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In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.
Assuntos
Humanos , Sequência de Aminoácidos , Cromatografia Líquida , Quimotripsina/metabolismo , Digestão , Proteínas de Membrana , Espectrometria de Massas em Tandem , Tripsina , Vitamina K Epóxido RedutasesRESUMO
Objective To investigate the influence of risperidone on differentiation of 3T3‐L1 pre‐adipocytes .Methods 3T3‐L1 pre‐adipocytes were induced to differentiate into mature adipocytes by adopting the classic hormone cocktail method and observed by the oil red O staining .Meanwhile ,the inducing medium was added with risperidone for studying its influence on 3T3‐L1 pre‐adi‐pocytes differentiation .Results 3T3‐L1 pre‐adipocytes were successfully differentiated into the mature adipocytes ,0 .1 ,1 ,10μmol/L risperidone all could inhibit the differentiation of 3T3‐L1 pre‐adipocytes .Conclusion Risperidone can inhibit the differentiation of 3T3‐L1 pre‐adipocytes .
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Objective:To observe the effect of Gemcitabine ( GEM) on the viability and apoptosis of non-small cell lung cancer HCC827 in vitro. Methods:The cell viability,apoptosis and cell cycle of HCC827 cells induced by Gemcitabine were detected with cell counting kit-8 assay (CCK-8),Annexin V-FITC/PI staining and flow cytometry. The expression of Bcl-2 protein of cells treated with GEM was examined by Western blot assay. Results: There was significant inhibition effect on HCC827 cells treated with 0. 1-1 000 ng/ml of GEM,which can promote the occurrence of HCC827 cell apoptosis and arrest cell in the S phrase. The apoptosis induced by GEM was accompanied with the down regulation of Bcl-2 protein. Conclusion: GEM can inhibit the cell viability and induce the HCC827 cell apoptosis and S phrase arrest. Its cell dead type was apoptosis,which was related with the expression of Bcl-2 protein.
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[ ABSTRACT] AIM:To investigate the effect of HOX transcript antisense RNA ( HOTAIR) on the migration and invasion abilities of liver carcinoma HepG 2 cells.METHODS:The expression of phosphoinositide-3-kinase regulatory sub-unit 3 (PIK3R3) in the liver cancer and normal liver tissues was detected by immunohistochemistry .The efficiency of gene silencing of HOTAIR or PIK3R3 by LV3-shHOTAIR or LV3-shPIK3R3 was determined by qPCR and Western blot .The mi-gration and invasion abilities of HepG 2 cells after silencing of HOTAIR and PIK3R3 were measured by wound healing assay and Transwell Matrigel invasion assay .The expression of miR-214 after silencing of HOTAIR and PIK3R3 was analyzed by qPCR.The expression of HOTAIR and PIK3R3 in the HepG2 cells was also evaluated by qPCR after transfected with miR-214 mimics or miR-214 inhibitor .Dual-luciferase reporter assay system was used to determine the regulatory effect of miR-214 on HOTAIR and PIK3R3 expression.RESULTS:PIK3R3 expression increased significantly in the liver cancer tissues compared with normal liver tissues .The abilities of invasion and metastasis of hepatocellular carcinoma were reduced after silencing of HOTAIR and PIK3R3.miR-214 expression was increased when silencing of HOTAIR and PIK3R3 was per-formed.HOTAIR and PIK3R3 expression was reduced after transfection with miR-214 mimics.HOTAIR and PIK3R3 ex-pression was increased after transfection with miR-214 inhibitor.The results of dual-luciferase reporter assay test showed that miR-214 directly regulated HOTAIR and PIK3R3 transcription activity .CONCLUSION: HOTAIR regulates the ex-pression of PIK3R3 through miR-214, thus promoting the migration and invasion abilities in the liver cancer cells .
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Objective To prepare the schizophrenia mouse model by using peritoneal injection of dizocilpine maleate(MK‐801) , then to study the peripheral blood miRNA‐132 abnormal expression in schizophrenia mouse model .Methods Adult male Kunming mice(KM) were selected and randomly divided into the normal saline group(control group) and MK‐801 group(experimental group) .The behaviors changes were observed .Real‐time PCR method was used to detect the miRNA‐132 expression in peripheral blood .Results (1) Compared with the control group ,the stereotyped behavior score in the experimental group was increased sig‐nificantly(P<0 .05) ,the spontaneous activity was increased significantly(P<0 .05) ,showing that the up‐right number was signifi‐cantly decreased ,while the walking‐grid number was significantly increased(P< 0 .05) ,which was consistent with the manifesta‐tions of schizophrenia mouse model .(2)Compared with the control group ,the miRNA‐132 expressed in the experimental group was down‐regulated ,the difference was statistically significant(P<0 .05) .Conclusion miRNA‐132 is abnormally expressed in blood of schizophrenia mouse .