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

Adenocarcinoma, Papillary ; blood supply ; metabolism ; pathology ; Adult ; Aged ; Carcinoma, Pancreatic Ductal ; blood supply ; metabolism ; pathology ; Chemokine CXCL12 ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lymph Nodes ; metabolism ; Lymphatic Metastasis ; Male ; Microvessels ; pathology ; Middle Aged ; Neoplasm Staging ; Neovascularization, Pathologic ; metabolism ; pathology ; Pancreas ; metabolism ; Pancreatic Neoplasms ; blood supply ; metabolism ; pathology ; Receptors, CXCR4 ; metabolism

Porcine circovirus type 2 (PCV2) can cause immunosuppression on herds. PCV2, as an essential pathogen of PCV2-systemic disease (PCV2-SD), has caused considerable economic losses in pig industry worldwide. Here we review and address the evolution, viral protein and immunolesion of PCV2 and preventive techniques of PCV2-SD.
Animals ; Circoviridae Infections ; veterinary ; Circovirus ; genetics ; Phylogeny ; Swine ; Swine Diseases ; virology ; Viral Proteins ; genetics

Objective To investigate the changes of serum bilirubin level in elderly patients with acute cerebral infarction and its significance.Methods 164 hospitalized elderly patients,who suffered from acute cerebral infarction within 1 week after onset,were divided into 2 groups according to age:group A aged over 60 years(n=85) and group B aged 40-60 years(n=79),and 66 healthy subjects aged over 40 years were collected as controls(group C).Serum bilirubin levels in all subjects were determined.The ratio of pulse pressure over mean arterial pressure(PP/MAP) in group A and B was calculated.Nerve function scores in the three groups were detected before and after 2 weeks of treatment.Meanwhile,the data of risk factors including blood glucose,blood pressure,blood lipids,smoking and drinking in group A and B were collected.Results Compared with group C,serum total,direct,indirect bilirubin levels were increased in group A and B(both P＜0.01),and the change was smaller in group A than in group B(P＜0.05).The nerve function scores was lower in group A than in group B before and after treatment [(35.2±12.6) vs.(44.3±7.9),(40.7±9.1) vs.(51.3± 4.1),t=5.58,9.73,both P＜0.01],but PP/MAP and the numbers of risk factors were higher in group A than in group B [(0.46±0.06) vs.(0.38±0.06),93.01 vs.71.20,both P＜0.01].There were no significant correlations of serum total,direct and indirect bilirubin levels with nerve function scores in group A or B(all P＞0.05).Conclusions Serum bilirubin level is increased in patients with acute cerebral infarction,but the endogenous antioxidant capacity is decreased because of aging,multiple risk factors and more serious atherosclerosis in elderly patients,and the increment of bilirubin level is relatively smaller in acute cerebral ischemia,leading to the reduced protective effect against stress.Serum bilirubin level may influence the prognosis in patients with acute cerebral infarction.

BACKGROUND: The particular bystander effect of suicide gene can remarkably kill tumor cells. Meanwhile, it can be used together with radiotherapy as well as immune gene therapy, and overcome the defect of low gene transduction efficiency.Cytosine deaminase (CD) can generate a powerful bystander effect.OBJECTIVE: To observe the effect of a eukaryotic expression plasmid plRES2-AcGFP1-CD mediated by liposome transfected into bone marrow mesenchymal stem cells (BMSCs) and its gene expression.DESIGN, TIME AND SETTING: A cytologic experiment at genetic level was performed at Research and Development Center of Stem Cell and Tissue Engineering, Dalian University of Technology from May to December 2007.MATERIALS: A total of 6 C57BL mice of SPF degree and weighing 18-20 g were used for separation and culture of BMSCs.Escherichia coli DH5a was provided by Research and Development Center of Stem Cell and Tissue Engineering, Dalian University of Technology. Lipofectamine~(TM) 2000 was provided by Invitrogen, China.METHODS: The DNA plasmids were extracted from transformed Escherichia coli DH5a. plRES2-AcGFP1-CD plasmid was identified by BamHI/Xhol double digestion. The BMSCs derived from mouse femur and tibia were cultured and purified by adhesion method in vitro. Signal cell suspension prepared by BMSCs of the third generation was cultured by adding fluorescence-labeled CD44, CD45, CD90 and CD105 antibodies. BMSCs of the third generation were transfected by LipofectamineTM 2000 mediation.MAIN OUTCOME MEASURES: Identification of recombinant plasmids; the expressions of surface markers on BMSCs were detected by flow cytometry; the expressions of cytosine deaminase gene were observed at 36 and 48 hours after transfection under fluorescent inverted microscope.RESULTS: After agarose gel electrophoresis, a band appeared at 1.0-1.5 kb of the digested products of plRES2-AcGFP1-CD plasmids, and the band was accorded with the length of cytosine deaminase gene in the length. Flow cytometry demonstrated that the cells were negative for CD45 but positive for CD44, CD90 and CD105. Fluorescent inverted phase contrast microscope suggested that at 36 hours after plRES2-AcGFP1-CD gene transfection an expression of green fluorescent protein was found in the BMSCs; additionally, at 48 hours after transfection, the expression of green fluorescent protein remained and the intensity was remarkably increased.CONCLUSION: The liposome-mediated plRES2-AcGFP1-CD gene successfully expressed in BMSCs, and the expression peaked at 48 hours after transfection.

