The present study was designed to establish a suitable assay to explore CCR2 b receptor antagonists from the natural products of Artemisia rupetris and Leontopodium leontopodioides. An aequorin assay was developed as a cell-based assay suitable for 384-well microplate and used for screening CCR2 b receptor antagonists from natural products. Through establishing suitable conditions, the assay was shown to be suitable for screening of CCR2 b receptor antagonists. Seven compounds were identified in preliminary screening. Five of them showed evident dose-response relationship in secondary screening. The structure–activity relationship study suggested that 7-position hydroxyl group of flavonoids was necessary, a polar group should be introduced on the 3-position, and the substituents on 2-position benzene ring of flavonoids have little influence on the potentency of the inhibition activity on CCR2 b receptor. The ortho-position dihydroxyl structure in quinic acid compounds may be important. In conclusion, Compounds HR-1, 5, 7, and AR-20, 35 showed activity as antagonist of CCR2 b receptor, which shed lights on the development of novel drugs as CCR2 b receptor antagonists for preventing inflammation related diseases.
[Objective]This study aimed to investigate the mutations of OmpF from an isolated antibiotic resistant Escherichia coli strain.[Methods]The mutant OmpF(mOmpF)from antibiotic resistant E.coli was amplified by PCR with Pfu DNA polymerase and ligated into the expression vector pET28a.Subsequently,the expression vector pET28-mOmpF was sequenced and analyzed by DNAMAN software and Swiss-Model online.[Result]Sequence analysis revealed that the open reading fragment of mOmpF was 903 bp long,which was mutated dramatically compared to that of the 1 020 bp long model OmpF.The DNA sequence shared only54.5%homology with OmpF.mOmpF was 44.6%identical to that of OmpF.Protein structure predication and analysis through Swiss-Model online suggested that the structure of mOmpF changed dramatically compared to OmpF.[Conclusion]The present study provided basis for further analyzing the relationships between the structure and functions of mOmpF from antibiotic resistant E.coli.
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