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Objective:To analyze the effects of silver ion on the integration frequency of the class 1 integron in Escherichia coli ( E. coli) BL21(DE3) host. Methods:Two recombinant plasmids, pUCINT and pACINAD, were successively transformed into E. coli BL21 (DE3) to construct HS2 strains. Three experimental groups were set up using 0.3 μg/ml, 0.6 μg/ml and 0.8 μg/ml silver ion LB liquid medium, while control group used common LB liquid medium. Silver ion was supplied by silver nitrate and HS2 strains were cultured at 37℃ for 24 h. The copy number of cointegrates and the total copy number of integrons in each group were detected by real-time polymerase chain reaction (qPCR), and the ratio of them was the integration frequency. Changes in the integration frequency were analyzed by three independent phenotypic screening method and the protein expression in HS2 strains was analyzed by mass spectrometry. Results:The integration frequency in HS2 strains in the control group and three experimental groups (0.3 μg/ml, 0.6 μg/ml and 0.8 μg/ml silver ion) was 1.79×10 -5 (1.44×10 -5, 3.13×10 -5), 2.07×10 -5 (1.49×10 -5, 2.67×10 -5), 2.25×10 -6 (1.47×10 -6, 4.54×10 -6) and 1.69×10 -6 (0.22×10 -6, 3.08×10 -6), respectively. The integration frequency in the 0.6 μg/ml and 0.8 μg/ml silver ion groups was significantly lower than that in the control group ( P<0.01), but there was no significant difference between the 0.3 μg/ml silver ion group and the control group. Results of three independent phenotypic screening method were consistent with those obtained by qPCR. Mass spectrometry analysis showed that there were differences in protein expression in HS2 strains between the control group and the experimental groups. Conclusions:Silver ion at a certain concentration had an inhibitory effect on the frequency of drug resistance gene cassette captured by bacterial integron.
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Objective:To investigate the effect of total saponins of Panax notoginseng (TSPN) on learning and memory of post-stroke depression (PSD) rats and its mechanism.Methods:Four-vessel occlusion method was used to build the rat stroke model and 7 days later these rats were given solitary breeding with chronic unpredictable mild stress (CUMS) to make depression model. Rats were randomly divided into Sham group ( n=10), Model group ( n=10), PSD group ( n=10) and TSPN group ( n=10). The rats in the Model group and PSD group were injected administered with equal volume of 0.9% saline 30 min post-brain ischemia, one injection per day for 30 days. while TSPN group were treated with TSPN. The dose of TSPN (75 mg/kg) was dissolved in 0.9% saline 10 g/L, once per day for 30 days. Then the learning and memory of rats were tested by Morris water maze.The protein levels of DCX and Nestin in the hippocampus were detected by Western blot. Furthermore, the DCX/Ki67 co-labeled cells in the SGZ of hippocampus were observed by the immunofluorescence. Results:The escape latency at the fifth day of PSD group((31.8±3.8)s) was longer than that in the Sham group((10.4±3.2)s) and Model group((19.8±3.7)s) ( t=9.23, 5.15; both P<0.05). The escape latency ((14.2±2.8)s) of TSPN group was shortened significantly than PSD group ( t=8.56, P<0.05). The times across the platform in the Sham group was (10.3±1.7), and the PSD group was (4.1±1.1), difference was statistically significant between two groups( t=11.24, P<0.05). The times across the platform (8.4±1.6) of TSPN group statistically increased compared with PSD group ( t=5.77, P<0.05). The protein levels of DCX and Nestin in the PSD group were (0.60±0.02), (0.58±0.03) respectively, and in the TSPN group were (1.07±0.07), (0.95±0.11) correspondingly, there were significant differences of the DCX, Nestin protein level between the two groups( t=20.22, 7.68, both P<0.01). Moreover, there was significant difference in the number of the DCX/Ki67cells in the hippocampus SGZ between the PSD group((16.2±2.8) /mm 2) and TSPN group ((21.2±3.1) /mm 2)( t=2.42, P<0.05). Conclusion:TSPN could improve the learning and memory of the rats with post-stroke depression through enhancing the hippocampus neurogenesis.
