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Objective:To investigate whether KtrA was a binding protein of c-di-AMP, the second messenger in Leptospira, and to explore the function and regulatory mechanism of the c-di-AMP-KtrA/B system. Methods:KtrA gene was amplified by PCR and cloned into pET42a plasmid to construct the pET42a ktrA prokaryotic expression vector. Then the vector was transferred into E. coli BL21DE3 to construct an engineering bacterium E. coli BL21DE3 pET42a-ktrA for the expression of recombinant KtrA (rKtrA). The expressed rKtrA was purified by affinity chromatography. BIAcore technology was used to detect the binding ability of rKtrA to c-di-AMP. Bacterial two-hybrid analysis was used to analyze the interaction between KtrA and KtrB in the leptospiral Ktr system with or without exogenetic c-di-AMP. The above genes were then complemented into the potassium transport-deficient E. coli mutants to analyze the function of the c-di-AMP-KtrA/B pathway. Results:An prokaryotic engineering bacterium for the expression of ktrA gene of Leptospira was constructed successfully. The purified rKtrA could specifically bind to c-di-AMP. There was interaction between KtrA and KtrB, but the interaction could be dissociated by c-di-AMP. The KtrA/B system was involved in potassium ion uptake and it was negatively regulated by c-di-AMP. Conclusions:Leptospiral KtrA was a c-di-AMP-binding protein and the c-di-AMP-KtrA/B system was involved in potassium ion transport.
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Objective: To investigate the effect of extracellular potassium (K+) concentration on the activities of sodium chloride co-transporter (NCC) and large conductance Ca2+-activated K+ channel (BK) in distal renal tubule of mice. Methods: Six specific pathogen free (SPF) C57BL/6 mice aged 8 to 10 weeks were sacrificed,and the kidney slices were made with previously reported method. Then,these slices were incubated randomly in normal K+,high K+,BaCl2 and RbCl solutions,respectively. The abundance and phosphorylation level of NCC in kidney slices at different K+ concentrations and different time courses were detected by Western blotting. The overall and membrane expressions of BK in kidney slices were also detected after incubation with different K+ solutions for 2 h. Results: Compared with normal K+ solution,NCC phosphorylation level was significantly decreased after incubation with high K+ solution for 5,15,30 min (all P<0.05),and NCC phosphorylation level was also decreased after intervention with K+ channel inhibitor Ba2+ or Rb+ (both P<0.05). After the treatment with high K+ solution for 2 h,neither the overall cell expression of BKα subunit and β4 subunit,nor membrane expression of BKα subunit was found significant changes compared with normal K+ incubation. Conclusion: High K+ can directly down-regulate NCC phosphorylation level,which may be preparation for kaliuresis of the downstream tubule of distal convoluted tubule.
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@#Objective To explore the expression and the changes of microtubule, aquaporin-4 (AQP4) and potassium ion channel 4.1 (Kir4.1) after spinal cord injury in rats.Methods Ninety female adult Sprague-Dawley rats were randomly divided into sham operation group (n=30) and injury group (n=60). The injury group was divided into six hours, one day, three days, five days and seven days subgroups, with twelve rats in each subgroup. Spinal cord injury at T10 was established with modified Allen's method (20 g×25 mm) in the injury group. The water content of spinal cord was measured at each time point after injury. Then, the pathology was observed with HE staining, the expression of α-Tubulin, AQP4 and Kir4.1 was detected and analyzed with immunohistochemical staining and Western blotting.Results The water content of the spinal cord was higher in the injured group than in the sham operation group (P<0.05), and was highest on the fifth day. HE staining showed that the gray matter hemorrhage at six hours after injury; one day after injury, the gray matter bled seriously, and neuron swelling was aggravated; three days after injury, the area of gray matter necrosis increased, and the edema phenomenon was obvious; five days and seven days after injury, the gray matter necrosis and the edema phenomenon were more serious. Western blotting and immunohistochemistry showed that the expression of AQP4 gradually increased after injury, and raised at peak on the fifth day; the expression of α-Tubulin and Kir4.1 was similar, and the expression gradually decreased after injury, especially on the fifth day.Conclusion The expression of α-Tubulin and Kir4.1 is similar after spinal cord injury, and is contrary to the expression of AQP4. α-Tubulin, AQP4 and Kir4.1 may be related after injury and may participate in the formation of spinal cord edema.
