Objective To observe the activities of ChAT + neurons in subventricular zone (SVZ) after ischemic stroke and their effects on angiogenesis in peri-infarction region and related signaling pathways. Methods C57BL/6 mice were randomly assigned into sham group,middle cerebral artery occlusion (MCAO) group and atropine group. Ischemic models were made by permanent coagulation of the distal middle cerebral artery. The expression of ChAT,AChE in SVZ and VEGF,VEGFR2,pERK in peripheral regions of ischemic injury was evaluated by Western blotting and immunofluorescence. 5-bromodeoxyuridine(BrdU)/CD31 double-labeled cells were also tested by immunofluorescence. Results At 14 d after the surgery,the ratio of ChAT/AChE in SVZ increased after stroke(P < 0.05). Compared with those in Sham group,the levels of VEGF,VEGFR2 and pERK were higher in MCAO group(P<0.05)and VEGFR2-positive and BrdU/CD31-positive cells increased significantly. However,lower expression of VEGF,VEGFR2 and pERK and less VEGFR2-positive and BrdU/CD31-positive cells were found in atropine group when compared with that in MCAO group. Conclusions The activities of ChAT +neurons in SVZ are enhanced after ischemic injury and they can promote angiogenesis in peripheral region of ischemic injury via upregulating VEGF-VEGFR2 signaling pathway and improving the brain function restoration.

Objective To improve the conventional method of quantitative assessment of regional cerebral blood flows(rCBF)by a perfusion CT study based on maximal slope model at the general infusion rate(

OBJECTIVETo investigate the role of Stat3 (one of the signal transductant and activator) signal transduction pathway in proliferation of vascular smooth muscle cells (VSMCs).

METHODSVSMC was transfected with Stat3 antisense oligonucleotide. ELISA analysis was performed on Stat3 expression in cultured rat thoracic VSMC. Cell proliferation and toxicity assay by Methyl Thiazole (MTT) was used to measure the cell proliferation. The expression of Stat3, p-Stat3, Cyclin D1 and Bcl-x(L) were measured by Western blot.

RESULTSStat3 protein expression was positive in VSMC. Targeting of Stat3 using antisense oligonucleotide directed against the translation site, resulted in significant growth inhibition and downregulation of Stat3, p-Stat3 and Cyclin D1.

CONCLUSIONSThe increase of Stat3 correlates significantly with cell proliferation of VSMC. Cyclin D1 may be the critical target gene in mediating proliferation. Blocking Stat3 signal transduction pathway may inhibit the growth of VSMCs.

Animals ; Cell Line ; Cell Proliferation ; Cyclin D1 ; metabolism ; Male ; Muscle, Smooth, Vascular ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; Rats ; Rats, Sprague-Dawley ; STAT3 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Transfection

OBJECTIVETo summarize the experience of surgical resection of 103 hilar cholangiocarcinoma.

METHODSOne hundred and three consecutive cases of hilar cholangiocarcinoma who underwent surgical resection at our hospital over the past ten years were reviewed retrospectively. The clinical data and long-term outcome were analyzed.

RESULTSOut of 103 cases, 43 patients underwent radical resection (41.7%), and 60 patients underwent palliative resection. There were 34 patients developed postoperative complications and 8 patients died in hospital. For the radical resection group, the median survival time was 29.9 months and 1-year, 3-year, 5-year survival rate was 69.6%, 42.0%, 20.9%, respectively, which was significant greater than 34.1%, 10.2%, 0 of the palliative resection group (P < 0.05). Over the past five years, 42 cases underwent pre-operative drainage of bile and the rate of combined liver resection reached 53.8%. The tumor radical resection rate has increased to 45.7%, the median survival time have reached 24.7 months (P < 0.05).

CONCLUSIONSImprovement of pre-operative management, intraoperative pathology for resection margin, and combined liver resection may help in increasing the radical resection rate. Radical resection can improve postoperative survival, and produce a satisfactory outcome for patient with hepatic hilar cholangiocarcinoma.

