ABSTRACT
The protective effect of an alpha-glucan (designated here as MT-α-glucan) from fruit body of maitake (Grifola frondosa) on NIT-1 pancreatic ß-cells damaged by streptozotocin (STZ) in vitro was investigated. The cell viability, insulin secretion, the activity of superoxide dismutase (SOD), glutathione peroxidase (GSHpx) and the content of reduced glutathione (GSH) increased significantly when the cells were incubated with MT-α-glucan (400, 200 µg ml(-1)). The content of malondialdehyde (MDA), nitric oxide (NO) production, and the activity of NO synthase (NOS), inducible NOS (iNOS) decreased significantly when the cells were incubated with MT-α-glucan. The destructive changes of NIT-1 cells ameliorated when incubated with MT-α-glucan under microscopic observation. These data suggested that MT-α-glucan had obvious protective effect on NIT-1 pancreatic ß-cells damaged by STZ, which might be related to its effects of decreasing levels of ß-cell-destroying factors such as oxidative stress and NO synthesis.
Subject(s)
Cytoprotection/drug effects , Glucans/pharmacology , Grifola/chemistry , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Streptozocin/toxicity , Animals , Cell Line , Cell Survival/drug effects , Fruiting Bodies, Fungal/chemistry , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/metabolism , Malondialdehyde/metabolism , Mice , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/metabolism , Oxidative Stress/drug effects , Superoxide Dismutase/metabolismABSTRACT
Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic arrest and induce apoptosis of tumor cells. A series of novel tetrahydro-beta-carboline derivatives were synthesized as kinesin spindle protein inhibitor and evaluated as potential antitumor agents. All compounds showed promising KSP inhibitiory activity. Compounds 8 and 9 exhibited better antitumor activity (Lung/A549, Stomach/AGS) than CK0106023 with GI50/IC50 values (1.07/1.62 and 1.46/3.27 micromol x L(-1), 1.09/>10 and 1.22/6.33 micromol x L(-1), respectively).
ABSTRACT
Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic arrest and induce apoptosis of tumor cells. A series of novel tetrahydro-beta-carboline derivatives were synthesized as kinesin spindle protein inhibitor and evaluated as potential antitumor agents. All compounds showed promising KSP inhibitiory activity. Compounds 8 and 9 exhibited better antitumor activity (Lung/A549, Stomach/AGS) than CK0106023 with GI50/IC50 values (1.07/1.62 and 1.46/3.27 micromol x L(-1), 1.09/>10 and 1.22/6.33 micromol x L(-1), respectively).
ABSTRACT
The potential effects of recombinant shark hepatical stimulator analogue (r-sHSA) in liver disease have been revealed in our previous studies. In order to further evaluate its clinic application, we carried out high cell-density fermentation in 5 L fermentor to get enough products. Based on the trials in shaking flask, we optimized the parameters for 5 L fermentor, including medium composition, medium supplement, inducer concentration and induction time, etc. In detail, the improved LB medium (0.97% glycerol, 0.91% yeast extract, 0.72% tryptone, 0.782% KH2PO4, 0.267% K2HPO4.3H2O, 0.062% MgSO4.7H2O, 0.5% NaCl, pH 7.0) is chosen to cultivate the engineering bacteria with the constant fermentation condition (pH 7.0, and the dissolved oxygen concentration is about 25%-30%). When bacterial culture reaches exponential phase, the modified feeding medium (620 g/L glycerol, 94.8 g/L tryptone, 3.3 mL/L trace elements, and 7.5 g/L MgSO4.7H2O) is then supplied through the method of exponential fed-batch mode. After the optical density (OD600) of engineering bacterial culture reaches to 23, the ultimately concentration of 0.5 mmol/L IPTG is added to induce the expression of r-sHSA for 6 h. Results show that the amount of r-sHSA production is (2.662 +/- 0.041) g/L, which is about 13.7 folds of the one optimized before.
Subject(s)
Animals , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Fermentation , Liver , Chemistry , Peptides , Genetics , Metabolism , Recombinant Proteins , Genetics , Sharks , MetabolismABSTRACT
With the development of the research on biotechnology and modern pharmacy, the application of enzyme drugs have grown rapidly and enzyme drugs have become an important branch of biopharmaceutics. In this article, some new varieties of therapeutic enzymes, enzyme targets, mechanisms and new technologies of application in therapeutic enzymes were reviewed, and the direction of development of therapeutic enzymes were discussed.
