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Objective To establish a rat model of cardiac hypertrophy induced by isoproterenol(ISO),and to study its basic characteristics . Methods Cardiac hypertrophy was induced in rats with ISO. The model rats received subcutaneous injections of 5 mg/kg ISO every day for 14 days. Results The heart weight/body weight and left ventricular weight/body weight ratios in model rats were significantly increased. The serum hydroxyproline level was significantly increased ,the superoxide dismutase level was significantly decreased ,and the malondialdehyde level was sig?nificantly increased in model rats. Conclusion The rat model of cardiac hypertrophy is successfully created by subcutaneous injection of ISO for 14 days. This model can be used in study of the mechanism of cardiac hypertrophy.
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Objective To investigate a method for the purification of the N?terminal peptide fragment(NT)of the myocardial calcium channel Cav1.2,and characterize its interaction with calmodulin(CaM). Methods EscherichiacoliBL?21 cells were transformed with plasmid pGEX?6p?3/NT harboring the NT?GST fusion gene. The cells harboring pGEX?6p?3/NT were cultured and protein expression was induced with isopropyl?β?D?thiogalactoside(IPTG). Then,the GST?NT fusion protein was purified by using glutathione Sepharose 4B(GS?4B)beads. GST was cleaved off with the PreScission protease,and SDS?PAGE was performed to detect the purity and relative molecular weight of the purified peptide. Further, GST pull?down assay was performed to characterize the interaction of the NT peptide with CaM. Results SDS?PAGE analysis showed that the NT peptide was successfully purified,with high purity. Results of the GST pull?down assay showed that the NT peptide could interact with CaM. Conclusion This study establishes a method for the purification of the NT peptide and lays the foundation for further research on the interaction partners and biological functions of NT.
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Objective To construct plasmid vectors of calmodulin(CaM)Mg2+binding site mutants,and to express,purify and identify the mutant proteins. Methods Three kinds of cDNAs coding for the mutated CaM were cloned into pGEX?6P?3 plasmid vectors. These recombinant plasmids were transfected into Escherichia coli BL21 to express GST fusion proteins of CaM mutants. The fusion proteins were purified with Glutathione?Sep?harose 4B beads and PreScission protease. Results Both enzyme digestion analysis and DNA sequence identification proved the successful con?struction of the CaM mutant plasmids. SDS?PAGE results showed the high purity of each CaM mutant protein. The concentrations of three CaM mu?tants were around 1.0 mg/mL. Conclusion Prokayotic expression vectors of CaM Mg2+binding site mutants were successfully developed,and the eli?gible CaM mutant proteins were obtained. This study provided an important basis for further study on CaM’s biological function.
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Objective To observe the effects of nonylphenol(NP)on the intracellular calcium concentration changes and cell proliferation,and the involvement of GPR30 receptor in H9c2 cell. Methods The intracellular calcium concentration changes were recorded by using intracellular calcium determination method and cell proliferation was observed by MTT method in H9c2 cell. Results NP(1×10-10 mol/L)increased the intra?cellular calcium concentration changing amplitude and promoted the proliferation of H9c2 cells,while NP(1×10-6 mol/L)decreased intracellular calcium concentration changing amplitude and suppressed cell proliferation. G15 could block the promoting effect of 1×10-10 mol/L NP on the intracel?lular calcium concentration and cell proliferation,but could not block the inhibition of 1×10-6 mol/L NP on the intracellular calcium increase and cell proliferation. Conclusion The results indicate that NP affect rapid calcium signal changes and cell proliferation in non?monotonic dose dependent manner,and its mechanism may be due to the different involvement of GPR30 receptor in different concentrations.
