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Objective To construct a CaME141G fusion protein-expressing plasmid,and to express,purify,and identify the activity of the recombinant protein. Methods The 141st site of the wild type CaM,E (GAG),was mutated to G (GGG),using site-specific mutagenesis technology. Escherichia coli BL-21 was transformed with the mutant plasmid. The GST-CaME141G fusion protein was mass-cultured and induced for expression. Subsequently,the GST-CaME141G fusion protein was purified using GS-4B beads. PreScission protease was applied to remove the GST,the Bradford method used to determine the concentration of purified protein,and SDS-PAGE used to detect its relative molecular weight and purity. The GST pull-down assay was used to study the protein's biological activity. Results The CaME141G protein was successfully purified at a high concentration and purity. The protein could interact with PreIQ protein fragments from the myocardial CaV1. 2 calcium channel C terminal,in a CaME141G concentration-dependent manner. Therefore,CaME141G has the ability to bind with the CaV1. 2 calcium channel. Conclusion This study successfully constructed a CaME141G fusion protein-expressing plasmid and purified the CaME141G protein. This lays a foundation for regulating the function of CaM mutations in the myocardial CaV1. 2 calcium channel,and for the study of its relationship with diseases of the cardiovascular system.
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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 expression vectors of calmodulin(CaM)mutants N2 and C2,and to express,purify,and identify the mutant proteins,in order to study the interactions between CaM and calcium channels. Methods The cDNA of N?lobe and C?lobe of CaM were used to prepare the cDNA of N2 and C2. Next,the recombinant cDNAs were cloned into a pGEX?6p?3 plasmid,and the recombinant plasmids were trans?ferred into E.coli BL21 cells. The transfected BL21 cells were stimulated with IPTG. The fusion proteins were extracted by ultrasonication and puri?fied by using GS?4B beads. Finally,protein activity was identified by the pull?down assay. Results Both the restriction digestion map and the DNA sequence identification results confirmed that the recombinant plasmids were successfully constructed. SDS?PAGE results showed high purity and concentration of N2 and C2 proteins. Their activities and binding abilities with the calcium channel fragment were confirmed by the pull?down assay.Conclusion In this study,expression vectors of N2 and C2 are successfully constructed,and physiologically active N2 and C2 CaM mutant proteins are obtained.
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Objective Many studies have suggested that cardiovascular risk factors seem to be more common in psoriasis patients than in general population.In this study we aimed to investigate the prevention and treatment of cardiovascular risk in psoriasis patients by Framingham cardiovascular risk assessment in patients with psoriasis vulgaris and normal people using Framingham score.Methods We conducted a prospective study including 90 outpatients with psoriasis vulgaris and 137 controls without psoriasis from October 2015 to October 2016 in our hospital.All psoriasis patients were diagnosed clinically and histopathologically.The severity of psoriasis was assessed according to the psoriasis area and severity index (PASI) score.Along with a thorough medical history and physical examination, serum lipid profile, blood pressure and fasting plasma glucose tests were carried out.The 10-year Framingham risk score (FRS) for general cardiovascular disease included indexes concerning age, gender, total cholesterol, HDL-cholesterol, systolic blood pressure and smoking history.Results We found the 10-year FRS was significantly higher in patients with psoriasis vulgaris than in controls (P0.05).No significance was found in the 10-year FRS of patients with mild and severe psoriasis (P>0.05).FRS was significantly higher in male patients and in patients above 50 years old (P<0.05).Conclusion Psoriasis patients, especially the older male patients, tend to have high risks of cardiovascular disease.Therefore, risk assessment for cardiovascular diseases should be conducted in psoriasis patients, and complications should be actively prevented and treated.
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Aim To construct 3 D structure model of cardiac Cav1.2 channel and check its accuracy and re-liability.Methods Homology model of Cav1.2 chan-nel α1 subunit was constructed using SWISS-MODEL server.The model was submitted to an online testing server built by University of California and scored by it.The binding of Cav1.2 channel with blocker or drug was simulated by MOE software molecular docking pro-gram to check the model′s accuracy and reliability.Re-sults Both the target sequence Cav1.2 α1 C and the template sequence Cav1.1 α1 S searched by SWISS-MODEL server belonged to L-type Ca2+channel.Since the homology was 7 1.5% revealed by sequence align-ment,homology modeling was performed using automa-ted mode.L-type Ca2+ channel blockers Verapamil, Nifedipine and Diltiazem could bind to the 3 D structure model of Cav1.2 channel,while sodium channel bloc-ker TTX could not.Furthermore,active ingredient of traditional Chinese drug Praeruptorin A and Berberine could also bind to the 3D structure model of Cav1.2 channel.Conclusion The 3 D structure model of Cav1.2 channel was constructed successfully,which provides reliable materials for further studies and estab-lishes the foundation for the application of homology modeling in the study of 3 D structure prediction of ion channels.
