This study is designed to obtain recombinant human acetylcholinesterase (rhAChE) and apply it in screening acetylcholinesterase inhibitors. The rhAChE was overexpressed in HEK293 cells transfected by plasmid of pCMV-AChE with the cationic liposome and rhAChE was found to be secreted into cell culture medium. AChE activity was assayed according to modified Ellman method to obtain kinetic parameters. IC so50 values for donepezil compounds of rhAChE were calculated to determine their activities of inhibition. The results showed that Km value was 151.9 micromol.L-1 donepezil inhibited rhAChE in a mixed competitive-noncompetitive way (Ki= 16.03 nmol.L-1, Ki = 18.36 nmol.L-1) and that most new compounds tested exhibited high activities of inhibition on rhAChE. The study suggests that rhAChE is available to be applied in screening AChE inhibitors in vitro.
Acetylcholinesterase ; genetics ; metabolism ; Cholinesterase Inhibitors ; analysis ; pharmacology ; HEK293 Cells ; Humans ; Indans ; analysis ; pharmacology ; Inhibitory Concentration 50 ; Kinetics ; Piperidines ; analysis ; pharmacology ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

Aim To evaluate the effects of pantoprazole on various experimental acute ulcer inrats and mice. Methods The model of a gastric ulcer of rats or mice was caused bystree- induced ulcer and ligatel pylurus-induced ulcer. Results & Conclusions At adose of 5, 10, 20 mg? kg-1 of Pantoprazole can markedly decrease the ulcer index ofstree-induced ulcer. Pantoprazole(4, 8, 16 mg? kg -1 ) significantly decrease the areaof ligated pylorus-induced gastric ulcer. It was also found that pantoprazole caninhibit the output of basic gastric acid.

Objective To find out the present situation of hepatitis B infection or immunity in Tianjin city,and to provide the scientific basis for the hepatitis B control and prevention.Methods 2 594 samples were selected with the methods of different proportionate stratified and cluster sampling,and the hepatitis B infection markers were detected by ELISA.Results The positive rates of HBsAg,anti-HBs,anti-HBc and HBV were 2.62％,46.72％,10.60％ and 51.54％.Conclusion Compared with the results in 1992,the positive rates of HBsAg,anti-HBc and HBV were decreased significantly,while the positive rate of anti-HBs increased significandy,which ascribed to the comprehensive measure with the vaccination against hepatitis B as a main strategy for control hepatitis B.

In order to investigate the angiogenic effect of intercellular adhesion molecule-1 (ICAM-1), two parts of experiment were performed. Chick embryo chorioallantoic membrane (CAM) assay was used for in vivo angiogenic research. The chick embryos were divided into 4 groups: ICAM-1 group (divided into 3 subgroups, I, II and III) for screening the angiogenic effect of ICAM-1 by adding different concentrations of ICAM-1 (0.1, 0.2 and 0.3 microg/microL) 5 microL into the chick embryo CAMs on the day 10 after incubation for every subgroup; Anti-ICAM-1 group A (divided into 2 subgroups, I and II) by adding different concentrations of Anti-ICAM-1 (1:100, 1:50) 5 microL into the chick embryo CAMs on the day 10 after incubation for every subgroup to evaluate the effect of ICAM-1 on the survival of microvessels through observing whether Anti-ICAM-1 could induce involution of the microvessels on CAMs; Anti-ICAM-1 group B (divided into 2 subgroups, I and II) by adding different concentrations of Anti-ICAM-1 (1:100, 1:50) 5 microL into the chick embryo CAMs on the day 6 after incubation for every subgroup to evaluate whether ICAM-1 involved in embryonic angiogenesis through observing the growth of microvessels on CAMs; Control group: ICAM-1 or Anti-ICAM-1 was substituted by PBS 5 muL on the day 10 or day 6 after incubation. Three days later, the CAMs were photographed in vivo, excised, sectioned and the number of microvessels was counted. In ICAM-1 group, there was increased number of microvessels arranged radially with "spoked-wheel" pattern around the gelatin sponges. The new microvessels growing perpendicularly to gelatin sponges were observed. The number of the microvessels growing in the CAM mesenchymes around the sponges in 3 subgroups was higher than that in control group (P<0.01), however, there was no significant difference among the 3 subgroups (P>0.05). In anti-ICAM-1 group A, the radially arranged microvessels were very unclear around the sponges contrast to that of ICAM-1 group. Few new microvessels were detected in the center of the sponges. The number of the microvessels growing in the CAM mesenchymes around the sponges in subgroup II was lower than that in control group (P<0.01). There was no significant difference in the number of the microvessels around the sponges between subgroup I and control group (P>0.05). In anti-ICAM-1 group B, the radially arranged microvessels were very unclear around the sponges contrast to that of control group. New microvessels were very scarce in the center of the sponges. The number of the microvessels growing in the CAM mesenchymes around the sponges in the 2 subgroups were less than that in control group (P<0.01), and there was significant difference between the 2 subgroups (P<0.05). It was suggested that ICAM-1 could induce angiogenesis and support the survival of microvessels, and ICAM-1 was involved in embryonic angiogenesis.