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Objective To explore the effect of the total saponins of panax notoginseng ( TSPN) on depression-like behavior following global cerebral ischemia depression in rats and its mechanism. Method-s Using four-vessel occlusion method to build the global cerebral ischemia model,then the cerebral ischemi-a rats were given solitary breeding with chronic unpredictable mild stress ( CUMS) to prepare depression model. Seventy rats were divided into sham group (n=10),model group ( n=20),PSD group ( n=20) and TSPN group (n=20). The rats in the TSPN group were administered TSPN intraperitoneally 30 min post-brain ischemia. The dose of TSPN (75mg/kg) was suspended in 0. 9% saline 10g/L,once per day for 30 days after reperfusion. While rats in the vehicle group and PSD group was treated with equal volume of 0. 9% saline,one injection per day until the rats were sacrificed at 30 days after brain ischemia. The BrdU,dou-blecortin (DCX) expression in the hippocampus was assessed by immunohistochemistry. Results In com-parison with the model group,the sucrose preference percentage in the PSD group was significantly lower ((46. 2±9. 2)%,(61. 2±7. 6)%;t=3. 18,P<0. 05),then the PSD rats were administered TSPN intraperito-neally,the sucrose preference percentage increased significantly ((62. 4±3. 4)%,(46. 2±9. 2)%;t=3. 43, P<0. 05). During the forced swimming test,the immobility time of PSD group was significantly increased compared with the model group ((119. 4±9. 7)s,(88. 0±15. 6)s ;t=4. 30,P<0. 01),while after PSD rats administering TSPN intraperitoneally,the immobility time was shorten remarkably ((97. 4±6. 7)s,(119. 4± 9. 7)s;t=3. 01,P<0. 05). Compared with the Model group( BrdU+:( 12. 6± 2. 2)/mm2,DCX+:( 38. 6± 4. 2)/mm2),the number of BrdU+ and DCX+ cells in the SGZ of hippocampus in PSD group decreased (BrdU+:(8. 8±1. 5)/mm2,DCX+:(27. 2±2. 8)/mm2;t=3. 25,4. 29,both P<0. 01). And compared with PSD group,the number of BrdU+ and DCX+ cells in the SGZ of hippocampus in TSPN group increased sig-nificantly (BrdU+:(14. 8±2. 8)/mm2,DCX+:(37. 0±3. 3)/mm2;t=4. 68,3. 69,both P<0. 05). Conclu-sion TSPN can improve the depression-like behavior of rats following global cerebral ischemia,which may be related with promoting hippocampal nerve regeneration.
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Objective@#To observe the integration frequency of aadA2 resistance cassette at attI site of the integron under different concentration of streptomycin. @*Methods@#Class 1 integron with known gene sequence was cloned into plasmid pACYC184 to produce recombinant plasmid pACIDA, meanwhile the integrase gene was cloned into plasmid pET28a to construct recombinant plasmid pETINT. These two recombinant plasmids were consecutively transformed into E. coli BL(DE3). These transformed bacteria was cultured in the LB medium at 37 ℃ overnight with addition of different concentration of streptomycin. The copy number of total integrons and the copy number of integrated aadA2 at attI site of integrons were determined by using real-time PCR. and the integration frequency is the result of the former divided by the latter. @*Results@#The resulting frequencies were (1.97±0.24)×10-3, (3.23±1.77)×10-3, (3.27±0.67)×10-3, 0.45±0.13 and 1.32±0.11, with respective streptomycin concentrations of 0, 20, 30, 40 and 50 μg/ml. The background frequency of integration without integrase overexpression was less than (1.75±0.33)×10-7. @*Conclusion@#These findings indicate that antibiotic concentration significantly increase recombination frequency of aadA2 resistance cassette at attI site of the integron, catalyzed by integron integrase.(Chin J Lab Med, 2018, 41: 531-535)
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Objective To investigate transcriptional expression and promoter CpG methylation status of A-kinase anchoring protein 12 (AKAP12) gene and analyze their correlation with clinical pathological stage in bladder transitional cell carcinoma. Methods AKAP12 mRNA expression level and promoter CpG metbylation status was measured by fluorescent quantitative RT-PCR (FQ-RT-PCR) and methylation specific PCR (MSP) in 30 bladder transitional cell carcinoma and adjacent normal tissues. The products of PCR were cloned and bisulfite sequenced. Results Decreased AKAP12 mRNA expression was demonstrated in 22 carcinomas (73. 3% ) and was significantly associated with turnout grade (P =0. 02).The frequency of promoter methylation of AKAP12 gene was 53. 3 % (16/30) and correlated with the tumor stage(r =0.52,Pn =0.03)and grade(r =0.61,Pn =0.01). Conclusion Aberrant promoter methylation of AKAP12 can result in the loss of gene expression and may association with bladder transitional cell carcinoma.
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Objective To establish a system for detecting integration frequency of antibiotic resist-ante integron.Methods We cloned integron and aadA2 gene cassette into different sites of plasmid pACYC 184,and the plasmid was transformed into E.coli BL21(DE3)containing plasmid overexpressing integrase.The positive clone was cultured overnight and then was spread on LB agar plate with or without streptomycin respectively,and with appropriate amount of bacteria.Clones after cultured overnight were counted to detect the integration frequency.Meanwhile we used positive clones in LB agar plate containing streptomycin as templates to carry out PCR.The purified PCR products were sequenced to identify the integration sites.Re-suits The integration frequency of integron capturing aadA2 gene cassette in BL21(DE3) host was 1.1 x 10-3 mainly at attI site.Conclusion This system can be used to detect integration frequency.
