Objective To dynamically observe changes of IgG, its subclasses and IgE in sera of mice by immunization with mixed recombinant of BCG-Em Ⅱ/3 and BCG-Em14-3-3 vaccine of Echinococcus multilocularis (Era). Methods Forty Balb/c mice of 12-14 week old and 20-25 g weight were intranasally vaccinated by the vaccine, 4 mice were killed randomly by the weight on 0,2,4,6,8,10,12,14,16 and 18 weeks of immunization respectively, sera were gathered from the eyeball to measure IgG, its subclasses and IgE by routine ELISA. Results Levels of IgG, IgG2a and IgG2b in the sera of mice increased obviously on 2-18 weeks, reached the highest level on 10, 4 and 4 weeks respectively, the value was 0.095±0.033,0.022±0.001,0.023±0.003 respectively, as compared with the value on 0 week(0.030±0.013,0.012±0.004,0.013±0.004), the difference being statistically significant(q=2.95,4.87,2.81 respectively, P < 0.01 or P < 0.05); levels of IgG1, IgG3 and IgE in the sera of mice decreased remarkably on 2-18 weeks,came to the lowest level on 4,2,6 weeks respectively, the value was 0.031±0.004,0.136±0.002,0.114±0.002 respectively, as compared with the value on 0 week(0.192±0.007, 0.175±0.013,0.024±0.003), the difference being statistically significant (q =5.16,4.93,5.32 respectively, P < 0.01 or P < 0.05). Conclusion Helper T cell(TH) Ⅰ response is induced in mice by mixed recombinant of BCG-Em Ⅱ/3 and BCG-Em14-3-3 vaccine on early immunization.

Objective To investigate the changes of cytokines of splenocytes in mice immunized with recombinant BCG-Em Ⅱ/3 vaccine of Echinococcus multilocularis(Em)and consequently challenged with Em protoscoleces.Methods Balb/c mice were subcutaneously or intranasally vaccinated and challenged with Em were separated and cultured with EmAg,ConA or PHA,respectively.The supematants were gathered to measure the levels of IL-2,IFN-γ,TNF-α.and IL-4 by ELISA Kits.Results The levels of IL-2,IFN-γ,TNF-α and IL-4 in the subcutaneous group were(34.6±2.7),(34.5±2.8),(265.0 ±0.0)and(9.8±2.6)ng/L respeetively:those in the intranasal group were(32.5±2.2),(33.6±2.7),(130.0±0.0)and(10.4±27)ng/L respectively;those in the control were(25.0±1.9),(30.0±0.0),(10.0±0.0)and(12.5±2.7)ng/L,respectively:there were statistical differences between the immunized groups and control group(P＜0.01 or＜0.05);The level of TNF-α in the subcutaneous group was higher than that in the intranasal group.Conclusion Th1 response has been induced in mice vaccinated with rBCG-Em Ⅱ/3 vaccine and challenged with Em protoscoleces.

Objective To dynamically observe the changes of subsets of splenocytes in mice by immunization with recombinant BCG-Eg95 vaccine of Echinococcus granulosus(Eg).Methods Balb/c mice were divided randomly into 3 groups according to their weishts:intranasal group.per os group and PBS control.The mice were vaccinated intranasally or orally by the vaccine respectively in experimental groups.and the control mice were given phosphate buffer saline intranasally.These mice were killed to get spleen on 0,2,4,6,8,10,12, 14,16 and 18 week of immunization,respectively.Splenocytes were separated to measure subsets of CD4+ and CD8+T ceUs by FACsort.Results There were marked differences in ratio of CD4+ and CD8+ subsets among the different groups(F value were 21.56 and 22.08 respectively,P＜0.05).There were very marked differences in ratio of CD4+ and CD8+ subsets in different weeks(F value were 5.75 and 6.29 respectively.P＜0.01).In the intranasal group,CD4+ and CD8+ T subsets increased obviously in 6～18 weeks and 12 weeks,and reached the highest level on 10 and 12 week,espectively.Their values were 0.348±0.013 and 0.090±0.003.respectively.There were marked or very marked differences(q value were 7.32 and 5.32 respectively,P＜0.01 or＜0.05)in comparison with 0 week(0.230±0.022 and 0.069±0.015).In the oral group,CD4+and CD8+ subsets rose reinarkablv on 6-16 weeks and 8-18 weeks,achieved the hishest level on 10 and 16 weeks,respectively.Their vahes were 0.405± 0.006 and 0.096±0.004,respectively.There were marked or very marked difference(q value were 7.53 and 5.35 respectively,P＜0.01 or＜0.05)in comparison with week 0(0.230±0.022 and 0.069±0.015).Conclusion CD4+and CD8+T subsets may play an important role in immune response induced in mice by rBCG-Eg95 vaccine.