Adult ; Aged ; Bile Duct Neoplasms ; mortality ; surgery ; Bile Ducts, Intrahepatic ; Cholangiocarcinoma ; mortality ; surgery ; Digestive System Surgical Procedures ; methods ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Survival Rate

OBJECTIVETo investigate the specific cytotoxity of tumor infiltrating lymphocytes (TIL) transfected with chimeric T cell receptor (CTCR) on cells which express KDR.

METHODSA recombinant retroviral plasmid (pMSCVneo-Vhgamma) was constructed by cloning VEGF121-hinger-FcRgamma (Vhgamma) into retroviral vector pMSCVneo. After packaging by PT67, the virus with high titer was used to infect TIL isolated from liver cancer tissues, and then MSCVneo-Vhgamma-TIL was generated. TIL infected with MSCVneo was used as a control. The cytotoxicty of the transgenic TIL on NIH3T3 and HepG2 expressing no KDR and on ECV304 and A375 expressing KDR was detected with MTT colorimetric assay.

RESULTSThe sequences of VEGF121 and hinger-FcRgamma were different from those reported, but the deduced amino sequences were identical to the reported ones. The cytotoxity of TIL infected with MSCVneo on target cell was similar to that of the control TIL; both only had mild cytotoxity on cancer cell line. No significant cytotoxity was found in TIL infected with MSCVneo-cTCR on NIH3T3, but its cytotoxity on ECV304 was significant. The cytotoxity on HepG2 was similar to that of MSCVneo-TIL and uninfected TIL, but cytotoxity on A375 was significantly higher.

CONCLUSIONChimeric T cell receptor permanently grafts TIL cell with predefined new specificity. TIL expressing Vhgamma can selectively recognize and kill vascular endothelial cell and tumor cells which express VEGF receptor KDR.

Animals ; Cytotoxicity, Immunologic ; Humans ; Lymphocytes, Tumor-Infiltrating ; immunology ; Mice ; NIH 3T3 Cells ; Plasmids ; Receptors, Antigen, T-Cell ; physiology ; Retroviridae ; genetics ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; physiology

Objective To investigate the effects of cholinergic signal on neural stem cell(NSC)differenti-ation in peri-infarction region after ischemic stroke. Methods Mice were randomly assigned into sham + vehicle group,middle cerebral artery occlusion(MCAO)+ vehicle group,MCAO + donepezil group and MCAO + atro-pine group(n = 25). MCAO was induced by thread-occlusion method. Modified neurological severity score (mNSS)was used to evaluate neurological function recovery,and the brain water content was measured by dry-wet weight method. NeuN/5-bromodeoxyuridine(BrdU),CNPase/BrdU,GFAP/BrdU double-labeled cells were tested by immunofluorescence. Results Brain water content of MCAO + vehicle group was significantly higher than that of sham operation group(P < 0.05). Donepezil-treated MCAO mice had lower neurologic deficit scores and brain water content than of MCAO + vehicle group(P < 0.05). On day 14 and day 28 after MCAO,the NeuN/BrdU, CNPase/BrdU and GFAP/BrdU immune-positive cells of MCAO + vehicle group were markedly increased as com-pared with that of sham+vehicle group(P<0.05).Compared with that of MCAO+vehicle group,the number of NeuN/BrdU-positive cells,CNPase/BrdU-positive cells and GFAP/BrdU-positive cells was higher in MCAO+done-pezil group,and the number of NeuN/BrdU-positive cells and CNPase/BrdU-positive cells of MCAO + atropine group was lower(P < 0.05). Conclusions Cholinergic signal could promote NSCs differentiation in peri-infarc-tion region,a lleviate cerebral edema,and improve the brain function restoration after stroke.