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Objectives: To investigate the effects of potassium supplementation after coronary artery bypass grafting (CABG) in patients with reduced left ventricular ejection fraction (EF < 50%). Methods: Present study retrospectively analyzed data from 200 CABG patients with cardiopulmonary bypass from January 2015 to June 2017. Patients were divided as heart failure with high ideal potassium supplement group (low EF<50%, potassium supplement to 5.0 mmol/L, n=41); heart failure normal potassium supplement group (low EF<50%, potassium supplement to 4.5 mmol/L, n=46); and normal potassium supplement group (normal EF>50%, potassium supplement to 4.5 mmol/L, n=113). The differences on related parameters during intraoperative and postoperative period were compared among the 3 groups. The primary endpoint was the incidence of atrial and ventricular arrhythmias requiring the use of lidocaine and amiodarone hydrochloride. Results :Atrial and ventricular arrhythmias requiring lidocaine and amiodarone hydrochloride were found in 13 patients (31.7%) in the ideal hyperkalemia group, while 28 (62.2%) in the normal hyperkalemia group needed both drugs (P=0.005). The blood pressure of ideal high potassium supplementation group was significantly higher than that of normal potassium supplementation group ([135.87±11.32] mmHg vs [111.21±31.31] mmHg, P<0.001). In the ideal hyperkalemia group, the time in the ICU was shorter ([3.92±0.98]days vs [5.12±1.12]days, P<0.001) and the hospitalization time was shorter ([10.54±2.14] days vs [13.54±2.01]days, P<0.001). There was no significant difference between the two groups in postoperative 24-hour urine volume, oxygen partial pressure, postoperative 24-hour bleeding volume and postoperative complications (P>0.05). Conclusions:The ideal potassium target of 5.0 mmol/L for patients with LVEF<50% CABG can reduce the incidence of arrhythmias that require the intervention of lidocaine and amiodarone hydrochloride during the perioperative period and shorten the stay time in the ICU.
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AIM:To observe the effects of granulocyte colony-stimulating factor (G-CSF) on calcium, sodium and potassium ion channel currents of the ischemic atrial myocytes in guinea pig by whole-cell patch clamp technique.METHODS:The guinea pig atrial myocytes were obtained by enzymolysis.Under ischemia and hypoxia condition, whole-cell patch clamp was used to observe the effects of G-CSF at various concentrations on the changes of the I-V curve, activation curve and availability of L-type calcium channel current (ICa,L) and voltage-dependent sodium channel current (INa), as well as I-V curve of delayed rectifier potassium channel current (IK).RESULTS:Under ischemic condition, the I-V curves of ICa,L were changed by acute G-CSF intervention in a dose-dependent fashion.Except for G-CSF at dose of 300 μg/kg, the other concentrations of G-CSF did not change the activation curve and availability of ICa,L, indicating that the effects of G-CSF on ICa,L were in a voltage-independent fashion.The I-V curves of ICa,L under ischemic condition were gradually approaching the normal levels by the higher dose of G-CSF, while the effect of 300 μg/kg G-CSF on ICa,L was similar to 100 μg/kg G-CSF.Acute G-CSF intervention at different doses did not change I-V curve, activation curve, and availability or steady-state availability of INa.As a part of IK, the rapid activating component (IKr) was improved by 100 μg/kg and 300 μg/kg G-CSF intervention with the similar effects, while the slowly activating component (IKs) was not changed by G-CSF.CONCLUSION:G-CSF affects ion channel electrophysiological properties of ischemic atrial myocytes in a voltage-independent but concentration-dependent manner, thus reducing the incidence of atrial arrhythmia.
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Objective:To study the effects of a potassium channel blocker 4-Amino pyridine(4-AP)on the proliferation of human tongue squamous cell carcinoma Tca8113 cells.Methods:CCK-8 assay was used to detect the proliferation of Tca8113 cells cultured with 4-AP at the concentration of 1,5,10,20,50 and 100 mmol/L for 12,24 and 48 h respectively,the cell proliferation inhibition rate was calculated,the cell cycle distribution was examined by flow cytometry.Data were analyzed by SPSS19.0 software.Results:With the increase of 4-AP concentration and culture time,cells showed some morphologic changes.4-AP at 5 -100 mmol/L dose and time dependently inhibited the proliferation,with 24 h exposure dose dependenty decreased S-phase population and increased G0 /G1 phase population of Tca8113 cells.Conclusion:4-AP may inhibit Tca8113 cell proliferation by regulation of the cell cycle distribution.
