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- Westergren-Thorsson, Gunilla, et al.
(författare)
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Proteomics - the protein expression technology to study connective tissue biology
- 2001
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Ingår i: Journal of Pharmaceutical and Biomedical Analysis. - 0731-7085. ; 24:5-6, s. 815-824
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Tidskriftsartikel (refereegranskat)abstract
- During the formation of peribronchial fibrosis in asthma, remodeling of connective tissue is due to an increase in deposition of extracellular matrix components like that of specific types of collagens and proteoglycans. By taking bronchial biopsies, we were able to isolate cell cultures derived from asthmatic patients and healthy volunteers, which provides a good model system to study differences regarding cell morphology and key connective tissue proteins in the remodeling process. Proteomics, utilizing two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. By using this powerful tool, it is possible to quantitatively study protein regulation and to obtain increased knowledge about the mechanism behind the inflammatory process and formation of peribronchial fibrosis. We have optimized a proteomic protocol enabling detailed investigation of the protein expression pattern in human lung cells. An increased expression pattern was obtained, whereby 20 protein spots could be detected by image analysis in the < 45 kDa region. Out of by MALDI TOF-MS. This protocol enables us to study 1000 2000 proteins simultaneously and the possibility to correlate protein expression to the physiological status of the cell culture investigated. We have found that two proteins, actin and tropomyosin, are increased in expression due to transforming growth factor- stimulation. These proteins are correlated to the transformation of normal fibroblasts to myofibroblasts which are involved in the remodeling processes observed in asthma.
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- Salas, Emir, et al.
(författare)
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A heterogeneous enzymatic assay for quantification of Plasmepsin II activity and the evaluation of its inhibitors
- 2004
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Ingår i: Journal of Pharmaceutical and Biomedical Analysis. - 0731-7085 .- 1873-264X. ; 34:4, s. 833-840
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Tidskriftsartikel (refereegranskat)abstract
- The emergence and worldwide spreading of Plasmodium falciparum strains that shown to be resistant to traditional drugs is considered a very serious health problem, given the high mortality and morbidity rate of Malaria. In the search for new drugs against this parasite, Hb hydrolyzing enzymes, such as Plasmepsin II (Plm II), have been classified as very promising targets for therapeutic attacks. In this work, it is developed a cheap and high-throughput heterogeneous enzymatic assay for measuring Plasmepsin II activity in order to use it as a tool in the discovery of new inhibitors of this enzyme. In this assay, Plasmepsin II acts upon a solid-phase bound synthetic peptide (DU2) whose sequence comprises the cleavage site F(33)-L(34) present in Hb alpha-chain. The peptide surface density is quantified by means of a classical ELISA-based procedure. In order to estimate the kinetic constants of the system and to quantify both, enzymatic and inhibitory activity, it was used a model for the kinetics of enzyme quasi-saturable systems previously developed by our group, that fitted very well to the experimental data. It was used Pepstatin as a model inhibitor of Plasmepsin II and the resulting dose-response relation agreed with the expected behavior for the Pepstatin-Plasmepsin II pair under the employed experimental conditions.
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