Objective To explore the molecular regulation mechanism of asporin in the matrix synthesis and secretion of the intervertebral disc,and to clarify its role in degenerative lesions of intervertebral disc.Methods There were 8 cases of intervertebral disc tissue in patients with severe intervertebral disc herniation (including typical clinical symptoms,signs and Pfirrmann's grade Ⅲ).There were 6 male and 2 female with an average age of (20.25 ± 3.37) years old (ranged from 11 to 28 years).After primary culture and redifferentiation in alginate beads,cells were reseeded and treated with different concentrations of TGF-β1 for 6,12,18 and 24 h.Total RNA extracted from the cells and was subjected to real-time PCR analysis to examine the expression of asporin.After silencing the expression of endogenous asporin by siRNA,the cells stimulated 24 h with TGF-β1.Total RNA extracted from the cells and was subjected to real-time PCR analysis to examine the expression of asporin,collagen Ⅱ and proteoglycans.After treatment of specific p38 inhibitor or ERK inhibitor for 12 h,cells were stimulated with TGF-β1 for 24h.Protein extracted from the cells by protein extraction kit to examine the level of asporin.Results In the primary intervertebral disc cell experiment,TGF-β1 stimulation induced asporin transcription significantly in a dose and time dependent manner.After 24 h stimulation,a significant difference between different concentration groups (5,10 and 15 ng/ml) was observed,2.754±0.24,3.651 ±0.319 and 4.583±0.38,respectively (F=24.782,P=0.001).Knockdown of endogenous asporin led to the upregulated expression of aggrecan and collagen]Ⅱ (aggrecan:t=7.387,P=0.002,collagen Ⅱ:t=4.443,P=0.0113).Specific p38 inhibitor was used to block p38 phosphorylation,and TGF-β1 on asporin induction was significantly inhibited.Conclusion Our results have verified a functional feedback loop between TGF-β1 and asporin in human intervertebral annulus cells indicating that TGF-β1 can increase asporin expression,whereas asporin inhibits TGF-β 1 signaling pathway by negative feedback,thereby inhibiting TGF-β1 mediated synthesis of extracellular matrix,and TGF-31 can increase asporin expression by p38 in human intervertebral disc cells.

