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

OBJECTIVETo explore etiology, clinical manifestation and immunological changes of infectious pneumonia of neonates in Chengdu area.

METHODSSerum specimens were collected from 111 infants with infectious pneumonia. Eight viral and mycoplasmal specific serum IgM antibodies were detected by enzyme linked immunosorbent assay (ELISA); C reactive protein (CRP), total IgG and its subclasses, IgA and IgM were determined by rate scattered nephelometry; T lymphocyte subpopulations were detected by biotin-streptavidin-peroxidase method, and clinical and other laboratory data were analyzed.

RESULTS(1) Etiological agents: specific serum IgM antibodies were positive in 40 of 111 cases (36.0%) with pneumonias. All the 30 control infants were negative for the specific serum IgM antibodies. Among 111 infants with infectious pneumonia, 20.7% had single viral or mycoplasmal infection, 40.5% had bacterial infection, 15.3% had viral and mycoplasmal infection with bacterial infection; 23.4% had infection with unknown agents. (2) The most common clinical manifestations were tachypnea and cyanosis. The next were cough, milk choking, rales, retractions of the supraclavicular, intercostal and subcostal areas. Roentgenographic examination commonly revealed vague opacities, increased density and patchy infiltration. (3) Immune status: (1) CD(3), CD(4) cell counts of infants with pneumonias were lower than those of the controls while their serum IgA, IgM concentrations were higher than those of the control. (2) The CD(3) and CD(4) cell counts of the group with bacterial infection were lower than those of the control group. (3) The serum IgA concentration of the group with viral and mycoplasmal infection was higher than those of the control group and the group with unknown infection. (4) The serum IgM concentration of the group with bacterial infection was higher than those of the control group. (5) There were no significant differences in CD(8) cell counts, CD(4)/CD(8), concentration of serum IgG and IgG(1 - 4) between pneumonia group and the control group, and among various infectious groups and the control.

CONCLUSIONPathogens of neonatal infectious pneumonia in Chengdu area included single viral or mycoplasmic infection or bacterial infection, viral and mycoplasmal infection with bacterial infection, and unknown infection. Immunological changes of newborn infants suffered from infectious pneumonia included declined CD(3) and CD(4) cell counts, particularly in bacterial infection.

Antibodies, Bacterial ; blood ; Antibodies, Viral ; blood ; Bacterial Infections ; complications ; C-Reactive Protein ; analysis ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin M ; blood ; Infant, Newborn ; Male ; Pneumonia ; diagnosis ; etiology ; immunology ; T-Lymphocyte Subsets ; immunology ; metabolism ; Virus Diseases ; complications

OBJECTIVETo determine the plasma DNA level and investigate its clinicopathological significance in women with cervical cancers.

METHODSBlood samples were collected from 42 cervical cancer patients, 20 patients with cervical intraepithelial neoplasia grade III (CINIII) and 20 healthy women. The plasma DNA was extracted using a commercial kit and detected by a fluorescentmeter.

RESULTSThe mean plasma DNA level in stage I cervical cancer patients was 12.78-/+5.58 ng/ml, significantly higher than that in CINIII patients (8.10-/+3.06 ng/ml) and normal controls (7.60-/+3.87 ng/ml) (P=0.001). The mean DNA level in stage II-III patients was 17.99-/+7.81 ng/ml, significantly higher than that in stage I patients (P=0.02). No significant difference was found in plasma DNA level between CINIII patients and the normal controls (P>0.05). When the cut-off for diagnosis of cervical cancer was 15.70 ng/ml, the sensitivity, specificity, positive predictive value and negative predictive value were 38.10%, 92.50%, 84.21% and 58.73%, respectively.

CONCLUSIONPlasma DNA level is closely related with malignant transformation and development of cervical cancer, and may become a useful means for differential diagnosis of cervical cancer.

Adult ; Aged ; Carcinoma, Squamous Cell ; blood ; diagnosis ; Cervical Intraepithelial Neoplasia ; blood ; diagnosis ; DNA, Neoplasm ; blood ; Female ; Humans ; Middle Aged ; Sensitivity and Specificity ; Uterine Cervical Neoplasms ; blood ; diagnosis

OBJECTIVETo investigate the effect of fluvastatin on lysophosphatidylcholine (LPC)-induced ventricular arrhythmias and its mechanism.

