According to the sequence of P450 cDNA of Eleutherococcus senticosus, specific primers were designed. Frokaryotic ex pression vector pET30a-P450 was constructed and the prokaryotic expression conditions were optimized. Results showed that the BL21 after being transformed with the recombinant expression vector accumulated the high amount of recombinant protein. SDS-PAGE analysis showed that the recombinant protein was about 53 kDa. The recombinant accumulated the highest amount of recombinant protein af ter IPTG (1 mmol x L(-1)) at 27-37 degrees C for 24 h. Consequently P450 gene of E. senticosus could be expressed successfully by prokaryotic expression vector pET30a-P450. Induction temperature, IPTG concentration, medium type and amount of induction time could all influence the expression of target protein, but the impact strength was different.
Cloning, Molecular ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Eleutherococcus ; enzymology ; genetics ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plasmids ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

In order to find the characteristics of two members of gene family of squaleneexpoxidase (SE) , a quantitative real time PCR method was developed to analyze the expression of Eleutherococcus senticosus SE1 and SE2 gene from different growth periods and in different organs. The result indicated that all the expression of SE2 more than SE1 in the whole growth period and organs of E. senticosus. And in the whole growth period, expression of SE1 showed a low-high-low characteristic. Both expression of SE2 and growth period showed the same trend. The lowest content of the expression was in the roots. SE1 expression have been improved more than SE2 when treated with MeJA. The expression of E. senticosus SE1 and saponins content had significantly positive correlation (P < 0.05) and the correlation coefficients was 0. 858, while the correlation was not significant for SE2. That indicated that SE1 played a key enzyme gene in the biosynthesis of triterpenoidsaponins
Eleutherococcus ; chemistry ; enzymology ; genetics ; growth & development ; Gene Expression Regulation, Plant ; Peroxidase ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Saponins ; analysis ; metabolism ; Transcriptome

Objective To investigate the role of plasmid pYC on proteome of Yersinia pestis. Methods Two dimensional electrophoresis was performed to strains of Yersinia pestis with and without the pYC plasmid, and differential proteins were identified by mass spectrometry. Results More than 500 protein spots of Yersinia pestis with and without plasmid pYC were recognized,and their protein profiles were generally similar. The chaperone GroEL was highly expressed in strains with plasmid pYC, whereas the protein GroEL was not encoded by plasmid pYC. ConclusionsPlasmid pYC has an impact on proteome of Yersiniapestis. The proteins of pYC-p10 and pYC-p11 encoded by plasmid pYC may regulate the expression of GroEL.

Objective To discuss methods of decoration reconstruction for finger defect in emergency and to observe the elinical effects.Methods Of the 41 cases of finger injuries of different degrees,15 were repaired with part of the skin flaps of the big toenails or skin flaps of the second toenalis,8 were repaired with part of the skin flaps of the big toenails,7 were reeonstructed with the second tiptoes,11 were repaired with the abdominal skin flaps of the big toes or lateral flaps of the second toes.Results All the 41 fingers sur- vived.One skin flap of the big toe was somewhat swelling and a decorating operation was performed.The 4～18 months of follow-up visitation of the rest cases revealed good function and shapes.No obvious functional ab- norality was found in the donating feet.Conclusion Various kinds of decoration reeonstruetion for finger defects are available to recover the hand shape and function as much as possible.

Objective To investigate the sensitivity and variability factors that were assessed on the forced swimming test (FST) using BALB/C and Kunming mice. Methods The immobility time of FST was compared using Kunming and BALB/C mice in different experimental conditions including circadian rhythm ( day and night) ,gender and water temperature ( 12,22 and 32℃ ) . Results (①) The immobility time of BALB/C during the daytime( ( 142.42 ± 33.58) s) was significantly increased than that at night ( ( 104.89 ± 34.33 ) s). (② The immobility time of Kunming mice( (91.95 ± 40.32) s) was significantly decreased than that of BALB/C mice ( ( 142.42 ± 33.58 ) s). (③)The immobility time under the water temperature of 22 C ( ( 92.24 ± 25.81 ) s) was significant longer than that under the water temperature of 32C ( (60.72 ± 11.11 ) s). Conclusion BALB/C stain,male mice,daytime and water temperature of 22℃ should be chosen in the FST.

0.05)and significantly delayed reaction time,(305?109)ms vs(212?70)ms(P

OBJECTIVETo observe MET-associated alteration during the trans-differentiation from MSCs to neuron-like cells, and to explore the possible molecular mechanism.

METHODSBone marrow MSCs were isolated from rat femur and purified in continuous cell culture. After induced differentiation to neuron-like cells by the combination of butylated hydroxyanisole (BHA) and dimethyl sulfoxide (DMSO), cells were tested by comparative polymerase chain reaction (PCR) for the relative expression of MET biomarkers and transcription factors, and for cell cycle by flow cytometry. Meanwhile, target genes of Wnt/β-catenin pathway were also analyzed by comparative PCR to determine the possible involvement.

RESULTSIn MSC-induced neuron-like cells, MET-associated transcription factors such as Snail, Slug, ZEB1, ZEB2, and Twist were significantly attenuated in expression level. The Mesenchymal marker Vimentin expression level was increased. Membrane protein E-cad was slightly down-regulated, while N-cad level was marginally elevated. Percentage of proliferating cells (S phase in cell cycle) markedly shrank from 40.42% for MSCs to 6.76% for MSC-derived neuron. Additionally, Wnt/β-catenin target genes β-catenin and c-myc were decreasingly expressed.

CONCLUSIONChemically induced trans-differentiation from MSC to neuron caused similar MET-featured alteration in gene expression and proliferation to known MET, which might be underlied by deactivation of Wnt/β-catenin pathway.

Animals ; Epithelial-Mesenchymal Transition ; Mesenchymal Stromal Cells ; cytology ; Neurons ; cytology ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Rats ; Wnt Signaling Pathway ; beta Catenin ; metabolism

Objective To construct a prokaryotic expression vector in BL21 to secretorily expressα-Cyclodextrin Glycosyltransferase(α-CGTase). Methods α-CGT gene was amplified from Bacillus macerens genome by PCR.pET26b and α-CGT gene were connected after digested with Nco I, Xho I respectivly, and then transformed into Escherichia coli BL21 strain.α-CGTase was expressed in fermentation culture medium and AA-2G was prepared by using α-CGTase, VC and starch.Results α-CGTase was expressed secretorily and the enzyme activity was up to 120 U/mL.AA-2G was prepared by the biotransformation of VC and starch using α-CGTase which proved to be correct by HPLC.Conclusion AA-2G was prepared by using self-madeα-CGTase, after optimized the preparation conditions the yield of AA-2G was 17.46 g/L, and the conversion rate reached 58.2%(mg/mg).