Otub1 stabilizes MDMX and promotes its proapoptotic function at the mitochondria.

Otub1 regulates p53 stability and activity via non-canonical inhibition of UbcH5, the MDM2 cognate ubiquitin-conjugating enzyme (E2). However, whether Otub1 regulates MDMX stability and activity is not clear. Here we report that Otub1 also suppresses MDM2-mediated MDMX ubiquitination in cells and in vitro, independently of its deubiquitinating enzyme activity. Consequently, overexpression of Otub1 markedly stabilized MDMX and increased its levels, whereas knockdown of Otub1 reduced the levels of MDMX. Interestingly, MDMX induced by Otub1 can localize to mitochondria in addition to the cytosol, enhance p53 phosphorylation at S46 (p53S46P) and promote mitochondria-mediated apoptotic pathway. Knockdown of MDMX reduced Otub1-induced p53S46P, which was shown to be critical for p53's mitochondrial function and apoptotic activity. Furthermore, Otub1 promotes UV-irradiation-induced p53S46P and apoptosis, which can be significantly inhibited by MDMX depletion. Together, these results suggest that Otub1 stabilizes MDMX and promotes p53S46P and mitochondria-mediated apoptosis, providing an alternative mechanism of Otub1's role in apoptosis.

Detection of coronary stenosis with myocardial contrast echocardiography using regadenoson, a selective adenosine A2A receptor agonist.

AIMS: Regadenoson is comparable to adenosine in pharmacologic radionuclide stress tests but has not been studied with stress myocardial contrast echocardiography. This study assessed the haemodynamic profile and ability of regadenoson, a novel selective A(2A) receptor agonist, to detect coronary artery stenosis during myocardial contrast echocardiography. METHODS AND RESULTS: Myocardial contrast echocardiography was performed to measure myocardial blood volume, myocardial blood flow velocity, and total regional myocardial blood flow before and after administration of regadenoson (5 µg kg(-1), 10 s bolus) in 10 open-chest dogs with mild-to-moderate coronary stenosis that was not flow limiting at rest. Regadenoson decreased blood pressure but did not change heart rate. It increased coronary blood flow significantly (P < 0.05) for 30 min, which was attenuated in proportion to coronary stenosis severity. Whereas myocardial blood volume maximally increased by 0.5-0.75-fold in the control region, it did not change in the region supplied by the non-flow limiting stenosis. Perfusion defects were visually and quantitatively detectable for as long as 10 min after administration of regadenoson. No arrhythmias were noted with regadenoson either prior to or during myocardial contrast echocardiography. CONCLUSION: Regadenoson can be used as a vasodilator stress agent with myocardial contrast echocardiography to detect the presence of physiologically significant coronary stenosis. The optimum time for image acquisition was 3-10 min after drug administration.

[Characterization of the biological activities of lyophilized recombinant adenovirus expressing the triple mutant of hypoxia inducible factor-1α].

OBJECTIVE: To investigate the influence of lyophilization on the biological activity of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1α (Ad-HIF-1α-564/402/803). METHODS: Ad-HIF-1α-564/402/803 was amplified from HEK293A cells and purified by ultracentrifugation in CsCl gradient solutions. The infection efficiency was observed by X-gal staining. The lyophilized adenovirus was prepared under appropriate conditions. Before and after lyophilization, the effect of Ad-HIF-1α-564/402/803 on hMVEC proliferation was evaluated by MTS assay. The recombinant adenovirus was confirmed by PCR and DNA sequence analysis before and 1 day, 6 months and 12 months after lyophilization, and hMVECs infected with Ad-HIF-1α-564/402/803 at these time points were examined for HIF-1α protein expression using Western blotting. RESULTS: No significant changes were observed in the effect of lyophilized Ad-HIF-1α-564/402/803 on hMVECs proliferation at the optimal multiplicity of infection of 100 pfu/cell (P>0.05). At the 4 time points, the recombinant adenovirus HIF-1α showed no structural alterations or significant changes in the expression level of HIF-1α protein in the transfected hMVECs (P>0.05). CONCLUSION: Lyophilized Ad-HIF-1α-564/402/803 can maintain its biological activities for a long time.

Perturbation of 60 S ribosomal biogenesis results in ribosomal protein L5- and L11-dependent p53 activation.

