RESUMEN
Most G protein-coupled receptors (GPCRs) do not generate membrane currents in response to ligand-receptor binding (LRB). Here, we describe a novel technique using endocytosis as a bioassay that can detect activation of a GPCR in a way analogous to patch-clamp recording of an ion channel in a living cell. The confocal imaging technique, termed FM endocytosis imaging (FEI), can record ligand-GPCR binding with high temporal (second) and spatial (micrometer) resolution. LRB leads to internalization of an endocytic vesicle, which can be labeled by a styryl FM dye and visualized as a fluorescent spot. Distinct from the green fluorescence protein-labeling method, FEI can detect LRB endocytosis mediated by essentially any receptors (GPCRs or receptors of tyrosine kinase) in a native cell/cell line. Three modified versions of FEI permit promising applications in functional GPCR studies and drug screening in living cells: 1) LRB can be recorded in "real time" (time scale of seconds); 2) internalized vesicles mediated by different GPCRs can be discriminated by different colors; and 3) a high throughput method can screen ligands of a specific GPCR.
Asunto(s)
Endocitosis , Ganglios Espinales/metabolismo , Ligandos , Microscopía Confocal/métodos , Imagen Molecular/métodos , Neuronas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Colorantes Fluorescentes/metabolismo , Ganglios Espinales/citología , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Ratas , Ratas Wistar , Receptores Adrenérgicos/metabolismo , Receptores Colinérgicos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de GABA-B/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Receptores de Serotonina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Liensinine and neferine, a kind of isoquinoline alkaloid, can antagonize the ventricular arrhythmias. The human ether-a-go-go-related gene (hERG) is involved in repolarization of cardiac action potential. We investigated the effects of liensinine and neferine on the biophysical properties of hERG channel and the underlying structure-activity relationships. The effects of liensinine and neferine were examined on the hERG channels in the stable transfected HEK293 cells using a whole-cell patch clamp technique, western blot analysis and immunofluorescence experiment. The pharmacokinetics and tissue distribution determination of liensinine and neferine in rats were determined by a validated RP-HPLC method. Liensinine and neferine induced decrease of current amplitude in dose-dependent. Liensinine reduced hERG tail current from 70.3±6.3 pA/pF in control group to 56.7±2.8 pA/pF in the 1 µM group, 53.0±2.3 pA/pF (3 µM) and 17.8±0.7 pA/pF (30 µM); the corresponding current densities of neferine-treated cells were 41.9±3.1 pA/pF, 32.3±3.1 pA/pF and 16.2±0.6 pA/pF, respectively. Neferine had binding affinity for the open and inactivated state of hERG channel, liensinine only bound to the open state. The inhibitory effects of liensinine and neferine on hERG current were attenuated in the F656V or Y652A mutant channels. Neferine distributed more quickly than liensinine in rats, which was found to be in higher concentration than liensinine. Both liensinine and neferine had no effect on the generation and expression of hERG channels. In conclusion, neferine is a more potent blocker of hERG channels than liensinine at low concentration (<10 µM), which may be due to higher hydrophobic nature of neferine compared with liensinine. Neferine may be safety even for long-term treatment as an antiarrhythmic drug.
Asunto(s)
Bencilisoquinolinas/farmacología , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Isoquinolinas/farmacología , Fenoles/farmacología , Animales , Antiarrítmicos/farmacocinética , Antiarrítmicos/farmacología , Bencilisoquinolinas/farmacocinética , Sitios de Unión , Membrana Celular/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Relación Dosis-Respuesta a Droga , Fenómenos Electrofisiológicos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Isoquinolinas/farmacocinética , Potenciales de la Membrana , Técnicas de Placa-Clamp , Fenoles/farmacocinética , Bloqueadores de los Canales de Potasio/administración & dosificación , Bloqueadores de los Canales de Potasio/farmacocinética , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Wistar , Relación Estructura-Actividad , Factores de Tiempo , Distribución Tisular , TransfecciónRESUMEN
Action potential (AP) patterns and dopamine (DA) release are known to correlate with rewarding behaviors, but how codes of AP bursts translate into DA release in vivo remains elusive. Here, a given AP pattern was defined by four codes, termed total AP number, frequency, number of AP bursts, and interburst time [N, f, b, i].. The 'burst effect' was calculated by the ratio (γ) of DA overflow by multiple bursts to that of a single burst when total AP number was fixed. By stimulating the medial forebrain bundle using AP codes at either physiological (20 Hz) or supraphysiological (80 Hz) frequencies, we found that DA was released from two kinetically distinct vesicle pools, the fast-releasable pool (FRP) and prolonged-releasable pool (PRP), in striatal dopaminergic terminals in vivo. We examined the effects of vesicle pools on AP-pattern dependent DA overflow and found, with given 'burst codes' [b=8, i=0.5 s], a large total AP number [N = 768, f = 80 Hz] produced a facilitating burst-effect (γ[b8/b1] = 126 ± 3%), while a small total AP number [N=96, 80 Hz] triggered a depressing-burst-effect (γ[b8/b1] = 29 ± 4%). Furthermore, we found that the PRP (but not the FRP) predominantly contributed to the facilitating-burst-effect and the FRP played an important role in the depressing-burst effect. Thus, our results suggest that striatal DA release captures pre-synaptic AP pattern information through different releasable pools.
