OBJECTIVETo establish an HPLC method for the content determination of baicalin, wogonin, chlorogenic acid, caffeic acid, cichoric acid, corynoline and adenosine in Pudilan Xiaoyan oral liquid.

METHODThe analysis was performed on a Phenomenex Luna C18 column (4.6 mm x 250 mm, 5 µm) with a gradient mobile phase of methanol-0.1% trifluoroacetic acid solution system at flow rate of 1.0 mL · min(-1). The detective wavelength was at 280 nm. The column temperature was 30 °C.

RESULTThe standard curves of seven studied components show good linearity in their concentration ranges with r ≥ 0.999 6. The average recovery was 98.73%-102.1% with RSD less than 2.6%.

CONCLUSIONThe method is rapid, simple and accurate, and can be applied for the quality control of Pudilan Xiaoyan oral liquid.

Caffeic Acids ; analysis ; Chlorogenic Acid ; analysis ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; analysis ; Flavanones ; analysis ; Flavonoids ; analysis ; Succinates ; analysis

Objective: Hepatocarcinoma cell line was transfected with anti-vascular endothelial growth factor (VEGF) hairpin ribozyme gene to observe the effect of hairpin ribozyme on VEGF expression and the growth of the xenografted tumors. Methods: The artificial anti-VEGF hairpin ribozyme gene was transfected into hepatocarcinoma SMMC-7721 cells via lipofectin mediation. The blank vector and the cell controls were prepared simultaneously. Then, positive clones were screened by genticin (G418). The transcription of ribozyme was confirmed by RT-PCR. The effects of the ribozyme on VEGF expression of SMMC-7721 cells were detected by semi-quantitative RT-PCR and immunohistochemical method. Cells in each group were inoculated into nude mice. The tumor volume and weight were recorded. The change in microvessel density and expression of VEGF was determined by immunohistochemistry. Results: Ribozyme gene was successfully transferred into tumor cells. The proliferation rate of ribozyme-transfected SMMC-7721 cells was significantly slower (P < 0.01). The expression of VEGF significantly decreased in ribozyme-transfected SMMC-7721 cells. After rebozyme transfection, the tumor formation rate significantly decreased and the growth speed of xenografted hepatocarcinoma markedly slowed down. The microvessel density and angiogenesis of the xenografted hepatocarcinoma were obviously reduced. Conclusion: Anti-VEGF hairpin ribozyme gene significantly inhibited the VEGF expression of hepatocarcinoma in vitro and in vivo by inhibiting angiogenesis of tumor cells. This study provided an experimental evidence for anti-angiogenesis gene therapy for hepatocarcinoma.

Apoptosis ; drug effects ; Cell Division ; drug effects ; Esophageal Neoplasms ; pathology ; Humans ; Selenomethionine ; pharmacology ; Tumor Cells, Cultured

Objective To investigate the intelligence standard for diagnose the sub-cretin children and children with mental retardation of socio-cultural type.Methods The full intelligence quotient(IQ),verbal intelligence quotient(VIQ)and performance intelligence quotient(PIQ)was tested by Wechsler scale(C-WISC)for mild iodine deficiency disordem children,children living in abnormal socio-cultural condition and normal children aged 7～14 years old in Qinba mountain area.The test results had been compared between the groups.Results There were no significant difference between psychomotor functioning well children and children living normal sociocuhural condition in VIQ,PIQ and full IQ(89.24±18.44 vs 90.75±17.58,87.58±15.78 vs 88.95±15.56,87.42±17.84 vs 89.02±17.18,t=1.14,1.19 and 1.24,respectively,all P＞O.05).PIQ and full IQ were significantly lower in mild iodine deficiency disorders children than in children with abnormal socio-cultural background (65.81±10.22 vs 72.33±13.23,62.42±12.31 vs 68.13±14.54,t=3.26,2.55,P＜0.01 or＜0.05,respectively).But the VIQ was not significantly different between these two groups.The average difference of VIQ and PIQ among mild iodine deficiency disorders children wag-0.32 without significant difierence(t=0.28,P＞0.05),however it was-2.91 among children under abnormal socio-cultural condition with significant difierenee(t=-3.59,P＜0.01).Conclusions IQ for iodine deficiency disorders children is characterized by that VIQ is damaged in parallel with PIQ,while that in children under abnormal soeio-cuhural condition is marked by that VIQ is retarded more severely than PIQ,which ean be used as an intelligence standard for differentiating the sub-cretin children from children wjth socio-cuhural mental retardation.

