Objective:Establish mass-scale purification technology of cell-derived influenza virus. Methods: A microcarrier-based process was used to produce human influenza virus in serum free-adapted Vero cells. The virus was purified in a sequence of downstream processing steps including inactivation, clarification, anion exchange chromatography, affinity chromatography and size-exclusion chromatography. Results: The recovery of HA reached 102%. 95.3% total protein, including 99.77% host cell protein, and 99.99% host cell DNA were removed during downstream processing. Conclusion: Providing a high effective purification technology for cell-derived influenza virus.

Objective To develop a real-time polymerase chain reaction(PCR)system to determine transcriptional level of MexAB-OprM multidrug efflux pump gene and to investigate the impact of androgra- pholide on MexAB-OprM gene transcription in Pseudomonas aeruginosa.Methods The fragments of mexB gene of mexAB-oprM operon and 30S rRNA gene rpsL were amplified and cloned into two plas- mids respectively.These plasmids were used as external standards for real-time PCR.Real-time PCR was applied to measure the mRNA transcripition of mexB and rpsL gene in Pseudomonas aeruginosa growing in medium with different concentrations of andrographolide.Results The plasmids for standard curve were constructed successfully.The relative mexB mRNA expressions in 50,100,150 and 200?g/mL andrographolide were 0.04?0.03,0.06?0.07,0.09?0.03 and 0.04?0.03 respectively, which were significantly lower than that in the control(0.24?0.04,P0.05).Conclusion Andrographolide can reduce the transcriptional level of MexAB-OprM,which may he one mechanism for its anti-infection effect.

Objective To explore the temporal and spatial expression pattern of tenascin-c(tnc) and tenascin-w(tnw) in zebrafish early development,to further explore the role of tenacsin in zebrafish embryo development,and the association between them.Methods Zebrafish embryos at 2 hours post fertilization(hpf),4 hpt,8 hpt,10 hpf,24 hpt,48 hpr,72 hpf and 7 days post fertilization(dpf) were collected to extract RNA for reverse transcription-polymerase chain reaction(RT-PCR) and fix the embryos at different stages for in situ hybridization.Temporal and spatial expression pattern of tnc and tnw on different stages of zebrafish early development was observed.Results tnc and tnw all expressed in zebrafish from 24 hpf to 7 dpf,but did not expressed from 2 hpf to 10 hpf.Tnc expressed at pharyngeal arch,notochord,somite in 24 hpf,then weakly expressed at somite,but highly expressed at otic vesicle,pectoral fin and hindbrain in 48 hpf,and tnc was expressed at hindbrain,pharyngeal and notochord and disappeared at somite and pectoral.tnw expressed at hindbrain,midbrain and otic vesicle in 24 hpf,expressed at somite,notochord,hindbrain,otic vesicle and pharyngeal in 48 hpf.In 72 hpf,tnw expressed weakly at somite and notochord.Conclusions Zebrafish tnc and tnw have special temporal and spatial expression pattern,and share partial overlapping expression pattern.

Objective To explore the expression pattern of tenascin-c(tnc)gene in zebrafish embryo abnormal development which was induced by ethanol,and to further understand the function of tnc gene in embryo develepment.Methods Zebrafish were treated with ethanol at different concentration from 100 to 500 mmol/L,and embryos at 24 and 48 hours were collected and fixed,then tnc expression pattern was observed by in situ hybridization and reverse transcriptase-polymerase chain reaction(RT-PCR).Results The result of RT-PCR showed that ethanol at 100 and 200 mmol/L could increase the expression of tnc,while the result of in situ hybridization showed that,while ethanol at 300 mmol/L and above decrease the expression of tnc in presumptive position at 24 hours,and ethanol at 100 mmol/L and above caused increase expression of tnc in zebrafish heart.Conclusions tnc is increased when treated with 100 and 200 mmol/L ethanol and is presented in the abnormal development of hearts of zebrafish,which can promote the normal development of embryos in some degrees.The expression pattern of tnc in pathologic state is highly conserved in all vertebrate,and in adult and embryos as well.

Objective To explore the effects of thiamine and riboflavin on H2O2-induced DNA oxidative damage in human umbilical vein endothelial cell line ECV304.Methods ECV304 cells were incubated with 10,100,500,1000 mg/L of thiamine or 20,100,300,500 nmol/L of riboflavin for 24 h,and then oxidative damage of cells were induced by 25 mol/L H2O2 for 30 min.DNA damage was detected with single cell gel electrophoresis(SCGE)assay.ECV304 cells incubated without H2O2,thiamine and riboflavin were served as negative controls,and those incubated with H2O2 and without thiamine and riboflavin were served as positive controls.Results H2O2 induced DNA damage,and the indices of percent of DNA damage cells,percent of tail DNA,tail length and Olive tail moment were increased.The indices of cells pretreated with 10,100,500 mg/L of thiamine or 20,100,300 nmol/L riboflavin were significantly decreased(P0.05).Conclusion Proper supplementation of thiamine and riboflavin may decrease H2O2-induced DNA oxidative damage,while excess thiamine and riboflavin supplementation may be harmful to DNA and enhance the susceptibility to H2O2 potentially.

