This study was aimed to construct a pEGFP-N1 vector of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motifs 13) so as to pave the way for further studying its synthesis and secretion. Human full-length cDNA sequence of ADAMTS13 was acquired by polymerase chain reaction (PCR) with Phusion(®) High-Fidelity (NEB), then the PCR product was double digested with EcoRI and XhoI. After digestion, the ADAMTS13 cDNA sequence was purified and recombined with the pEGFP-N1 vector. The DNA sequence analysis showed that ADAMTS13 was ligated to the pEGFP-N1 vector correctly. After transient expression in HeLa cells, the expression of EGFP could be detected by fluorescent microscopy, and the expression of ADAMTS13 protein could be detected by SDS-PAGE and Western blot. It is concluded that the ADAMTS13-pEGFP-N1 vector is successfully constructed, and it can be widely used in further research on the mechanism of the synthesis and secretion of ADAMTS13.
ADAM Proteins ; genetics ; ADAMTS13 Protein ; Gene Expression ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; HeLa Cells ; Humans ; Transfection

OBJECTIVETo probe the mechanism of EEG activation and Xingnao Kaiqiao, evaluate the actions of cinnabaris and realgar in Xingnao Kaiqiao of Angong Niuhuang pill, guess the significance of cinnabaris and realgar in specific indication treatment of Angong Niuhuang pill, and provide experimental bases for the rationality of Angong Niuhuang pill building-up.

METHODSeventy SD rats were divided into seven groups: the control, the model, the Angong Niuhuang pill (0.4 g x kg(-1)), the Angong Niuhuang pill without cinnabaris and realgar (0.32 g x kg(-1)) , the cinnabaris and realgar (0.08 g x kg(-1)), the realgar (0.04 g x kg(-1)), and the cinnabaris (0.04 g x kg(-1)). Rats in the control and model groups were given distilled water. After three days of administration, the brain damage model was made by Lipopolysaccharides (LPS) injection through caudal vein and the catecholamine (CA) and its metabolites levels in cerebral cortex, included noradrenaline (NE), adrenaline (E), 3-methocy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA), Homovanlic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC), were determined by high-performance liquid chromatography with electrochemical detector (HPLC-ECD). Influences of Angong Niuhuang pill, Angong Niuhuang pill without cinnabaris and realgar, cinnabaris and realgar on monoamine transmitters were observed in brain damage rats caused by LPS.

RESULTLPS could raise NE, 5-HT, 5-HIAA levels and reduce E, DOPAC levels, but had no influence on HVA, DA, MHPG levels. Angong Niuhuang pill had the trend of raising E, DOPAC levels and reducing NE level, and could reduce 5-HIAA level obviously comparing with models. But Angong Niuhuang pill without cinnabaris and realgar was different, NE level was significantly higher compared to models and Angong Niuhuang pill, DA level was also significantly higher compared to all groups. Cinnabaris and realgar had the same action trends with Angong Niuhuang pill, and separate realgar could obviously reduce 5-HT.

CONCLUSIONInfluence on CA and its metabolites levels in cerebral cortex may be one of the mechanisms of Angong Niuhuang pill's EEG activation, and cinnabaris and realgar have the same action on CA levels in cerebral cortex. The results of the present work allow us to put forward the hypothesis that cinnabaris and realgar are most likely one of the important material basis in Xingnao Kaiqiao of Angong Niuhuang pill.

Animals ; Arsenicals ; pharmacology ; Brain Injuries ; chemically induced ; metabolism ; physiopathology ; Catecholamines ; metabolism ; Cerebral Cortex ; metabolism ; physiopathology ; Drug Combinations ; Electroencephalography ; drug effects ; Lipopolysaccharides ; Male ; Medicine, Chinese Traditional ; Mercury Compounds ; pharmacology ; Norepinephrine ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Serotonin ; metabolism ; Sulfides ; pharmacology

BACKGROUNDIt is known that excessive release of glutamate can induce excitotoxicity in neurons and lead to seizure. Dexamethasone has anti-seizure function. The aim of this study was to investigate glutamate-dexamethasone interaction in the pathogenesis of epilepsy, identify differentially expressed genes in the hippocampus of glutamate-induced epileptic rats by mRNA differential display, and observe the effects of dexamethasone on these genes expression.