Asunto(s)
Potenciales de Acción/fisiología , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Vesículas Sinápticas/fisiología , Algoritmos , Animales , Simulación por Computador , Estimulación Eléctrica , Electroquímica , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Cinética , Masculino , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Vesículas Sinápticas/metabolismoRESUMEN
BACKGROUND/AIMS: Human ether-à-go-go-related gene (hERG) has an important role in the repolarization of the cardiac action potential. Our studies were to investigate the effects of oxymatrine (one of the natural constituents extracted from Chinese herb Sophora flavescens Ait) on hERG-encoded K(+) channels at different temperatures and its underlying mechanism. METHODS: The effects of oxymatrine were examined on hERG channels stably expressed in HEK293 cells using a whole-cell patch clamp technique. RESULTS: At the temperature 30°C, oxymatrine inhibited hERG current in a concentration-dependent manner and the IC(50) was â¼665 µM. However at the temperature of 20°C, low concentration oxymatrine C≤100 µM increased hERG current density. However, high concentration oxymatrine C>100 µM inhibited the hERG current density significantly. Oxymatrine only affected the activation kinetic of hERG channels at all temperatures and had a high binding affinity for open state of hERG channels except the 300 µM-20°C group which had a high binding affinity for inactive state of hERG channels. CONCLUSION: Oxymatrine is a low potency blocker of hERG K+ channels at 30°C, low concentration oxymatrine affect the hERG activation gating with accelerating hERG tail current at 20°C, oxymatrine is a potential hERG activator at low temperatures.
Asunto(s)
Alcaloides/farmacología , Canales de Potasio Éter-A-Go-Go/fisiología , Quinolizinas/farmacología , Fenómenos Electrofisiológicos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/metabolismo , Células HEK293 , Humanos , Cinética , Técnicas de Placa-Clamp , Unión Proteica , TemperaturaRESUMEN
OBJECTIVE: To evaluate the mid-term efficacy of radiofrequency ablation of nucleus pulposus by intervertebral foramen endoscopy BEIS technique in the treatment of lumbar spine surgery failure syndrome over 60 years old. METHODS: The clinical data of 40 patients over 60 years old with lumbar spine surgery failure syndrome admitted from January 2010 to January 2015 were retrospectively analyzed. Among them, there were 34 males and 6 females, aged from 60 to 76 years old with an average of 66 years, the courses of disease ranged from 10 months to 4 years. The patients were divided into two groups (BEIS group and revision group) according to the different surgery. The intervertebral foramen endoscopy BEIS technique and the transforaminal lumbar interbody fusion (TLIF) were performed in BEIS group and revision group respectively. There was no significant difference in general data such as sex, age, course of disease, surgical segment between two groups(P>0.05). The operation time, intraoperative bleeding volume, bed rest time after operation and hospitalization time were observed between two groups. At preoperative, postoperative 1 month, 1 year, 3 years, visual analogue scale(VAS) and Japanese Orthopaedic Association Score(JOA) were used to compare the efficacy. RESULTS: The operation time, intraoperative bleeding volume, bed rest time after operation and hospitalization time in BEIS group were (60.2±10.3) min, (60.1±4.5) ml, (2.2±1.5) d, (4.04±1.40) d, respectively, which were significantly lower than those of revision group (P<0.05). The VAS and JOA scores of the two groups at different time after operation were significantly improved (P<0.05), and there was statistically significant difference between two groups (P<0.05). CONCLUSIONS: Radiofrequency ablation of nucleus pulposus by intervertebral foramen endoscopy BEIS technique is more effective than TLIF revision in the treatment of lumbar spine surgery failure syndrome over 60 years old. It has advantages of shorter operation time, less bleeding, shorter bed rest after operation and hospitalization time, and is worthy of clinical promotion.
