Nuclear apoptosis induced by isolated mitochondria

We isolated and purified mitochondria from mouse livers and spinach leaves. When added into egg extracts of Xenopus laevis, they caused nuclei of mouse liver to undergo apoptotic changes. Chromatin condensation, margination and DNA ladder were observed. After incubating isolated mitochondria in some hypotonic solutions, and centrifuging these mixtures at high speed, we got mitochondrial supernatants. It was found that in the absence of cytosolic factor, the supernatant alone was able to induce apoptotic changes in nuclei. The effective components were partly of protein. DNA fragmentation was partly inhibited by caspase inhibitors AC-DEVD-CHO and AC- YVADCHO. Meanwhile, caspase inhibitors fully blocked chromatin condensation. Primary characterization of the nuclear endonuclease(s) induced by mitochondrial supernatants was also conducted. It was found that this endonuclease is different from endonuclease G, cytochrome c-induced nuclease, or Ca2+-activated endonuclease.

Novel derivative of Danshensu acts as anti-thrombotic agent for modulation of protein disulfide isomerase family member ERp57

OBJECTIVE To identify the specific targets of a novel derivative of Danshensu ADTM as the protein disulfide isomerase(PDI)family proteins including ERp57.To further investigate the underlying mechanism of ADTM to modulate ERp57 to regulate platelet function with direct interaction withαⅡbβ3 integrin.METHODS To isolate the protein targets that bound to ADTM,a biotin-conjugated ADTM analogue(BAA)was designed and synthesized.BAA(300μmol·L-1)was incubated with rat blood platelet lysates and the BAA-protein complexes were pulled down with NeutrAvidin-agarose followed by protein profiling using LC-MS/MS.To determine platelet aggregation in vitro,rabbit platelets were incubated with the indicated concentrations of compounds and aggregation was induced by ADP(10μmol·L-1)or AA(200μmol·L-1)and measured using a platelet aggregometer.To determine platelet aggregation-induced by ADP in rat in vivo,ADTM(5-20mg·kg-1)in comparison with DSS(10mg·kg-1)and clopidogrel(18mg·kg-1)were administered daily by i.v.injection for 5d,respectively.To determine the action of ADTM on the ERp57/αⅡbβ3 interaction,it was examined by immunoprecipitation with anti-αⅡbβ3antibody,followed by detection of ERp57 immunoreactivity using immunoblotting.RESULTS BAA could bind to various proteins involved in platelet function.In particular,platelet aggregation-associated proteins were identified with>95% protein identification probability including ERp72,ERp57ERp5 and PDI,which are members of the protein disulfide isomerase(PDI)family related to platelet function and redox homeostasis.ADTM exhibited potent inhibition on the redox activity of ERp57 in a concentration-dependent manner(IC50=100 300μmol·L-1).In in vitro studies,ADTM exhibited concentration-dependent inhibition on ADP-induced and AA-induced platelet aggregation with comparable effects to aspirin and clopidogrel.In vivo study showed that ADP-induced platelet aggregation was significantly compromised(>40%reduction)in rats treated with ADTM(20mg·kg-1).Similarly,ADTM also exhibited significant anti-thrombotic effect in vivo as shown in the ferric chloride(FeCl3)-induced venous thrombosis.Immunoprecipitation with anti-αⅡbβ3antibody,followed by detection of ERp57 immunoreactivity using immunoblotting showed that ADTM disrupted the interaction of ERp57 with αⅡ bβ3.CONCLUSION These results demonstrated that ADTM exhibited broad-spectrum anti-platelet activities and ERp57 is a potential therapeutic target for anti-platelet therapy.