OBJECTIVETo discuss the possible mechanism of drug resistance transmission between Staphylococcus and Escherichia coli.

METHODSThe chloramphenicol resistance plasmid of Staphylococcus aureus was extracted to transform the sensitive Escherichia coli, and the drug-resistant Escherichia coli were screened by drug sensitivity test.

RESULTSThe drug-resistant Escherichia coli were successfully obtained.

CONCLUSIONStaphylococcus may have a natural shuttle plasmid of drug resistance, which can transform Escherichia coli under specific conditions.

Drug Resistance, Bacterial ; genetics ; Escherichia coli ; drug effects ; genetics ; Plasmids ; Staphylococcus ; genetics ; Transformation, Bacterial

Bacteria ; drug effects ; genetics ; Drug Resistance, Bacterial ; genetics ; Plasmids ; Quinolones ; pharmacology

OBJECTIVETo investigate the proliferation of mouse splenic lymphocytes after shRNA mediated BTLA gene silence.

METHODSThree specific shRNAs and one nonspecific shRNA (scrambled) were ligated to pSilencer3.1-H1-neo plasmid and then subcloned into lentiviral vector pLB. Recombinant viral particles were harvested post-transduction to 293T cells. Mouse lymphocytes were infected with viral supernatant after 24 h incubation and continuously cultured till 4 days. Expression of eGFP was detected by fluorescence microscopy, efficiency of infection and expression of BTLA on lymphocyte cell by FCM. CCK-8 assay was used to detect the proliferation of lymphocytes.

RESULTSLentiviral expression vectors pLB-shRNA/BTLA were successfully generated. The lentiviral particles were correctly packaged. Expression of BTLA protein in specific shRNA group was significantly decreased comparing to those in control group. O.D. value at A(450) of lymphocytes stimulated by anti-CD3 antibody showed significant difference compared with normal BTLA group (P < 0.05), while there was no difference between ConA stimulated group and control (P > 0.05).

CONCLUSIONGene-specific shRNA can knockdown the expression of BTLA. The proliferation of lymphocytes stimulated by anti-CD3 antibody after RNAi demonstrates significant enhancement as compared to the unstimulated lymphocytes, while stimulated by ConA showed no difference compared to normal lymphocytes.

Animals ; Genetic Vectors ; Lentivirus ; genetics ; Lymphocytes ; drug effects ; Mice ; Plasmids ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVESTo investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells.

METHODSHBx gene fragment was amplified from subtype adr HBV plasmid by PCR, and inserted into Hind III and Kpn I sites of green fluorescent protein (GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP/HBx. The pEGFP-C1 and pGFP-HBx were introduced into HepG2 cells by Lipofectamine 2000 to obtain HepG2 cells expressing GFP. GFP-HBx fusion protein was selected using G418. The expression of HBx gene was demonstrated by RT-PCR analysis. HepG2, HepG2/GFP and HepG2/GFP-HBx cells were treated with adriamycin (2.5 microg/ml), and apoptosis of the cells was determined by their morphological changes, trypan blue exclusion, and flow cytometry analysis.

RESULTSUnder a fluorescence microscope, visible expression of GFP and GFP-HBx fusion proteins were observed in HepG2/GFP and HepG2/GFP-HBx cells, even after growing over 70 generations. RT-PCR analysis showed that HBx gene was expressed in HepG2/GFP-HBx cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in HepG2 and HepG2/GFP cells while no significant cell death was observed in HepG2/GFP-HBx cells. Flow cytometry analysis showed that apoptosis rates in HepG2/GFP-HBx (3.94%) cells were significantly lower than those in HepG2 (59.03%) and HepG2/GFP cells (61.38%) at 36 hours after the adriamycin treatment (P < 0.01). No significant differences of apoptosis rates of HepG2/GFP-HBx (3.94%) and of the untreated cells (2.12%, 2.78%, 2.55%) (P > 0.05) were observed.

CONCLUSIONA HepG2 cell line expressing GFP and GFP-HBx fusion proteins was successfully established. HBV X protein blocks adriamycin-induced apoptosis of these HepG2 cells.