In this study,nearly 200 endophytic bacteria were isolated from different part of Huperzia serrata, over 60 bacterium with clear antifungal activity were selected from those cultures.Among them,strain H-6 exhibited the highest antifungal activity which was strongly inhibits the growth of many plant pathogenic fungi such as Sclerotinia scleroliorum,Fusarium graminearumt,Sclerotinia libertiana,Phytophthora capsici Leonia and Sesame fusarium wilt.According to the characteristics of morphology,physiology and biochem- istry tests and the comparison of 16S rDNA sequence,the strain H-6 was similar to the Burkholderia.So strain H-6 was identified as Burkholderia sp.H-6.The results also showed that Burkholderia sp.H-6 was markedly different from Burkholderia cepacia that was applied widely in agriculture as antagonistic bacteria. The medium and culture conditions of the strain all were optimized by single factor and orthogonal experiments.In the investigation of the culture condition,growth was carried out in a basal medium(potato juice)and gradually supplemented with the various ingredients to be investigated.The major ingredients be- ing investigated included carbon sources and nitrogen sources.The optimal antifungal activity production condition is growth in a medium(potato juice with 2.5%mannitol and 0.1%NaNO3),initial pH 4.0 at 28℃.

Objective To investigate the importance of plasmid-mediated quinolone resistance in the development of quinolone resistance in clinical isolates of gram-negative bacteria.Methods A total of 541 consecutive clinical isolates of gram-negative ba- cilli resistant or intermediate to ciprofloxacin were screened for the qnrA gene by PCR.Conjugation experiments were carried out with azide-resistant E.coli J53 as a recipient.The aac(6')-Ib-cr gene was detected.The mutations in the quinolone-resist- ance-determining region (QRDR) of the gyrA and parC genes were identified in qnrA positive strains.Results qnrA was identi- fied in 7 of the 541 strains.Among the qnrA positive strains,5 were Enterobacter cloacae.No qnrA was detected in nonfer- menters.Quinolone resistance was transferred in 4 of 7 qnrA positive strains.Transconjugants had 12-to 125-fold increases in MIC of ciprofloxacin relative to that of the recipient.Seven strains contained qnrA with a nucleotide sequence identical to that originally reported.Two transconjugants with higher ciprofloxacin MICs contained aac(6')-Ib-cr gene.Mutations occurred in the QRDR of the gyrA and parC genes in 5 PCR-positive clinical strains.Conclusions Transferable plasmid-mediated quinolone resistance associated with qnrA is highly prevalent in clinical strains of Enterobacter spp.aac(6')-Ib-cr gene and mutations in the quinolone targets may co-exist with qnrA,which may contribute to the further increase of resistance to quinolones.

The type of basic media and the contents of plant growth substances were investigated by orthogonal design experiment,and also the effects of different culture conditions on the growth of suspension cells and the accumulation of total flavonoids in Eucommia ulmoides were studied.The results showed that B5 medium supplemented with 0.5mg/L NAA,0.6mg/L 6-BA and 30g/L sucrose,at initial pH 5.0～5.5,20g(FW)/L inoculation quantity and 110 r/min of rotation speed was a preferable culture conditions for E.ulmoides suspension cells growth and flavonoids synthesis.The results of metabolic kinetics analysis for E.ulmoides cell suspension culture showed that the logistic and Luedeking-Piret equations can be used for describing the kinetics of cell growth,sucrose consumption and flavonoids production during the process.The maximum specific growth rate(?m),the actual growth yield based on sucrose(YG) and maintenance coefficient(m) were 0.417/d,0.619g/g and 0.0206g/(g?d-1) respectively.All these outcomes could give a basis for establishing the suspension cell culture of E.ulmoides and production of the natural active components in large-scale.