Asunto(s)
Fusión Vertebral , Anciano , Endoscopía , Femenino , Humanos , Vértebras Lumbares , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
INTRODUCTION: Prognostic biomarkers are highly needed to properly manage patients with cancer and improve their clinical courses. The relationship between lymphocyte-to-monocyte ratio (LMR) at diagnosis and ovarian cancer prognosis has been extensively studied, but little consensus has been reached regarding its utility as a biomarker of poor outcome. Thus, this study aimed to investigate the potential prognostic value of pretreatment LMR in such patients to shed light on this issue. METHODS: We searched the scientific databases of MEDLINE, Embase, Cochrane Library, and WangFang for relevant studies about the inflammatory prognostic factor LMR in ovarian cancer, based on specific inclusion and exclusion criteria. The following parameters were analyzed among others: LMR values and respective cut-offs, patient's overall survival (OS) and progression-free survival (PFS), and clinicopathological features. RESULTS: Eight studies, including 2259 patients, were eligible for inclusion in this meta-analysis. We found that low LMR was associated with both poor OS [Hazard ratio (HR): 1.92; 95% confidence interval (CI): 1.58-2.34; p < 0.001] and PFS (HR: 1.70; 95% CI: 1.54-1.88; p < 0.001). Moreover, our findings revealed that low LMR was correlated with high G2/G3 histological grade (OR: 1.67; 95% CI: 1.26-2.20; p < 0.001) and late III-IV FIGO stage tumors (OR: 3.55; 95% CI: 2.68-4.70; p < 0.001), high serum CA-125 level (OR: 2.18; 95% CI: 1.71-2.77; p < 0.001), and presence of malignant ascites (OR: 1.87; 95% CI: 1.11-3.14; p = 0.02) and lymph node metastases (OR: 1.70; 95% CI: 1.13-2.54; p = 0.01). CONCLUSION: Pretreatment LMR is a potential prognostic marker of poor outcome in ovarian cancer patients and may thus be important in clinical care and disease control.
Asunto(s)
Linfocitos/citología , Monocitos/citología , Neoplasias Ováricas/sangre , Biomarcadores de Tumor/sangre , Femenino , Humanos , Recuento de Leucocitos , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Pronóstico , Supervivencia sin Progresión , Tasa de SupervivenciaRESUMEN
BACKGROUND: Circular RNAs (circRNAs) have displayed dysregulated expression in several types of cancer. Nevertheless, their function and underlying mechanisms in cervical cancer remains largely unknown. This study aimed to describe the regulatory mechanisms in cervical cancer. METHODS: We downloaded the circRNAs expression profiles from Gene Expression Omnibus database, and RNAs expression profiles from The Cancer Genome Atlas database. We established a circRNA-miRNA-mRNA and circRNA-miRNA-hubgene network. The interactions between proteins were analyzed using the STRING database and hubgenes were identified using MCODE plugin. Then, we conducted a circRNA-miRNA-hubgenes regulatory module. Functional and pathway enrichment analyses were conducted using R packages "Clusterprofile". RESULTS: Six circRNAs, 15 miRNAs, and 158 mRNAs were identified to construct the ceRNA network of cervical cancer. PPI (protein-protein interaction) network and module analysis identified 7 hubgenes. Then, a circRNA-miRNA-hubgene subnetwork was constructed based on the 1 DEcircRNAs, 3 DEmiRNAs, and 3 DEmRNAs. The KEGG pathway analysis indicated DEmRNAs are involved in progesterone-mediated oocyte maturation, cell cycle, and oocyte meiosis. CONCLUSION: These ceRNAs are critical in the pathogenesis of cervical and may serve as future therapeutic biomarkers.
