ABSTRACT
Myosin II plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin II ATPase activity based on molybdenum blue method, using a known myosin II ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate (ATP) and calcium chloride, pH, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin II ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin II ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin II ATPase inhibitors in vitro.
Subject(s)
Animals , Rabbits , Biological Products , Chemistry , Drug Evaluation, Preclinical , Methods , Enzyme Inhibitors , Chemistry , Kinetics , Molybdenum , Chemistry , Myosins , Chemistry , MetabolismABSTRACT
Previously, an mAb 10F7 was developed against H5N1 hemagglutinin, which was highly specific to 34 different H5N1 strains and showed good neutralizing activity. In the present study, the single-chain fragment of the antibody was cloned into a prokaryotic vector and then expressed in E. coli. The activity of the scFv was tested in hemagglution inhibition and neutralization experiment. Two H5N1 virus strains were inhibited to bind erythrocyte cells by the scFv while the H9 virus was not. Also, five H5N1 virus strains were neutralized during infecting MDCK cells. These results showed an approachable method for developing therapeutic antibody to H5N1 virus.
Subject(s)
Animals , Amino Acid Sequence , Antibodies, Viral , Genetics , Allergy and Immunology , Metabolism , Antibody Specificity , Allergy and Immunology , Birds , Virology , Cell Line , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel , Influenza A Virus, H5N1 Subtype , Allergy and Immunology , Influenza in Birds , Virology , Neutralization Tests , Recombinant Proteins , Allergy and Immunology , Metabolism , Single-Chain Antibodies , Genetics , Allergy and Immunology , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To compare therapeutic effects of Shu-Mu point combination, routine needling method and western medicine treatment on endometriosis (EM).</p><p><b>METHODS</b>Ninety cases were randomly divided into a Shu-Mu point combination group (n = 30) treated with acupuncture at Ganshu (BL 18), Pishu (BL 20), Shenshu (BL 23), Qimen (LR 14), Zhangmen (LR 13), Jingmen (GB 25); a routine needling group (n = 30) treated with acupuncture at Hegu (LI 4), Zhongji (CV 3), Guanyuan (CV 4), Sanyinjiao (SP 6) and a western medicine group treated with oral administration of Danazol. Clinical symptoms and signs, tumor marker serum CA125 values and adverse effects in the three groups were investigated before and after treatment.</p><p><b>RESULTS</b>The total effective rate was similar in the three groups. The Shu-Mu point combination group was superior to other two groups in improvement of dysmenorrhea, irregular menstuation, lumbago and sacrodynia, anus engorge, etc. (P < 0.01). After treatment, serum CA125 in the Shu-Mu point combination group significantly decreased (P < 0.01).</p><p><b>CONCLUSION</b>Shu-Mu point combination needling method has an obvious therapeutic effect on endometriosis, with lower adverse effect than that of the western medicine group.</p>