Turbidimetric Determination of Fecal Calprotectin Using Two Table Top Chemistry Analyzers: Mindray BS-200E and Cobas® c111.

BACKGROUND: Fecal calprotectin assays are widely used in diagnosis and monitoring of inflammatory bowel disease (IBD) in patients with suspected IBD. The most frequently used technique is ELISA and microtiter plates. Turbidimetric assays for analysis of fecal calprotectin can significantly reduce turnaround time. Many laboratories may be reluctant to run fecal samples on their large chemistry analyzers. The aim of this study was to evaluate fecal calprotectin particle enhanced turbidimetric immunoassay (PETIA) on smaller chemistry analyzers that could be dedicated for fecal samples. METHODS: The BÜHLMANN fCAL® turbo assay was validated on two table top chemistry analyzers, Mindray BS-200E and cobas® c111. RESULTS: The assay was linear in the range between 20 and 1,900 µg/g with a limit of quantification around 20 µg/g on both instruments. The total coefficient of variation was < 7% in the range between 50 and 1,300 µg/g on both instruments. No antigen excess hook effect was observed up to 18,000 µg/g on the Mindray BS-200E and up to 20,000 µg/g on cobas® c111. The BÜHLMANN fCAL® turbo assay showed a high correlation with the BÜHLMANN fCAL® ELISA. CONCLUSIONS: Running the BÜHLMANN fCAL® turbo on Mindray BS-200E or cobas® c111 chemistry analyzers can provide rapid test results without exposing large routine chemistry analyzers to stool samples.

Kinetic instability of p53 core domain mutants: implications for rescue by small molecules.

Oncogenic mutations in the tumor suppressor protein p53 are found mainly in its DNA-binding core domain. Many of these mutants are thermodynamically unstable at body temperature. Here we show that these mutants also denature within minutes at 37 degrees C. The half-life (t(1/2)) of the unfolding of wild-type p53 core domain was 9 min. Hot spot mutants denatured more rapidly with increasing thermodynamic instability. The highly destabilized mutant I195T had a t(1/2) of less than 1 min. The wild-type p53-(94-360) construct, containing the core and tetramerization domains, was more stable, with t(1/2) = 37 min at 37 degrees C, similar to full-length p53. After unfolding, the denatured proteins aggregated, the rate increasing with higher concentrations of protein. A derivative of the p53-stabilizing peptide CDB3 significantly slowed down the unfolding rate of the p53 core domain. Drugs such as CDB3, which rescue the conformation of unstable mutants of p53, have to act during or immediately after biosynthesis. They should maintain the mutant protein in a folded conformation and prevent its aggregation, allowing it enough time to reach the nucleus and bind its sequence-specific target DNA or the p53 binding proteins that will stabilize it.

Two sequence motifs from HIF-1alpha bind to the DNA-binding site of p53.

There is evidence that hypoxia-inducible factor-1alpha (HIF-1alpha) interacts with the tumor suppressor p53. To characterize the putative interaction, we mapped the binding of the core domain of p53 (p53c) to an array of immobilized HIF-1alpha-derived peptides and found two peptide-sequence motifs that bound to p53c with micromolar affinity in solution. One sequence was adjacent to and the other coincided with the two proline residues of the oxygen-dependent degradation domain (P402 and P564) that act as switches for the oxygen-dependent regulation of HIF-1alpha. The binding affinity was independent of the hydroxylation state of P564. We found from NMR spectroscopy that these sequence motifs bind to the DNA-binding site of p53c. Because the two sequences are homologous and separated by 120 residues, and one is in a largely unstructured transactivation domain, we speculate that each sequence motif in HIF-1alpha binds to a different subunit of the p53 tetramer, leading to very tight binding. The binding data support the proposal that p53 provides a route for the degradation in hypoxic tumor cells of HIF-1alpha that is not hydroxylated at the two proline residues.

A peptide that binds and stabilizes p53 core domain: chaperone strategy for rescue of oncogenic mutants.

Conformationally compromised oncogenic mutants of the tumor suppressor protein p53 can, in principle, be rescued by small molecules that bind the native, but not the denatured state. We describe a strategy for the rational search for such molecules. A nine-residue peptide, CDB3, which was derived from a p53 binding protein, binds to p53 core domain and stabilizes it in vitro. NMR studies showed that CDB3 bound to p53 at the edge of the DNA binding site, partly overlapping it. The fluorescein-labeled peptide, FL-CDB3, binds wild-type p53 core domain with a dissociation constant of 0.5 microM, and raises the apparent melting temperatures of wild-type and a representative oncogenic mutant, R249S core domain. gadd45 DNA competes with CDB3 and displaces it from its binding site. But this competition does not preclude CDB3 from being a lead compound. CDB3 may act as a "chaperone" that maintains existing or newly synthesized destabilized p53 mutants in a native conformation and then allows transfer to specific DNA, which binds more tightly. Indeed, CDB3 restored specific DNA binding activity to a highly destabilized mutant I195T to close to that of wild-type level.