Distribution of drug inactive enzyme genes in bacterial isolates and mechanism of its induction and inhibition
WU Yi-Fei, SUN Ai-Hua, ZHAO Jin-Fang, GE Yu-Mei, YAN Jie
Journal of ZheJiang University(Medical Science), 2013, 42(2): 131-140.
Objective： To determine the distribution and the predominant gene carrying model of drug inactive enzyme genes in bacterial isolates，and the mechanism of its induction and inhibition.
Methods： The β-lactam，aminoglycosides and macrolides inactive enzyme genes were detected by PCR and sequencing in S.aureus, E.coli，K.pncumoniae, A.baumanii and E.cloacae isolates.The expression of inactive enzyme genes were examined by real-time fluorescent quantitative RT-PCR when the bacterial isolates were treated with antibiotics or a histidine kinase blocker closantel.
Results： In 63 isolates of E.coli，4 kinds of β-lactam，2 aminoglycosides and 1 macrolides inactive enzyme-encoding genes were detected and the predominant gene-carrying models were [TEM+CTX-M]+aac(3)-Ⅱ+mphA(25.4%) and [TEM+CTX-M]+ aac(6′)-Ⅰb(20.6%).In 24 isolates of S.aureus，2 kinds of β-lactam and 3 aminoglycosides inactive enzyme-encoding genes were detected and the predominant gene-carrying models were aph(3′)(41.7%) or aac(6)-Ⅰe-aph(2)-Ⅰa(25.0%).In 28 isolates of K.pncumoniae，4 kinds of β-lactam and 2 aminoglycosides inactive enzyme-encoding genes were detected and the predominant gene-carrying models were [TEM+SHV]+[aac(6′)-Ⅰb+aac(3)-Ⅱ](28.6%) and [TEM＋SHV]+[aac(6′)-Ⅰb+aac(3)-Ⅱ]+ mphA(17.8%).The
isolates of A.baumanii and E.cloacae also had a predominant model to carry 2 or 3 kinds of inactive enzyme-encoding genes.1/4 MIC of penicillin，cefotaxime or streptomycin induced the up-regulation of expression of 3 β-lactam or 4 aminoglycosides inactive enzyme-encoding genes(P<0.05)，and this effect was inhibited by closantel(P<0.05).
Conclusions： The bacterial isolates frequently carry multiple kinds of inactive enzyme-encoding genes with different predominant gene-carrying models.Low concentration antibiotics can induce the up-regulation of inactive enzyme gene expression，which can be inhibited by histidine kinase blocker.
Role of phospholipase C in cytoskeleton rearrangements of dendritic cells invaded by Mycobacterium tuberculosis
XU Shui-Ling, XU Yan, HUANG Jia, FAN Hong-Yan, JIN Meng-Mei
Journal of ZheJiang University(Medical Science), 2013, 42(2): 184-191.
Objective： To investigate the role of phospholipase C(PLC）in cytoskeleton rearrangement of mouse dendritic cells invaded by Mycobacterium tuberculosis.
Methods： Mouse dendritic DC2.4 cells were co-cultured with Mycobacterium tuberculosis H37Rv.F-actin of DC2.4 cells were strained with palloidin-TRITC，the microtubule was stained with anti-β-tubulin monoclonal antibody and FITC-conjugated affnipure anti-mouse IgG.The changes of cytoskeleton in DC2.4 cells induced by Mycobacterium tuberculosis H37Rv were determined by fluorescence microscopy and the rates of F-actin rearrangements were calculated.The expressions of PLC in cytoplasm and cytomemberane of DC2.4 cells were measured by ELISA.DC2.4 cells were pretreated with PLC inhibitor U73122，then F-actin rearrangements induced by invasion of Mycobacterium tuberculosis were observed.
Results： Bacterial invasion was observed while DC2.4 cells were co-incubated with Mycobacterium tuberculosis H37Rv for 2 h.The rates of invasion were（26.1±4.5）%,（39.9±5.6）%,（51.2±5.9）%,（57.9±6.1）% and（63.9±6.8）% at 4，6，8，10 and 12 h of co-culture，respectively; while those were（13.6±3.1）%,（14.2±3.9）%,（15.1±4.3）%,（16.8±4.0）% and（18.3±5.2）% after blocked by PLC，respectively.The rates of the F-actin rearrangements at 2，4，6，8，10 and 12 h after DC2.4 cells were invaded by H37Rv were（26.9±1.5）%,（59.3±2.8）%,（72.7±4.8）%,（78.2±5.9）%,（63.3±2.9）% and （43.2±2.6）%，respectively; while those were（18.5±1.2）%,（22.3±1.7）%,（23.6±2.5）%,（24.8±2.3）%,（22.3±1.3）% and （23.8±1.8）% after blocked by PLC，respectively.There were no changes of the microtubule observed in DC2.4 cells at the same time points.The rates of the F-actin rearrangements before blocked by PLC were higher than those after PLC blockade at 4，6，8 and 10 h（P＜0.05）.The expressions of PLC in cytomembrane in DC2.4 cells increased after 2 h and reached its highest level at 8 h.The PLC inhibitor U73122 inhibited the expressions of PCL in cytomembrane of DC2.4 cells，but not in cytoplasm.
Conclusions： Mycobacterium tuberculosis can provoke to F-actin rearrangements through PLC molecule，which would further lead to Mycobacterium tuberculosis invasion of DC2.4 cells.
Effects of ginsenosides Rb1 on learning and memory and expression of somatostatin in sleep deprivation rats
DONG Jing-Yin, WANG Jun-Bo, FANG Jie, YUAN Zhang-Gen, LU Ke-Jie, JIN Yi, ZENG Ling-Hui
Journal of ZheJiang University(Medical Science), 2013, 42(2): 197-204.
Objective： To determine the effects of ginsenosides Rb1(GSRb1) on learning and memory and expression of somatostatin (SS) in the hippocampus and the frontal cortex in rat model of sleep deprivation (SD).
Methods： Rats were randomized into groups of SD 2 d,SD 4 d,SD 6 d,and SD 0 d,while each group was sub-divided into GSRb1 group and normal saline (NS) sub-groups.Rats were intraperitoneal administered with 30 mg/(kg·d） of GSRb1 or NS for 7 d,then the learning and memory abilities were examined by measuring average swimming speed and mean escape latency using Morris maze.Expression of somatostatin was detected with immunohistochemical method and image analysis in the hippocampus and the frontal cortex.
Results： Compared with SD 0 d rats,SD rats exhibited significant decrease in the average swimming speed and increase in the escape latency (P<0.01).The expression of somatostatin in the hippocampus was decreased significantly in SD 2 d,SD 4 d and SD 6 d rats (P<0.05).However,decrease was only observed in SD 4 d and SD 6 d rats in the frontal cortex (P<0.05).Parallel comparison between NS control and GSRb1 treated rats demonstrated that rats treated with GSRb1 in each subgroup exhibited faster swimming speed and shorter escape latency (P<0.05).Meanwhile,the expression of somatostatin was increased in SD 2 d,SD 4 d and SD 6 d rats in the hippocampus and in SD 4 d and SD 6 d rats in the frontal cortex (P<0.05),respectively.
Conclusion： GSRb1 enhances the expression of somatostatin in sleep deprivation rats and subsequently may improve learning and memory abilities of rats.