Mechanisms involved in PERK-dependent autophagy under arsenite exposure / 军事医学

Objective To explore whether PERK is involved in the regulation of arsenite-induced autophagy.Methods Human hepatoma cells HepG2 were cultured and treated with arsenite.The expression level of autophagic hallmarks and the activation status of PERK were detected by Western blotting.The transactivation of p53 and the induction of its downstream target genes expression were also detected by Western blotting after knockdown of PERK expression.Transactivity of p53 was detected by dual luciferase reporter assay after knockdown of PERK expression.Results An increase in the LC3BII:I ratio,the induction of Beclin-1 expression and the degradation of p62 were readily observed in arsenite-treated HepG2 cells,but the effects were abolished after knockdown of PERK expression.Furthermore,phosphorylation of p53 at Ser15 and Ser392,transactivation of p53 and the induction of its downstream target gene DAPK1 expression were effectively inhibited under the same PERK knockdown conditions.Conclusion PERK regulates arsenite-induced autophagy by activating p53-dependent DAPK1 upregulation.

PM2.5 induces VEGF expression and inflammatory responses by trans-activating AP-1 in human bronchial epithelial cells / 军事医学

Objective To explore the role of the transcriptional factor activator protein (AP)-1 in mediating vascular endothelial growth factor ( VEGF) expression in human bronchial epithelial cells exposed to PM 2.5.Methods Beas-2B cells was treated with PM2.5.Luciferase assay was used to detect the activation status of AP-1 and transcription of VEGF in the Beas-2B cells.The induced activation of c-Jun, ATF2 and VEGF expression was tested by Western blotting assay.Results PM2.5 induced transactivation of the transcriptional factor AP-1, accompanied by phosphorylation of the AP-1 components, c-Jun and ATF2 in Beas-2B cells.Moreover, when AP-1 activation was inhibited by knocking down c-Jun or ATF2 expressions, induction of VEGF expression was partially attenuated in Beas-2B cells.Conclusion AP-1 is a critical transcriptional factor in mediating PM2.5-induced VEGF expression and inflammatory responses in human bronchial epithelial cells.

IKKαregulates ultraviolet radiation-induced activation of p53 in a p38K-dependent manner / 军事医学

Objective To explore the signal transduction mechanism of inhibitor kappa B kinase α( IKKα) , one of the catalytic subunits of IKK complex , for regulating p53 transactivation in the cellular ultraviolet radiation ( UVB) repsonse. Methods The transactivation of p53 was determined by dual-luciferase reporter gene analysis system while the expression and activation of IKKα, IKKβ, p53 and p38K was detected by Western blotting assay .Results UVB exposure induced activation and transactivation of p 53 in the wild type mouse fibroblasts ,but the effect was blocked by IKKa deficiency and recovered by reconstitution of IKKαexpression.Under the same conditions , IKKαregulated p38K activation, while inhibi-ting p38K activation down-regulated p53 transactivation under UVB exposure .Conclusion IKKαregulates UVB-induced phosphorylation and activation of p 53 in a p38K-dependent manner .

Deficiency of Erbin in MDA-453 human breast cancer cells induces trastuzumab resistance in vitro / 军事医学

Objective To explore the effect of Erbin deficiency in MDA-453 cells on trastuzumab(Herceptin) resist-ance .Methods The specific short hairpin RNA ( shRNA) targeting Erbin was designed and cloned into plasmid pSuppres-sor, which was subsequently transfected into MDA-453 human breast cancer cells .After being selected by G418, MDA-453 cells stably expressing Erbin shRNA were obtained and nominated as MDA-453-Erbin sh.The MDA-453 cells express-ing control shRNA ( MDA-453-NC) were used as control cells .The expression of Erbin at the protein level in MDA-453-NC and MDA-453-Erbin sh cells was analyzed by Western blotting .Cell proliferation and colony formation assays were em-ployed to investigate the effect of Erbin knockdown on the sensitivity of MDA -453 cells to trastuzumab in vitro.The levels of Erbin expression in human breast cancer tissue and normal breast tissue samples were evaluated by immunohistochemistry . Results MDA-453 cells, in which Erbin expression was stably knocked-down, were established.Deficiency of Erbin in MDA-453 cells could antagonize the anti-proliferation effects of trastuzumab in vitro.The level of Erbin expression was de-creased in some breast cancer tissue samples compared with normal breast tissue samples .Conclusion Erbin deficiency may induce the resistance of breast cancer cells to trastuzumab therapy .

