Effect of recombinant human annexin A5 on expression of p-PKCα and p120-catenin during endotoxin-induced damage to cardiomyocytes / 中华麻醉学杂志

Objective To evaluate the effect of recombinant human annexin A5 on the expression of phosphorylated protein kinase C alpha (p-PKCα) and p120-catenin during endotoxin-induced damage to cardiomyocytes.Methods H9c2 cells cultured in vitro were randomly divided into 3 groups (n=18 each) using a random number table:control group (group C),endotoxin group (group L),and recombinant human annexin A5 group (group A).Recombinant human annexin A5 (final concentration 5 ng/ml) was added,and the cells were incubated for 2 h in group A,and then lipopolysaccharide (final concentration 1 μg/ml) was added,and the cells were incubated for 4 h in L and A groups.At 4 h of incubation,cell apoptosis was detected using the cell apoptosis detection kit,the intercellular space was measured using the confocal microscopy,and the expression of p-PKCα and p120-catenin was determined by Western blot.The apoptosis rate was calculated.Results Compared with group C,the apoptosis rate was significantly increased,the intercellular space was significantly widened,the expression of p120-catenin was significantly down-regulated,and the expression of p-PKCα was significantly up-regulated in group L (P＜0.05).Compared with group L,the apoptosis rate and intercellular space were significantly decreased,the expression of p120-catenin was significantly up-regulated,and the expression of p-PKCα was significantly down-regulated in group A (P＜0.05).Conclusion Recombinant human annexin A5 can inhibit phosphorylation of PKCα and up-regulate the expression of p120-catenin,thus attenuating endotoxin-induced damage to cardiomyocytes.

4.4 Å Resolution Cryo-EM structure of human mTOR Complex 1

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates signals from growth factors, cellular energy levels, stress and amino acids to control cell growth and proliferation through regulating translation, autophagy and metabolism. Here we determined the cryo-electron microscopy structure of human mTORC1 at 4.4 Å resolution. The mTORC1 comprises a dimer of heterotrimer (mTOR-Raptor-mLST8) mediated by the mTOR protein. The complex adopts a hollow rhomboid shape with 2-fold symmetry. Notably, mTORC1 shows intrinsic conformational dynamics. Within the complex, the conserved N-terminal caspase-like domain of Raptor faces toward the catalytic cavity of the kinase domain of mTOR. Raptor shows no caspase activity and therefore may bind to TOS motif for substrate recognition. Structural analysis indicates that FKBP12-Rapamycin may generate steric hindrance for substrate entry to the catalytic cavity of mTORC1. The structure provides a basis to understand the assembly of mTORC1 and a framework to characterize the regulatory mechanism of mTORC1 pathway.

Starvation-induced autophagy in cultured non-small cell lung cancer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe autophagy induced by starvation in non-small cell lung cancer A459 and 95D cells.</p><p><b>METHODS</b>A549 and 95D cells in logarithmic growth in 1640 medium were cultured in Earle's balanced salt solution (EBSS) for 0, 1, 2, 3, 4 or 5 h. Autophagosome formation in the cell culture was observed by MDC fluorescent staining, and the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 in the cells were detected using Western blotting.</p><p><b>RESULTS</b>Compared with the control cells, the cells with prolonged starvation showed increased MDC-positive cells and autophagosome formation. The expression of Beclin-1 and the LC3-II/LC3-I ratio also increased as the starvation prolonged, reaching the peak levels at 3 h and 4 h, respectively.</p><p><b>CONCLUSION</b>Autophagy can be induced by starvation in A549 and 95D cells in correlation with the expression of autophagy-related proteins LC3 and Beclin-1. These cell models of nutritional deficiency-induced autophagy may allow for a better understanding of the role of autophagy in the development of non-small cell lung cancer.</p>

Role of Src tyrosine kinase in damage to alveolar epithelial cells caused by mechanical stretch / 中华麻醉学杂志

Objective To evaluate the role of Src tyrosine kinase in damage to alveolar epithelial cells caused by mechanical stretch.Methods MLE-12 cells cultured in vitro were randomly divided into 3 groups using a random number table:mechanical stretch group (group S),dimethyl sulfoxide control group (group D),and Src tyrosine kinase inhibitor PP2 group (group P).In D and P groups,dimethyl sulfoxide 30 μl/ml and PP2 100 μmol/L were added to the culture medium,respectively,and the cells were then cultured for 30 min.The cells underwent mechanical stretch for 8 h with frequency of0.5 Hz and amplitude of 20％ in the three groups.At 0,2,4 and 8 h of mechanical stretch,MLE-12 cells in 3 wells of each group were collected for determination of cell apoptosis with flow cytometry and expression of occludin using Western blot.The apoptosis rate was calculated.Results Compared with S group,no significant changes were found in the apoptosis rate and expression of occludin at each time point in group D,and the apoptosis rate was significantly decreased,and the expression of occludin was up-regulated at 2,4 and 8 h of mechanical stretch in group P.Conclusion The activation of Src tyrosine kinase is involved in damage to alveolar epithelial cells caused by mechanical stretch.

Role of protein kinase C in mechanical ventilation-induced lung injury in rats / 中华麻醉学杂志

Objective To investigate the role of protein kinase C (PKC) in mechanical ventilation-induced lung injury in rats.Methods Thirty healthy male Wistar rats,weighing 250-300 g,were randomly divided into 5 groups (n =6 each):control group (group C),small tidal volume group (group S),small tidal volume and PKC inhibitor group (group S + P),large tidal volume group (group L),and large tidal volume and PKC inhibitor group (group L + P).VT =42 ml/kg,RR =40 bpm,I∶E =1∶ 2,PEEP =0,FiO2 =21％ in groups L and L + P,while VT=7 ml/kg,RR=40 bpm,I∶E=1∶2,PEEP=0,FiO2 =21％ in groups S and S+P.The rats were only tracheostonized in group C,while the rats were mechanically ventilated for 4 h in the other four groups.PKC inhibitor bisindolylmaleinide Ⅰ 0.12 mg/kg was injected intramuscularly 1 h before anesthesia in groups S + P and L + P.The animals were sacrificed immediacy after tracheotomy in group C,and at 4 h of ventilation in the other four groups and lungs were removed for calculation of wet/dry lung weight ratio (W/D ratio) and for microscopic examination.The expression of occludin was determined in the lung tissues by Western blot.Results Compared with group C,W/D ratio was significantly increased and the expression of occludin was down-regulated in the other four groups (P ＜ 0.05).Compared with group S,W/D ratio was significantly increased and the expression of occludin was down-regulated in group L,and W/D ratio was decreased and the expression of occludin was up-regulated in group S + P (P ＜ 0.01).W/D ratio was significantly lower and the expression of occludin was higher in group L + P than in group L (P ＜ 0.01).The pathological changes were attenuated in groups S + P and L + P as compared with groups S and L.Conclusion PKC is involved in mechanical ventilation-induced lung injury in rats.