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Objective: To identify the chemical constituents of leaf essential oil of Forsythia koreana (F. koreana) and evaluate its effects on bacterial strains. Methods: The essential oil of leaf of F. koreana was extracted by using hydrodistillation process and the volatile components investigated with the help of gas chromatography coupled with mass spectrometry. The antibacterial study was carried out with the help of agar disc diffusion method, MIC, MBC and viable count. The mode of action was determined with help of potassium ion flux, cellular material release and scanning electron microscopy. The antioxidant activity was determined with the help of 2, 3-diphenyl-2-picrylhydrazyl method, nitric oxide scavenging activity and superoxide anion radical scavenging assay. Results: Total ten compounds were identified as trans-phytol (42.73%), cis-3-hexenol (12.95%), β-linalool (10.68%), trans-2-hexenal (8.86%), trans-2-hexenol (8.86%), myrcenol (3.86%), 4-vinylphenyl acetate (3.86%), (4Z)-4,6-heptadien-1-ol (3.18%), lemonol (2.73%) and benzeneacetaldehyde (2.27%) by gas chromatography coupled with mass spectrometry. The antibacterial study was demonstrated that leaf essential oil of F. koreana act against foodborne and other pathogenic bacteria. The mode of action revealed that this essential oil acted on the cytoplasmic membrane, resulting in loss of integrity and increased permeability. In addition, leaf essential oil of F. koreana was shown to be rich in linalool, which contributes to improved antioxidant activity. Conclusions: These results show that leaf essential oil of F. koreana has great potential as a natural food preservative, antibacterial and antioxidant agent.
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OBJECTIVE@#To identify the chemical constituents of leaf essential oil of Forsythia koreana (F. koreana) and evaluate its effects on bacterial strains.@*METHODS@#The essential oil of leaf of F. koreana was extracted by using hydrodistillation process and the volatile components investigated with the help of gas chromatography coupled with mass spectrometry. The antibacterial study was carried out with the help of agar disc diffusion method, MIC, MBC and viable count. The mode of action was determined with help of potassium ion flux, cellular material release and scanning electron microscopy. The antioxidant activity was determined with the help of 2, 3-diphenyl-2-picrylhydrazyl method, nitric oxide scavenging activity and superoxide anion radical scavenging assay.@*RESULTS@#Total ten compounds were identified as trans-phytol (42.73%), cis-3-hexenol (12.95%), β-linalool (10.68%), trans-2-hexenal (8.86%), trans-2-hexenol (8.86%), myrcenol (3.86%), 4-vinylphenyl acetate (3.86%), (4Z)-4,6-heptadien-1-ol (3.18%), lemonol (2.73%) and benzeneacetaldehyde (2.27%) by gas chromatography coupled with mass spectrometry. The antibacterial study was demonstrated that leaf essential oil of F. koreana act against foodborne and other pathogenic bacteria. The mode of action revealed that this essential oil acted on the cytoplasmic membrane, resulting in loss of integrity and increased permeability. In addition, leaf essential oil of F. koreana was shown to be rich in linalool, which contributes to improved antioxidant activity.@*CONCLUSIONS@#These results show that leaf essential oil of F. koreana has great potential as a natural food preservative, antibacterial and antioxidant agent.
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The purpose of this study is to investigate the expression of major potassium channel subtypes in the brain of chronical mild stress (CMS) rats and reveal the effects of fluoxetine on the expression of these channels. Rats were exposed to a variety of unpredictable stress for three weeks and induced anhedonia, lower sucrose preference, locomotor activity and lower body weight. The protein expressions were determined by Western blot. CMS significantly increased the expression of Kv2.1 channel in frontal cortex but not in hippocampus, and the expression level was normalized after fluoxetine treatment. The expression of TREK-1 channel was also obviously increased in frontal cortex in CMS rats. Fluoxetine treatment might prevent this increase. However, the expression of Kv3.1 and Kv4.2 channels was considerably decreased in hippocampus after CMS, and was not affected by fluoxetine. These results suggest that different subtypes of potassium channels are associated with the pathophysiology of depression and that the therapeutical effects of fluoxetine may relate to Kv2.1 and TREK-1 potassium channels.
