Intestinal Oxalobacter formigenes colonization in calcium oxalate stone formers and its relation to urinary oxalate.

BACKGROUND AND PURPOSE: Oxalobacter formigenes is an anaerobic commensal colonic bacterium capable of degrading oxalate through the enzyme oxalyl-CoA decarboxylase. It has been theorized that individuals who lack this bacterium have higher intestinal oxalate absorption, leading to a higher urinary oxalate concentration and an increased risk of calcium oxalate urolithiasis. We performed a prospective, controlled study to evaluate O. formigenes colonization in calcium oxalate stone formers and to correlate colonization with urinary oxalate and other standard urinary stone risk factors. PATIENTS AND METHODS: Thirty-five first-time calcium oxalate stone formers were compared with 10 control subjects having no history of urolithiasis and a normal renal ultrasound scan. All subjects underwent standard metabolic testing by submitting serum and 24-hour urine specimens. In addition, all subjects submitted stool samples for culture and detection of O. formigenes by Xentr(ix) O. formigenes Monitor. RESULTS: Intestinal Oxalobacter was detected in only 26% of the stone formers compared with 60% of the controls (p < 0.05). Overall, the average urinary oxalate excretion by the two groups was similar (38.6 mg/day v 40.8 mg/day). Among stone formers, however, there were statistically higher urinary oxalate concentrations in O. formigenes-negative patients compared with those testing positive (41.7 mg/day v 29.4 mg/day) (p = 0.03). Furthermore, all 10 stone formers with hyperoxaluria (>44 mg/day) tested negative for O. formigenes (p < 0.05). CONCLUSIONS: Calcium oxalate stone formers have a low rate of colonization with O. formigenes. Among stone formers, absence of intestinal Oxalobacter correlates with higher urinary oxalate concentration and an increased risk of hyperoxaluria. Introduction of the Oxalobacter bacterium or an analog of its enzyme oxalyl-CoA decarboxylase into the intestinal tract may be a treatment for calcium oxalate stone disease.

Oxalobacter formigenes and its potential role in human health.

Oxalate degradation by the anaerobic bacterium Oxalobacter formigenes is important for human health, helping to prevent hyperoxaluria and disorders such as the development of kidney stones. Oxalate-degrading activity cannot be detected in the gut flora of some individuals, possibly because Oxalobacter is susceptible to commonly used antimicrobials. Here, clarithromycin, doxycycline, and some other antibiotics inhibited oxalate degradation by two human strains of O. formigenes. These strains varied in their response to gut environmental factors, including exposure to gastric acidity and bile salts. O. formigenes strains established oxalate breakdown in fermentors which were preinoculated with fecal bacteria from individuals lacking oxalate-degrading activity. Reducing the concentration of oxalate in the medium reduced the numbers of O. formigenes bacteria. Oxalate degradation was established and maintained at dilution rates comparable to colonic transit times in healthy individuals. A single oral ingestion of O. formigenes by adult volunteers was, for the first time, shown to result in (i) reduced urinary oxalate excretion following administration of an oxalate load, (ii) the recovery of oxalate-degrading activity in feces, and (iii) prolonged retention of colonization.

A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP).

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP trade mark, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.

Effect of rhinovirus challenge on antioxidant enzymes in respiratory epithelial cells.

The host inflammatory response appears to be an important contributor to the pathogenesis of human viral respiratory illness. Virus-induced oxidative stress appears to mediate an early phase of elaboration of the proinflammatory cytokine interleukin-8 by respiratory epithelial cells. The purpose of these studies was to determine if virus-induced alterations in either the expression or function of antioxidant enzymes contributes to the cellular oxidative stress following rhinovirus challenge. The activities of Mn superoxide dismutase (MnSOD), catalase and glutathione peroxidase (GPX) were not significantly changed by rhinovirus challenge. CuZn superoxide dismutase (CuZnSOD) activity six hours after challenge was 2.55 +/- 0.56 U/mg protein in rhinovirus-challenged cells compared to 1.16 +/- 0.54 U/mg protein in control cells (p = 0.029). This increased activity was associated with a concomitant increase in CuZnSOD mRNA and protein concentration. These data suggest that rhinovirus-induced changes in the host cell redox state that result in the early elaboration of interleukin-8 are not mediated by inhibition of either the expression or function of these antioxidant enzymes.