assay conditions. The mean (SD, range) XT-I activity values in 30 blood donor sera were 18.4 (3.0, 8.7-24.8) mU/L. The limit of detection and lower limit of quantification were 8.5 microg/L (0.05 mU/L) and 163 microg/L Bio-BIK-F-Xyl (0.93 mU/L XT-I activity), respectively. Interassay imprecision (CV) was 5.4%-26.1% in the range of 0.64 to 129 mU/L, and mean recovery was 107% (range, 96%-129%). Method comparison with the radiochemical assay showed a moderate correlation (r = 0.79). The Passing-Bablok regression line was: radiochemical assay = 0.045 LC-MS/MS + 0.061 mU/L, S(y/x) = 0.186. CONCLUSIONS: This simple and robust LC-MS/MS assay permits the rapid and accurate determination of XT-I activity in human serum.

[A change of tumor PyNPase level at intraperitoneal and intravenous administrations of paclitaxel].

A level of PyNPase activity was measured after intraperitoneal (ip) and intravenous (i.v.) administrations of paclitaxel on the animal model. Nude mice received the subcutaneous implantation of WiDr cells. About 3 weeks later, the ip and i.v. administrations of paclitaxel were performed 2 times at 20 mg/kg and 15 mg/kg, respectively. About 1 week later, the mice were sacrificed. The level of PyNPase activity was measured by the ELISA method. The level of PyNPase of ip and i.v. was higher than that of the control group, but the level of PyNPase revealed no significant difference between ip and i.v.. This result suggested that intraperitoneal administration of paclitaxel enhanced an efficiency of 5'-DFUR and capecitabine as much as intravenous administration of paclitaxel.

[Clinicopathological significance of pyrimidine nucleoside phosphorylase (PyNPase) and dihydropyrimidine dehydrogenase (DPD) in advanced colorectal cancer].

We examined clinicopathological characteristics and prognoses of seventy advanced colorectal cancer cases by measuring pyrimidine nucleoside phosphorylase (PyNPase) and dihydropyrimidine dehydrogenase (DPD) in tumor and normal tissue. PyNPase activities in cancerous tissue obtained from resected were 82.7 +/- 41.9 U/mg protein, which were significantly higher than 37.2 +/- 24.0 U/mg protein in normal tissue (p < 0.001). On the other hand, DPD activities in cancerous tissue were significantly lower in normal tissue (p < 0.05). In cases with lymphnode metastases, PyNPase activities of cancerous tissue were significantly higher than that of no lymphnode metastases cases (p < 0.05). In cases with grade 2 side-effects or higher by oral adjuvant chemotherapy, DPD activities in normal tissue were significantly lower than that of other cases (p < 0.05). With regard to Dukes' B and C cases that were resected curatively, PyNPase activities of cancerous tissue of higher group's prognosis were worse than that of the lower group. In the group received 5'-DFUR as adjuvant chemotherapy, non-recurrent survival rate of the group exhibiting higher PyNPase activities was better than that of the lower group.

A genetic and structural analysis of the N-glycosylation capabilities.

The recent draft sequencing of the rice (Oryza sativa) genome has enabled a genetic analysis of the glycosylation capabilities of an agroeconomically important group of plants, the monocotyledons. In this study, we have not only identified genes putatively encoding enzymes involved in N-glycosylation, but have examined by MALDI-TOF MS the structures of the N-glycans of rice and other monocotyledons (maize, wheat and dates; Zea mays, Triticum aestivum and Phoenix dactylifera); these data show that within the plant kingdom the types of N-glycans found are very similar between monocotyledons, dicotyledons and gymnosperms. Subsequently, we constructed expression vectors for the key enzymes forming plant-typical structures in rice, N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101), core alpha1,3-fucosyltransferase (FucTA; EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) and successfully expressed them in Pichia pastoris. Rice GlcNAc-TI, FucTA and xylosyltransferase are therefore the first monocotyledon glycosyltransferases involved in N-glycan biosynthesis to be characterised in a recombinant form.

Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose.

The plant glycosyltransferases, beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT), are responsible for the transfer of beta1,2-linked xylose and core alpha1,3-linked fucose residues to glycoprotein N-glycans. These glycan epitopes are not present in humans and thus may cause immunological responses, which represent a limitation for the therapeutic use of recombinant mammalian glycoproteins produced in transgenic plants. Here we report the genetic modification of the N-glycosylation pathway in Arabidopsis thaliana plants. Knockout plants were generated with complete deficiency of XylT and FucT. These plants lack antigenic protein-bound N-glycans and instead synthesise predominantly structures with two terminal betaN-acetylglucosamine residues (GlcNAc(2)Man(3)GlcNAc(2)).