METHODSTwenty male SD rats were randomly allocated into two equal groups, namely LPC treatment group and fluvastatin pretreatment group. Langendorff apparatus was used for cardiac perfusion ex vivo with 5 µmol/L LPC for 5 min followed by washing for 30 min in LPC treatment group, and in fluvastatin pretreatment group, a 30-min perfusion with 10 µmol/L fluvastatin was administered before LPC perfusion. The LPC-induced nonselective cation current (I(NSC)) in the ventricular myocytes was recorded using the whole-cell voltage-clamp method.

RESULTSFluvastatin significantly inhibited LPC-induced ventricular tachyarrhythmia/fibrillation and I(NSC). The small G-protein Rho inhibitor (C3) and Rho-kinase inhibitor (Y-27632) in the pipette solution also suppressed LPC-induced I(NSC).

CONCLUSIONFluvastatin offers cardiac protection against LPC by inhibiting LPC-induced I(NSC). LPC induces fatal arrhythmia via a Rho/Rho-kinase-mediated pathway.

Animals ; Arrhythmias, Cardiac ; chemically induced ; metabolism ; Drug Antagonism ; Fatty Acids, Monounsaturated ; pharmacology ; Indoles ; pharmacology ; Ion Channels ; drug effects ; Lysophosphatidylcholines ; adverse effects ; Male ; Myocytes, Cardiac ; metabolism ; Rats ; Rats, Sprague-Dawley ; rho-Associated Kinases ; metabolism

ObjectiveTo investigate the effect of the Yap1 gene and tanshinone ⅡA on the proliferation, migration, and invasion abilities of Huh-7 hepatoma cells. MethodsA total of 10 pairs of human hepatocellular carcinoma (HCC) samples and adjacent tissue samples were collected in Zhongnan Hospital of Wuhan University from June 1 to December 1, 2019. Quantitative real-time PCR and Western blotting were used to measure the expression of the Yap1 gene and phenotype-related molecules. MTT cell proliferation detection reagent was used to measure the inhibition rate of cell proliferation after the treatment with different concentrations of tanshinone ⅡA. Western blotting was used to measure the changes in the expression of apoptosis-and migration-related markers after different interventions. Flow cytometry and Transwell assay were used to measure apoptosis and cell migration and invasion abilities. The data of 375 cases of liver cancer and 50 cases of relatively normal liver tissue samples were downloaded from The Cancer Genome Atlas, including clinicopathological information. The t-test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups. ResultsIn 8 of the 10 pairs of HCC samples and adjacent tissue samples, HCC samples had significantly higher expression of Yap1 than the adjacent tissue samples. Compared with the normal human liver epithelial cells L02, the Huh-7 and HCCL-M3 hepatoma cells had a significant increase in the expression of Yap1. The silencing efficiency of si-Yap1-3 transfection reached 87.004% at the protein level. MTT results showed that tanshinone ⅡA effectively inhibited the proliferation of Huh-7 cells, with a half inhibitory concentration of 8.683 μmol/L. After the cells were treated with si-Yap1-3 and tanshinone ⅡA, there was an increase in the expression of the downstream marker for proliferation and migration E-cadherin and a reduction in the expression of vimentin, and the results of Transwell assay showed that compared with the si-NC group, the tanshinone ⅡA+si-Yap1-3 group had significant reductions in the migration and invasion abilities of Huh-7 cells (migration: 43.19±2.88 vs 132.20±10.03, t=8.527, P=0.001; invasion: 53.95±4.20 vs 179.10±11.11, t=4.484, P=0.011). The group treated with si-Yap1-3 and tanshinone ⅡA had an increase in the expression of the apoptosis-related marker Bax and a reduction in the expression of Bcl-2, as well as a significantly higher early apoptosis rate than the si-NC group (2598% vs 9.21%, χ2=4.078, P＜0.05). ConclusionOncogene Yap1 silencing combined with tanshinone ⅡA can promote the apoptosis of Huh-7 hepatoma cells and inhibit their migration and invasion, which can provide certain guiding significance for clinical medication.

Objective To explore the application of modified seldinger technique (MST) in advanced cancer patients and its superiority.Methods The MST was used in 20 patients who required peripherally insert central catheter (PICC) and they were divided into experimental group (n=40) and control group (n=40). The experimental group was treated with seldinger technique and the control group was treated with traditional PICC. The intubation success rate, complication rate and clinical application advantages were accounted and compare between the two groups.Results The intubation success rate in experimental group was 97.5% and that in control group was 80.0%,there were statistical significance (χ2=6.135,P<0.05); the incience of postoperative complications between experimetal group and control group were statistical significance (χ2=12.468,16.970,2.051;P<0.05).Conclusions Modified Seldinger technique with ultrasound guided have a useful effect, it should be widely carried out in clinical.

