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As a classic fluorescent detect technique, fluorescence resonance energy transfer (FRET) has been widely used in biological researches. Researchers have developed a series of fluorescence detect probes which were based on FRET. Caspase family plays an important role in apoptosis pathway, especially Caspase-8 which located, at the initial of death receptor mediated apoptosis pathway, whose its activation can trigger subsequent precaspases' activation and lead to apoptosis. So it is of great significance to detect the activation of Caspase-8 in apoptosis assay. In this study, a fluorescent probe based on FRET has been designed which can detect the activity change of Caspase-8 in cells. To identify the effectiveness and specificity of the probe, we measure the Caspase-8 activity under the Caspase-8 specifically activated apoptosis inducer RGD-TRAIL with the flow cytometry FRET detection platform. The results show that the probe can respond to the activity change of Caspase-8 in apoptotic cells, and the change can be quantified rapidly by flow cytometry. The study provides a more efficient and convenient detection method of Caspase-8 activity in living cells.
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Objective To construct pET21a-sBAFF by cloning the extracellular regions of 134-285 amino acids of BAFF, a member of human TNF family, and then express the gene in prokaryotic cells and purify the expressed product.Methods cDNA of K562 cell line was used as the template to amplify sBAFF gene to construct pET21a-sBAFF.Expression of sBAFF in E.coli BL21 was induced by IPTG, and the expressed proteins were assayed by SDS-PAGE.The bacteria were analyzed by sonication, and the target proteins mainly existed as inclusion bodies.Then sBAFF was purified by Ni2 +-IDA affinity chromatography.SDS-PAGE electrophoreses displayed that the expressed sBAFF migrated with a relative molecular weight of 18000.ResuIts The induction parameters such as temperature and inducing time were optimized.The target protein accounted for 38.59%of the total bacterial proteins.After refolding, 38.14% of sBAFF proteins were polymerized as an active trimer.The dimer form of sBAFF, which is representative of wrongly refolded product, accounted for very few.ConcIusion The expression and purification of BAFF which formed active trimer after refolding pave the way for its further function study and provide a novel approach for the development of BAFF-targeted therapeutics for autoimmune diseases.
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Objective:To study the biological characteristics of condylar chondrocytes isolated from mechanical stress stimulated TMJ condylar cartilage of rats.Methods:The rat models of mechanical stress stimulated condylar cartilage were made by class III orthope-dic force for 14 days.The cartilage from control and experimental rats were observed in gross view.The cell harvest rate and viability were examined,proteomic analysis was performed.Results:After stress stimulation the thickness,the elasticity of condylar cartilage and number of chondrocytes were significantly reduced.The weight of mandibular cartilage was decreased(P<0.01).The harvest rate and the viability rate of the cells were decreased(P<0.01).The chondrocytes in the experimental group appeared to be elongated. Proteomic analysis showed that stress-related proteins,signal transduction proteins were up-regulated;cytoskeleton proteins,cell prolif-erative proteins were down-regulated.Conclusion:Mechanical stress stimulation of condylar cartilage may result in biological activity and protein changes of the condylar cartilage chondrocytes.