High level concentration of pyrimidine nucleoside phosphorylase in esophageal squamous cell carcinoma but no correlation with clinicopathological parameters.

Pyrimidine nucleoside phosphorylase (PyNPase) converts 5'-deoxy-5-fluorouridine to 5'-fluorouracil, which exerts an anticancer effect before being catabolized by dihydropyrimidine dehydrogenase (DPD). Recently, PyNPase has been shown to be identical to a potent angiogenic factor, platelet-derived endothelial cell growth factor. We analyzed the concentration of PyNPase and DPD in 33 patients with esophageal squamous cell carcinoma in fresh-frozen samples by enzyme-linked immunosorbent assay. In addition, we evaluated the clinical significance and prognostic value of PyNPase expression in esophageal carcinoma. The PyNPase concentration of tumor tissue was statistically higher than that of normal tissue of the esophagus (248 +/- 146 U/mg protein vs 73 +/- 63 U/mg protein, P = 0.0001), whereas DPD showed no difference (90 +/- 62 U/mg protein vs 88 +/- 62 U/mg protein, P = 0.825). The ratio of PyNPase to DPD of tumor tissue was statistically higher than that of normal tissue of the esophagus (3.3 vs 0.95, P = 0.0001). There were no significant differences between the group with high tumor to normal tissue ratios of PyNPase concentration and the low-ratio group in terms of the tumor length, depth, lymph node metastasis, lymph vessel invasion, vascular invasion, stage and survival. In conclusion, 5'-deoxy-5-fluorouridine may be effective on esophageal carcinoma and PyNPase concentration in esophageal carcinoma may not be a useful prognostic marker for patients with esophageal squamous cell carcinoma.

[Effect of preoperative oral 5'-DFUR on PyNPase level in gastrointestinal malignant tumor tissues].

BACKGROUND & OBJECTIVE: Pyrimidine nucleoside phosphorylase (PyNPase) exists mainly in tumor tissues.5'-deoxy-5-fluorouridine(5'-DFUR) can decrease its level in tumor tissues. However, the effect of preoperative oral 5'-DFUR on PyNPase level in the different time after administration has not been reported. This study was designed to investigate the suitable duration of preoperative chemotherapy through observing the changes of PyNPase levels in gastrointestinal malignant tumors after preoperative oral administration of 5'- DFUR in different duration. METHODS: Seventy-three patients with gastrointestinal malignant tumors were divided into four groups by the duration of preoperative oral 5'-DFUR (600-1,200 mg x d(-1)): group A, three days, 27 cases; group B, one week, 22 cases; group C, two weeks, 15 cases; group D, two months, 9 cases. Meanwhile, group E, control group, had 24 inpatients with gastrointestinal malignant tumors at the same term. All the above-mentioned patients did not receive the other chemotherapy or radiotherapy. The changes of PyNPase levels in tumor tissues of different groups were tested using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), etc. RESULTS: (1)Under electron microscope, there were many irrecoverable, lethal changes in tumor cells of group C. The outlines of the tumor cells were normal under light microscope, and more fibroconnective tissues were seen only in the stroma of group D. (2)The expressing levels of PyNPase mRNA and protein production in tumor tissues reduced obviously in group A (0.79+/-0.08, 19.26+/-1.65), and decreased most obviously in group C (0.43+/-0.07,5.91+/-1.45) comparing with group E (0.95+/-0.09, 29.34+/-1.82). However, there was no significant difference between group C and group D (0.42+/-0.04, 5.36+/-1.19) for the levels of PyNPase mRNA and protein production. The correlation coefficient between the levels of PyNPase mRNA and protein in tumor tissues of different group was r=0.92(P< 0.0001). CONCLUSION: 5'-DFUR by oral administration before operation might destroy gastrointestinal malignant tumor cells. As the duration prolonged, the content of PyNPase in tumor tissues decreased progressively, being lowest level in two weeks after chemotherapy. So two-week duration might be suitable for the treatment.

Xylosyltransferase activity in seminal plasma of infertile men.

BACKGROUND: Proteoglycans play an important role during the mammalian conception process and are functionally involved in the preservation of the sperm motility and velocity. Human xylosyltransferase (EC 2.4.2.26, XT) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan chains in proteoglycans. XT activity in body fluids was shown to be an indicator for the actual proteoglycan biosynthesis rate. METHODS: Xylosyltransferase activity was determined in seminal plasma and serum samples of 50 healthy semen donors, 20 infertile men with oligo-, astheno- or teratozoospermia and men after vasectomy. For identification of the XT secreting glands split ejaculates were analyzed. RESULTS: XT activity in seminal plasma samples of infertile men was significantly reduced (mean 1.53 mU/l, 90% range 0.95-2.52 mU/l, p<0.05) in comparison to healthy donors (mean 3.21 mU/l, 90% range 1.82-4.65 mU/l). The analysis of the fractionated ejaculates revealed that the highest XT activity was found in portions secreted by the seminal vesicle glands. CONCLUSIONS: In view of the increasing interest for in vitro fertilization, monitoring the seminal plasma XT activity of men with unexplained infertility is proposed to be an advantageous additional biochemical parameter that could be useful for improving the methods producing pregnancy in some couples.