METHODSSeizure models were established by injecting 5 microl (250 microg/microl) monosodium glutamate (MSG) into the lateral cerebral ventricle in rats. Dexamethasone (5 mg/kg) was injected intraperitoneally at 30 minutes after MSG inducing convulsion. The rats' behavior and electroencephalogram (EEG) were then recorded for 1 hour. The effects of dexamethasone on gene expression were observed in MSG-induced epileptic rats at 1 hour and 6 hours after the onset of seizure by mRNA differential display. The differentially expressed genes were confirmed by Dot blot.

RESULTSEEG and behaviors showed that MSG did induce seizure, and dexamethasone could clearly alleviate the symptom. mRNA differential display showed that MSG increased the expression of some genes in epileptic rats and dexamethasone could downregulate their expression. From more than 10 differentially expressed cDNA fragments, we identified a 226 bp cDNA fragment that was expressed higher in the hippocampus of epileptic rats than that in the control group. Its expression was reduced after the administration of dexamethasone. Sequence analysis and protein alignment showed that the predicted amino acid sequence of this cDNA fragment kept 43% identity to agmatinase, a member of the ureohydrolase superfamily.

CONCLUSIONSThe results of the current study suggest that the product of the 226 bp cDNA has a function similar to agmatinase. Dexamethasone might relax alleviate seizure by inhibiting expression of the gene.

Animals ; Base Sequence ; Dexamethasone ; pharmacology ; Electroencephalography ; drug effects ; Epilepsy ; chemically induced ; drug therapy ; genetics ; Gene Expression Profiling ; Gene Expression Regulation ; drug effects ; Male ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; Rats ; Rats, Sprague-Dawley ; Sodium Glutamate ; pharmacology

OBJECTIVETo construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E. coli, and to explore their values in measuring ADAMTS13 activity as substrates.

METHODSThe DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1, a Schistosoma japonicum GST fusion expression vector. The expression of GST-vWF73-H and GST-vWF114-H was induced in liquid culture, followed by purification with Ni-NTA agarose column. The cleavage of two GST fusion proteins by recombinant ADAMTS13 (rADAMTS13) or plasma from normal individuals and thrombotic thrombocytopenic purpura (TTP) patients were identified by Western blot. Based on an enzyme-linked immunosorbent assay (ELISA) with anti-GST and anti-His monoclonal antibodies, GST-vWF73-H and GST-vWF114-H were used to measure plasma ADAMTS13 activity as substrates.

RESULTSTwo small molecular substrates of ADAMTS13, GST-vWF73-H and GST-vWF114-H, are expressed and purified, which could be specifically cleaved by rADAMTS13 or plasma from healthy individuals, but not by plasma from congenital or idiopathic TTP patients. An ELISA assay was established to detect plasma ADAMTS13 activity using GST-vWF73-H and GST-vWF114-H as substrates.

CONCLUSIONSTwo GST fusion proteins in vWF A2 domain, vWF73 and vWF114, were expressed effectively using a prokaryotic expression system and could be used to detect ADAMTS13 activity as substrates.

ADAM Proteins ; blood ; genetics ; ADAMTS13 Protein ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Glutathione Transferase ; metabolism ; Humans ; Male ; Purpura, Thrombotic Thrombocytopenic ; blood ; genetics ; metabolism ; von Willebrand Factor ; genetics ; metabolism