Apoptosis ; drug effects ; Doxorubicin ; pharmacology ; Hep G2 Cells ; Humans ; Plasmids ; Trans-Activators ; genetics

Expression strain of des-pGlu1-brazzein was constructed and the conditions using lactose as inducer was also optimized. The Influences of three factors which were lactose concentration, induction time and inducing temperature on the growth of strain and on the yield of des-pGlul-Brazzein was analyzed in detail. The result indicated that high lactose concentration inhibit the growth of strains (P < 0.01) but made no difference on expression of target protein between 0.5%-5% (P > 0.05), Biomass would be improved as time passed (P < 0.01), but the yield of target protein didn't increase obviously at 30 degrees C compared with at 37 degrees C. Further result showed that the greater expressed level of des-pGlul-Brazzein, as high as about 20% of total cell protein, could be achieved after the strain had been induced with 0.5% lactose under 28 degrees C - 30 degrees C for 4 h.
Dose-Response Relationship, Drug ; Escherichia coli ; drug effects ; genetics ; Isopropyl Thiogalactoside ; pharmacology ; Lactose ; pharmacology ; Plant Proteins ; genetics ; Plasmids ; genetics ; Temperature ; Time Factors ; Up-Regulation ; drug effects

OBJECTIVESTo investigate whether antisense human telomerase reverse transcriptase (hTERT) could inhibit the activity of telomerase and the proliferation of K562 cells.

METHODSThe antisense plasmid was constructed by reverse insertion of hTERT PCR product into plasmid pLNCX-neo. Then the constructed plasmid was introduced into K562 cells by liposomes-mediated DNA transfection. The inhibition effects of telomerase on the proliferation of K562 cells were analyzed by MTT and colony formation assay, the telomerase activity of K562 cells by TRAP-PCR ELISA methods.

RESULTSThe growth rate of antisense hTERT transfected K562 cells was significantly lower than those of the controls, and the colony formation capacity of the transfected cells decreased significantly (P < 0.01), the colony number is (100.33 +/- 7.57)/10(3) cells, (92.67 +/- 5.86)/10(3) cells and (50.33 +/- 6.11)/10(3) cells for control K562 cells, K562 neo cells and antisense hTERT transfected HL60 cells, respectively. The telomerase activity of antisense hTERT transfected K562 cells was significantly inhibited.

CONCLUSIONThe expression of an antisense sequence to the mRNA sequence of telomerase protein subunit can inhibit the activity of telomerase, slow the cell growth and inhibit the capacity of colony formation of K562 cells.

Cell Division ; drug effects ; Humans ; K562 Cells ; Plasmids ; genetics ; RNA, Antisense ; genetics ; pharmacology ; RNA, Messenger ; genetics ; Telomerase ; drug effects ; genetics ; metabolism ; Transfection

A gene replacement/disruption system of Amycolatopsis mediterranei U32 was developed based on the established electroporation conditions as well as appropriate selective markers. Through two-step selection, ahbas gene in U32 was replaced by a promoterless alpha-amylase gene constructed on the plasmid pDK110 of E. coli. The first single-crossover and the second double-crossover frequencies were approximately 0.5%-0.7% and 2%, respectively. Denaturation of the plasmid pDK110 increased the integration frequency about 7-10 folds, while electric shock treatment of the single-crossover recombinants increased the frequency of second crossover recombination about 5 folds. Employing denatured DNA fragments containing an apramycin-resistance gene flanked with regions of the respective genes, One-step disruption of rifO and amrA genes of U32 was also achieved with an efficiency of 30-50 transformants per microgram of DNA.
Actinomycetales ; drug effects ; genetics ; growth & development ; DNA, Bacterial ; genetics ; Drug Resistance, Microbial ; genetics ; Genes, Bacterial ; genetics ; Mutagenesis ; Nebramycin ; analogs & derivatives ; pharmacology ; Plasmids ; genetics ; Recombination, Genetic