Pre-B-cell colony-enhancing factor, whose expression is up-regulated in activated lymphocytes, is a nicotinamide phosphoribosyltransferase, a cytosolic enzyme involved in NAD biosynthesis.

The murine homologue of the previously identified human "pre-B-cell colony-enhancing factor" (PBEF) gene coding for a putative cytokine has been identified by screening a subtractive library enriched in genes expressed in activated T lymphocytes. Unlike most cytokine genes known to date, the PBEF gene is ubiquitously expressed in lymphoid and non-lymphoid tissues and displays significant homology with genes from primitive metazoans (marine sponges) and prokaryotic organisms. Recently, a bacterial protein encoded by nadV, a gene from the prokaryote Haemophilus ducreyi displaying significant homology with PBEF, has been identified as a nicotinamide phosphoribosyltranferase (NAmPRTase), an enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis. Using a panel of antibodies to murine PBEF, we demonstrate in this work that, similarly to its microbial counterpart, the murine protein is a NAmPRTase, catalyzing the condensation of nicotinamide with 5-phosphoribosyl-1-pyrophosphate to yield nicotinamide mononucleotide, an intermediate in the biosynthesis of NAD. The role of PBEF as a NAmPRTase was further confirmed by showing that the mouse gene was able to confer the ability to grow in the absence of NAD to a NAmPRTase-defective bacterial strain. The present findings are in keeping with the ubiquitous nature of this protein, and indicate that NAD biosynthesis may play an important role in lymphocyte activation.

Pyrimidine nucleoside phosphorylase activity in normal tissues of the uterus and ovary and in benign and malignant lesions of these organs.

BACKGROUND: Pyrimidine nucleoside phosphorylase (PyNPase) is identical to the protein, platelet-derived endothelial cell growth factor (PD-ECGF), which has angiogenic activity. The physiological roles of PyNPase activity in the uterus and ovary are not known. In this study, we measured PyNPase activity in normal tissues of the uterus, ovary, and lymph nodes, and in benign and malignant lesions of these organs, and we considered the clinical implications of PyNPase activity in the uterus and ovary. METHODS: Tissue samples were obtained from 163 patients (whose diseases are listed below) during surgery. PyNPase activity was measured spectrophotometrically, by monitoring the formation of 5-fluorouridine. RESULTS: Mean PyNPase activity in tissues from the lesions of patients with cervical cancer (n = 20), uterine endometrial cancer (n = 26), leiomyoma (n = 23), ovarian cancer (n = 46), ovarian endometriosis (n = 21), and benign epithelial ovarian tumor (n = 27) was significantly greater than that in the corresponding normal tissues. The PyNPase activity in the normal endometrium was significantly higher in the secretory phase than in the proliferative phase. The activity in normal or metastatic lymph nodes was significantly greater than that in normal tissues of the uterus and ovary. Mean PyNPase activity in cancerous cervical tissues was significantly greater than that in cancerous endometrial tissues or cancerous ovarian tissues. There were no significant differences in PyNPase activity in cervical cancer, endometrial cancer, and ovarian cancer tissues according to tumor stage. The enzyme activity appeared to be greater in histopathological G3 grade endometrial cancer than in G1 and G2 endometrial cancer. The enzyme activity in mucinous adenocarcinoma of the ovary was significantly lower than that in serous, endometrioid, and clear cell adenocarcinomas. All patients with cervical squamous cell carcinomas with PyNPase activity greater than 500 nmol/min per mg protein exhibited lymph node metastasis. CONCLUSION: Increased PyNPase activity consistently reflected neoplastic growth, and varying levels of activity were seen in different histologic cell types. This enzyme activity may be involved in cervical squamous cell carcinomas, in normal and metastatic lymph nodes, and in the normal, secretory phase in the endometrium.

[Significance of pyrimidine nucleoside phosphorylase (PyNPase) and dihydropyrimidine dehydrogenase (DPD) in advanced gastric cancers].