Ribosomal proteins play an important role in p53 activation in response to nucleolar stress. Multiple ribosomal proteins, including L5, L11, L23, and S7, have been shown to bind to and inhibit MDM2, leading to p53 activation. However, it is not clear whether ribosomal protein regulation of MDM2 is specific to some, but not all ribosomal proteins. Here we show that L29 and L30, two ribosomal proteins from the 60 S ribosomal subunit, do not bind to MDM2 and do not inhibit MDM2-mediated p53 suppression, indicating that the ribosomal protein regulation of the MDM2-p53 feedback loop is specific. Interestingly, direct perturbation of the 60 S ribosomal biogenesis by knocking down either L29 or L30 drastically induced the level and activity of p53, leading to p53-depedent cell cycle arrest. This p53 activation was drastically inhibited by knockdown of L11 or L5. Consistently, knockdown of L29 or L30 enhanced the interaction of MDM2 with L11 and L5 and markedly inhibited MDM2-mediated p53 ubiquitination, suggesting that direct perturbation of 60 S ribosomal biogenesis activates p53 via L11- and L5-mediated MDM2 suppression. Mechanistically, knockdown of L30 or L29 significantly increased the NEDDylation and nuclear retention of L11. Knocking down endogenous NEDD8 suppressed p53 activation induced by knockdown of L30. These results demonstrate that NEDDylation of L11 plays a critical role in mediating p53 activation in response to perturbation of ribosomal biogenesis.

[Effect of different doses of recombinant adenovirus mediated hypoxia-inducible factor-1alpha on angiogenesis in ischemic lower hind limb of rabbits].

OBJECTIVE: To investigate the effect of recombinant adenovirus-mediated hypoxia-inducible factor-1alpha (Ad-HIF-1alpha) at different doses on angiogenesis in a rabbit model of hind limb ischemia. METHODS: Left hind limb ischemia was induced in 45 Zealand white rabbits by ligation of the left femoral artery. The rabbits were randomly divided into 5 groups (n=9) to receive intramuscular injections of 0.5 ml saline, 2x10(10) PFU empty vector (Ad-null), or different doses of Ad-HIF-1alpha (2x10(9), 2x10(10) or 2x10(11) PFU) immediately after the operation. On the 7th day after the operation, real-time PCR was used to detect the expression of HIF-1alpha mRNA in the skeletal muscles. Immediately and on the 14th and 28th days after the operation, contrast enhanced ultrasound (CEU) was used to observe the blood perfusion of the hind limb. On the 28th day postoperatively, immunohistochemistry for CD31 was performed to evaluate the microvascular density (MVD). RESULTS: Real-time PCR showed that Ad-HIF-1alpha significantly increased the expression of HIF-1alpha mRNA (P<0.01) in a dose-dependent manner as compared with that in the saline and Ad-null groups (P<0.01). CEU revealed greater blood perfusion in the hind limb of rabbits in association with increased dose of Ad-HIF-1alpha (P<0.05 or P<0.01); similar changes in the MVD was observed following Ad-HIF-1alpha injections as shown by immunohistochemistry (P<0.05 or P<0.01). No significant differences were found either in the blood perfusion or MVD between saline and Ad-null groups (P>0.05). CONCLUSION: Ad-HIF-1alpha can dose-dependently promote the angiogenesis in the ischemic limb of rabbits.

[Targeted delivery of bone marrow mesenchymal stem cells by ultrasound-mediated microbubble destruction].

OBJECTIVE: To investigate the feasibility of bone marrow mesenchymal stem cell (MSC) transplantation with ultrasound-targeted microbubble destruction. METHODS: Twenty-one Wistar rats were divided into MSCs-iv group (MSCs-iv), ultrasound+MSCs-iv group (US+MSCs-iv), ultrasound+microbubble+MSCs-iv group (US+MB+MSCs-iv) with intravenous MSC transfer, ultrasound and microbubble treatment as indicated. The skeletal muscles were obtained from the rats for microscopic examination with HE staining. The hindlimb gracilis and semimembranosus muscles were sampled 7 days after MSC transplantation, and the transplanted MSCs were detected by immunohistochemistry. The vital organs were collected from rats in US+MB+MSCs-iv group for immunohistochemistry. RESULTS: In US+MB+MSCs-iv group, HE staining demonstrated the presence of red blood cell leakage into the tissue space in the gracilis and semimembranosus muscles, and immunohistochemistry identified large numbers of transplanted MSCs in the the gracilis and semimembranosus muscles and the spleen, whereas no labeled cells were detected in the skeletal muscles in other groups. CONCLUSION: Ultrasound-targeted microbubble destruction provides a useful means for enhancing the efficiency of stem cell transplantation.