Objective To investigate the effect of sarcopenia and vitamin D deficiency on osteoporosis (OP) in patients with rheumatoid arthritis (RA).Methods Six hundred and forty-eight patients with RA were enrolled into the study,while 158 normal subjects were recruited as the control group.Bone mineral density (BMD) at total hip and lumbar vertebra 2-4 were measured by dual energy X-ray absorptiometry (DEXA),limbs skeletal muscle mass was determined in 267 patients with RA and 156 normal subjects by bioelectrical impedance method.Serum 25-hydroxy vitamin D [25(OH)D] levels were determined by electrochemiluminescence in 234 RA patients and 68 normal subjects.Clinical and laboratory features,daily dosage and duration of glucocorticoid (GC) were recorded.Numerical data and categorical data comparisons were analyzed using t test,x2 test,linear correlation analysis,single factor analysis of variance test and Logistic regression analysis test.Results ① Incidence of OP in RA (37.8％,245/648) was significantly higher than that in the controls (13.9％,22/158)(x2=32.712,P＜0.01).Incidence of sarcopenia was evidently higher in RA,compared with normal subjects [55.8％(149/267),9.0％(14/156),x2=91.176,P＜0.01].Percentage of sarcopenia was higher in RA with OP compared with RA without OP group [76.6％(49/64),39.3％(35/89),x2=20.848,P＜0.01].②Compared to control group,serum 25 (OH)D levels were significantly lower in RA group [(13.4±9.7) ng/ml,(22.4±6.3) ng/ml,t=9.063,P＜0.01].Rate of vitamin D deficiency iu RA was also higher than that in controls [80.8％(189/234),36.8％(25/68),x2=49.412,P＜0.01].③The differences of serum 25 (OH)D levels among different bone metabolic state groups at lumbar vertebra in RA (normal bone mass,osteopenia,OP) were statistically significant (F=6.263,P=0.003),whichrepresented a clearly decreasing trend along with the decreasing of serum 25 (OH)D levels (P=0.001).④Linear correlation analysis found that skeletal muscle mass indexes at limbs in RA were positively correlated with BMD and serum 25 (OH)D levels (P＜0.05).⑥Logistic Regression analysis revealed that sarcopenia (OR=4.373,P=0.002),age (OR=1.083,P=0.001) and duration of disease (OR=1.074,P=0.029) were the risk factors for occurrence of OP in patients with RA.Conclusion Sarcopenia generally exists in patients with RA,which is correlated with decreasing of serum 25 (OH)D levels,and also is the risk factor for the occurring of OP in RA.

Objective To analyze and compare the clinical and laboratory features between patients with ankylosing spondylitis (AS) and nonradiographic axial spondyloarthritis (nr-axSpA).Methods One hundred and forty-one patients with AS and 73 cases with nr-axSpA were recruited.Clinical and laboratory indexes of individuals were recorded in detail,Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Ankylosing Spondylitis Disease Activity Score (ASDAS) crp were calculated.Spondyloarthritis research consortium of Canada (SPARCC) score standard was used to evaluate the degree of bone marrow edema in sacr-oiliac joint under magnetic resonance imaging scanning.T test,rank test and x2 test were used for statistical analysis.Results The average age of patients with AS was obviously higher than that of patients with nr-axSpA (t=4.962,P＜0.01).Patients with AS were more often male,and those with nr-axSpA were more often female (82.0％ of the AS patients were men and 49.3％ of the nr-axSpA patients were men (x2=24.079,P＜0.01).Disease duration of AS was significantly longer than that of the nr-axSpA (Z=6.396,P＜0.01).The incidence of human leukocyte antigen (HLA)-B27 positive in AS was 89.4％,which was similar to that in patients with nr-axSpA (84.9％) (x2=0.884,P＞0.05).21.6％ (21 cases) of patients with AS had peripheral swollen joints,which was higher than that in nr-axSpA (2.2％,x2=8.861,P=0.003).Forty cases in AS had tender joints (41.2％),only 6 patients in nr-axSpA had tender joints (13.3％,x2=11.458,P＜0.01).Serum erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) levels in patients with AS were clearly higher than those in nr-axSpA (P＜0.01) patients.In AS,the patient global assessment (PGA),BASDAI and ASDAScrp value was significantly higher than that in nr-axSpA (P＜0.01).There were no differences in SPARCC score or incidence of bone marrow edema in sacroiliac joint in magnetic resonance imaging scanning between AS and nr-axSpA (P＞0.05).Percentage of patients with occipit-to-wall distance higher than 0 cm in AS was higher than that in nr-axSpA,and the mean distance of fingers to ground in AS was also higher than that in nr-axSpA (x2=19.844,P＜0.01;Z=5.724,P＜0.01).Chest expansion degree and Schboer's test in AS was much lower than that in nr-axSpA,respectively (Z=3.083,P=0.002;Z=5.103,P＜0.01).BASFI in AS was higher than that in nr-axSpA (Z=5.840,P＜0.01).The ratio of joint function in AS was obviously worse than that in nr-axSpA (x2=1 1.369,P=0.01).Conclusion Compared to patients with nr-axSpA,AS patients are male predominant,and have severer inflammation in clinical and laboratory findings and are worse in functional status.