Subject(s)
Adenosine Deaminase , Genetics , Therapeutic Uses , Antineoplastic Agents , Therapeutic Uses , Enzyme Replacement Therapy , Methods , Fibrinolytic Agents , Therapeutic Uses , Protein C , Genetics , Therapeutic Uses , RNA, Catalytic , Genetics , Therapeutic Uses , Streptokinase , Genetics , Therapeutic Uses , Urokinase-Type Plasminogen Activator , Genetics , Therapeutic UsesABSTRACT
We have evaluated the anti-diabetic effect of a alpha-glucan (MT-alpha-glucan) from the fruit body of maitake mushrooms (Grifola frondosa) on KK-Ay mice (a kind of genetical type 2 diabetes animal model). The effects of MT-alpha-glucan (450 or 150 mg kg (-1)) on diabetic mice were investigated by observing the changes in body weight, the level of fasting plasma glucose, glycosylated serum protein (GSP), hepatic glycogen, serum insulin, triglycerides, cholesterol, free fatty acid, liver superoxide dismutase (SOD), glutathione peroxidase (GSHpx), reduced glutathione (GSH) and malondialdehyde (MDA). Moreover, the binding capacity of insulin receptors on liver crude plasma membranes was assayed and histopathological changes in the pancreas were observed. Treatment with MT-alpha-glucan significantly decreased the body weight, level of fasting plasma glucose, GSP, serum insulin, triglycerides, cholesterol, free fatty acid and MDA content in livers. Treatment with MT-alpha-glucan significantly increased the content of hepatic glycogen, GSH and the activity of SOD and GSHpx. Moreover, the insulin binding capacity to liver crude plasma membranes increased and histopathological changes in the pancreas were ameliorated in the treatment group. These data suggest that MT-alpha-glucan has an anti-diabetic effect on KK-Ay mice, which might be related to its effect on insulin receptors (i.e., increasing insulin sensitivity and ameliorating insulin resistance of peripheral target tissues).
Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Glucans/pharmacology , Grifola/chemistry , Hypoglycemic Agents/pharmacology , Receptor, Insulin/drug effects , Animals , Blood Glucose/drug effects , Blood Proteins/drug effects , Body Weight/drug effects , Cell Membrane/drug effects , Cell Membrane/metabolism , Cholesterol/blood , Disease Models, Animal , Fatty Acids, Nonesterified/blood , Female , Fruit , Glucans/isolation & purification , Glutathione/drug effects , Glutathione Peroxidase/drug effects , Glycoproteins/drug effects , Hypoglycemic Agents/isolation & purification , Insulin/blood , Insulin/metabolism , Liver/drug effects , Liver/metabolism , Liver Glycogen/analysis , Malondialdehyde/metabolism , Mice , Pancreas/drug effects , Pancreas/pathology , Receptor, Insulin/metabolism , Superoxide Dismutase/drug effects , Triglycerides/blood , Glycated Serum ProteinsABSTRACT
A strategy, termed alanine-scanning mutagenesis, was used to identify the amino acid residues which are critical to the antigenicity of Escherichia coli L-asparaginase (L-ASP). Three continuous alkaline residues, 195RKH197, were mutated to Ala selectively. Four mutant recombinant L-ASPs were constructed and expressed in E. coli, and then purified. The purified mutants showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were more than 95% pure by reverse high-performance liquid chromatography. The activities of wild-type and mL-ASPs in the fermentative medium were all about 130 U/mL. The change from 195RKH197 to 195AAA197 reduced the antigenicity of the enzyme greatly as shown in competition enzyme-linked immunosorbent assay using polyclonal antibodies raised against the wild-type L-ASP from rabbits. The results show that residues 195RKH197 of E. coli L-ASP are critical to its antigenicity.
Subject(s)
Antigens/immunology , Asparaginase/genetics , Asparaginase/immunology , Escherichia coli/enzymology , Escherichia coli/immunology , Mutagenesis, Site-Directed/methods , Antibodies/immunology , Asparaginase/isolation & purification , Asparaginase/metabolism , Cell Extracts , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Mutation/genetics , Recombinant Proteins/geneticsABSTRACT
AIM:To construct nine novel L-asparaginase mutants and study their enzyme-activity.METHODS:The mutants were constructed using overlap extension PCR according to the principle of alanine-scanning mutagenesis. The enzyme-activity was detected by Nessler's method. RESULTS:The DNA sequencing showed that the mutagenesis was consistent with the theoretical prediction. The enzyme-activity assay demonstrated that each mutant possessed enzyme activity equal to the original enzyme. CONCLUSION:Through gene modification,epitop of L-asparaginase was changed without activity loss.These results provide foundation for further study of the structure-function relationship of L-asparaginase.
ABSTRACT
This study was intended to establish a method of preparation of recombinant human insulin, with (His)6-Arg-Arg-human proinsulin (RRhPI) expressed by Escherichia coli. After DEAE-Sepharose Fast Flow ion-exchange chromatography, Sephadex G-25 chromatography and refolding, enzyme cleavage and Superdex 75 size exclusion chromatography,the RRhPI expressed by Escherichia coli in inclusion body form was converted to human insulin. The obtained recombinant human insulin was analyzed by SDS-PAGE, HPLC, amino acid composition analysis and bioidentity test (mouse convulsion test). The results indicate that our obtained preparation is highly purified, active recombinant human insulin.
Subject(s)
Humans , Chromatography, High Pressure Liquid , Escherichia coli , Genetics , Metabolism , Insulin , Genetics , Proinsulin , Genetics , Recombinant Proteins , GeneticsABSTRACT
The ocean is the treasure house by which human being live. The variety and particularity of marine creature provide people many physiological active substances, which are novel and unique in structure and effect. The recent research of marine active antitumor proteins and peptides was summarized in this article.