We measured pyrimidine nucleoside phosphorylase (PyNPase), a known angiogenetic factor, and dihydropyrimidine dehydrogenase (DPD) in advanced gastric cancers. PyNPase was expressed in cytoplasm of the cancer cells and surrounding interstitial cells. The levels of PyNPase and DPD were significantly higher in cancer tissue. With respect to tumor factors, the level of PyNPase was significantly higher in cases positive for venous invasion. We divided patients into two groups, with high and low activities of IAP and MMP-9. The level of PyNPase was significantly higher in the high IAP activity group. A correlation was suggested between the level of PyNPase and the activity of IAP. 5'-Deoxy-5-fluorouridine (5'-DFUR) is transformed into 5-FU by PyNPase and manifests antitumor effects. DPD is a rate-limiting enzyme in the process of degradation of 5-FU. In the present study, the level of PyNPase/DPD was significantly higher in cancer tissue. PyNPase/DPD suggests not only the malignant potential of the tumor but also the efficiency of chemotherapy using 5-FU, especially 5'-DFUR.

Intratumoral pyrimidine nucleoside phosphorylase (PyNPase) activity predicts a selective effect of adjuvant 5'-deoxy-5-fluorouridine (5'DFUR) on breast cancer.

BACKGROUND: Pyrimidine nucleoside phosphorylase (PyNPase) is the enzyme that converts 5'-deoxy-5-fluorouracil (5'DFUR) to 5-fluorouracil (5FU). Its activity in cancer tissue may correlate with the selective antitumor activity of 5'DFUR in breast cancer. METHODS: Two hundred and sixteen T2 breast cancer patients were treated consecutively with surgery followed by 5'DFUR (600 mg/body/day) + tamoxifen (20 mg/body/day) for 2 years. PyNPase activity in breast cancer tissue, determined by high-performance liquid chromatography, ranged from 4.2-626.0 micrograms FU/mg protein/hr (mean +/- SD, 203.5 +/- 122.4), and the examined patients were divided into two groups: group A (high PyNPase group), cases with the PyNPase activity equal to or more than the mean value of 203.5 micrograms FU/mg protein/hr, and group B (low PyNPase group), cases with activity less than the mean value. RESULTS: Although there was no difference in relapse-free survival (RFS) between groups A and B, among node-positive patients (n = 83) those in group A tended to have a longer RFS. When divided into subgroups according to estrogen receptor (ER) status, among node-positive and ER-positive tumors (n = 49), the RFS was significantly better in group A than in group B (p < 0.05). CONCLUSION: Intratumoral PyNPase activity might be of use as a predictor of the effect of adjuvant 5'DFUR on breast cancer.

Identification of two genetic groups in Bacteroides fragilis by multilocus enzyme electrophoresis: distribution of antibiotic resistance (cfiA, cepA) and enterotoxin (bft) encoding genes.

Ninety-three Bacteroides fragilis strains of different origin were analysed by multilocus enzyme electrophoresis (MLEE). Fourteen of the 15 genetic loci analysed were polymorphic, whilst nucleoside phosphorylase was monomorphic. There was a mean of six alleles per locus and a mean genetic diversity of 0.393. Cluster analysis identified 90 electrophoretic types (ETs) separated into two major phylogenetic divisions at a genetic distance of 0.70. Division I consisted of 81 ETs carrying the endogenous class A beta-lactamase gene cepA, whereas division II comprised 9 ETs carrying the class B beta-lactamase gene cfiA, but not cepA. The presence of these two genes was assessed by PCR and the expression of the cfiA gene was investigated by determining the level of resistance to the antibiotic imipenem. MLEE showed a smaller genetic distance among the genotypes of the imipenem-resistant than among the imipenem-susceptible strains. No other particular cluster was observed. The enterotoxin gene (bft) was detected by PCR: DNA sequencing of the products obtained showed that the different bft alleles (bft-1, bft-2 and bft-3) were scattered randomly troughout the phylogenetic tree. No association between distinct clones and clinical manifestations (sepsis, abscesses, diarrhoea), geographical origin or host origin (human or animal) could be found.

Reversed-phase high-performance liquid chromatography of nicotinic acid mononucleotide for measurement of quinolinate phosphoribosyltransferase.

A system has been developed for the determination of quinolinate phosphoribosyltransferase (QPRT) activity in liver and kidney homogenates using HPLC. A product, nicotinic acid mononucleotide (NaMN), is separated by reversed-phase chromatography (a Tosoh ODS 80TS was used as an analytical column) using a mixture of 10 mM KH2PO4-K2HPO4 buffer (pH 7.0) containing 1.48 g/l tetra-n-butylammonium bromide-acetonitrile (9:1, v/v) as a mobile phase. The flow-rate was 1.0 ml/min, the detection wavelength was 265 nm. The column temperature was maintained at 40 degrees C. Under these conditions, NaMN was eluted at about 8.1 min. Sample preparation was very straightforward. The reaction mixture of QPRT assay was stopped by immersing the tube into a boiling water bath, the resulting supernatant was filtered, and the filtrate was directly injected into a HPLC system. The total HPLC analysis time was approximately 20 min.