This study is to investigate the effect of osthol on osteoclasts' activity, bone resorption as well as apoptosis in vitro, and explore the mechanism of osthol in preventing osteoporosis. Osteoclasts were separated from long-limb bones of new born rabbits, cultured in 24-well plate with glass slices and bone slices, and treated by 1 x 10(-5) mol x L(-1) osthol. Osteoclasts were identified by observing live cells with phase contrast microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits. The numbers of bone resorption pits were counted as well as the surface area of bone resorption on bone slice. Osteoclasts were stained with acridine orange to detect the cell apoptosis. The ratio of apoptotic osteoclasts was observed under fluorescence microscope. The gene expression of RANKL, OPG, TRAP and p-JNK1/2 protein expression were examined using real time PCR and Western blotting, respectively. Comparing with the control group without osthol, the rates of apoptotic osteoclasts increased obviously and the number and area of bone resorption pits decreased evidently with 1 x 10(-5) mol x L(-1) osthol. There is significant difference between control group and experiment group treated by 1 x 10(-5) mol x L(-1) osthol. Therefore, the osthol through RANK+RANKL/TRAF6/Mkk/JNK signal pathway inhibits the osteoclasts activity, enhances osteoclasts apoptotic and inhibits the bone resorption.
Acid Phosphatase ; metabolism ; Animals ; Apoptosis ; drug effects ; Bone Resorption ; Cells, Cultured ; Cnidium ; chemistry ; Coumarins ; isolation & purification ; pharmacology ; Gene Expression ; Isoenzymes ; metabolism ; Mitogen-Activated Protein Kinase 8 ; metabolism ; Mitogen-Activated Protein Kinase 9 ; metabolism ; Osteoclasts ; metabolism ; pathology ; Osteoprotegerin ; metabolism ; Phosphorylation ; Plants, Medicinal ; chemistry ; RANK Ligand ; metabolism ; Rabbits ; Seeds ; chemistry ; Signal Transduction ; Tartrate-Resistant Acid Phosphatase

Objective To determine whether cleavage developmentally retarded embryos have not cleaved during a 24 hour period could develop into blastocysts and produce hESC cell lines.Methods A total of 120 such embryos were cultured to blastocyst stage by sequential culture.Blastocysts formation rate and quality of blastocyst were detected under microscope.The relation between blastocyst formation rate and blastomere number,the fragment of blastomere and blastomere symmetry were analyzed by stepwise Logistical regression analysis.Inner cell masses(ICMs)were isolated by immunosurgery.Colonies derived from the ICMs were passed every 4-7 days and the derivatives were passaged and identified.Results A total of 22 blastocysts were obtained from 120 embryos.The blastulation rate was 18.7%.Early blatocyst, blastocyst,full blastocyst,expanded blastocyst,hatching blastoeyst and hatched blastocyst accounted for 5.9%,23.5%,35.3%,23.5%,5.9%,and 5.9% respectively.The grade of ICM and trophoblast was mostly scored C or B.Blastocyst formation rate was related to cell number and blastomere symmetry but not fragment.Immunosurgery resulted in the formation of 7 ICMs and 3 primary colonies,which produced 2 cell lines.The cell lines satisfied the criteria that characterize pluripotent hESC cells.Undifferentiated cells were positive for AKP,SSEA-4,TRA-1-60,and TRA-1-81.It could continue to proliferate in vitro and form embryoid bodies when cultured in suspension.It had capability to form teratoma in SCID mice.Both cell lines had normal karyotypes after 45 and 34 passages respectively.Conclusions Our results suggest that a subset of developmentally retarded embryos can form blastocysts and give rise to hESC cell lines.

