A European Pathogenic Microorganism Proteome Database: Construction and Maintenance

A relational database structure based on MS-Access and MySQL to store and manage proteomics data was established. This system may be used to publish two-dimensional electrophoretic proteomics data, and also may be accessed by external users who want to compare their own data with those in the databases. The maintenance of the database is managed centrally. The producers of proteomics data do not need to construct a database themselves. Users can introduce mass spectra into the database, which allows the searching of peptide mass fingerprints against their own protein sequence databases. The first release published in January 2002 contains data from Mycobacterium tuberculosis, Helicobacter pylori, Borrelia garinii, Francisella tularensis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Jurkat T-cells and mouse mammary gland projects (http://www.mpiib-berlin. mpg.de/2D-PAGE/)

Hyaluronan Export through Plasma Membranes Depends on Concurrent K+ Efflux by Kir Channels

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K+ channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl2 which all belong to ATP-sensitive inwardly-rectifying Kir channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K+ channels Kir3.4 and Kir6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K+ efflux

Assembling proteomics data as a prerequisite for the analysis of large scale experiments

<p>Abstract</p> <p>Background</p> <p>Despite the complete determination of the genome sequence of a huge number of bacteria, their proteomes remain relatively poorly defined. Beside new methods to increase the number of identified proteins new database applications are necessary to store and present results of large- scale proteomics experiments.</p> <p>Results</p> <p>In the present study, a database concept has been developed to address these issues and to offer complete information via a web interface. In our concept, the Oracle based data repository system SQL-LIMS plays the central role in the proteomics workflow and was applied to the proteomes of <it>Mycobacterium tuberculosis</it>, <it>Helicobacter pylori</it>, <it>Salmonella typhimurium </it>and protein complexes such as 20S proteasome. Technical operations of our proteomics labs were used as the standard for SQL-LIMS template creation. By means of a Java based data parser, post-processed data of different approaches, such as LC/ESI-MS, MALDI-MS and 2-D gel electrophoresis (2-DE), were stored in SQL-LIMS. A minimum set of the proteomics data were transferred in our public 2D-PAGE database using a Java based interface (Data Transfer Tool) with the requirements of the PEDRo standardization. Furthermore, the stored proteomics data were extractable out of SQL-LIMS via XML.</p> <p>Conclusion</p> <p>The Oracle based data repository system SQL-LIMS played the central role in the proteomics workflow concept. Technical operations of our proteomics labs were used as standards for SQL-LIMS templates. Using a Java based parser, post-processed data of different approaches such as LC/ESI-MS, MALDI-MS and 1-DE and 2-DE were stored in SQL-LIMS. Thus, unique data formats of different instruments were unified and stored in SQL-LIMS tables. Moreover, a unique submission identifier allowed fast access to all experimental data. This was the main advantage compared to multi software solutions, especially if personnel fluctuations are high. Moreover, large scale and high-throughput experiments must be managed in a comprehensive repository system such as SQL-LIMS, to query results in a systematic manner. On the other hand, these database systems are expensive and require at least one full time administrator and specialized lab manager. Moreover, the high technical dynamics in proteomics may cause problems to adjust new data formats. To summarize, SQL-LIMS met the requirements of proteomics data handling especially in skilled processes such as gel-electrophoresis or mass spectrometry and fulfilled the PSI standardization criteria. The data transfer into a public domain via DTT facilitated validation of proteomics data. Additionally, evaluation of mass spectra by post-processing using MS-Screener improved the reliability of mass analysis and prevented storage of data junk.</p

Inhibition of hyaluronan export reduces collagen degradation in interleukin-1 treated cartilage

BACKGROUND: Osteoarthrosis is characterized by cartilage erosion, proteolysis of aggrecan and collagen, and disturbed rates of synthesis of aggrecan and hyaluronan by chondrocytes, with hyaluronan over-production being an early reaction. We considered that inhibition of hyaluronan export might prevent subsequent proteoglycan loss and collagen degradation. METHODS: To test this hypothesis, we studied a tissue culture model using bovine cartilages explants activated with IL-1α to induce osteoarthritic reactions using the phosphodiesterase-5 inhibitors tadalafil, zaprinast and vardenafil. RESULTS: These drugs inhibited hyaluronan export, but they did not inhibit hyaluronan synthase activity. Simultaneously, they inhibited proteoglycan loss and collagen degradation, but not their synthesis. They also reduced the release of gelatinases into the culture media and diffusion of the indicator protein horseradish peroxidase through the cartilage explants. The mechanism of action of these compounds may be through inhibition of hyaluronan exporter multidrug resistance-associated protein 5 (MRP5), because the effective drug concentrations were much higher than required for phosphodiesterase-5 inhibition and intracellular cGMP accumulation. CONCLUSION: Inhibition of hyaluronan over-production may be an appropriate target to attenuate IL-1-induced reactions in osteoarthritic cartilage

Recovery of ΔF508-CFTR Function by Citrate

Treatment of cystic fibrosis relies so far on expensive and sophisticated drugs. A logical approach to rescuing the defective ΔF508-CFTR protein has not yet been published. Therefore, virtual docking of ATP and CFTR activators to the open conformation of the CFTR protein was performed. A new ATP binding site outside of the two known locations was identified. It was located in the cleft between the nucleotide binding domains NBD1 and NBD2 and comprised six basic amino acids in close proximity. Citrate and isocitrate were also bound to this site. Citrate was evaluated for its action on epithelial cells with intact CFTR and defective ΔF508-CFTR. It activated hyaluronan export from human breast carcinoma cells and iodide efflux, and recovered ΔF508-CFTR from premature intracellular degradation. In conclusion, citrate is an activator for ΔF508-CFTR and increases export by defective ΔF508-CFTR into the extracellular matrix of epithelial cells

Mutations in the Fc-region of IgG from synovial fluids of patients with rheumatoid arthritis

Rheumatoid arthritis an antigen-driven autoimmune disease and the eliciting antigens are unknown. They should be present in immune complexes of synovial fluids. The immune complexes were isolated from synovial fluids of patients with rheumatoid arthritis and analyzed by polyacrylamide gel electrophoresis and Western blotting. Immune complexes contained IgG as major protein. The antibodies dissociated at pH 2.5 and self-aggregated upon neutralization. In ELISA assays, they had the highest specificity to homologous IgG, less to normal IgG and lowest of heterologous arthritic IgG confirming earlier results that altered IgG structures were antigenic. Sequencing of the Fc-regions revealed mutations in all patients analyzed. Therefore, mutations within the Fc-regions of IgG could be antigens in rheumatoid arthritis. We propose that methods should be developed to prove this hypothesis