The expression of ESBLs genes in Escherichia coli isolates induced by β-lactam antibiotics and inhibited by histidine kinase inhibitors / 中华微生物学和免疫学杂志

Objective To investigate the genotypes of extended spectrum β-lactamases (ESBLs) and their carrying modes in Escherichia coli (E.coli) isolates,and to analyze the mechanism of protein phosphorylation and ESBLs gene expression induced by β-lactam antibiotics or inhibited by histidine kinase inhibitors.Methods The predominant genotypes of ESBLs (KPC,TEM,SHV and CTX-M) and their carrying modes were identified by PCR and sequencing analysis.E-test and micro-tube dilution method were applied to measure minimal inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs).Immobilized metal ion affinity chromatography,bacterial protein phosphorylation detection kit and real-time fluorescent quantitation RT-PCR were performed to analyze the enhancing effects of 1/4 MIC penicillin or cefotaxime or the inhibitory effects of histidine kinase inhibitors (closantel,bromized or iodized methylimidazol) on protein phosphorylation and the expression of ESBLs at mRNA level in E.coli isolates.Results In 183 β-lactam antibiotics-resistant E.coli isolates,TEM and CTX-M genes (83.1％ and 77.1％) were highly expressed than other two ESBLs genes with a prevalent carrying mode of coexisting (65.0％) (P＜0.05).Penicillin or cefotaxime at 1/4 MIC induced the protein phosphorylation and promoted the expression of TEM,SHV and CTX-M at mRNA level (P＜0.05).Closantel (200 μmol),bromized methylimidazol (2 or 10 μmol) or iodized methylimidazol (20 or 50 μmol) could neither kill E.coli isolates nor inhibit their growth,but could inhibit the protein phosphorylation induced by above mentioned antibiotics and enhance the expression of ESBLs at mRNA level (P＜0.05).Moreover,the susceptibility of antibioticresistant E.coli strains to penicillin and cefotaxime were increased (P＜0.05).Conclusion TEM and CTX-M were the predominant genotypes of ESBLs carried by β-lactam antibiotics-resistant E.coli strains isolated from Zhejiang province,which were mostly found in a TEM plus CTX-M carrying mode.Sublethal dose of β-lactam antibiotics could up-regulate the expression of ESBLs genes in E.coli isolates via TCSS,but it could be inhibited by histidine kinase inhibitors.

BQ123 Alleviates Lung Injury Following Limb Ischemia-Reperfusion in Rats / 医药导报

Objective To observe BQ123 on lung tissue injury following ischemia-reperfusion of hind limbs in rats.Methods A total of 30 male SD rats were randomly divided into control,I-R,and BQ123 groups,with 10 rats in each group. After 4- hour of ischemia and 4-hour of reperfusion to the hind limbs, ET-1 was measured by radioimmunoassay, levels of myeloperoxidase (MPO) and malondialehyde (MDA) were examined by biochemical method,and the content of P-selectin was examined by ELISA. Immunohistochemical method was used to detect the expressions of Fas,Bcl-2 and Caspase-3 in lung tissues. Pulmonary apoptosis was examined by means of TdT-mediated dUTP nick end labeling ( TUNEL) . Results Compared with the control group,the levels of ET-1,MPO,MDA and P-selectin in lung tissues were all increased significantly in I-R group ( P<0. 01). The expressions of Fas,Bcl-2 and Caspase-3 were 0. 294±0. 003,0. 108±0. 005,and 0. 174±0. 003,significantly up-regulated in the I-R group. The apoptosis rate [(18. 83±2. 86)%] was significantly increased in the I-R group (P<0. 01). Compared with I-R group,the tissue contents of ET-1,MPO,MDA and P-selectin were significantly lowered in BQ123 group (P<0. 01). The expression levels of Fas (0. 115±0. 007) and Caspase-3 (0. 159±0. 006) were decreased,but the expression of Bcl-2 was increased (0. 128±0. 005). The apoptosis rate in BQ123 group was significantly lower [(10. 67±2. 16)%,P<0. 01].Conclusion BQ123 may have protective effects on lung tissue after limb ischemia-reperfusion in rats by means of improving neutrophil aggregation and reducing the expression of proteins related to cell apoptosis.

Prokaryotic expression of Leptospira interrogans groEL gene and immunoprotection of its products in hamsters / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai, and to determine the immunoprotective effect of recombinant GroEL protein (rGroEL) in LVG hamsters.</p><p><b>METHODS</b>The groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced. A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroEL protein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL. The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L.interrogans strain Lai. The cross agglutination titers of sera from immunized hamsters with different L.interrogans serogroups were detected using MAT.</p><p><b>RESULTS</b>The nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank. The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL. The immunoprotective rates of 100 and 200 μg rGroEL in LVG hamsters were 50.0 % and 75.0%, respectively. The sera from the rGroEL-immunized LVG hamsters agglutinated all the L.interrogans serogroups tested with different levels.</p><p><b>CONCLUSION</b>The GroEL protein is a genus-specific immunoprotective antigen of L.interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.</p>

Apoptosis in liver injury following limbs ischemia reperfusion and the protective effects of taurine / 中国综合临床

Objective To observe the apeptosis in liver injury following limbs ischemia-reperfusion(IR) in rats and the protective effects of taurine.Methods The model of limbs ischemia-reperfusion injury was established.30 Wistar rats were randomized into 3 groups: control group,IR group and tanrine + reperfusion group (TR group) (n = 10 for each group).The levels of malondialdehyde (MDA) and xanthineoxidas (XOD), calcium and myeloper-oxidase (MPO) in the liver tissue were measured.DNA fragmentation was observed and analyzed by agarose gel e-lectrophoresis.Apoptosis was detected by TUNEL methods.The morphologic changes were observed with HE stai-ning.Results Compared with control group,the values of MDA, XOD, MPO, calcium in liver tissue were increased significantly in IR group (P<0.01), but the values of those in TR group were lower than in IR group (P<0.01).The percentage of apeptosis cell was higher in IR group than in control group(P<0.01).Compared with IR group, the percentage of apoptosis cell was lower in TR group (P<0.01).IR group presented DNA ladder pattern, while TR group showed no specific DNA ladder pattern in agarose gel electrophoresis.Conclusion Apoptosis participates in the liver injury after limb ischemia-reperfusion.Taurine can mitigate the liver injury and apoptosis after limb is-chemia-reperfusion injury in rats.