Objective To investigate the binding interaction between the calmodulin(CaM)mutant D130V and the IQ motif of the car-diac Cav1.2 channel.Methods The binding of mutant CaM-D130V to the IQ motif was predicted by fold recognition modeling,homology modeling,and protein docking.The plasmid was transformed into Escherichia coli BL-21 sensory cells via heat shock at 42 ℃ to induce the expression of glutathione S-transferase(GST)fusion protein.The protein was extracted by ultrasonic fragmentation and purified using GS-4B beads.PreScission protease was applied to remove the GST.SDS-PAGE was performed to detect the purity of protein.A GST pull-down assay was conducted to detect the interaction between CaM-D130V and IQ motif.Results Protein docking results showed that both CaM-WT and CaM-D130V could bind to the IQ motif of the cardiac Cav1.2 channel,but the binding sites of the mutant CaM-D130V to the IQ motif were reduced,and its binding conformation was changed compared with the CaM-WT,with decreased binding energy(|S|reduced from 48.086 6 kcal/mol to 47.309 5 kcal/mol).The GST pull-down assay indicated that the binding of CaM-D130V to IQ motif significantly decreased(P＜0.01),and the affinity was significantly reduced at 2 mmol/L Ca2+concentration compared with CaM-WT.Conclusion The reduced binding ability of CaM-D130V to the IQ motif of the cardiac Cav1.2 channel may contribute to functional alterations in the channel.These findings provide a theoretical basis for understanding the pathogenesis of CaM mutant-associated cardio-vascular diseases as well as targeted therapies.

Objective To investigate the binding interaction between the calmodulin(CaM)mutant D130V and the IQ motif of the car-diac Cav1.2 channel.Methods The binding of mutant CaM-D130V to the IQ motif was predicted by fold recognition modeling,homology modeling,and protein docking.The plasmid was transformed into Escherichia coli BL-21 sensory cells via heat shock at 42 ℃ to induce the expression of glutathione S-transferase(GST)fusion protein.The protein was extracted by ultrasonic fragmentation and purified using GS-4B beads.PreScission protease was applied to remove the GST.SDS-PAGE was performed to detect the purity of protein.A GST pull-down assay was conducted to detect the interaction between CaM-D130V and IQ motif.Results Protein docking results showed that both CaM-WT and CaM-D130V could bind to the IQ motif of the cardiac Cav1.2 channel,but the binding sites of the mutant CaM-D130V to the IQ motif were reduced,and its binding conformation was changed compared with the CaM-WT,with decreased binding energy(|S|reduced from 48.086 6 kcal/mol to 47.309 5 kcal/mol).The GST pull-down assay indicated that the binding of CaM-D130V to IQ motif significantly decreased(P＜0.01),and the affinity was significantly reduced at 2 mmol/L Ca2+concentration compared with CaM-WT.Conclusion The reduced binding ability of CaM-D130V to the IQ motif of the cardiac Cav1.2 channel may contribute to functional alterations in the channel.These findings provide a theoretical basis for understanding the pathogenesis of CaM mutant-associated cardio-vascular diseases as well as targeted therapies.

OBJECTIVE: To investigate the impact of renal denervation on the blood pressure, plasma renalase content and expression of renalase and tyrosine hydroxylase (TH) in the idney of spontaneous hypertensive (SH) rats and to explore the role of renal denervation in lowering the blood pressure. METHODS: SH rats were randomly assigned into a baseline group, a surgery (renal denervation) group, a sham group and a control group (n=48). WKY rats matched in age (n=12) served as a baseline control group. All rats were housed until 12 weeks old. Then, the rats in the baseline group and the WKY group were sacrificed whose blood and kidney were collected for examination. In the renal denervation group, the sham group and the control group, the blood pressure was monitored continuously. One week and 6 weeks after the renal denervation, 6 rats in each group were sacrificed whose blood and kidney were collected. ELISA was employed to measure the plasma renalase and Western blot assay done to detect the expression of TH and renalase in the kidney. RESULTS: Compared with WKY rats, blood pressure significantly increased and TH protein expression markedly elevated (P<0.05) in SH rats in the baseline group, but plasma renalase content and protein expression of renalase in the kidney dramatically reduced (P<0.05). One week after the surgery, the mean arterial pressure and TH protein expression in the surgery group were lowered compared with the baseline group and dramatically reduced compared with the sham group and the control group (P<0.05). In the surgery group, the renalase level was markedly increased compared with the baseline group, the sham group, and the control group (P<0.05). Six weeks after the renal denervation, the mean arterial pressure and TH level in the surgery group were significantly increased but the renalase content and expression markedly reduced compared with those 1 week, but there were no marked differences among the surgery group, the sham group, and the control group (P>0.05). No pronounced differences in the above variables were found between the sham group and the control group at any time point (P>0.05). CONCLUSION Renal denervation can lower the blood pressure, which may attribute to the suppression of sympathetic nerves, increase in plasma renalase content and renalase expression in the kidney.
Animals ; Blood Pressure ; physiology ; Hypertension ; surgery ; Kidney ; enzymology ; innervation ; Male ; Monoamine Oxidase ; blood ; metabolism ; Rats ; Rats, Inbred SHR ; Sympathectomy ; methods ; Sympathetic Nervous System ; physiopathology ; Tyrosine 3-Monooxygenase ; metabolism

Objective Isolating and purifying the major allergens from Metapenaeus ensis and identifying its immunoreactivity for diagnostic application. Methods The proteins extracted from Metapenaeus ensis were purified by DEAE-52 ion-exchange chromatography and Sephadex G-100 gel filtration. The eluted peaks were detected by dot-ELISA,and the immunoreactivity of the purified allergens was detected by ELISA with the sera from patients allergic to shrimp. The molecular weights of the purified allergic proteins were detected by SDS-PAGE. Results Two major allergic proteins which have highly immunoreactivity in ELISA detection were purified from Metapenaeus ensis. The molecular weights of which were 36 and 68kDa,respectively. Conclusion The major allergens of Metapenaeus ensis purified by ion-exchange chromatography and gel filtration have effective immunoreactivity and can be used as specific antigen in ELISA reagent.