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Objective To construct a recombinant plasmid vector containing the distal fragment of the distal C-terminus (dDCT) of the Cav 1.2 channel,and express,extract,and purify dDCT protein and characterize its biological activity.Methods dDCT cDNA was ligated into the pGEX-6p-1 vector to create a recombinant plasmid that was subsequently transformed into Escherichia coli BL21 competent cells.Expression of GST-dDCT fusion protein from this plasmid was induced with isopropy-β-D-thiogalactoside,and the resulting protein was purified using glutathione-sepharose 4B beads.The biological activity of dDCT was analyzed by GST pull-down assay.Results The recombinant plasmid was verified by restriction enzyme digestion and sequencing.The concentration and purity of the dDCT protein,which was extracted by ultrasonication,were high enough to detect dDCT activity.The binding of dDCT to CT1 was determined to be concentration-dependent.Conclusion The recombinant dDCT plasmid was successfully constructed,providing the fundamental basis for future studies on mechanisms of Cav 1.2 channel autoregulation.
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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 explore whether dithiothreitol(DTT)is helpful for PreScission Protease to cut off the GST from GST?CT3 protein. Meth?ods The pGEX?6P?3/CT3 recombinant plasmid was transfected into Escherichia coli BL21,and the GST?CT3 fusion protein was purified by B?PER method. PreScission Protease was applied with 10 mmol/L DTT to cut off the GST,then the SDS?PAGE was performed for identification of the CT3 protein. Results Without DTT,it was very difficult for PreScission Protease to cut off the GST from GST?CT3 protein. However,in the pres?ence of 10 mmol/L DTT,PreScission Protease could cut off the GST easily as identified by SDS?PAGE. Conclusion 10 mmol/L DTT can help Pre?Scission Protease to cut GST from GST?CT3 protein,so as to achieve high concentration of CT3.
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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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<p><b>OBJECTIVE</b>To determine the pesticide residues in Shengmaiyin.</p><p><b>METHOD</b>The residues were simultaneously identified and quantified by capillary gas chromatography. The capillary column was OV-1701(0. 32 mm x 30 m, 0.5 microm), splitless injection and Agilent 63Ni electron capture detector ECD were adopted. The carrier gas were high-pure nitrogen, the flow rate were 60 mL x min(-1), the injector temperature was 260 degrees C, and the detector temperature was 300 degrees C.</p><p><b>RESULT</b>It showed a good linear relationship between peak area and pesticide concentration for 11 kinds of pesticides in the detection concentration range. All correlation coefficients were higher than 0.996 2. The average recovery was 83.1% -114.7%.</p><p><b>CONCLUSION</b>A simple and efficient monitoring method of pesticide residues is established which provides the foundation for the safe use and analysis of Shengmaiyin.</p>
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Chromatography, Gas , Methods , Drug Contamination , Drugs, Chinese Herbal , Chemistry , Pesticide Residues , Reproducibility of ResultsABSTRACT
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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With patch clamp method,the effects of taurine (Tau) on slow inward calcium current (Ica) were investigated in single ventricular cells of guinea pigs. Tau decreased Ica in a dose- and voltage-dependent mode,but had little effect on reversal potential. Tau 0. 1 umol - L-1 decreased Ica by 17% and its maximal effective concentration was near 100 umol -L '. The initial block of Tau had already reached by 47% after a rest out of stimulation. Tau facilitated the reduction of lea through increasing clamp pulse frequency. The obtained results suggested that it decreased Ica in a use-dependent manner.
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The effect of 5.8 ////mol L-1 propafeone on 0 phase depolarization of action potential were studed in meonatal and adult canine Purkinje fibres. Results showed: 1. in both groups, propafenone caused a reduction if APA and a decrease of Vmax; and the neonatal fibres were less sensitive to propafenone; 2. the inhibition of Vmax by propafenone showed frequency-dependent manner in both groups; and with theincrease of frequencies, the extent to which Vmax was inhibited became similiar in both groups. This sudy suggests that neonates were less sensitive to propafenone, but at higher frequencies neonates showed similiar responses as adults.