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Objective To synthesize 99Tcm-TP5-3 and evaluate its biodistribution and kinetics as a molecular probe for the detection of apoptosis,and evaluate tumor apoptosis after a single dose of paclitaxel chemotherapy in MDA-MB-231 breast tumor model.Methods TP5-3 was labeled with 99Tcm directly,and analyzed with HPLC.The radioactivity in tissues was measured and expressed as ％ID/g and T/NT (tumor/muscle).The mice bearing MDA-MB-231 breast tumor were divided into two groups:the treatment group which was given a single dose of paclitaxel (40 mg· kg-1,via tail vein),and the control group which was injected with the same volume of normal saline.After therapy,99Tcm-TP5-3 was injected via tail vein in both groups (100 μ1 for each mouse).MicroSPECT/CT was performed at 3 h postinjection.Radioactivity in different tissues was determined after imaging.Apoptotic cells were measured with flow cytometry.The morphological changes of the apoptotic cells were observed by light microscopies.One-way analysis of variance,two-sample t test and linear correlation analysis were used to analyze the data.Results The radiolabeling efficiency was ＞ 95％ and the radiochemical purity of 99Tcm-TP5-3 was (96.0± 1.5)％ at room temperature for 4 h.The predominant uptake was found in the kidneys at 30 min postinjection ((8.48± 1.07) ％ID/g),with rapid tracer clearance from the circulation.By comparison with activity at 5 min postinjection ((13.74± 4.21) ％ID/g),85％ of the initial activity reduced in blood at 4 h ((2.07±0.35) ％ID/g; F=11.310,P＜ 0.05).99Tcm-TP5-3 was mainly accumulated in the kidneys,liver and stomach,and excreted via the kidneys.T/NT in the treated group was 4.21±0.06,which was significantly higher than that of the control group (1.57±0.67; t =12.820,P＜0.05).The radioactivity of tumor tissue in the treatment group was much higher than that in the control group (4.82±0.54) ％ID/g vs (1.44±0.38) ％ID/g,t=0.679,P＜0.05).The tumor uptake of 99Tcm-TP5-3 in the treatment group positively correlated well with the apoptotic cells (r =0.985,P＜0.05).Histopathology further confirmed that a large number of apoptosis had occurred in the tumor after paclitaxel treatment.Conclusion 99Tcm-TP5-3 appears to have potential to be a useful molecular probe for imaging tumor cell apoptosis.
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To explore the structure and function of thioredoxin glutathione reductase (TGR) from Schistosoma j aponi-cum ,the homologous model of TGR in Schistosoma j aponicum was constructed by Swiss-Pdbviewer based on sequence and structure alignment .The potential substrates binding sites of TGR were analyzed and these sites of various TGRs were also as-sessed .Our results showed that the homologous model of Schistosoma japonicum TGR based on Schistosoma mansoni TGR structure was proved to be reasonable by PROCHECK program .Analysis of binding sites showed that NADPH and GDS bind-ing sites were conservative sites and GSH binding site was a specific site for parasite .Our data suggested that inhibitors which work in NADPH and GDS binding sites of other various TGRs may also interact with TGR form Schistosoma j aponicum .GSH binding region might be one of the potential targets for design of specific inhibitors of parasite TGRs .In addition ,C-terminal of TGR plays an important role in electron transfer and may participate in the binding of the substrate .Thus compound inhibiting swing of C-terminal could effectively restrain Schistosoma j aponicum TGR activity .
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This study is to investigate the apoptotic induction effect of the combination of diosgenin and TNF-related apoptosis-inducing ligand (TRAIL) on non-small-cell lung cancer cell line A549 by using the Chou-Talalay method, and observe the mechanism of the combination. The apoptotic effect of diosgenin or TRAIL alone and their combination on A549 and normal cell line 293T proliferation was measured by MTT assay. Chou-Talalay method was used to evaluate the combination effect. Apoptosis was examined by Hoechst 33342 staining and flow cytometry assay. Western blotting detects the expression of apoptosis-associated proteins. Diosgenin or TRAIL alone can inhibit proliferation ofA549 in a concentration-dependent manner. According to the Chou-Talalay method, when f(a) = 0.1, CI > 1, when f(a) > 0.1, CI < 1. Combined with TRAIL, the IC50 of diosgenin decreases from 21.864 to 14.810 micromol x L(-1) (P < 0.05) on A549 cells. But for 293T cells, IC50 of diosgenin does not change significantly. As with Hoechst 33342 staining and flow cytometry assay, the apoptosis ratios also increased in the combination group. At protein expression level, combination-treated group displays increased Caspase-8, Caspase-9, Bid, Caspase-3 activation and PARP cleavage, significantly decreased Bcl-2 and increased Bax expression, and MAPK pathways were activated. The combination of diosgenin and TRAIL has synergistic effect on A549 cells.