Fifteen known compounds were isolated from Swertia delavayi by silica gel, Sephadex LH-20 and Rp-18 column chromatographies. Based on extensive spectroscopic analysis (MS, 1H, 13C-NMR), their structures were identified aserythrocentaurin (1), erythrocentaurindimethylacetal (2), sweroside (3), swertiamarin (4), gentiopicroside (5), swertiakoside A (6), 2'-O-acetylswertiamarin (7), 4'-O-[(Z) -coumaroyl] swertiamarin (8), 1,5,8-trihydroxy-3-methoxyxanthone (9), 8-O-β-D-glucopyranosyl-1-hydroxy-2,3, 5-trimethoxyxanthone (10), 8-O-[β-D-xyl- opyranosyl-(1 --> 6)-β-D-glucopyranosyl]-7,8-dihydroxy-3-methoxyxanthone (11), isovitexin (12), β-sitosterol (13), daucosterol (14), and oleanolic acid (15). Among them, ten ones (14, 7-11, 13) were obtained from S. delavayi for the first time. The isolates were evaluated for their anti-HBV activities in HepG 2. 2. 15 cell line in vitro. The results showed that compound 1, 2, 6, 7, 9 and 12 exhibited significant inhibitory activity on HBV DNA replication with IC50 values from 0.05 to 1.46 mmol x L(-1).
Antiviral Agents ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Hepatitis B virus ; drug effects ; genetics ; Magnetic Resonance Imaging ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization ; Swertia ; chemistry

OBJECTIVETo investigate the susceptibility of von Willebrand factor (VWF) type 2A mutant A1500E to proteolysis by metalloprotease ADAMTS13 and to provide the direct supports for the pathogenesis of VWF mutation A1500E responsible for von Willebrand disease (VWD) type 2A.

METHODSRecombinant wild-type VWF (WT-VWF) and A1500E mutant VWF transiently expressed on transfected HeLa cell lines. Expression media were collected and concentrated, then cleaved directly by recombinant ADAMTS13 (rADAMTS13). Compared with WT-VWF, the susceptibility of A1500E mutant VWF to proteolysis by ADAMTS13 was analyzed using SDS-agarose gel VWF multimers analysis.

RESULTSIn vitro the expression of VWF:Ag in the supernatants of WT-VWF and A1500E mutant VWF were 1.10 U/ml and 0.78 U/ml, respectively, while VWF:Ag in cells lysates of A1500E mutant VWF was 90.6% of that of WT-VWF. The SDS-agarose gel VWF multimers analysis showed that there were no differences between WT-VWF and A1500E mutant VWF. The A1500E mutant VWF could be efficiently cleaved by ADAMTS13 under static condition without denaturants such as urea and guanidine HCl. VWF multimeric analysis showed that high and intermediate molecular weight multimers dramatically decreased while low molecular weight multimers obviously increased. Conversely, WT-VWF could not be cleaved by ADAMTS13 under the same condition.

CONCLUSIONThe A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.

ADAM Proteins ; genetics ; metabolism ; ADAMTS13 Protein ; Genotype ; HeLa Cells ; Humans ; Hydrolysis ; Mutation ; Recombinant Proteins ; genetics ; metabolism ; von Willebrand Disease, Type 2 ; genetics ; metabolism ; von Willebrand Factor ; genetics

The ethyl acetate extract of the flower of Albizia julibrissin was isolated and purified by silica gel, Sephadex LH-20 and MCI GEL CHP-20P column chromatography to yield 29 compounds. Their structures were elucidated as 8-hydroxy-2, 6-dimethyl-2E, 6Z-octadienoic acid (1), 8-O-formyl-2, 6-dimethyl-2E, 6Z-octadienoic acid (la), 8-hydroxy-2, 6-dimethyl-2E, 6E-octadienoic acid (2), 8-O-formyl-2, 6-dimethyl-2E, 6E-octadienoic acid (2a), (2E, 6S)-2, 6-dimethyl-6-O-beta-D-xylpyranosyloxy-2, 7-menthia-folic acid (3), clovan-2beta, 9alpha-diol (4), 2beta-O-formyl-clovan-9alpha-ol (4a), 2beta, 9alpha-O-diformyl-clovan (4b), vomifoliol (5), (6S, 9R)-roseoside (6), vanillin (7), 4-O-ethylgallic acid (8), 3-ethoxy4-hydroxy-benzoic acid (9), p-hydroxybenzaldehyde (10), gallic acid (11), protocatechoic acid (12), stearic acid (13), palmitic acid (14), 2, 3-dihydroxypropyl hexadecanoate (15), linoleic acid (16), scopoletin (17), indole-3-carboxaldehyde (18), 2-furoic acid (19), 5-(hydroxymethyl)-2-furaldehyde (20), (22E, 24R)-5alpha, 8alpha-epidioxy-ergosta-6, 22-dien-3beta-ol (21), (22E, 24R)-5alpha, 8alpha-epidioxy-ergosta-6, 9, 22-trien-3beta-ol (22), (+)-lariciresinol 9'-stearate (23), formononetin (24) and uridine (25). Compounds 1a, 2a, 4a and 4b were new artifacts from the separation process, and others were obtained from A. julibrissin for the first time.
Albizzia ; chemistry ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Flowers ; chemistry ; cytology ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVETo explore the regulatory effect of arginine on the secretion of hepatic insulin-like growth factor-1 (IGF-I), and the mechanism of enhancing the immune function by arginine.

