Comparative molecular analysis of ovine and bovine Streptococcus uberis isolates.

Streptococcus uberis causes clinical and subclinical mastitis in cattle and sheep, but it is unknown whether the composition of Strep. uberis populations differs between host species. To address this, we characterized a collection of bovine and ovine Strep. uberis isolates with shared geographical and temporal origins by means of an expanded multilocus sequence typing scheme. Among 14 ovine and 35 bovine isolates, 35 allelic profiles were detected. Each allelic profile was associated with a single host species and all but one were new to the multilocus sequence typing database. The median number of new alleles per isolate was higher for ovine isolates than for bovine isolates. None of the ovine isolates belonged to the global clonal complexes 5 or 143, which are commonly associated with bovine mastitis and which have a wide geographical distribution. Ovine isolates also differed from bovine isolates in carriage of plasminogen activator genes, with significantly higher prevalence of pauB in ovine isolates. Isolates that were negative for yqiL, one of the targets of multilocus sequence typing, were found among ovine and bovine isolates and were not associated with a specific sequence type or global clonal complex. One bovine isolate carried a gapC allele that was probably acquired through lateral gene transfer, most likely from Streptococcus salivarius. We conclude that ovine isolates are distinct from bovine isolates of Strep. uberis, and that recombination between isolates from different host species or bacterial species could contribute to changes in virulence gene profiles with relevance for vaccine development.

The metabolism of 16-fluoroestradiols in vivo: chemical strategies for restricting the oxidative biotransformations of an estrogen-receptor imaging agent.

16 alpha-Fluoro-17 beta-, 16 alpha-fluoro-17 alpha-, and 16 beta-fluoro-17 beta-[6,7-3H]estradiol were prepared from [6,7-3H]estrone via fluorination of 3,17-bis(tert-butyldimethylsilyloxy)-[6,7-3H]estratetraene with N-fluoropyridinium triflate and reduction of 16 alpha/beta-fluoro[6,7-3H]estrone with NaBH4. The three isomers were separated by silica-phase high-performance liquid chromatography. They were administered intravenously (4 mumol/kg to anaesthetized male rats. Their biliary metabolites (90-97% of dose over 6 h) were characterized by high performance liquid chromatography-mass spectrometry and compared with those of [6,7-3H]17 beta-estradiol. The four estrogens and their hydroxylated and methoxylated metabolites were excreted as glucuronides. C-16 fluorination blocked C-16 hydroxylated and also the dehydrogenation of the C-17 hydroxyl group. The 16 alpha-17 beta isomer was extensively glucuronylated at C(O)3 but also underwent aromatic hydroxylation and methoxylation before conjugation. Its C-17 epimer was subject to much greater aromatic hydroxylation but the catecholestrogen was O-methylated to a greater relative extent. The 16 beta-17 beta derivative underwent alicyclic as well as substantial aromatic hydroxylation and yielded numerous isomeric glucuronides of O-methylated catechols. Thus, the fluorine exerted complex effects (inhibitory and enhancing) on both localized (D-ring) and distal (A-ring) biotransformations of the estradiol molecule; the direction and magnitude of the effects being dependent upon the stereochemistry at C-16 and C-17. These findings provide structural guidelines for restricting the metabolism of tumor-imaging fluoroestrogens and thereby enhancing their delivery to the target tissue.

Synthesis and physicochemical properties of the furan dicarboxylic acid, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, an inhibitor of plasma protein binding in uraemia.

The furan dicarboxylic acid, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (5-propyl FPA) accumulates in the plasma of patients with chronic renal failure and is a major contributor to the drug binding defect of uraemic plasma. This acid has also been implicated in several other aspects of the uraemic syndrome: anaemia, irregularities of thyroid function, neurological symptoms and inhibition of active tubular secretion. The acid is not commercially available and its synthesis, starting with Meldrum's acid and methyl succinyl chloride, is described. The pKa values were measured by titration and values of 3.2 and 3.6 respectively were assigned to the carboxylic acid groups attached directly to the ring at position 3 and at position 2 (on the side-chain). The partition coefficient (log P) between hydrochloric acid and octanol was 1.2 and the distribution coefficient (log D; octanol-phosphate buffer pH 7.4) was -0.59. The pKa values and the degree of hydrophobic character of 5-propyl FPA are consistent with those of other protein-bound acids which undergo active tubular secretion by the kidney and this substance may serve as an endogenous marker for the effects of drugs and disease on this process.