Objective To establish a rapid assay with high sensitivity and specificity based on the sequences for group specific gene (GS) and pathogenicity island pag A gene.Methods The PCR primers and probes were designed after the whole sequence was systemically analyzed with bio-informafion tools and blasted with Genebank database.The amplicons were inserted into plasmids so that they could be used as the standard templates to evaluate the sensitivity of the diagnostic system.This assay was based on TaqMan probes and portable Smartcycle PCR machine.Results The detection level was approximately 100 copies per reaction.There was no cross-reaction with other species of Bacillus.This assay could be completed in one hour in laboratory.Conclusion The duplex TaqMan PCR assay could be used to detect Bacillus anthracis rapidly with high sensitivity and specificity.

OBJECTIVETo characterize DNA-PKcs and Ku70 expressions in hepato- and cholangio-neoplastic tissues and the association with the degree of malignancy and invasiveness of the tumors.

METHODSThe expression of DNA-PKcs and Ku70 was examined in 47 cases of hepato- or cholangio-neoplasm by immunohistochemistry.

RESULTSKu70 was expressed in all of the neoplastic tissues examined and with a little variation in levels. The highest expression was observed in adenocarcinomas and adenomas. There was no statistically significant association between Ku70 expression level and the degree of their malignancy extent or invasiveness. In contrast to Ku 70, a wide variation in expression levels of DNA-Pkcs was observed among different types of neoplastic tissues. The highest ratio of positive expressing cells was detected in hepatocellular carcinomas (92.1%), which was significantly higher than that in cholangioadeno carcinomas (65.3%) and biliary cystadenocarcinomas (51.9%). Low or no expression level was detected in papillary adenoma cases. DNA-PKcs expression of invasive adenomas and adeno-carcinomas (61.2%) was significantly higher than that of non-invasive adenomas and adeno-carcinomas (30.4%). There was no expression observed in the normal tissues adjacent to the tumors.

CONCLUSIONDNA-PKcs is expressed in hepato- and cholangio-neoplasms and its variable level of expression is associated with the types of the tumor and their degree of malignancy and invasiveness. DNA-PKcs could be recognized as a new biomarker for liver neoplasm.

Adenocarcinoma ; enzymology ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, Nuclear ; biosynthesis ; genetics ; Bile Duct Neoplasms ; enzymology ; Bile Ducts, Intrahepatic ; enzymology ; Biomarkers, Tumor ; biosynthesis ; genetics ; Carcinoma, Hepatocellular ; enzymology ; DNA-Activated Protein Kinase ; biosynthesis ; genetics ; DNA-Binding Proteins ; biosynthesis ; genetics ; Female ; Humans ; Ku Autoantigen ; Liver Neoplasms ; enzymology ; Male ; Middle Aged

Objective To compare the influences of Yiqihuoxue Formula ( using Naoluoxintong, YF) and Bushenshengsui Formula (BF) on the expressions of Nestin in the subventricular zone (SVZ) of ischemic lateral ventricle, neuron specific enolase ( NSE) and glial fibrillary acidic protein ( GFAP) in the ischemic half-dark zone in rats with focal cerebral ischemia-reperfusion (I/R). Method The rat model of focal cerebral I/R was established by using thread approach, and then the rats were randomly divided into the sham-operation group, model group, YF group and BF group. After cerebral ischemia for 2 hours the reperfusion was given to the rats for 1 week, 2 weeks or 3 weeks respectively. The positive cell quantity and the value of average absorbance ( A value) in the expressions of Nestin, NSE and GFAP were detected by using immunofluorescence staining method. Result In SVZ the positive cell quantity in Nestin expression was higher significantly in YF group after I/R for 1 week and 2 weeks, and in BF group after I/R for 2 and 3 weeks than that in the model group (P <0. 05 or P <0.01) , that in YF group after I/R for 1 week was higher significantly than that in BF group (P <0.01) , and that in BF group after I/ R for 2 and 3 weeks was higher significantly than that in YF group ( P <0.05 ). In the ischemic half-dark zone the positive cell quantity in NES expression was higher significantly in YF group after I/R for 1 week and in BF group after I/R for 2 weeks than that in the model group (P <0.01) , that in YF group after I/ R for 1 week was higher significantly than that in BF group (P <0.01) , and that in BF group after I/R for 2 weeks than that in YF group (P <0.01) . In the ischemic half-dark zone the average absorbance of GFAP expression was higher significantly in YF group after I/R for 1 week and 2 weeks and in BF group than that in the model group (P<0.05 or P<0.01)). There was no significant difference between YF group and BF group at all time points (P >0.05). Conclusion YF and BF can promote the proliferation and differentiation of neural stem cells after focal cerebral ischemia through increasing Nestin quantity in SVZ and NSE quantity, and improving GFAP expression in the ischemic half-dark zone. These two formulas have difference in the increase of Nestin and NSE quantity.