OBJECTIVEEnvironmental estrogens at an elevated concentration are known to produce adverse effects on human and animal life. However, the majority of researches have been focused on industrial discharges, while the impact of livestock wastes as a source of endocrine disrupters in aquatic environments has been rarely elucidated. In order to investigate the contribution of environmental estrogens from livestock, the estrogenic activity in water samples from a farm wastewater treatment plant was analyzed by a recombinant yeast screening method.

METHODSThe extracts prepared from 15 selected water samples from the farm wastewater treatment plant, among which 6 samples were from pre-treatment section (influents) and 9 from post-treatment section (effluents), were analyzed for estrogenic activity by cellar bioassay. Yeast cells transfected with the expression plasmid of human estrogen receptor and the Lac Z reporter plasmid encoding beta-galactossidase, were used to measure the estrogen-like compounds in the farm wastewater treatment plant.

RESULTSThe wastewater samples from influents showed a higher estrogenic potency than the effluent samples showing a low induction of beta-galactossidase relative to solvent control condition. By comparison with a standard curve for 17 beta-estradiol (E2), estrogenic potency in water samples from the influents was calculated as E2-equivalent and ranged from 0.1 to 150 pM E2-equivalent. The estrogenic potency in water samples from the effluents was significantly lower than that in the influents, and 7 water samples had less detectable limit in the total of 9 samples.

CONCLUSIONYeast bioassay of estrogenic activity in most of the samples from the farm wastewater after disposal by traditional sewage treatment showed negative results.

Agriculture ; Biological Assay ; methods ; Dose-Response Relationship, Drug ; Estrogens ; chemistry ; toxicity ; Genetic Engineering ; Industrial Waste ; analysis ; Plasmids ; genetics ; Saccharomyces cerevisiae ; drug effects ; genetics ; Waste Disposal, Fluid ; methods ; Water Pollutants, Chemical ; analysis

Omega-3 polyunsaturated fatty acids (PUFAs) have been broadly investigated and shown to exert many preventive and therapeutic actions besides their important role in maintenances human health and normal development. In mammals, the level of omega-3 PUFAs is relatively too low compared with omega-6 PUFAs, which metabolically and functionally distinct from omega-3 PUFAs and often have important opposing physiological functions. Either the inefficiency of omega-3 PUFAs or the excess of omega-6 PUFAs will cause many healthy problems. So methods have been sought to increase the amount of omega-3 PUFAs and to improve the omega-6/omega-3 ratio in body. In this study, the sFat-1 gene, which putatively encodes a omega-3 fatty acid desaturase, was chemically synthesized according to the sequence from Caenorhabditis briggssae (with codon usage modified), and constructed into a mammal expression vector pcDNA3. 1-sFat1-EGFP. This vector was introduced into CHO cells by lipid-mediated transfection, and it's expression quickly and effectively elevated the cellular omega-3 PUFAs (from 18-carbon to 22-carbon) contents and dramatically improved the ratio of omega-6/omega-3 PUFAs. Cellular lipids extracts from stably selected cells were analyzed with GC-MS and the results showed that amount of total omega-6 PUFAs dropped from 48.97% (in GFP cells)to 35.29% (in sFat-1 cells), whereas the amount of total omega-3 PUFAs increased from 7.86% to 24.02%, respectively. The omega-6/omega-3 ratio also dropped from 6.23 to 1.47. These data demonstrates the Caenorhabditis briggssae omega-3 Fatty Acid Desaturase gene, sFat-1, was synthesized successfully and can produce omega-3 PUFAs by using the corresponding omega-6 PUFAs as substrates, which shows its potential for use in the production of omega-3 PUFAs in transgenic animals.
Animals ; CHO Cells ; Caenorhabditis ; enzymology ; genetics ; Cricetinae ; Cricetulus ; Fatty Acid Desaturases ; genetics ; physiology ; Fatty Acids ; analysis ; Plasmids ; Polymerase Chain Reaction