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Regulación Neoplásica de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , ARN/genética , Neoplasias del Cuello Uterino/genética , Biomarcadores de Tumor/genética , Femenino , Humanos , Mapas de Interacción de Proteínas , ARN/metabolismo , ARN CircularRESUMEN
The novel quaternary ammonium modified magnetic carboxyl-carbon nanotubes (QA-Mag-CCNTs) have been synthesised and characterized. QA-Mag-CCNTs were applied in magnetic dispersive solid phase extraction (Mag-dSPE) for preconcentration of perchlorate from tea leaves prior to liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) analysis. The Mag-dSPE procedure for preconcentration of perchlorate succeed in overcoming the flaw (containing target analyte randomly) of commercially available SPE cartridge. Under optimal conditions, the results showed higher extraction efficiency of QA-Mag-CCNTs, with recoveries between 85.2% and 107%. And the satisfactory precision with inter-day and intra-day RSD values were lower than 8.0%. Furthermore, QA-Mag-CCNTs were evaluated for reuse up to 20 times. The limit of quantification (LOQ) for perchlorate was 8.21â¯ngâ¯kg-1. The developed method was successfully applied in tea leaves for food-safety risk monitoring in Zhejiang province, China. The results showed the concentrations of perchlorate in 229 out of 240 collected samples were in the range of 0.082-988⯵gâ¯kg-1. It was confirmed that QA-Mag-CCNTs were highly effective materials used for preconcentration of perchlorate.
Asunto(s)
Nanotubos de Carbono/química , Percloratos/análisis , Compuestos de Amonio Cuaternario/química , Té/química , Cromatografía Liquida , Campos Magnéticos , Estructura Molecular , Hojas de la Planta/química , Espectrometría de Masas en TándemRESUMEN
A series of ethylenediamine (EDA)-functionalized magnetic polymers (EDA-MPs) have been prepared via suspension polymerization with the usage amount of the functional monomer glycidylmethacrylate (GMA) varied during the suspension polymerization procedure. The EDA-MPs were characterized by transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), X-ray diffractometer (XRD), thermogravimetry and differential thermogravimetry analysis (TG-DTA), Fourier-transformed infrared spectroscopy (FTIR) and elementary analyzer (EA). The adsorption properties of the EDA-MPs for the removal of Cr(VI) in wastewater were deeply studied. The results showed the adsorption efficiency was highly pH dependent and decreased with the increasing of initial concentration of Cr(VI). The adsorption data taken at the optimized condition, i.e., 35 degrees C and pH of 2.5 were well fitted with the Langmuir isotherm. The maximum adsorption capacities (q(m)) of EDA-MPs to Cr(VI) were highly related to the contents of EDA-MPs, i.e., the q(m) of EDA-MPs to Cr(VI) calculated from the Langmuir isotherm increased from 32.15 to 61.35 mg g(-1) with the increasing of the usage amount of GMA. The adsorption kinetic data were modeled by the pseudo-second-order rate equation, and the adsorption of Cr(VI) by all the present EDA-MPs reached equilibrium in 60 min.
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Cromo/aislamiento & purificación , Etilenodiaminas/química , Compuestos Férricos/química , Magnetismo , Polímeros/química , Contaminantes Químicos del Agua/aislamiento & purificación , Microscopía Electrónica de Transmisión , Espectroscopía Infrarroja por Transformada de Fourier , Termogravimetría , Difracción de Rayos XRESUMEN
We studied the effects of isoquinoline alkaloid neferine (Nef) extracted from the seed embryo of Nelumbo nucifera Gaertn on Human ether-à-go-go-related gene (HERG) channels stably expressed in human embryonic kidney (HEK293) cells using whole-cell patch clamp technique, western blot analysis and immunofluorescence experiment. Nef induced a concentration-dependent decrease in current amplitude according to the voltage steps and tail currents of HERG with an IC(50) of 7.419 microM (n(H) -0.5563). Nef shifted the activation curve in a significantly negative direction and accelerated recovery from inactivation and onset of inactivation, however, slowed deactivation. In addition, it had no significant influence on steady-state inactivation curve. Western blot and immunofluorescence results suggested Nef had no significant effect on the expression of HERG protein. In summary, Nef can block HERG K(+) channels that functions by changing the channel activation and inactivation kinetics. Nef has no effect on the generation and trafficking of HERG protein. A blocked-off HERG channel was one mechanism of the anti-arrhythmic effects by Nef.