This study was aimed to explore the role and mechanism of IFN-γ in the regulation of hemopoiesis in mice. Murine IFN-γ fragment was amplified from murine splenic cells with RT-PCR and plasmid pCDH1-mIFN-γ-EF1-copGFP (pCDH-mIFN-γ-GFP) was constructed. Plasmids pCDH-mIFN-γ-GFP and pCDH1-EF1-copGFP (pCDH-GFP) together with packaging plasmids pPACK-A, pPACK-B and pPACK-C were respectively transfected into 293T cells by using a method of calcium phosphate precipitation to produce lentivirus. Bone marrow mononuclear cells (BMMNC) from male C57BL/6J mice were transfected with the lentiviral vector pCDH expressing mIFN-γ and green fluorescent protein (GFP). The cells were cultured in M3434 semi-solid medium for colony formation assay and transplanted into lethally-irradiated mice through caudal vein injection, and the peripheral blood cell counts and GFP were monitored regularly after transplantation. The results showed that lentiviral vector pCDH-mIFN-γ-GFP was constructed successfully and 293T cells transfected with mIFN-γ secreted mIFN-γ. Transfection of mIFN-γ into BMMNC decreased colony formation, colony number of the mIFN-γ group was significantly less than that of the control group. The recovering of circulating blood cell parameters in mIFN-γ transplantation group was significantly later than control group. GFP positive cells could be detected in the peripheral blood at 8 weeks after transplantation. It is concluded that mIFN-γ may inhibit the colony-forming capacity of transduced BMMNC and delay the hematopoietic reconstitution.
Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Line ; Genetic Vectors ; Hematopoiesis ; drug effects ; genetics ; Interferon-gamma ; genetics ; pharmacology ; Lentivirus ; genetics ; Male ; Mice ; Mice, Inbred C57BL ; Plasmids ; Transfection

OBJECTIVETo construct a lamivudine-resistant plasmid containing 1.2 unit genome of duck hepatitis B virus and identify its replication and drug-resistance in avian LMH hepatica cells.

METHODSThe recombinant plasmid PBS-DHBV1.2 was constructed using the 1.2-genome length DHBV DNA sequence from a dimer DHBV genome with pcDNA3.1 as the template. With site-directed mutagenesis, we obtained PBS-DHBV1.2-M512V plasmids with polymerase gene mutation from PBS-DHBV1.2. Two constructed plasmids were transiently transfected into LMH cells using FuGENETM6 transfection reagent and cultured in the medium containing different concentrations of lamivudine. Southern blot hybridization was performed to detect DHBV replication intermediates.

RESULTSPCR amplification, restriction digestion and plasmid sequencing all confirmed successful construction of PBS-DHBV1.2-M512V recombinant plasmid. Southern blot analysis identified the presence of all the expected DHBV replication intermediates in LMH cells. The replication capacity of the mutant plasmid was decreased by 2.7 times compared with that of the wild plasmid. The IC(50) of lamivudine was 37.12∓8.81 ng/ml for the mutant, greater than that of the wild plasmid (10.90∓4.80 ng/ml).

CONCLUSIONCompared with the wild plasmid, the mutant plasmid has a lower replication capacity and sensitivity to lamivudine in vitro.

Antiviral Agents ; pharmacology ; Drug Resistance, Viral ; drug effects ; genetics ; Hepatitis B Virus, Duck ; drug effects ; genetics ; Lamivudine ; pharmacology ; Mutagenesis, Site-Directed ; Plasmids

Resistant gonococci are very prevalent in many countries, particularly in Asia. This study was conducted to determine the trend of resistance, the effect of decreasing the ciprofloxacin susceptibilities of gonococci on the prevalence of penicillinase-producing N. gonorrhoeae (PPNG), and to compare the epidemiology of strains with the previous studies. A total of 602 strains of gonococci were isolated from prostitutes in 1997-1999. Antimicrobial susceptibility was tested by NCCLS disk diffusion and agar dilution methods. For epidemiologic analysis, vplasmid analysis and pulsed-field gel electrophoresis (PFGE) were performed. The proportion of PPNG remained high (79%), and the strains with decreased susceptibility to ciprofloxacin increased significantly from 67% in 1997 to 84% in 1999. Compared to our previous study, the PFGE patterns were similar, while the proportion of strain with the 3.2-MDa plasmid markedly decreased. In conclusion, a rapid increase in ciprofloxacin-nonsusceptible strains may suggest difficulties in the treatment of gonococcal infections in the near future with the drug. The recent decrease of PPNG with the 3.2-MDa plasmid may suggest that there is an epidemiological change in gonococcal infections, and the prevalence of related PFGE patterns suggests the dissemination of a few clones among the high risk populations.
DNA/genetics* ; DNA/drug effects ; Drug Resistance, Microbial/genetics* ; Electrophoresis, Gel, Pulsed-Field ; Endonucleases/pharmacology ; Female ; Human ; Neisseria gonorrhoeae/genetics* ; Plasmids/genetics*