Plasma ghrelin level was measured by RIA in 49 grids with idiopathic central precocious puberty(ICPP)and 35 girls with simple premature thelarche(PT).The results showed that the plasma ghrelin level was negatively correlated with Tanner stage,bone age,Lid at 15,30 and 60 min after GnRH activation, uterus and ovary volumes.The plasma ghrelin level in the ICPP group was remarkably lower than that in the PT and control groups(both P

Wistar rats with different levels of iodine nutrition were killed after 3,6 and 12 months of experiments.Serum thyroid hormones were assayed with RIA.The activity of typeⅠdeiodinase(DⅠ)and typeⅡdeiodinase(DⅡ)was measured based on the release of radioiodide from the ~(125)Ⅰ-labeled substrate.The result showed that hypothyroidism reflected by decreased T_4 happened during the initial phase of iodine deficiency.The activity of DⅠand DⅡin rats was raised significantly in iodine deficiency groups.An excess of iodine inhibited DⅠactivity resulting in decreased serum TT_3 and FT_3.However,DⅡactivity increased in rats with iodine excess, attributing to the inactivation of T_3 and T_4 to the substrate of DⅡenzyme.

This study is to establish an HPLC-DAD-ELSD method for simultaneous determination of 5 flavones and saponins in Rhizoma Anemarrhenae including neo-mangiferin, mangiferin, timosaponin B II, timosaponin B III and timosaponin A III. Samples were analyzed on a Merck Purospher STAR column(4.6 mm x 250 mm, 5 μm). The mobile phase consisted of acetonitrile( A) and 0. 1% formic acid (B) with gradient elution at a flow rate of 1.0 mL · min(-1). The column temperature was set at 40 °C. The DAD detector wavelength was set at 254 nm. The ELSD conditions were as follows: the nebulizing gas flow rate was 2.0 L · min(-1) and temperature of drift tube was 105 °C. The volume was 10 μL. The five compounds were well separated with good linear correlations. The mean recoveries were between 102.0%-104.0%. This method was quick and reliable which provides a foundation for quality control of R. Anemarrhenae.
Anemarrhena ; chemistry ; Chromatography, High Pressure Liquid ; instrumentation ; methods ; Drugs, Chinese Herbal ; analysis ; Flavones ; analysis ; Rhizome ; chemistry ; Saponins ; analysis

Nine compounds were isolated from the leaves of Ilex latifolia. Their structures were respectively identified as 5-hydroxy-6, 7, 8, 4'-tetramethoxyflavone (1), tangeretin (2), nobiletin (3), 5-hydroxy-6, 7, 8, 3', 4'-pentamethoxyflavone (4), 5, 6, 7, 8, 4'-pentamethoxyflavonol (5), 5, 6, 7, 8, 3', 4'-hexamethoxy-flavonol (6), 5-hydroxy-3', 4', 7-trimethoxyflavanone (7), soyacerebroside I (8), and soyacerebroside II (9) by their physicochemical properties and spectroscopic data Compounds 1-9 were isolated from this plant for the first time.
Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Ilex ; chemistry ; Plant Leaves ; chemistry

OBJECTIVETo develop a method to obtain and identify human coronary artery endothelial cells obtained during percutaneous coronary interventions (PCI).

METHODSCoronary guide wires were used to obtain endothelial cells from coronary arteries in 28 patients undergoing PCI. The cells were eluted from the wire tips and then purified by magnetic beads coated with anti-CD146 antibody. von Willebrand factor (vWF) was used as an immunocytochemical marker for endothelial cells. The cellular viability was evaluated by observing cell membrane integrity and energy-dependent uptake of DiI-labeled acetylated low-density lipoprotein.

RESULTSAn average of 96 coronary artery endothelial cells with good viability per patient were obtained by one guide wire. vWF identification showed their endothelial morphology and immunoreactivity.

CONCLUSIONThe viable coronary endothelial cells could be obtained during routine percutaneous coronary interventions combined with magnetic beads isolation technique. These cells may be used for further cellular functional analyses (such as immunocytochemistry and molecular biology) and expand our understanding on mechanisms of coronary artery diseases.

Biopsy ; methods ; Coronary Vessels ; cytology ; pathology ; Endothelium, Vascular ; cytology ; pathology ; Female ; Humans ; Male ; Middle Aged