METHODSWistar rats were randomly divided into normal control (NC), wound control (WC), and wound with arginine (Arg) groups, with 8 rats in each group. The rats in WC and Arg groups were inflicted with soft tissue trauma on the back. The rats in Arg group were fed a diet supplemented with 5% arginine for one week, while those in NC and WC groups were fed with glycine. The serum contents of arginine, ornithine, growth factor (GH), NO and IGF-I were determined 7 days after feeding. T cell proliferation and IGF-I mRNA expression in hepatic tissue were also measured. Meanwhile, the rat hepatocytes were cultured in serum-free medium containing different concentrations of arginine. The supernatant was collected for the determination of IGF-I level.

RESULTS1). There was no obvious difference of the serum level of arginine and ornithine between NC and WC groups (P > 0.05), but the contents of them were obviously higher in the Arg group compared with other two groups (P < 0.01). 2). No difference in the serum GH level was found among all the groups (P > 0.05), but the serum NO content in WC and Arg groups was significantly lower than that in NC group (P < 0.01), and the serum IGF-I content in WC group decreased obviously compared with that in NC group (P < 0.01). 3). The thymocyte proliferation rate in WC group was also markedly lower than that in NC group (P < 0.01), but that in Arg group was improved compared with WC group (P < 0.01). 4). The expression of hepatic IGF-I mRNA: The relative value of IGF-I mRNA was 1.19 +/- 0.06, 1.08 +/- 0.06 and 1.29 +/- 0.06 in NC, WC and Arg, respectively, while the value in WC was lower than that in NC (P < 0.05) group, and that in Arg group was much higher than that in WC group (P < 0.01). 5). The IGF-I level in the supernatant of cultured hepatocytes: When Arg concentration was 0.0750, 0.7500, 7.5000 mmol/L in the culture medium, the IGF-I level in the supernatant of hepatic cell medi-um was obviously higher than that in the medium without arginine (P < 0.01). Although IGF-I level decreased in the culture medium with arginine in the dose of 37.5000 mmol/L, it was still obviously higher than that in the medium without arginine (P < 0.01).

CONCLUSIONArginine could also produce the immune enhancing effect by stimulating hepatic IGF-I secretion.

Animals ; Arginine ; pharmacology ; Enteral Nutrition ; Insulin-Like Growth Factor I ; metabolism ; Liver ; drug effects ; secretion ; Male ; Rats ; Rats, Wistar ; Soft Tissue Injuries ; metabolism ; therapy

OBJECTIVETo summarize the surgical effect and clinical application value of esophagectomy with extended 2-field lymph node dissection for patients with esophageal carcinoma.

METHODSFrom June 1987 to December 2008, 1690 patients with esophageal cancer underwent esophagectomy with extended 2-field (thoracic and abdominal) dissection of lymph nodes. Patients with the middle and lower thoracic esophageal cancer underwent Ivor-Lewis esophagectomy, and patients with upper thoracic esophageal cancer underwent Akiyama esophagectomy. 2-field (thoracic and abdominal) lymph node metastases information and the 1, 3, 5, 10-year survival rates were analyzed retrospectively.