Catecholestrogens as mediators of carcinogenesis: correlation of aromatic hydroxylation of estradiol and its fluorinated analogs with tumor induction in Syrian hamsters.

17 beta-Estradiol is known to induce kidney tumors in male Syrian hamsters when administered chronically, whereas 4-fluoroestradiol does so only after an extended induction period and 2-fluoroestradiol is not carcinogenic; both fluorinated analogs are hormonally active. Because C-4 and C-2 hydroxylations are, respectively, minor and major routes of estrogen metabolism in vivo, these observations suggest mediation of tumorigenesis by catecholestrogen metabolites. However, the analogs were reported to undergo oxidative defluorination in vitro. We have determined the metabolic fates of estradiol, 2-fluoroestradiol, and 4-fluoroestradiol in male hamsters. [6,7-3H]Estradiol was principally C-2 hydroxylated when given intravenously at either 0.1 mumol/kg or 50 mumol/kg; 2-hydroxyestradiol was eliminated in bile and urine, largely as a glucuronide, without undergoing extensive deactivation via O-methylation. Alicyclic alcohol metabolites were minor products. [6,7-3H]2-Fluoroestradiol underwent either glucuronylation or sequential dehydrogenation and alicyclic hydroxylation followed by glucuronylation but neither oxidative defluorination nor compensatory C-4 hydroxylation. [6,7-3H]4-Fluoroestradiol was also considerably dehydrogenated to the keto form and glucuronylated. Nevertheless, only 4-fluoroestradiol yielded appreciable quantities of C-2 hydroxylated metabolite at the lower dose; methylation was an insignificant pathway. No defluorinated products were observed. Dehydrogenation of both analogs and alicyclic hydroxylation of the 2-fluoroestrone metabolite were less extensive at the higher dose; all of the polar metabolites of 2-fluoroestradiol in bile, although not those in urine, declined to trace amounts. C-2 hydroxylation of 4-fluoroestradiol was greater at this dose. Thus, the rank order of catechol formation from estradiol and its fluoro analogs observed in vivo, unlike that found in microsomal incubations, was consistent with the hypothesis that catechols mediate estrogen-dependent renal carcinogenesis in hamsters.

Phase I and plasma pharmacokinetic study of infusional fluorouracil combined with recombinant interferon alfa-2b in patients with advanced cancer.

PURPOSE: Enhanced fluorouracil (FUra) cytotoxicity caused by recombinant interferon alfa-2b (rIFN-a) has been reported, but the mechanism, optimal dose, and schedule remain unknown. Therefore, a phase I and pharmacokinetic study of FUra with escalating doses of rIFN-a was initiated. PATIENTS AND METHODS: FUra (750 mg/m2/d) was given by continuous intravenous (IV) infusion for 5 days. rIFN-a (0.1 to 15 x 10(6) U/m2/d) was given subcutaneously (SC) daily for 5 days concurrent with FUra. Courses were repeated every 14 to 21 days. Forty-four patients were enrolled; 39 received at least two courses. During the first course of therapy, FUra levels before and after administration of rIFN-a were quantitated in 26 patients by high-pressure liquid chromatography. RESULTS: The maximum-tolerated dose of rIFN-a was 10 x 10(6) U/m2/d. Stomatitis was dose-limiting. Three partial and five minor responses occurred. Interpatient pharmacokinetics showed that rIFN-a did not alter steady-state plasma concentration (Css; range, 0.77 +/- 0.35 mumol/L to 1.85 +/- 0.48 mumol/L), elimination half-life (t1/2; mean, 9.7 +/- 4.3 minutes), area under the concentration-versus-time curve (AUC; range, 93 to 224 mumol/L x hours), total-body clearance (CI; range, 1,172 to 3,236 mL/min), or volume of distribution (range, 11.9 to 49.2 L) of FUra. Intrapatient data evaluation revealed a dose-independent effect of rIFN-a. The mean FUra Css after rIFN-a administration (1.31 mumol/L) was greater than that before rIFN-a administration (1.02 mumol/L, P < .0001). FUra Cl after rIFN-a administration was reduced by 20% to 35% compared with use of FUra alone (P < .0001). Patients with a greater than 20% decrease in FUra Cl had a fourfold greater incidence of diarrhea. CONCLUSION: rIFN-a reduces FUra Cl and, consequently, increases FUra-associated toxicity. Phase II studies of FUra and rIFN-a seem to be warranted.