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Objective To explore the feasibility of adding a flexible linker between two-pore-domain potassium channel TREK-1 (TWIK related K + channel 1)monomers to construct a tandem-linked dimer.Methods PCR was used to add a flexible linker between the two TREK-1 monomers.The cRNA obtained from in vitro transcription using the above vector was injected into Xenopus oocytes.After 24 -48 h,currents were recorded from these oocytes using a two-electrode voltage clamp.The effects of extracellular Ba2 + and pH on TdTREK-1 were observed and compared with those of native dimeric TREK-1.Results The tandem-linked dimeric TdTREK-1 was highly expressed in Xenopus oocytes.The currents through these channels were inhibited by extracellular Ba2 +and acidification.Furthermore,the responsiveness of the concatenated dimers to these extracellular stimuli was similar to that of native dimers.Conclusion Adding a flexible linker between the two monomers to construct the tandem-linked dimer does not affect the expression and gating properties of TREK-1, suggesting that the method be feasible.Such a method will allow the manipulation of a single subunit,which will help basis study the structure and function of TREK-1.
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OBJECTIVE To investigate the effect of clopidogrel(Clog),a platelet aggregation inhibitor,on the development of colitis-associated colon cancer(CAC)and its possible mechanism. METHODS To establish a CAC model,male BALB/c mice were treated with single azoxymethane(AOM) 10 mg · kg-1 by ip. One week later,the mice drank 2.5% dextran sulfate sodium(DSS)for one week and water for two weeks,which lasted three cycles. From the first day mice received 2.5%DSS water, Clog 12.5,25.0 and 50.0 mg · kg-1 was ig administered once a day. Body mass,clinical symptoms,the number of colon tumor and tumor size in colon tissue were recorded. Hyperplasia of tumors was analyzed by HE staining. In the early inflammatory phase of the CAC model,the length of colons was measured, histological structure and epithelium cell proliferation of colon tissues were evaluated by HE staining and Ki67 staining,respectively. In the tumorigenesis and progression phase of the CAC model,epithe?lium cell proliferation of colon tissues was evaluated by Ki67 staining. The mRNA expression of tumor necrosis factor-α(TNF-α)was detected by real-time quantitative PCR. The expression of chemokine(C-X-C motif)ligand 2(CXCL2)and its receptor 2(CXCR2)in colon tissues was detected by PCR and immu?nohistochemistry. RESULTS Compared with model group,clinical symptoms of mice in Clog 12.5 mg · kg-1 group were alleviated,the size of colon tumors was decreased(P<0.05),and hyperplasia of tumors was reduced(P<0.05). During the inflammatory phase,the clinical symptoms of mice in Clog 12.5 mg·kg-1 group were significantly alleviated(P<0.05),the decrease of body mass was reduced(P<0.01),the colon shrinkage was ameliorated(P<0.01),the inflammatory injury and epithelium cell proliferation in colon tissues were reduced(P<0.05). During the tumorigenesis and progression phase,epithelium cell prolif?eration in colon tissues in Clog 12.5 mg·kg-1 group was reduced(P<0.01),and the mRNA and protein expression of TNF-α,CXCL2 and CXCR2 of colon tissues was decreased(P<0.05). CONCLUSION Clog can alleviate inflammation during the CAC early inflammatory phase and inhibit the formation of CAC. The antitumor effect of Clog may be related to the decrease in expression of CXCL2 and CXCR2.
