[L-VDCC autoregulation abnormality contributes to calcium overload in myocardial ischemia-reperfusion injury].

Calcium overload is a vital mechanism of myocardial ischemia-reperfusion injury, which is a hot therapeutic target in cardiovascular research. It has been well recognized that the dysfunction of calcium relevant proteins, including L-type voltage- dependent calcium channel (L-VDCC), sarco/endoplasmic reticulum ATPase 2a (SERCA2a)/phospholamban (PLB), RyR2, Na+/Ca2+ exchanger, Na+/H+ exchanger, etc. contributes to calcium overload in cardiomyocytes during ischemia-reperfusion injury, in which the diastolic calcium concentration is increased and the amplitude of calcium transients is decreased. There are two phases in calcium increase. The early phase is partially mediated by calcium channels, and the latter one is mainly mediated by Na+/Ca2+ exchanger. L-VDCC, a main subtype of calcium channels in myocardium, is involved in calcium overload, but the underlying molecular mechanism is not well elucidated yet. L-VDCC is regulated by intrinsic and extrinsic pathways. PKG and PKA as extrinsic regulators are not proper candidates to increase L-VDCC activity of cardiomyocyte in vitro, whereas the myocardial ischemia-reperfusion injury is highly possible to enhance L-VDCC activity by delaying calcium-dependent inactivation (CDI), advancing calcium-dependent facilitation (CDF), and weakening distal carboxy terminus (DCT) inhibition. Therefore, it is rational to propose that the L-VDCC autoregulation abnormality may play an important role in calcium overload during myocardial ischemia-reperfusion injury.

[Construction of eukaryotic expression vector of siRNA specific for MAD2 and its effect on the growth of gastric cell line SGC7901].

AIM: To construct the RNA interference eukaryotic expression vector specific for human MAD2 gene and to observe its effect on the growth of gastric cancer cell line SGC7901. METHODS: The expression vectors of pSilencer3.1/MAD2-siRNA1 and pSilencer3.1/MAD2-siRNA2 were constructed by gene recombination and then were stably transfected into the gastric carcinoma cell line SGC7901 by liposome mediation. The expression of MAD2 on the levels of protein and mRNA was detected by Western blot and RT-PCR, and the monoclone with the highest inhibition efficiency was selected. The growth of the transfected cells was assessed by MTT. And the cells treated with 1.0 mg/L vincristine (VCR) for 24 h were analyzed by FCM for cell cycle. RESULTS: Sequence-specific siRNAs targeting MAD2 significantly down regulated the expression of MAD2 in SGC7901 cells. In MAD2-siRNA transfected cells, the rate of cell growth increased markedly and cell cycle couldn't be arrested in M phase induced by VCR, while the cells transfected with the mock vector could. CONCLUSION: Down regulation of MAD2 expression of SGC7901 bv sequence-specific siRNA could accelerate the cell growth and impair the mitosis arrest of SGC7901 induced by VCR.

[Inhibition of the in vitro interaction between hypoxia inducible factor 1alpha and p300 by mitogen-activated protein kinase phosphatase 1].

OBJECTIVE: To investigate the effects of mitogen-activated protein kinase phosphatase-1 (MKP-1) on the interaction between hypoxia-inducible factor (HIF)-1alpha and coactivator p300. METHODS: Prokaryotic expression vector PGEX-4T1 was constructed based on DNA recombination technology. GST fusion proteins were purified by glutathione-agarose beads and transfected into Escherichia coli BL-21. Effects of MKP-1 or PD98059 on in vitro interaction between HIF-1alpha and p300 was determined by Pull-down assay coupled with Western bloting. Effects of MKP-1 on the p300 expression level was detected using Western bloting. RESULTS: (1) GST fusion proteins were expressed in BL-21 Escherichia coli and purified proteins were obtained. (2) Less amount of p300 was pulled down by GST fusion protein from SGC7901-MKP-1 cells than from control cells after 12 hours of exposure to hypoxia. (3) 12 hours after hypoxia, Less amount of p300 was pulled down by GST fusion protein from PD98059 treated cells than from DMSO treated cells or SGC7901 cells. (4) There were no significant differences in p300 expression levels between recombinant MKP-1 transfected cells and control cells after 12 hours of exposure to hypoxia. CONCLUSION: MKP-1 inhibits the in vitro interaction between HIF-1alpha and p300.

[A study on the regulatory mechanisms of mitogen-activated protein kinase phosphatase-1 in hypoxia inducible factor-1 trans-activation].

OBJECTIVE: To investigate the regulatory mechanisms of mitogen-activated protein kinase phosphatase-1 (MKP-1) in hypoxia inducible factor (HIF)-1 trans-activation. METHODS: (1) Gastric cancer cells of the line SGC7901 were cultured, then continued to be cultured in hypoxic environment, and was lysed. The supernatant was collected. Western blotting was used to detect the content of total extracellular signal-regulated kinase (ERK) and phosphorylated ERK. (2) Another SGC7901 cells were cultured with PD98059, inhibitor of ERK passway, or SB203580, inhibitor of p38 passway, in the same manner as above-mentioned. Dual luciferase reporter (DLR) was used to detect the luciferase activity so as to measure the HIF-1 trans-activation. (3) siRNA vector U6M2 plasmid against MKP-1 mRNA was constructed. In another experiment SGC7901 cells were cultured and U6M2 and blank vector U6 were transfected into the cells respectively. 24 hours later, the cells were cultured in hypoxic environment with added PD98059 of different concentrations for 12 hours. Dual luciferase reporter (DLR) was used to detect the luciferase activity HIF-1 trans-activation. (4) Another SGC7901 cells were co-transfected with U6M2, pGL-3SV40HRE vector containing promoter SV40, and pRL-TK (internal control vector). Then PD98059 was added, the cells were lysed, and the activity of fluorescein was tested. (5) SGC7901 cells were cultured, transfected with UdM2 or U6 respectively, and 24 hours later cultured under hypoxia with PD98059 of different concentrations for 12 hours. ELISA was used to examine the VEGF protein concentration in the culture fluid. RESULTS: (1) The content of phosphorylated ERK in the SGC7901 cells increased along with the time of hypoxia, peaked at the 12th hour, and then decreased. However, there was no difference in total ERK expression. (2) After 12 hours of hypoxia, different concentrations of PD98059 inhibited the luciferase activity, however, SB203580 of different concentrations had no effect. (3) 24 hours after transfection, the expression of phosphorylated form of ERK in the SGC7901cells transfected with siRNA plasmid against MKP-1 mRNA was higher compared with that in cells transfected with blank vectors after 12 hour of exposure to hypoxia. (4) PD98059 inhibited the luciferase activity either in U6 cells or in U6M2 cells. Notably, when the PD98059 concentration was above 50 micromol/L, there was no difference in HIF-1 activity between the U6 and U6M2 cells. (5) PD98059 of different concentrations all inhibited the VEGF expression either in U6 cells or in U6M2 cells, and when the concentration of PD98059 was over 50 micromol/L there was no difference in VEGF expression level between the U6 cells and the U6M2 cells. CONCLUSION: In SGC7901 cells, the function of MKP-1 is involved in regulation of HIF-1 trans-activation via inactivation of the ERK pathway.