ABSTRACT
Objective To construct mutant strains of methicillin resistant Staphylococcus epidermi-dis (MRSE) with psm-mec gene deletion and to investigate the function of psm-mec gene.Methods The drug sensitivity test and DNA sequence analysis were performed to screen out the tetracycline and chloram -phenicol sensitive clinical strains of MRSE , whose upstream and downstream sequences of psm-mec gene were identical to those of the Staphylococcus epidermidis reference strain RP62A.The recombinant plasmid pBT2-Δpsm-mec was constructed by using the fusion PCR and a temperature sensitive shuttle plasmid .After being identified , the plasmid was transformed into the Staphylococcus aureus RN4220 strain by electropora-tion, and then transformed into the selected clinical isolates of MRSE .The mutant strains of MRSE with psm-mec deletion were screened out and identified after homologous recombination .The differences in biofilm formation between the mutant and wild-type strains were analyzed for further elucidation the relationships be-tween the psm-mec gene and biofilm formation in MRSE strains .Results Three clinical MRSE isolates for the construction of mutant strains with psm-mec gene deletion were screened out and identified by using drug sensitivity test and sequence alignment analysis .The mutants constructed via homogenous recombination were screened out and identified .Compared with the corresponding wild-type strains, the three mutants with psm-mec gene deletion showed significantly decreased ability of biofilm formation , demonstrating that the psm-mec genes strains induced the biofilm formation of MRSE .Conclusion The Δpsm-mec mutant strains were successfully constructed .The psm-mec gene played an important role in the biofilm formation of Staphy-lococcus epidermdis.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of hydroxycamptothecin (HCPT) on the proliferation, cell cycle and apoptosis of human lung carcinoma cell line A549.</p><p><b>METHODS</b>The growth of A549 cells exposed to HCPT was observed by staining with acridine orange/ethidium bromide dye. Agarose gel electrophoresis was performed to detect DNA fragmentation of the apoptotic cells. The cell cycle distribution of the exposed cells was analyzed using flow cytometry, and cell apoptosis was examined with annexin V-FITC/PI staining. RT-PCR was used to investigate Bcl-2 gene expression changes in the exposed cells.</p><p><b>RESULTS</b>Agarose gel electrophoresis of the DNA from HCPT-treated cells showed a DNA ladder, and typical apoptotic appearance of the exposed cells was observed under fluorescence microscope. Treatment of A549 cells with 1 µmol/L HCPT for 24 h resulted in a cell apoptosis rate of 18.11%, significantly higher than the rate in control cells (0.09%, P<0.05). The treatment also caused a significant reduction of Bcl-2 mRNA expression by 70% (P<0.05).</p><p><b>CONCLUSION</b>HCPT can significantly inhibit the proliferation, induce apoptosis, and down-regulate Bcl-2 gene expression in human lung carcinoma cell line A549, suggesting the involvement of Bcl-2 gene in the inhibitory effect of HCPT on A549 cells.</p>