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Objective To examine the expression of potassium channel mRNA in myocardial tissue of mice with low selenium.Methods Forty C57BL/6 mice were randomly divided into four groups according to body weight(18-22 g),10 mice in each group,half male and half female.The low-selenium treatment groups were fed with low-selenium diet(selenium content was 0.004 mg/kg) for 4,12 and 24 weeks,respectively,and the control group was fed with normal diet(selenium content was 0.256 mg/kg).The mice were killed by cutting neck method,hearts were taken out and RNA was extracted by Trizol method.The expressions of potassium ion channel genes (KCNA4,KCND2,KCND3,KCNE1,KCNE2,KCNJ2,KCNJ12 and KCNQ1) at the mRNA level in heart were determined using real-time polymerase chain reaction.Results In low-selenium 4 weeks group,the mRNA expressions of KCNA4 gene(25.3 ± 0.09) and KCND2 gene(4.85 ± 0.05) were higher than that of the control group (1.00 ± 0.00,1.00 ± 0.00,all P ＜ 0.05); in low-selenium 24 weeks group,the mRNA expression of KCNJ12 gene (22.7 ± 0.10) was higher than that of the control group(1.00 ± 0.00,P ＜ 0.05); the mRNA expressions of KCND3,KCNE1,KCNE2,KCNJ2,KCNQ1 genes were compared with the corresponding control groups,the differences were not statistically significant(all P ＞ 0.05).Conclusions Low selenium can affect the mRNA expression of mouse cardiac potassium ion channel genes.
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Aim To investigate the effect of 15-ketoeicosatetraenoic acid(15-KETE)and its mechanism through ion channels on rat isolated pulmonary arterial rings by using organ bath technique.Methods Sixteen healthy Wistar rats weighing 220?20 g were divided into two groups(n=8): normoxia group breathing fresh air(FiO_2=21%) and hypoxia group breathing hypoxic air(FiO_2=10%) in a hypoxic box.Pulmonary arteries(PA)were extracted after 9 d and cut into rings(0.5~1.0 mm in diameter and 3 mm in length) for organ bath experiments.Results(1) With increasing concentration from 0 to10~(-6) mol?L~(-1),15-KETE increased PA rings tension gradually in a dosedependent fashion;(2) 4-aminopyridine(2 mmol?L~(-1)),a Kv channel blocker significantly decreased constriction of rat isolated PA rings induced by 15-KETE,and results were similar in two groups;(3) The K_(ATP) channel blocker glyburide(10~(-6)6 mol?L~(-1)) and the BK_(Ca) channel blocker tetraethylammonium(10 mmol?L~(-1)) did not affect constriction of rat isolated PA rings induced by 15-KETE;(4) The BKCa channel blocker nifedipine(10~(-6) mol?L~(-1)) and Ca~(2+)-free Krebs solution significantly decreased constriction of rat isolated PA rings induced by 15-KETE.Conclusion Kv channels play a role in constriction of PA induced by 15-KETE;L-type Ca~(2+) channel blocker and extracellular calcium ion also influence constriction of isolated PA rings induced by 15-KETE.
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Objective To investigate the effects of midazolam on the delayed rectifier outward potassium channel current (Ik) using the whole-cell patch clamp technique. Methods Pyramidal neurons were isolated acutely from 5-15 d SD rat hippocampus. We measured amplitudes of the delayed outward rectifier potassium currents by activating depolarizing pulse from - 50mV to 30mV. Different concentrations of midazolam were added and potassium channel currents were measured. Results Delayed outward rectifier potassium channel currents were inhibited by midazolam in a dose-dependent manner. EC50 was (8.31 ?2.78) ? 10-8 mol/L and Hill constant was 0.90? 0.16.Conclusions Our results suggest that block of the outward rectifier potassium channel current by midazolam may contribute to the mechanism of midazolam anesthesia action.