Objective To conduct serological investigation on H5N1/H9N2/H7N7 infection among people occupied in poultry fields. Methods Serum samples were collected from people working in live poultry and none-poultry retailing food markets, poultry wholesaling, large-scale poultry breading factories and in small-scale farms, wide birds breeding, swine slaughtering houses and from normal population. Antibodies of H5, H9 and H7 with hemagglutination inhibition and neutralization tests were tested and analyzed. Logistic regression and χ2 test were used. Results Among 2881 samples, 4 were positive to H5-Ab(0.14%), 146 were positive to H9-Ab (5.07%) and the prevalence of H9 among people from live poultry retailing (14.96%) was the highest. Prevalence rates of H9 were as follows: 8.90% in people working in the large-scale poultry breading factories, 6.69% in the live poultry wholesaling business, 3.75% in the wide birds breeding, 2.40% in the swine slaughtering, 2.21% in the non-poultry retailing, 1.77% in the rural poultry farmers and 2.30% in normal population. None was positive to H7-Ab among 1926 poultry workers. Conclusion The H5 prevalence among people was much lower than expected, but the H9 prevalence was higher. None of the populations tested was found positive to H7-Ab. There was a higher risk of AIV infection in live poultry retailing, wholesaling and large-scale breading businesses, with the risk of live poultry retailing the highest. The longer the service length was, the higher the risk existed.

The last essential enzyme in the biosynthetic pathway of trilobatin, phloretin-4'-O glycosyltransferase (P4'-OGT), catalyzes the conversion of trilobatin to phloretin in vitro. However, only a few P4'-OGTs have been found in plants. This study used Malus domestica phloretin-4'-O glycosyltransferase (MdPh-4'-OGT) as a query to identify and clone two UDP-glucuronosyltransferase (UGT) genes, designated UGT74L2 and UGT74L3, from the transcriptome of Andrographis paniculata. According to a phylogenetic tree analysis, UGT74L2 and UGT74L3 belonged to the UGT74 family, which has been linked to several activities in other species. The in vitro enzymatic reaction demonstrated that UGT74L2 could particularly catalyze the formation of trilobatin from phloretin, but UGT74L3 had no effects. By using Ni-NTA affinity chromatography to extract the soluble UGT74L2 recombinant protein, the enzymatic kinetics of the activity was investigated using phloretin as the substrate. The results showed that the optimal temperature and pH for UGT74L2 enzymatic reaction were 40 ℃ and 8.0 (Tris-HCl system), respectively. Three metal ions (Ca²⁺, Mn²⁺ and Co²⁺) showed inhibitory effect on the activity of UGT74L2, while Mg²⁺ could improve the activity of UGT74L2. Other tested metal ions have no significant effect on UGT74L2. The results of enzymatic kinetic parameters that the K_m value was 29.84 μmol·L^-1, the k_cat was 0.02 s^-1, and the k_cat·K_m^-1 was 572.6 mol^-1·s^-1. By homology modeling, molecular docking and mutation experiments, we found that multiple amino acids residues around the substrate binding pocket play quite an important role during catalytic process, In summary, we identified a novel P4'-OGT gene from medicinal plant Andrographis paniculata and provided a new efficient catalyst to synthesize trilobatin. Meanwhile, this study provides a reference for mining new efficient glycosylation modules from plants.

The lignan glycosyltransferase UGT236(belonging to the UGT71 B family) from Isatis indigotica can catalyze the production of phloridzin from phloretin in vitro. UGT236 shares high identity with P2'GT from apple. In this study, the recombinant plasmid pET28 a-MBP-UGT236 was transferred into Escherichia coli Rosetta(DE3) cells and induced by isopropyl-β-D-thiogalactoside(IPTG). The purified UGT236 protein was used for enzymatic characterization with phloretin as substrate. The results showed that UGT236 had the optimal reaction temperature of 40 ℃ and the optimal pH 8(Na_2HPO_4-NaH_2PO_4 system). The UGT236 activity was inhibited by Ni~(2+) and Al~(3+), enhanced by Fe~(2+), Co~(2+), and Mn~(2+), and did not affected by Mg~(2+), Ca~(2+), Li~+, Na~+, or K~+. The K_m, K_(cat), and K_(cat)/K_m of phloretin were 61.03 μmol·L~(-1), 0.01 s~(-1), and 157.11 mol~(-1)·s~(-1)·L, and those of UDPG were 183.6 μmol·L~(-1), 0.01 s~(-1), and 51.91 mol~(-1)·s~(-1)·L, respectively. The possible active sites were predicted by homologous modeling and molecular docking. By mutagenisis and catalytic activity detection, three key active sites, Glu391, His15, and Thr141, were identified, while Phe146 was related to product diversity. In summary, we found that the lignan glycosyltransferase UGT236 from I.indigotica could catalyze the reaction of phloretin into phloridzin. Several key amino acid residues were identified by structure prediction, molecular docking, and site-mutagenesis, which provided a basis for studying the specificity and diversity of phloretin glycoside products. This study can provide a reference for artificially producing glycosyltransferase elements with high efficiency and specific catalysis.
Glucosyltransferases/genetics* ; Glycosyltransferases/metabolism* ; Isatis ; Lignans/metabolism* ; Molecular Docking Simulation ; Phloretin/metabolism* ; Phlorhizin/metabolism*