Objective To investigate the clinical value of serum 14-3-3η protein levels in patients with rheumatoid arthritis (RA) and secondary osteoporosis (OP). Methods 259 RA patients and 80 healthy controls were recruited. Serum 14-3-3η levels were determined by ELISA and bone mineral density (BMD) were detected by the DEXA. Results Firstly, the levels and the positive rate of serum 14-3-3η protein were significantly high-er in RA patients than healthy controls (P < 0.000 1). Secondly, ROC curve revealed that the sensitivity of 14-3-3η protein for diagnosis of early RA was 91.7% and its specificity was 99.6% when the cut-off point was 0.879 ng/mL (AUC = 0.917, P < 0.000 1). Finally, 14-3-3η protein concentrations revealed significant differ-ence among the groups of bone mass normal, osteopenia and osteoporosis in early RA (χ2=7.974, P = 0.019). Conclusion Serum 14-3-3η protein levels increase significantly in RA , especially in early RA , which is relat-ed to clinical symptoms and osteoporosis.

Objective To sequence and analyze the complete nucleotide sequence of the mitochondrial genome of Oncomelania hupensis. Methods Four long fragments were amplified by long PCR using the primers designed based on mtDNA-COⅠ, Cytb, 16S rRNA and COⅢ gene sequences, and sequenced by conserved primer-walking. Rusults The mitochondrial genome (GenBank accession no. FJ997214) was a circular molecule of 15 182 bp with a total A+T content of 67.32%, and contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 tRNA genes, and an A+T-rich region of 72 bp. All 13 protein-coding genes of the O. hupensis mtDNA used ATG as start codon. Canonical TAA and TAG termination codons were found in 12 protein-coding genes, and the remaining one (ND1) had an incomplete termination codon (T). Two short gene overlaps were found with a length of 4 bp and 7 bp, respectively. The length of 21 total intergenic region of mtDNA was 145 bp ranging from 1-30 bp. A total of 22 transferring RNA were found, all of which were typical cloverleaf structure except for two tRNASer, one tRNAGln and one tRNAIle. Conclusion The complete sequence of O. hupensis mitochondrial genome has been determined.

Objective To investigate the species diversity and distribution of medical mollusca in Shanghai City. Methods From August 2012 to October 2013,all kinds of habitats in 8 districts and counties in Shanghai City,namely Jiading,Qingpu, Baoshan,Minhang,Songjiang,Jinshan,Chongming,Pudong,were selected for the field survey according to the distribution characteristics of the river system,and all the specimens of medical mollusca in the investigation sites were collected and classi?fied by morphological identification. Meanwhile,the species composition,habitats as well as the fauna of the medical mollusca collected were analyzed. Results A total of 5 211 specimens were collected,which belonged to 2 classes,14 families,18 gen?era and 25 species,including Oncomelania hupensis hupensis,Pomacea canaliculata,Parafossarulus striatulus,Alocinma longicornis,Physa acuta,Galba pervia,Hippeutis cantori,etc. The species numbers of medical mollusca in Chongming,Jin?shan,Pudong new area and Qingpu districts(counties)were 22,22,21 and 20,respectively,which were more than those of other areas. The habitat analysis suggested that the species numbers in the river and wetland were the most,both of which were 14 species. The main faunas of the medical mollusca in Shanghai were the cosmopolitan and oriental species. Conclusions The freshwater gastropod species are paucity in Shanghai City,but almost of them can be served as the intermediate hosts of certain parasites to transmit snail?related parasitic diseases,so the surveillance of medical mollusca should be strengthened.