OBJECTIVE To investigate the repressor SmeT and other sequences of efflux pump SmeDEF in the clinic isolates of Stenotrophomonas maltophilia to involve in antibiotic resistance. METHODS The mRNA level of smeD examined the resistance profile of the clinical strains to antimicrobials was by agar dilution and their reaction to pump inhibitor was investigated by RT-PCR,and the smeD-smeT fragment was amplified by PCR,and then sequenced.Adding poly(A) to 3′ end of RNA,applying 3′RACE,nested PCR,then sequence-analysis. RESULTS The amino acid sequences of the forepart of SmeT were conservative in all the 7 strains.In antibiotic-resistant strains with positive reaction to pump inhibitor,the intergenic sequences of smeD-smeT were different from those in sensitive ones.The variations in(-165 to-82)nt of smeT and several nucleic acids before the start codon of smeT,possibly involved in antimicrobial resistance.All isolates didn′t have Leu166Gln mutation within the SmeT protein of D457R,which was reported possibly associated with antibiotic resistance.The resistant strains with efflux positively inhibited had Asp218Glu substitution,different from the negatively-inhibited ones. CONCLUSIONS The expression level of SmeDEF is related with the antibiotic resistance of S.maltophilia.The possible resistance mechanism includes the variations in the intergenic smeD-smeT and/or smeT codon sequence.

To study the role of BAMBI in adipogenesis, we constructed lentivirus interfering vector targeting on porcine BAMBI, packaged and infected the porcine preadipocyte. The differentiation state of preadipocyte was detected by Oil Red O staining and Oil Red O extraction assay and the expression levels of adipogenic marker genes were detected by Real-time qPCR and Werstern bloting. Results show that BAMBI expression was significant decreased after lentivirus infection, which was repressed more than 60% by shRNA2. Moreover, knockdown BAMBI increased the lipid accumulation of porcine preadipocyte and improved the expression of PPARγ (peroxisome proliferator-activated receptorγ) and ap2 (adipocyte protein 2). In summary, these data indicated that BAMBI inhibited adipocyte differentiation by facilitating the phosphorylation of ERK1/2.
Adipocytes ; cytology ; Adipogenesis ; Animals ; Cell Differentiation ; Membrane Proteins ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; PPAR gamma ; metabolism ; Phosphorylation ; Swine

Objective To investigate the effects of levofloxacin,ciprofloxacin,ceftazidime,pip-eracillin,cefoperazone/sulbactam,erythromycin,sulfamethoxazole and gentamycin on the bacterial biofilms of Stenotrophomonas mahophilia.Methotis Biofilm and conventional susceptibilities were determined for S.maltophilia isolates from 42 patients.The model of S.maltophilia biofflms in vitro was developed in the Mueller-Hinton broth--micmtiter inoculator or silica films.After antibiotic challenge plate 20 h,each plate was sonicated and the absorbance value at 620 nm(A620)was measured on a microtiter plate colorimeter be-fore and after incubation for 6 h.Then the biofilm inhibitory concentrations were calculated.Finally,based on the acquired data.the experiments of combinafion effects of erythromycin with the 3 antibiotic agents on the formed biofilms of 5 picked strains were designed and worked out.Results The sensitive rate of 42 S.maltaphilia to levofloxacin.sulfamethoxazole and piperacillin were 83.33%,66.67%and 54.76%,re-spectively.The bilfilm inhibitory concentrations were much higher than the corresponding minimal inhibitory concentrateion after formed biofflms.Conclusion Forty-two S.maltophilia are multi-resistant to antibiotic agent.And levofioxacin may have a better effect against biofilms compared with others.The inhibition effect of combination erythromycin with levofloxacin is more obvious among all the 3 antibiotic agents.

To clarify the function of miR-103 in the differentiation of porcine preadipocyte, we carried out real-time PCR to detect the expression pattern of miR-103 during adipogenesis, and clarified its expression tendency through cell differentiation. Then we used adenovirus that overexpressed miR-103 to infect porcine preadipocyte. Subsequently, mRNA and protein expression of adipogenesis marker--PPARgamma and aP2 was analyzed by real-time PCR and Western blotting. At last, Oil-Red O staining was used to detect lipids accumulation in the 8th day after adipogenic inducement. The expression of miR-103 increased during adipocyte differentiation; compared with the control, the preadipocyte infected by pAd-miR-103 had an elevated expression level of adipocyte marker gene PPARgamma, aP2, and obvious lipid droplet was seen in the 8th day after adipogenic inducement. These results showed that miR-103 can enhance adipogenesis in primary cultured porcine adipocytes.
Adenoviridae ; genetics ; metabolism ; Adipocytes ; cytology ; metabolism ; Adipogenesis ; genetics ; Animals ; Base Sequence ; Cell Differentiation ; MicroRNAs ; genetics ; metabolism ; Molecular Sequence Data ; PPAR gamma ; genetics ; metabolism ; Primary Cell Culture ; RNA, Messenger ; genetics ; metabolism ; Swine ; Transfection

Retinol-binding protein 4 (RBP4) is adipocyte-derived secreted adipokines and elevated RBP4 expression level was closely related to insulin resistance and type II diabetes mellitus. However, the exact mechanisms are unknown. To clarify the mechanism, RBP4 lentivirus particles were packaged to infect porcine preadipocytes. Then porcine preadipocytes were activated by insulin or induced model of insulin resistance. RBP4 interference efficiency and the gene expression of each treatment groups in PI3K/Akt pathways were examined by QRT-PCR and Western blotting. The result shows that RBP4 mRNA and protein expressions were suppressed more than 60% (P < 0.01). Furthermore, no matter under insulin stimulation or insulin resistance, RBP4 knockdown significantly increased the mRNA expressions of AKT2, PI3K, GLUT4 and IRS1 compared with the control. The protein phosphorylate levels of AKT2, PI3K, IRS1 arised, meanwhile enhanced the AKT2, PI3K, GLUT4 total protein expressions. Collectively, knockdown of RBP4 increased the insulin sensitivity through upregulated PI3K/Akt pathways related factors' expression and phosphorylation in porcine adipocytes. This research will provide a new idea to treat insulin resistance related diseases.
Adipocytes ; metabolism ; Animals ; Gene Knockdown Techniques ; Insulin Resistance ; physiology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Retinol-Binding Proteins, Plasma ; genetics ; pharmacology ; Signal Transduction ; Swine