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Objective:To construct the eukaryotic expression plasmid carrying hTERT-P2A-EGFP, and to explore its expression and transfection efficiency in the HEK293FT cells.Methods:The recombinant plasmid was constructed by using pBABE-puro-hTERT and pRRLSIN-cPPT-MSCV-EGFP plasmids.The hTERT,P2A,and EGFP genes were obtained using pBABE-puro-hTERT as template by PCR.And the correct hTERT was inserted into pRRLSIN-cPPT-MSCV-EGFP vector.Then the recombinant plasmid containing hTERT-P2A-EGFP gene was obtained and identified.The HEK293FT cells were transfected by the recombinant plasmid, and the expression of green fluorescence protein(GFP) was observed by fluorescence microscope.Results:The PCR results showed that the fragments of hTERT, P2A, and EGFP were 3 400, 110 and 720 bp.And the length of gene fragment(hTERT-P2A-EGFP)was 4 300 bp by enzyme digestion.The results of sequencing showed that the 1 547 site of the target gene was mutated.Using site-directed mutagenesis, the 1 547 site was successfully mutated.And the target gene sequence was completely identical with the sequence published in GenBank.The recombinant plasmid was transfected into the HEK293FT cells, and GFP was observed in the cells.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%.Conclusion:The recombinant plasmid carrying hTERT-P2A-EGFP gene is successfully constructed, and it can be used for cell transfection.
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Objective To investigate the secondary metabolites of the South China Sea spine body sponge Acanthella cavernosa.Methods The acetone extract of A.cavernosa was isolated and purified by repeated column chromatography on sili-ca gel,sephadex LH-20,and high-performance liquid chromatography (HPLC).Structures were determined by spectroscopic analysis and comparison with reported data.Results Nine compounds were isolated.Their structures were determined as kali-hinol E (1),kalihinol A (2),10-epi-kalihinol X (3),10-epi-kalihinol I (4),1 H-indole-3-carboxylic acid methyl ester (5), 1H-indole-3-carboxylic acid (6),3-buten-2-one,4-(2,3,6-trimethylphenyl)-(3E)(7),aristolone (8),and 5α,8α-epidioxy-(22E,24R)-erost-6,22-dien-3β-ol (9).Conclusion Compounds 5 to 8 were isolated from the sponge of genus Acanthalla for the first time.
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Objective To compare health status of senior high school graduates living as urban vs suburban areas in Beijing.Methods Wilcoxon rank sun test,Ridit analysis and fuzzy mathematics synthesis were used to evaluate body shape index,physical function index and health defects indicator of 39 982 senior high school graduates in Beijing.Results A total of 9 117 urban boys,9 299 suburban boys,10 380 urban girls and 11 186 suburban girls were studied.For the boys,the urban was superior to the suburban areas in body shape index (Rurt=0.518,Rsub=0.482,P＜0.05) and physical function index (Rurt=0.520,Rsub=0.480,P＜0.05),and the urban was inferior to suburban areas in health defects indicator (Rurb=0.501,Rsub=0.554,P＜0.05) and the composite index (Ruurb=0.484,Rsub=0.516,P＜0.05).For the girls,the urban was superior to the suburban areas in physical function index (Rurb=0.562,Rsob=0.442,P＜0.05),and the urban was inferior to the suburban areas in health defects indicator (Rurb=0.473,Rsub=0.523,P＜0.05).The differences between urban and suburban areas tended to be of no statistical significance in body shape (U=5.79,P＞0.05) and composite index (U=5.73,P＞0.05).In fuzzy mathematics synthesis,the scores of urban boys,suburban boys,urban girls and suburban girls were 68.37,69.13,69.83 and 69.79,respectively.Conclusion Our investigation identified some differences in body shape index,physical function index and health defects indicator among senior high school graduates living at urban vs.suburban areas,although the differences of synthetical health were not statistically significant.
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To observe the chelation of GRP78 with lead (Pb) and its localization changes, astroglial cells from Wistar rat brain were primarily cultured in medium with acetate Pb. The processes were terminated at different time points. The immunoprecipitation (IP) and Western blotting were used for GRP78 purification and expression and the Pb concentration was determined by employing atomic absorption spectrophotometry (AAS). The localization change of GRP78 was observed with colloid gold immunoelectron microscopy. The results showed that the expression of GRP78 was increased significantly in the cells treated with 1.0 mumol/L acetate Pb for 24 h and peaked at 96-192 h (P<0.01), and at the 12th day, the expression of GRP78 began to decrease but was still higher than normal (P<0.05). Pb content started to increase when cells were treated by acetate Pb for 24 h, and the peak appeared at 8 day (P<0.01), and then Pb content decreased gradually, but was still higher than normal (P<0.05). GRP78 protein expression began to remarkably increase when it transferred from ER to the cytosol around the nuclei 24 h after treatment with Pb. It is concluded that GRP78 in astroglia could strongly chelate with Pb ions and it might be a target protein of Pb.
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BACKGROUND:Whether plumbum(Pb) exposure induced learning and memory disorder in animals is correlated with the changes of the activity of protein kinase C(PKC) in the brain tissue? OBJECTIVE:To investigate the alterative rules of PKC activity in the brain tissue of rat in development stage under chronic exposure of Pb and its effects on the memory development of the mice. DESIGN:A randomized controlled study employing experimental animals as subjects. SETTING: Cellbiology Key Laboratory of the Ministry of Health,China Medical University. MATERIALS:The study was conducted in the Laboratory of Biochemistry and Molecular Biology of China Medical University. Totally 72,5- 6 weeks old Kunming mice were selected. METHODS:Lead acetate of different concentration was used to feed female mice after copulation.Fetal mice contacted Pb through breast feeding and drinking water.Fetal mice were executed separately on 1st day(P1),8th day(P8),15th day(P15),22nd day(P22), and 30th day(P30) after birth for brain tissue harvesting.PCK activity in the brain tissue of the mice exposed to Pb was detected in vitro by[γ 32P] ATP.Budding mice were exposed to lead acetate of different concentration for the observation of the effects of Pb in difference concentration on the memory of the mice through memory behavioral training and test by passive avoidance response experiments. RESULTS:PKC activity detection indicated that PKC activity in brain of Pb exposed budding mice during initial growth stage was higher than normal,while it was lower than normal during advanced stage of growth.High concentration Pb had relative stronger inhibition on PKC activity.Memory behavioral training indicated that low concentration Pb would induce elevation of memory curve in the initial stage of the development in mice but reduction of memory curve in middle and long term.The increase of Pb concentration would reduce memory curve. CONCLUSION:Pb has inhibitive effects on the development of PKC activity in brain tissue of mice:the higher the Pb concentration is, the more significant the inhibition is.Low concentration Pb seems to have stimulatory effects on memory in short period while long term exposure to Pb would induce memory inhibition.The inhibitive effect of high concentration Pb is more significant.Pb has certain correlation with PKC activity in brain tissues of budding mice and memory function.
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In order to meet the requirements of medical teaching contents and curriculum system reform, we adopt the experience ofHarvard University School of Medicine to integrate Biochemistry and Cell Biology into a new one—— "Chemistry and Biology of theCell". We have carried out the integration of the two courses three times. In this new course, repeated contents have been reduced, thelinkage of the two courses has been increased, and the burden of students was lightened,which makes students contact the clinic earlierand enables to improve the students' ability to solve problems.