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OBJECTIVE To study the effect of epipolythiodioxopiperazine compound C87 on tumor cell proliferation and explore the potential mechanisms. METHODS Tumor cells were exposed to C87 0.05-1 μmol.L-1 for 24, 48 and 72 h, cell viability was determined by sulforhodamine B (SRB) assay and the half growth inhibition (Gl50 ) was calculated. After treatment with C87 0.1-2.5 μmol.L-1 for 6 h, or C87 2.5 μmol.L-1 for 0-6 h, the generation of reactive oxygen species (ROS) was measured using the compound 2′,7′-dichlorofluoresceindiacetate and flow cytometry analysis. After treatment with C87 2.5 μmol.L-1 , either alone or with antioxidant N-acetylcysteine (NAC), for 6 h, the generation of ROS was measured by flow cytometry analysis. Tumor cells were exposed to C87 0.05-1 μmol.L-1 , either alone or with NAC, for 24 and 48 h, while cell viability was determined by SRB assay. RESULTS The cell viability was significantly reduced following exposure to C87 0.05-1 μmol.L-1 for 24, 48 and 72 h in a concentration-dependent manner in A549, HCT116, HeLa and SMMC7721 cells(P<0.05). At 72 h, the value of r2 was 0.946, 0.989, 0.973 and 0.984(P<0.05), respectively. The cell viability was significantly reduced following exposure to C87 1 μmol.L-1 for 24 - 72 h in a time-dependent manner in A549, HCT116, HeLa and SMMC7721 cells(P<0.05). The value of r2 was 0.983, 0.956, 0.951 and 0.873(P<0.05), respectively. The generation of ROS was increased after exposure to C87 0.25-2.5 μmol.L-1 in a concentration-dependent manner in HCT116 and HeLa cells for 6 h (r2 = 0.760, P = 0.045: r2 = 0.987, P=0.001), and after exposure to C87 2.5 μmol.L-1 in a time-dependent manner in HCT116 and HeLa cells for 0.5-6 h (r2 = 0.886, P = 0.017: r2 = 0.994, P = 0.000).The C87-induced ROS generation could be blocked by NAC in HCT116 and HeLa cells(P<0.05). The C87 induced cell death could be blocked by NAC 5 and 10 mmol.L-1 , and the Gl50 value was 1.446 and 1.134 μmol.L-1 for 24 h (the Gl50 value of C87 group was 0.513 μmol.L-1 ), and 0.882 and 1.166 μmol.L-1 for 48 h (the Gl50 value of C87 group was 0.333 μmol.L-1 ). CONCLUSION The novel epipolythiodioxopiperazine derivative C87 exerts potent antitumor activity in vitro, possibly via triggering ROS production.
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Objective To establish a simple, feasible and precise quality control method for the determination of contents and related substances of demethyl levophencynonate hydrochloride (L-LPC)tablets.Methods The mobile phase consisted of methanol,acetonitrile and sodium acetate buffer solution(pH 5.0),at a ratio of 4∶3∶3,at a flow rate 1.0 ml/min and a detection wavelength of 220 nm.Samples were injected 100 μl and determined at room temperature.Results The calibration curves showed good linearity (R2 =1) within the test range of 0.1-50μg/ml.The recovery of the method was about (100.15 ±0.73)%, and the stability of working solutions was acceptable in 8 h (RSD=0.36%).Conclusion The results indicated that the developed method can be readily used as a quality control method.
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Stress exerts profound effects on cognitive and emotional functions. As a regulator of response to stress,the corticotropin releasing factor (CRF) during stress impacts organism function through hypothalamic-pituitary-adrenal axis. Recent research has shown that dendritic branches were abnormal and dendritic spine was lost under stress. In contrast,stressed mice with CRF receptor 1 (CRFR1) deficiency or CRFR1 antagonist exhibited normal dendritic morphology of hippocampal neurons .The structure and related factors expression of hippocampal neurons were affected by CRFR1 as a G protein-coupled receptor(GPCR), but the molecular mechanism about CRF-induced direct injury effect on hippocampal neurons has been poorly understood. The paper reviews the research progress on the contribution of stress activated CRF-CRF receptor signaling to the structure and related factors of hippocampal neurons.
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Aim ToconstructHEK293cellsstablyex-pressing corticotropin releasing factor receptor 1 ( CRFR1 ) , and evaluate its function by the cAMP as-say.Methods CulturedHEK293cellsweretransfect-ed with CRFR1-expressing vector by Lipofectamine 2000 and were selected by using G418 . CRFR1 ex-pression was detected by Western blot, RT-PCR and immunofluorescence.Results Westernblot,RT-PCR and immunofluorescence data revealed that the HEK293 cells expressed CRFR1 protein stably. The dose-responsive relationship experiment revealed that CRF induced a CRFR1-mediated cAMP production in HEK293 cells with EC50 =(5. 64 ± 0. 05) × 10 -10 mol ·L-1.Conclusion HEK293celllinesstablyex-pressing CRFR1 were constructed successfully, which would provide a cellular model to facilitate the research on the biological function of CRFR1 and CRFR1-targe-ted drug screening.
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Antitumor effects of erythromycin and the related mechanism were investigated in the present study. Neuroblastoma cells (SH-SY5Y) were exposed to erythromycin at different concentrations for different durations. Cell proliferation was measured by cell counting, and cell viability was examined by MTT assay. Cell cycle phase distribution and the cytosolic calcium level were detected by flow cytometry. Mitochondrial membrane potential was measured by the JC-1 probe staining and fluorescent microscopy. The expression of an oncogene (c-Myc) and a tumor suppressor [p21 (WAF1/Cip1)] proteins was analyzed by using Western blotting. Erythromycin could inhibit the proliferation of SH-SY5Y cells in a concentration- and time-dependent manner. The cell cycle was arrested at S phase. Mitochondrial membrane potential collapsed and the cytosolic calcium was overloaded in SH-SY5Y cells when treated with erythromycin. The expression of c-Myc protein was down-regulated, while that of p21 (WAF1/Cip1) protein was up-regulated. It was concluded that erythromycin could restrain the proliferation of SH-SY5Y cells. The antitumor mechanism of erythromycin might involve regulating the expression of c-Myc and p21 (WAF1/Cip1) proteins.