A novel designed single domain antibody on 3-D structure of ricin A chain remarkably blocked ricin-induced cytotoxicity.

Ricin A chain (RA), an N-glycosidase, is able to fatally disrupt protein synthesis by attacking the Achilles heel of the ribosome RNA (rRNA). As specific immunotoxins, emergence of inhibitors for RA may obtain access to antagonistics against ricin intoxication and contribute to ameliorate the concomitant side effects. Many experimental results showed that the engineered VHs, which possessed solubility, stability, small size and consequently easier to express, purify and manipulate in vitro, were self and long-lived molecules compared to synthetic peptides. In this study, based on the crystal structure of RA, a novel recombinant human single-domain antibody expressing a polypeptide against RA in the CDR3 loop (named rVH(PT)) was obtained using computer-guided molecular design method. Theoretically, rVH(PT) could penetrate deeply into the active cleft of RA and act as a potent antagonist analogue to block the RA-rRNA interaction. Followed results showed that the recombinant VH(PT) was easily expressed of high-yield production and in a partially soluble fusion form in Escherichia coli. In vitro cytotoxicity experiments demonstrated that it possessed remarkable ability to block ricin-induced cytotoxicity. This study highlights the potential of human VH to display biostructure and biofunction of peptides designed on RA functional domain and could be useful in developing new antidotes with potential therapeutic uses to neutralize unintended exposure to ricin.

Cleavage of Bcl-2 Protein by Activated Caspase-3 Is Associated with Inactivation of Lyn(p53/56) Kinase Activity in Human M-07e Leukemic Cells during Apoptosis / 中国实验血液学杂志

The growth of M-07e human megakaryocytic leukemia cells is strictly dependent on GM-CSF. In M-07e cells, the GM-CSF receptor (GM-CSF R) is composed of two subunits: a low affinity alpha subunit and a phosphorylated beta subunit, which is constitutively linked to lyn(53/56) protein tyrosine kinase. In this study, The role of lyn kinase in regulating TGF-beta 1-induced apoptosis in M-07e cells was examined. The removal of rhGM-CSF from the culture medium resulted in down-regulation of lyn kinase activity, followed by growth inhibition and programmed cell death. Apoptosis of M-07e cells was accompanied with a massive cleavage of Bcl-2 and Bax proteins into shortened fragments with molecular mass of 22 kD and 18 kD, respectively. Using specific inhibitors, the cleavage of Bcl-2, but not Bax, was found to be processed through activated caspase-3 (CPP32), which is abundantly expressed in M-07e cells. TGF-beta 1 inhibited rhGM-CSF-stimulated cell growth and promoted apoptosis in M-07e cells with a pattern identical to that induced by rhGM-CSF depletion, which included massive cleavage of both Bcl-2 and Bax proteins and inactivation of lyn kinase activity. TGF-beta 1 did not affect the levels of lyn protein or the beta-subunit, neither did it block the interaction between these two components. Also, TGF-beta 1 treatment did not diminish the expression of the alpha subunit in M-07e cells. Our results showed that TGF-beta 1 inhibits cell proliferation and promotes apoptosis in M-07e cells by inactivating the GM-CSF R-associated lyn kinase activity. Further, This study showed that Bcl-2 cleavage by activated CPP32 is a naturally occurring event associated with apoptosis, which is under the regulation of lyn kinase activation.

Gene cloning,expression and preliminary activity analysis of hBcl-2 / 中国免疫学杂志

Objective:Through gene cloning,expression and activity analysis of hBcl 2 to provide enough proteins for the development of new drugs on the basis of 3 D structue of hBcl 2 protein.Methods:Gene cloning using PCR amplification,identification with Western Blot and MALDI TOF MS.Results:hBcl 2 gene was cloned,inserted into pET28a(+) and solublely expressed in E.Coli.Its molecule weight was confirmed through MALDI TOF MS,which fits exactly with its theoretical value.After purification to reach electrophoresis homogeneity,it showed the ability of combining specifically with BH 3 domain of Bak,and then provide the basis for the further research on small chemical compounds which could specifically bind hBcl 2 protein.Conclusion:In this work hBcl 2 was successfully expressed and recovered its binding activity in a soluble form. [