OBJECTIVETo investigate the role of sulfated tyrosine in regulating the activity of tyrosylprotein sulfotransferases (TPST) 1 and TPST2.

METHODSConstructs of TPST 1 and TPST2 were amplified by polymerase chain reaction (PCR), then fused into immunoglobulin G1 Fc region. All the variants in which sulfated tyrosines were mutated to phenylalanine were made by the PCR-based Quick Change method and confirmed by sequencing the entire reading frame. Small hairpin RNA (shRNA) constructs-targeting nucleotides 259-275 of TPST1 and nucleotides 73-94 of TPST2 were generated and subcloned into pBluescript. Human embryonic kidney (HEK) 293T cells were transfected with these plasmids. One day later, cells were split: one part was labeled with 35S-cysteine and methionine or 35S-Na2SO3 overnight, the second part was used for 125I labeled binding experiment, and the third part was retained for binding and flow cytometry.

RESULTSTyrosines at position 326 of TPST1 and position 325 of TPST2 were sulfated posttranslationally. Tyrosine sulfation of TPSTs was effectively inhibited by sulfation inhibitors, including specific shRNAs and non-specific NaCIO3. shRNAs reduced the sulfation of C3a receptor and C5a receptor, and partially blocked the binding of these two receptors to their respective ligands.

CONCLUSIONSThe activities of TPSTs were regulated by tyrosine sulfation. Inhibition of sulfotyrosine decreases the binding ability of C3a receptor and C5a receptor to their respective ligands.

Cell Line ; Complement C3a ; metabolism ; Complement C5a ; metabolism ; Humans ; Protein Binding ; Protein Processing, Post-Translational ; Receptor, Anaphylatoxin C5a ; metabolism ; Receptors, Complement ; metabolism ; Sulfotransferases ; genetics ; metabolism ; Transfection ; Tyrosine ; analogs & derivatives ; metabolism

OBJECTIVEWe investigated the effects of pitavastatin on angiogenesis and perfusion in C3H/He mice with unilateral hind limb ischemia.

METHODSC3H/He mice treated with saline (n = 15) or pitavastatin (1 mg.kg(-1).d(-1), n = 15) per gavage for 1 week underwent unilateral hind limb ischemia surgery and were treated for another 5 weeks. Hind-limb blood flow was measured by Laser Doppler perfusion imager (LDPI, ischemic/nonischemic limb, %) at baseline, immediately after ischemia and weekly thereafter for 5 weeks. Endpoints included local vessel counts by immunofluorescence, phospho-Akt positive cell counts by immunoenzyme histochemical technique, vascular endothelial growth factors (VEGFs) expression in ischemic limbs by Western blot and serum nitric oxide metabolite (NOx) by chrome dioxide Griess method.

RESULTSLower extremity perfusion was significantly improved in pitavastatin treated mice vs. controls as measured by LDPI% at 1 week post ischemia and thereafter (P < 0.05). Pitavastatin treatment was associated with significantly increased capillary count [(47 +/- 11) vs. (26 +/- 14)/per high-power field (x 200), P < 0.05] and greater percentage of phospho-Akt positive cells [(6 +/- 1) vs. (2 +/- 0)/per high-power field (x 200), P < 0.05] in ischemic limbs. Serum NOx [(77.3 +/- 21.8) vs. (52.1 +/- 11.2) mol/L, P < 0.05) and VEGF protein expression in ischemic limbs were also significantly increased in pitavastatin group than those in control group.

CONCLUSIONSPitavastatin enhances angiogenesis and perfusion in CsH/He mice with limb ischemia.

Animals ; Disease Models, Animal ; Ischemia ; physiopathology ; Lower Extremity ; blood supply ; Male ; Mice ; Mice, Inbred C3H ; Neovascularization, Physiologic ; drug effects ; Nitric Oxide ; blood ; Quinolines ; pharmacology ; Vascular Endothelial Growth Factors ; metabolism

Animals ; History, 20th Century ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; isolation & purification ; Influenza, Human ; epidemiology ; history ; immunology ; virology ; Russia ; epidemiology