[Assessment of the cardiotoxicity of recombinant human endostatin using myocardial biochemical markers in cancer patients].

OBJECTIVE: To evaluate the value of the myocardial biochemical markers including creatine kinase MB isoenzyme (CK-MB), cardiac isoform of Tropnin-T (cTnT) and N-termimal pro-brain natriuretic peptide (NT-proBNP) and electrocardiogram (ECG) in monitoring the cardiotoxicity of recombinant human endostatin (rh-endostatin) in cancer patients. METHODS: Forty cancer patients were divided into two groups and received rh-endostatin in addition to chemotherapy (group A, n=24) or chemotherapy only (Group B, n=24). Serum CK-MB, cTnT levels and plasma NT-proBNP levels were measured and the ECG was recorded in all the patients before and after each of the two therapy cycles. RESULTS: In group A, serum CK-MB, cTnT and plasma NT-proBNP levels were significantly increased after the treatment in comparison with the baseline levels (P<0.05), but such increment was not observed in group B (P>0.05). With comparable baseline levels of CK-MB, cTnT and NT-proBNP before the treatment (P>0.05), patients in group A showed significantly higher levels of the indices than those in group B after each therapy cycle (P<0.05). Increased ECG abnormality were observed after rh-endostatin treatment in Group A (P<0.05) at a rate significantly higher than that of Group B after the second treatment cycle (P<0.05). CONCLUSION: Rh-endostatin has definite cardiotoxicity, and detection of the myocardial biochemical markers of CK-MB, cTnT and NT-proBNP may help predict the occurrence of cardiotoxicity.

[Mechanism of recombinant adenovirus-mediated mutations of hypoxia inducible factor 1alpha in modulation of cell apoptosis].

OBJECTIVE: To investigate the mechanism by which recombinant adenovirus (Ad)-mediated mutations of hypoxia inducible factor 1alpha (Ad-HIF-1alpha-Ala564-Ala803) regulates cell apoptosis. METHODS: LoVo cells were infected with recombinant Ad-HIF-1alpha-Ala564-Ala803 and control virus Ad-lacZ under normoxia condition. Real-time PCR was used to detect HIF-1alpha and p21WAF1/CIP1 mRNA expressions at different time points. Western blotting was employed to verify HIF-1alpha and p21WAF1/CIP1 protein expression. Hoechst 33342 flourescein staining was performed to observe the ratio of apoptotic LoVo cells. RESULTS: The expression levels of HIF-1alpha mRNA and protein increased after infection with Ad-HIF-1alpha- Ala564-Ala803, accompanied by an increase in p21WAF1/CIP1 mRNA and protein expressions. The apoptotic ratio was significantly higher in LoVo cells infected with recombinant Ad-HIF-1alpha-Ala564-Ala803 (16.2%) than in the control cells (5.5%, P=0.00). CONCLUSION: HIF-1alpha may induce cell cycle arrest by up-regulating p21WAF1/CIP1 expressions to promote LoVo cell apoptosis.

[Construction and identification of adenovirus vector for double-mutant human hypoxia-inducible factor-1alpha gene].

OBJECTIVE: To construct an adenovirus vector containing the double-mutant hypoxia-inducible factor-1alpha (HIF-1alpha) gene for exploring the therapeutic angiogenesis for coronary heart disease. METHODS: Human double-mutant HIF-1alpha cDNA was obtained from PCR of pShuttle-2-HIF-1alpha containing the mutant HIF-1alpha gene (564). The recombinant adenoviral plasmid containing mutant HIF-1alpha cDNA was constructed by ligation of recombinant pShuttle2 containing double-mutant HIF-1alpha cDNA and Adeno-X viral DNA, followed by its identification and transfection into adenoviral packaging cells HEK293 via lipofectamine 2000. RESULT AND CONCLUSION: The recombinant pAdeno-HIF-1alpha was correctly constructed and verified by restriction endonuclease and DNA sequencing analyseis. This recombinant adenovirus containing the double-mutant HIF-1alpha gene may facilitate further investigation of mutant HIF-1alphagene therapy for coronary heart disease.