BACKGROUNDWe have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B11, which mimics an ovarian carcinoma associated antigen OC166 - 9 and whose corresponding monoclonal antibody is COC166 - 9 (Ab1). In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment (scFv)/human granulocyte macrophage colony-stimulating factor (hGM-CSF) and 6B11scFv in BALB/c mice.

METHODSThe fusion protein 6B11scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSF. BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively.

RESULTSThe fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F (ab) 2' and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6B11ScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11scFv/hGM-CSF compared with the 6B11scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week.

CONCLUSIONThe fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11scFv/hGM-CSF for active immunotherapy of ovarian cancer.

Animals ; Antibodies, Anti-Idiotypic ; immunology ; Antibodies, Neoplasm ; blood ; Cancer Vaccines ; immunology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Immunization ; Immunoglobulin Fragments ; immunology ; Mice ; Mice, Inbred BALB C ; Ovarian Neoplasms ; immunology ; Recombinant Fusion Proteins ; immunology

Background We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B11, which mimics an ovarian carcinoma associated antigen OC166-9 and whose corresponding monoclonal antibody is COC166-9 (Ab1). In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment (scFv)/human granulocyte macrophage colony-stimulating factor (hGM-CSF) and 6B11scFv in BALB/c mice. Methods The fusion protein 6B11scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSF. BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively. Results The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F(ab)2' and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6B11ScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11scFv/hGM-CSF compared with the 6B11scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week. Conclusion The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11scFv/hGM-CSF for active immunotherapy of ovarian cancer.

OBJECTIVETo study the epidemic situation and dominant strain of influenza in children with acute respiratory infection (ARI) during Flu season from Oct. 2005 to Mar. 2006 in Taiyuan.

METHODSMadin-darby canine kidney (MDCK) cell culture and hemagglutination inhibition (HI) assay were used to isolate and identify type A influenza viruses (H1N1 and H3N2) and B influenza viruses from clinical samples collected from outpatients who visited the Department of Pediatric because of ARI from Oct. 2005 to Mar. 2006. Oct. 2005 and Mar. 2006, we collected 415 blood samples from children and adults to detect the influenza virus antibody titers by HI test to exclude respiratory diseases.

RESULTS7 strains of H1N1 were isolated from 87 clinical specimens, with a positive rate of H1N1 as 8.04%. Out of 415 blood samples being collected, the positive rates and the geometric mean titer of H1N1 antibody Mar. 2006 were significantly higher in 0-3, 3-7 and 7-18 year-olds than Oct.2005.

CONCLUSIONH1N1 epidemic influenza did occur among children in winter and spring of 2005--2006 in Taiyuan city.

Adolescent ; Animals ; Antibodies, Viral ; blood ; Cell Line ; Child ; Child, Preschool ; China ; epidemiology ; Dogs ; Hemagglutination Inhibition Tests ; Humans ; Infant ; Influenza A Virus, H1N1 Subtype ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; isolation & purification ; Influenza B virus ; isolation & purification ; Influenza, Human ; epidemiology ; Population Surveillance

OBJECTIVETo examine the urinary level of tissue factor (uTF) and its procoagulant activity (PCA) in patients with diabetes mellitus, and explore the relationship between uTF and renal damage in diabetes mellitus.

METHODSEighty-six patients with type 2 diabetes mellitus were divided into 3 groups according to urine albumin excretion (UACR), namely normal albuminuria group, microalbuminuria group and macroalbuminuria group. The levels of uTF, PCA, blood urea nitrogen (BUN), serum creatinine (CRE), serum cystatin C (CYSC), glycohemoglobin A1c (HbA1c), and high-sensitivity C-reactive protein (hs-CRP) were measured in all the patients and 21 healthy controls.

RESULTSCompared with normal control, the diabetic patients showed significantly increased levels of uTF and PCA. The urinary TF-PCA was positively correlated to BUN, CYSC, CRE, UACR, fasting glucose and hs-CRP, but not to uTF; only hs-CRP, UACR were positively correlated to uTF.

CONCLUSIONuTF is probably implicated in the development and progression of diabetic nephropathy.

Adult ; Albuminuria ; urine ; Blood Coagulation ; Case-Control Studies ; Creatinine ; urine ; Diabetes Mellitus, Type 2 ; physiopathology ; urine ; Female ; Humans ; Male ; Middle Aged ; Thromboplastin ; urine