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BACKGROUND: As a common anticoagulant, heparin is widely used in clinic, but it has remarkable side effects such as severe bleeding and heparin-induced thrombocytopenia, and it cannot inactivate fibrin-bound thrombin. Annexin Ⅴ derivative (AND) is inosculated with C-terminal of hirudin and annexin Ⅴ, and its anticoagulation and anti-thrombosis effects are compared with those of heparin. OBJECTIVE: To investigate the relationship between quantitative effectiveness and time effectiveness of AND on coagulation and thrombosis, and study its reliability. DESIGN: Completely randomized grouping design and controlled study. SETTING: Cardiac Department of amunicipal hospital. MATERIALS: The experiment was conducted at the Animal Laboratory of Jiangsu Provincial People's Hospital from July 2000 to April 2001. Totally 32 male New Zealand white rabbits were randomly divided into 4groups, namely, high dosage AND group, low dosage AND group, common heparin group and saline group with 8 in each group. METHODS: Heparin and AND were diluted with saline.①High dosage AND group: 0.7 mg/kg AND was injected intravenously and followed by intravenous dripping of 0.35mg/(kg ·h)for 2 hours.Low dosage AND group: 0.3 mg/kg AND was injected intravenously and followed by intravenous dripping of 0.15 mg/(kg·h) for 2 hours. Heparin group: 75 IU/kg heparin was injected intravenously and followed by intravenous dripping of 37.5 IU/(kg·h) for 2 hours. Saline group: The same volume of saline and medication were used as those in drug groups.② Blood sample was collected from the femoral vein before administration so as to test blood routine, activated partial thromboplastin time(APTT)and prothrombin time (PT) after 15-, 30- and 60-minute administration and 2-hour withdrawal.③Saccule was separated from endothelium of femoral artery to measure blood pressure of distal femoral artery at 15 minutes after administration.Time of pulse pressure equal to 0 mmHg was recorded when the vessel was occluded completely by a thrombus.Finally the injured femoral arteries whose vessel was stripped were collected to measure its length, wet weight and dry weight. ④Observation of AND toxicity and sideeffects:During the experiment,vital signs of the animals were measured,such as blood pressure,heart rate and breath;in addition,bowelhemorrhage was observed and the number of leucocytes was counted after dissection of some of the animals. MAIN OUTCOME MEASURES:①Effect of AND on blood coagulation system and arterial thrombosis.②AND toxicity and side effects. RESULTS: All the 32 white rabbits entered the final analysis. ① Anticoagulant effect: APTT: Fifteen minutes after administration, APTT in AND group was the longest,which was(136.86±39.46)s in high dosage AND group and (122.90±34.19) s in low dosage ANDgroup.Moreover, APTT was longer than that in saline group [(95.14±24.64) s], but shorter than that in common heparin group [(180.00±0.00) s, P ＜ 0.05, 0.01]. At 30 minutes after administration,AND in high dosage group still had coagulation,and APTT was (124.61±40.19) s in high dosage group, which was longer than that in saline group [(85.57±27.67) s], but APTT was (112.94±43.17) sin low dosage group,which was shorter than that in common heparin group [(85.57±27.67)s,P ＜ 0.05].APTT was shorter in high and low dosage groups than in common heparin group at 60 minutes after administration (P ＜ 0.05),and longer than that in saline group 2 hours after drug withdrawal,but there was not significant difference(P ＞ 0.05).PT:PT in common heparin group was longer than that in high and low dosage groups at 15,30 and 60 minutes after administration (P ＜ 0.05).② Effect on arterial thrombosis:Wet weight of thrombus:It was lighter in AND group than in common heparin group(P ＜ 0.05). Dry weight of thrombus:Thrombus was lighter in high and low dosage groups than in common heparin group, and was lighter in high dosage group than in low dosage group (P ＜ 0.05).Thrombus length:It was shorter in low dosage group than in saline group (P ＜ 0.05), and shorter in high dosage groupthan in common heparin group (P ＜ 0.05). Time of complete occlusion: It was longer in high and low dosage groups than in saline group(P ＜ 0.05).③ AND toxicity and side effects:The behavior of rabbits in high and low dosage groups was similar to that in other two groups. Obvious hemodynamic changes were not found, and bowel hemorrhage was not observed, either. CONCLUSION: AND is an effective anticoagulant and anti-thrombosis agent; the highest anticoagulation effect occurs at 15 minutes afteradminis tration. However, the anticoagulant effect is poor as compared to heparin.The effect is poorer after 60-minute administration. Effect of AND on thrombus is stronger than that of heparin,but the size of thrombus is smaller than that of heparin, and the dosage-dependence manner was found. In addition, the anti-thrombus effect of AND is stronger in high dosage group than in low dosage group.