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Objective To clone the coding region and 3′non?coding region of calmodulin 2(CaM2)in guinea pig,to provide the genetic informa?tion for studying the gene function of Calmodulin 2. Methods Total RNA was extracted from heart tissue of guinea pig,the coding region and 3′non?coding region of CaM2 were amplified by RT?PCR and 3′?RACE PCR methods,and the recombinant plasmid was constructed by inserting cDNA of the coding region and 3′non?coding region of CaM2 into the cloning vector by genetic engineering technology followed by DNA sequencing and se?quence analysis. Results The cloned coding region of CaM2 was 450 bp,and the 3′non?coding region of CaM2 was 660 bp. The amino acid se?quences of the coding region of CaM2 was consistent with those of other CaM subtypes,and the 3′non?coding region of CaM2 had low homology with those of other subtypes. Conclusion The cloning of CaM2 coding region and 3′non?coding region in guinea pig was the foundation for further study on the gene function of CaM2 and its role in related diseases.
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Objective To observe the effects of nonylphenol(NP) on L-type Ca~(2+) currents(I_(Ca-L)) in isolated guinea-pig ventricular myocytes.Methods The ventricular myocytes were isolated from guinea-pig hearts.The L-type Ca~(2+) currents in the ventricular myocytes treated with NP were measured with whole cell patch-clamp technique.Results Nonylphenol inhibited lew,at different potentials.Nonylphenol (10~(-6) mol·L~(-1),10~(-5) mol·L~(-1)) reduced the peak amplitude of I_(Ca-L) from-3.2±1.5 pA·pF~(-1) to-1.6±0.8 pA·pF~(-1)(P<0.01) and-1.4±0.7 pA·pF~(-1)(P<0.01),respectively.Nonylphenol did not influence the activation curve of I_(Ca-L) significantly.Conclusion Nonylphenol could in hibit the L-type calcium currents in isolated guinea-pig ventricular myocytes with a concentration-dependent manner.Objective To observe the effects of nonylphenol(NP) on L-type Ca~(2+) currents(I_(Ca-L))in isolated guinea-pig ventricular myocytes.Methods The ventricular myocytes were isolated from guinea-pig hearts.The L-type Ca~(2+) currents in the ventricular myocytes treated with NP were measured with whole cell patch-clamp technique.Results Nonylphenol inhibited lew,at different potentials.Nonylphenol hibit the L-type calcium currents in isolated guinea-pig ventricular myocytes with a concentration-dependent manner.
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Polysaccharide sulfate (PSS) is a new type of antiatherosclerotic medicine for its effects of anticoagulation, anti-thrombosis and modulation of dyslipidemia. However, it is still uncertain whether PSS could modulate the diabetic dyslipidemia or not. Here, the rat model of diabetic dyslipidemia was developed and the effects of PSS on glucose and lipid levels were investigated in this animal model. Wistar rats were iv injected with streptozotocin 20 mg·kg-1 after feeding with high fat diet for one and a half month. Then, rats received orally PSS (30, 90, and 180 mg·kg-1) for 1 month. After oral treatment with PSS (90 and 180 mg·kg-1) for 1 month, the levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) were significantly reduced and the level of high density lipoprotein-cholesterol (HDL-C) increased, compared with diabetic control rats. Moreover, PSS (30, 90, and 180 mg·kg-1) had a tendency to reduce glucose and insulin levels, and significantly increased insulin sensitivity index. Our results suggest that PSS could improve insulin sensitivity and relieve dyslipidemia in diabetic dyslipidemic rats.
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Aim To explore the pharmacological mechanism and the effects of polysaccharide sulfat(PSS) on cardiovascular diseases induced by type 2 diabetes mellitus(DM) through observing the risk factors.Methods Type 2 diabetic animal model was established by high-sugar and high-fat diets,combined with injection of small amount streptozotocin(STZ 20 mg?kg-1,iv).Adult male wistar rats were divided into five groups: normal control group,model group,polysaccharide sulfate group,metformin group and lovastatin group.They were treated with exact medicne for 8 weeks,but control group and model group were treated with 0.9% Nacl.During this process,FBG and serum lipid concentrations were measured.22 weeks later,the rats were sacrificed.The activity of tissue-type plasminogen activator(t-PA) and plasminogen activator inhibitor type-1(PAI-1) were detected by chemical methods.The aortas were collected for histopathlogical,immunohistochemical and Western blot studies.Results FBG concentrations and serum lipid(TC,TG,LDL) levels decreased in PSS group as compared from those of model group(P