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ASICs are H+-gated novel cation ion channels, which belong to the epithelial sodium channels (NaC/DEG) superfamily. As recent studies focus, ASICs are expected to be pharmacological targets on protecting the neuron from ischemia and damage, improving the ability of memory and study, curing epilepsy and analgesia. It is not until the most recentness that the subunits of ASICs have been cloned. Now, researchers have paid more attention to the distribution, expression, function and modulation of ASICs in the organism.
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Aim Series of compounds,which were considered to be the antagonists of ET-1 receptor,were synthesized by Beijing Institute of Pharmacology and Toxicology.The biological activity of these compounds was screened and some active compounds were selected for further pharmacological characterization on pulmonary hypertension.Methods Radioligand binding assay was performed to study the binding affinity of compounds for ETA and ETB receptors.The biological activity of compounds was evaluated in isolated rat aortic ring and in systemic arterial pressure(SAP)of anesthetized rat experiments.In addition,hypotensive effect of compounds was investigated on monocrotaline induced pulmonary hypertension in rats.Results Compounds bind to ETA receptor had over 10 000 fold higher affinity than to ETB receptor.Contraction induced by ET-1 in isolated rat aortic ring was inhibited by compounds,and 1 ?mol?L-1 ETP-508 shifted the cumulative concentration-contraction response curve to ET-1 to right with no change in the maximal response.In vivo,the increase in SAP induced by ET-1 〔3.7 ?g?(0.5 ml)-1?kg-1〕 was inhibited by 2 mg?kg-1 compounds by intravenous infusion.Furthermore,BQ-485 and ETP-508 by intravenous infusion(0.4 mg?h-1)significantly inhibited 80 mg?kg-1(sc)monocrotaline induced pulmonary hypertension in rats.Conclusions These results indicate that ETP-508 and BQ-485 are highly selective ETA receptor antagonists and significantly inhibite monocrotaline induced pulmonary hypertension in rats.
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Despite the important properties of GPCRs as drug targets,improving the subtype selectivity and efficiency of new drugs targeting at GPCRs is a predominant challenge.The study of GPCRs allosterism shows there exist great complexity and diversity in allosterism and allosteric sites,and it also provides a new opportunity for exploring new drugs with subtype selectivity and efficiency.This review summaries the development of GPCRs allosterism in recent years and shows GPCRs allosterism and its biological significance.
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AIM:To investigate the inhibitory effect of dihydro-?-erythroidine on nicotine induced-current in cultured superior cervical ganglion of neonatal rats.METHODS:Pneumatic pressure administration of drug and whole-cell recording techniques were performed to compare induced-current amplitude.RESULTS:Dihydro-?-erythroidine competitively antagonized nicotinic effect,and EC50 was about 0.015mmol/L.CONCLUSION:Dihydro-?-erythroidine is a competitive antagonist of nicotinic receptors in sympathetic neurons.
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Agmatine was first identified and characterized as a candidate for CDS (clonidine displacing substance) in the bovine brain in 1994. The following researches demonstrated that agmatine was a widely distributed endogenous substance and performed a lot of biological functions in the central nervous system. The evidences revealed its targets were diverse and its mechanisms were complicated. Now it is well accepted that agmatine is a new neurotransmitter and/or neuromodulator and it might be an endogenous ligand of imidazoline receptor.
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The fifth muscarinic receptor (M5), the last one of the mus ca rinic receptor family to be cloned, has the same basic formation characterizatio n as G-protein coupled receptor family. M5 transduces signals by coupling with G-proteins, which then modulate the activities of a number of effector enzymes and ion channels. As M5 also plays a variety of prominent physiological roles by regulating central transmitters NO and DA, it has been considered as a novel dr ug therapy target for drug addiction, dysfunction of dopamine-ergic nervous sys tem, Alzheimers disease and cerebral ischemia.
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Acid-sensing ion channels (ASICs), which are activated by extracelluar H+, are H+-gated cation channels. At present, seven subunits of ASICs family have been identified. Numerous studies revealed that ASIC1a plays important roles in synaptic transmission and plasticity,spatial learning and memory, nociception, as well as cerebral ischemia under physiological and pathological conditions. This review concerns the latest research about ASIC1a to promote the understanding of its physiological and pathological functions.