Objective:To explore the expression of MMP-3 and extracellular matrix metalloproteinase inducer(EMMPRIN)in benign and malignant breast lesions and its relevance.Methods:Using immunohistochemical method,we examined the expression of MMP-3 and EMMPRIN proteins in 58 breast cancer specimens,28 premalignant lesions,40 hyperplasia specimens,40 benign tumor specimens and 20 normal breast tissues.The expression of MMP-3 mRNA and EMMPRIN mRNA was examined using in situ hybridization in 20 breast cancer specimens,20 premalignant lesions,20 hyperplasia specimens,20 benign tumor specimens and 20 normal breast tissues.The values of integral optic density(IOD)of the expression was calculated by image analysis system.Results:The expression of MMP-3 and EMMPRIN proteins and mRNA was stronger in breast cancer and premalignant lesions than in the other lesions.The IODs of MMP-3 and EMMPRIN proteins and mRNA in breast cancer and premalignant lesion were significantly higher than those of hyperplasia,benign tumors and normal breast tissues(P

Objective To observe enhancing effect of nerve regeneration on peripheral nerve defect models bridged by silicone tube idled with valproic acid (VPA). Methods In present research we demon-strate the effect of VPA on peripheral nerve regeneration and recovery of motor function following sciatic nerve transaction in rats. An 8-mm sciatic nerve deficit was created in a rat mode land bridged by a 1-cm silicone tube.Then, 10 lad of 8% VPA were perfused into the silicone chamber in the VPA group. The same volume of normal saline was delivered in the control group. Results Each animal was observed sciatic nerve function index (SFI) at 2-week intervals and studied electrophysiology at 4-week intervals for 12 weeks. Histological and morphometrical analyses were performed at the end of the experiment, 12 weeks after operation. Using the digital image-analysis system, thickness of the myelin sheath was measured, and total numbers of regenerated axons were counted. There was a significant difference in SFI, electrophysiological index (motor-nerve conduct velocity, MCV), and morphometrical results (regenerated axon number and thickness of myelin sheath) in nerve regeneration between the VPA group and controls (P ＜ 0.05). Conclusion The results demonstrated that VPA is able to enhance sciatic nerve regeneration in rats, suggesting the potential clinical application of VPA for the treatment of peripheral nerve injury in humans.

Objective To explore the effect of sodium fluoride on DNA damage and apoptosis on ox peripheral blood lymphocyte cultured in vitro.Methods Using lymphoeytes separation medium lymphocytes were separated and different concentration of NaF(0,4,8,12,16 mg/L)were added into the cultual system of lymphocytes for 24 h.Cell viability was measured by MTT.nuclear changes stained by acridine orange-ethidium bromine staining (AO/EB)were observed under fluoroscope,DNA fragment was measured by agarose gel electrophoresis.DNA damage was detected by alkaline SCGE.The differences between each groups were compared.Results Cells were exposed to 4,8,16 and 24 mg/L NaF in 24 h,cell survival rates,respectively being(73.743±0.552)%,(69.184±0.724)%,(65.736±0.055)%and(63.651±0.287)%,decreased significantly compared to control group.There were distinctive cell apoptosis,evident DNA damage and visible dose-effect relation(R2=0.7456).Conclusions A certain concentration of sodium fluoride lcads to lymphocytes apoptosis and DNA damage.