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<p><b>OBJECTIVE</b>To explore the linkage relationship between specific genetic markers and arrhythmogenic right ventricular cardiomyopathy (ARVC) in Chinese pedigrees.</p><p><b>METHODS</b>The microsatellite genetic markers D2S152, D14S252, and D10S1664 were studied for their linkages to ARVC in five Chinese ARVC pedigrees and a normal population of 121 Chinese individuals. Genomic DNA of the pedigrees and normal population was amplified using PCR techniques. Denaturing polyacrylamide sequencing gel (4%) electrophoresis was used to detect microsatellite repeat polymorphisms. Gels were silver-stained. A classical linkage analysis program was used assuming models of autosomal dominance and recession.</p><p><b>RESULTS</b>The logarithm of the odds (LOD) scores of D2S152 with ARVC in LW, WD, DS, LC and TY pedigrees were 2.174, -0.589, -infinity, - (indicating that linkage is not supported in this mode), and -infinity respectively in autosomal dominant model (recombination fraction = 0.000 respectively)and were -infinity, -infinity, -infinity, -infinity, and 0.182 respectively in the autosomal recessive model. The LOD scores of D14S252 with ARVC in LW, WD, DS, LC and TY pedigrees were -, -, -infinity, -, and 0 respectively in autosomal dominant model, and were -infinity, -0.812, -infinity, -infinity, and 0.087 respectively in autosomal recessive model. The LOD scores of D2S152 with ARVC in LW, WD, DS, LC and TY pedigrees were -, -0.539, -, and 0.602 respectively in autosomal dominant model and were -, -infinity, -infinity, -infinity, and - infinity respectively in autosomal recessive model.</p><p><b>CONCLUSIONS</b>The LOD score for D2S152 in the LW pedigree was 2.174, indicating that the chance of linkage is about 150:1. This suggests that there is a possible ARVC-related gene near this marker. There were no clear linkage relationships between ARVC and D10S1664 and D14S252 in this family, and no linkages between ARVC and any of the three genetic markers in the other four families. These results also suggest that there is genetic heterogeneity in LW and in the other pedigrees.</p>
Subject(s)
Female , Humans , Male , Arrhythmogenic Right Ventricular Dysplasia , Genetics , Asian People , China , Genetic Linkage , Genetic Markers , Lod Score , Microsatellite RepeatsABSTRACT
Hirudin is one of the most potent anti-coagulant protein ever found, and its C-terminus is a key domain for inhibiting thrombin.In order to enhance its specificity,a novel anti-coagulant protein was constructed via fusing the C-terminus of hirudin to Annexin V, which was expected to sustain both anti coagulant activity and phorspholipid affinity. The structure of the designed protein was predicted with both molecular mechanics and dynamics. Molecular dynamics was adopted to simulate the docking interaction between the fusion protein and thrombin. The results showed the inhibitory activity of the fusion protein to thrombin.