Direkt zum Inhalt | Direkt zur Navigation

Eingang zum Volltext


Bitte beziehen Sie sich beim Zitieren dieses Dokumentes immer auf folgende
URN: urn:nbn:de:bsz:25-opus-6988
URL: http://www.freidok.uni-freiburg.de/volltexte/698/

García-Piñeres, Alfonso J.

Contribution to the elucidation of the anti-inflammatory activity of Sesquiterpene Lactones

Ein Beitrag zur Aufklärung der antiinflammatorischen Wirkung von Sesquiterpenlactonen

Dokument1.pdf (8.796 KB) (md5sum: 2e2391e451d99109e66969f5af83aa98)

Kurzfassung in Deutsch

To analyze the mechanism by which sesquiterpene lactones (SLs) inhibit DNA binding of the transcription factor NF-kB, the role of cysteine residues in its DNA binding domain was tested using single amino acid mutants of the p65 subunit. The results confirm that p65 is alkylated by SLs, and that the alkylation of Cys38 is sufficient to inhibit p65 binding. The amount of remaining IkB-a is too low to explain the observed NF-kB inhibition. No inhibition of the IKK was observed in an assay performed with immunoprecipitated kinase. Four different SLs were used in these experiments, and led to identical results, confirming that the inhibition of NF-kB by SLs follows a general mechanism. These results were confirmed in four different cell lines.
The effect of SLs and the alkylant N-ethyl maleimide (NEM) on DNA binding of the p65 and p50 subunits was compared. While NEM inhibited the binding of both, SLs only prevented binding of p65, being more selective. The inhibition by NEM relied on alkylation of Cys38 in p65, or its equivalent in p50.
A densitometric method for the quantification of EMSA analyses was also established. This allowed the determination of the concentration required to obtain a half-maximal inhibition of NF-kB (IC50). The method was used to calculate the IC50 values for 24 SLs.
Up to now, the anti-inflammatory activity of SLs has been studied using cell culture. To perform analyses that more closely resemble the in vivo conditions, flow cytometry was used to determine the expression of CD69, a marker of T-cell activation, and of interleukin-2 (IL-2), an inflammatory cytokine, in whole blood. Incubation with different concentrations of different SLs led to the inhibition of both. Surprisingly, the activity of bifunctional SLs was lower than that of monofunctional, in contrast to the activity determined using EMSA. Measurements of SL binding to serum protein suggest that this difference is due to a stronger binding of bifunctional SLs to the protein.

SWD-Schlagwörter: Sesquiterpenlactone , Transkriptionsfaktor , Nuklearfaktor Kappa B , Antiphlogistikum , FACS
Institut: Institut für Pharmazeutische Biologie
Fakultät: Fakultät für Chemie und Pharmazie (bis Sept. 2002)
DDC-Sachgruppe: Chemie
Dokumentart: Dissertation
Erstgutachter: Merfort, Irmgard (Prof. Dr.)
Sprache: Englisch
Tag der mündlichen Prüfung: 25.04.2003
Erstellungsjahr: 2003
Publikationsdatum: 21.05.2003