OBJECTIVETo observe X-ray features of before and after treatment of cervical imbalance syndrome with osteopathy and traction intervention in youth patients, then to investigate the clinical effect of the treatment of the cervical imbalances syndrome in youth by osteopathy.

METHODSFrom September 2007 to December 2010, one hundred and eighty-seven students of 19 to 22 years (means 21 years) with neck pain as the main symptom in Beijing university of Chinese medicine were selected and divide them into osteopathy group and traction groups randomly. In osteopathy group, there were 94 patients including 40 males and 54 females; in traction group, there were 93 patients including 42 males and 51 females. The treatment of osteopathy group lasted for 3 weeks,three times per week; traction group by traction treatment three weeks,three times a week, too. X-ray before treatment and 3 weeks after treatment were collected.

RESULTSThe osteopathy group:94 cases, before treatment, abnormal curvature in 57 cases, spinous position change in 45 cases and angular displacement in 44 cases, vertebral sliding in 15 cases, Ruth Jackson line intersect proneness change in 70 cases,stretch change in 47 cases; after treatment, abnormal curvature in 35 cases, spinous position change in 24 cases and angular displacement in 18 cases, vertebral sliding in 3 cases, Ruth Jackson line intersect proneness change in 41 cases, extension change in 33 cases; X-Ray measurement results: cervical curvature improved from (7.070 +/- 4.629) degrees before treatment to (7.660 +/- 4.156) degrees after treatment, angular displacement improved from (13.790 +/- 2.590) degrees before treatment to (11.050 +/- 2.560) degrees after treatment; vertebral sliding improved from (3.770 +/- 0.350) mm before treatment to (3.160 +/- 0.485) mm after treatment. The change of angular displacement and vertebral sliding were so significant before and after treatment in this group, there were statistical defferences between before and after the treatment (P < 0.01) in osteopathy group. The traction group: 93 cases, before treatment,abnormal curvature in 60 cases, spinous position change in 39 cases and angular displacement in 39 cases, vertebral sliding in 15 cases, Ruth Jackson line intersect proneness change in 70 cases, stretch in 47 cases; after treatment,abnormal curvature in 50 cases, spinous position change in 29 cases and angular displacement in 17 cases; vertebral sliding in 3 cases, Ruth Jackson line intersect proneness change in 41 cases, stretch in 33 cases; X-Ray measurement results: cervical curvature improved from (5.590 +/- 4.639) degrees before treatment to (5.990 +/- 4.330) degrees after treatment, angular displacement improved from (13.360 +/- 2.064) degrees before treatment to (11.210 +/- 1.872) degrees after treatment; vertebral sliding improved from (3.790 +/- 0.339) mm before treatment to (3.480 +/- 0.332) mm after treatment. The change of angular displacement and vertebral sliding were also so significant in traction group before and after treatment, there were statistical defferences between before and after the treatment (P < 0.01).

CONCLUSION(1) Pathological changes can be called youth cervical imbalance syndrome. (2) Stress points, angular displacement, cervical vertebral slip, curvature straightened, spinous position change are main X-ray performances. (3) Both osteopathy and traction intervention on are efficient in youth neck pain. (4) Youths cervical vertebra imbalance is early but reversible performance.

Cervical Vertebrae ; diagnostic imaging ; Female ; Humans ; Joint Instability ; diagnostic imaging ; therapy ; Male ; Radiography ; Spinal Diseases ; diagnostic imaging ; therapy ; Traction ; X-Rays ; Young Adult