Novel selective MMP-13 inhibitors reduce collagen degradation in bovine articular and human osteoarthritis cartilage explants.

OBJECTIVE: MMP-13 is highly upregulated in arthritis and therefore strongly implicated in the pathogenesis of osteoarthritis (OA). Selective inhibition of MMP-13 may provide the desired cartilage degradation protection, while overcoming the musculoskeletal toxicity seen with nonselective inhibition of MMPs. METHODS: Activity and selectivity of novel MMP-13 inhibitors were determined in enzymatic and collagenase assays. Inhibition kinetics and competitive binding experiments were performed. The inhibition of collagen degradation was studied in cartilage explants from OA patients and in bovine and human articular cartilage systems. RESULTS: We have identified a new class of very potent and highly selective non-zinc-binding MMP-13 inhibitors. Selective MMP-13 inhibitors completely blocked type II collagen degradation in bovine explants and showed up to 80% inhibition in human OA cartilage. CONCLUSIONS: These results indicate MMP-13 as the primary collagenase in the human OA cartilage and in the IL-1/OSM-induced cartilage degradation process and suggest that selective MMP-13 inhibitors may be a potential treatment of OA.

A high-throughput microtiter plate-based screening method for the detection of full-length recombinant proteins.

The Gram-negative bacterium Escherichia coli is an important host for the (heterologous) production of recombinant proteins. The development and optimization of a protocol to overproduce a desired protein in E. coli is often tedious. A novel high-throughput screening method based on the Luminex xMAP bead technology was developed allowing a rapid evaluation of a certain expression strategy. A variant of green fluorescent protein (GFPuv) from Aequorea victoria was used as a reporter to establish the methodology. The N-terminus and the C-terminus of GFPuv were engineered to contain a His(6)- and an HA-tag (YPYDVPDYA), respectively. The double-tagged protein was loaded onto Luminex-microspheres via its His(6)-tag, the presence of the HA-tag was verified using an anti-HA antibody. High-throughput detection of full-length proteins (containing both tags) on the beads was performed using an automated Luminex 100IS analyzer. The results were compared to results obtained by classical Western blot analysis. Comparison of the two methods revealed that the Luminex-based method is faster and more economical in detecting full-length (intact) soluble recombinant protein, allowing one to routinely screen a high number of parameters in gene expression experiments. As proof of concept, different protocols to overproduce double-tagged model eucaryotic proteins (human protein S6 kinase 1 and human tankyrase) in E. coli were monitored using the new approach. Relevant parameters for optimizing gene expression of the corresponding genes were rapidly identified using the novel high-throughput method.

Characterization of Erwinia chrysanthemi from a Bacterial Heart Rot of Pineapple Outbreak in Hawaii.

The first reported outbreak of bacterial heart rot of pineapple (Ananas comosus var. comosus) in Hawaii occurred in December 2003. Of immediate concern was the differentiation of heart rot caused by Erwinia chrysanthemi from a soft rot caused by E. carotovora subsp. carotovora because of regulatory issues. Presumptive identifications of the isolated bacteria were made using bacteriological tests (including reactivity with an Erwinia-specific monoclonal antibody, E2) and compared with identifications obtained by two general methods: carbon source utilization profiling (Biolog) and 16S rDNA sequence analysis. The panel of bacteriological tests consistently differentiated E. chrysanthemi from E. carotovora subsp. carotovora and other nonquarantine organisms. BOX-polymerase chain reaction fingerprint patterns further differentiated the pineapple-isolated E. chrysanthemi strains from those obtained from other plants and irrigation water. Pineapple leaf inoculations revealed that only E. chrysanthemi from pineapple produced watersoaking and rot similar to that observed on the original symptomatic plants, thus identifying these strains as the causal agents of the outbreak. In this situation, where rapid identification of an unknown pathogen was necessary, standard bacteriological tests then available in the laboratory provided reliable differentiation of E. chrysanthemi from E. carotovora subsp. carotovora. Additional strain characterization is needed before the pineapple-isolated E. chrysanthemi strains can be classified into a species of the new genus Dickeya.