RESULTSLymph node metastases were found in 713 patients. The lymph node metastases rate was 42.2% (713/1690).Thoracic lymph node metastasis rate was 39.3% (665/1690), among which in the right pleural apical para-tracheal triangle was 20.7% (349/1690), in the posterior upper mediastinum was 26.3% (444/1690), in the lower mediastinum was 18.2% (307/1690). Abdominal lymph node metastasis rate was 20.1% (339/1690). THE Postoperative complication rate was 16.4% (278/1690), among which the pulmonary complication rate ranking the first, was 43.6% (136/312). The operative mortality rate was 0.2%. The 1-year, 3-year, 5-year and 10-year survival rates were 88.2% (1388/1574), 63.5% (868/1367), 54.8% (705/1287) and 30.8% (232/754), respectively. The 5-year survival rate in patients without lymph node metastasis was 76.2% (448/588), but that in patients with lymph node metastases was 36.8% (257/669).

CONCLUSIONThe results of this study demonstrated that Ivor-Lewis and Akiyama esophagectomy with two-field lymph node dissection exposes the operation fields clearly and make radical lymphadenectomy thoroughly, especially the lymph nodes in the posterior upper mediastinum around the recurrent laryngeal nerve and in the right pleural apical para-tracheal triangle. It is essential that patients with esophageal carcinoma with lymph node metastases should undergo esophagectomy with extended 2-field dissection of lymph nodes. This can elevate the postoperative 5-year survival rate remarkably.

Adenocarcinoma ; mortality ; pathology ; surgery ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; mortality ; pathology ; surgery ; Esophageal Neoplasms ; mortality ; pathology ; surgery ; Esophagectomy ; adverse effects ; methods ; Female ; Humans ; Lymph Node Excision ; adverse effects ; methods ; Lymphatic Metastasis ; Male ; Middle Aged ; Respiratory Insufficiency ; etiology ; Retrospective Studies ; Survival Rate

BACKGROUNDA severe deficiency of ADAMTS13 activity contributes to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Measuring the activity of ADAMTS13 is helpful for the diagnosis of TTP and the prognostic monitor in TTP patients. Most available assays are cumbersome and costly, so not easily adapted to routine laboratories. ADAMTS13 cleaves von Willebrand factor (VWF) within the domain A2, located between domains A1 and A3. Therefore, specific assays for ADAMTS13 activity could be based on the different structures of VWF before and after the cleavage. Using this hypothesis we try to establish a new and simple method to determine ADAMTS13 activity.

METHODSFirst, plasma samples were exposed in denaturing condition to allow cleavage of VWF by ADAMTS13. Then, the ADAMTS13 activity was measured with two novel monoclonal antibodies, SZ-129 and SZ-125, which specifically recognize the VWF A1 and A3 domains by using a two-site sandwich ELISA. Compared with a residual-collagen binding assay (R-CBA), plasma ADAMTS13 activities in 161 samples were assessed, and the inhibitory activities of ADAMTS13 autoantibody in 24 TTP patients were determined. The relationship of these two assays was analyzed by linear correlation, and the sensitivity and specificity of the new assay was also evaluated.

RESULTSPlasma ADAMTS13 activities in normal people and TTP, acute myocardial infarction (AMI), and idiopathic thrombocytopenic purpura (ITP) patients determined by the new assay were (89.75 +/- 7.93)%, (17.63 +/- 18.71)%, (68.55 +/- 18.08)%, (85.83 +/- 9.84)%, respectively. Results were consistent with those of R-CBA, the squared correlation factor was 0.9183 of the two assays. The new assay can easily discriminate a TTP plasma sample from a non-TTP plasma sample (P < 0.01), and the coefficient of variation for the new assay was 6.17%. In 23 idiopathic TTP patients, the inhibitor activity of ADAMTS13 autoantibody ranged from 12% to 100%, while no inhibitory activity was detected in one hereditary TTP patient.

CONCLUSIONThis new and simple assay for ADAMTS13 activity could be used routinely in the clinic to determine the activity of ADAMTS13.

ADAM Proteins ; metabolism ; ADAMTS13 Protein ; Adolescent ; Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Purpura, Thrombotic Thrombocytopenic ; metabolism ; Young Adult ; von Willebrand Factor ; metabolism