Discovery of the ARP2 protein as a determining molecule in tumor cell death

Cancer is a multifactorial disease that constitutes a serious public health problem worldwide. Prostate cancer advanced stages are associated with the development of androgen-independent tumors and an apoptosis-resistant phenotype that progresses to metastasis. By studying androgen-independent lymphoid nodule carcinoma of the prostate (LNCaP) cells induced to apoptosis by serum elimination, we identified the activation of a non-selective cationic channel of 23pS conductance that promotes incoming Ca2+ currents, as well as apoptosis final stages. arp2cDNA was isolated and identified to be of the same cell type, and mRNA was expressed in Xenopus laevis oocytes, which was found to be associated with the activation of incoming Ca2+ currents and induction to apoptosis. cDNA, which encodes the ARP2 protein, was overexpressed in LNCaP cells and Chinese hamster ovary cells, which induced apoptosis. Our evidence suggests that protein ARP2 overexpression and transit to the cell membrane allows an increased Ca2+ incoming current that initiates the apoptosis process in epithelial-type cells whose phenotype shows resistance to programmed cell death.

Microvascular anatomy of olfactory and accessory olfactory (vomeronasal) organs in adult Xenopus laevis: scanning electron microscopy of vascular corrosion casts / Anatomía microvascular de órganos olfatorios y accesorios (vomeronasal) en adultos Xenopus laevis: microscopía electrónica de barrido de moldes de corrosión vascular

Microvascular anatomy and histomorphology of olfactory and vomeronasal organs in adult Xenopus laevis Daudin were studied by scanning electron microscopy of vascular corrosion casts and paraplast embedded stained serial tissue sections. Results show that the arterial supply is bilaterally by terminal arterioles of the medial branch of the nasal artery and by the palatal artery. Arterioles give rise to a capillary meshwork characteristic for respiratory surfaces in principal chambers and in dorsal and caudal areas of middle chambers. Anterior and inferior areas of the middle chambers own a distinctly different capillary network with conspicuous short capillary loops. Loops have a dilated tip and extend in acute angles towards the chamber lumen. The vomeronasal organ (VNO) locates beneath the olfactory organ. It has a medial to lateral extension and attaches with its caudal circumference to the medial nasal glands. Its capillary bed displays rectangular meshes which preferentially orientate along the long axis of the VNO. Locally, capillaries form short hairpin-like or strongly twisted loops with dilated tips which point towards the lumen of the VNO. These capillaries slow-down blood velocity and may lead to an increased exchange of oxygen, nutrients and water-borne odorants in the middle chambers and of pheromones in the VNO. In the latter vascular structures are present which might serve as a vascular pump.

Se estudiaron la anatomía microvascular e histomorfología de los órganos olfatorios y vomeronasales de Xenopus laevis Daudin adultos, mediante microscopía electrónica de barrido de moldes de corrosión vascular y secciones de tejido seriadas, teñidas e incluídas en paraplast. Los resultados muestran que el suministro arterial es bilateral por arteriolas terminales de la rama medial de la arteria nasal y por la arteria palatina. Las arteriolas dan lugar a un lecho capilar característico de las superficies respiratorias en las cámaras principales y en las áreas dorsal y caudal de las cámaras intermedias. Las áreas anterior e inferior de las cámaras centrales poseen una red capilar significativamente diferente con llamativos bucles capilares cortos. Los bucles tienen una punta dilatada y se extienden en ángulos agudos hacia la luz de la cámara. El órgano vomeronasal (VNO) se ubica debajo del órgano olfatorio. Se extiende de medial a lateral y se une con su circunferencia caudal a las glándulas nasales mediales. El lecho capilar muestra mallas rectangulares que se orientan preferentemente a lo largo del eje longitudinal del VNO. Localmente, los capilares forman bucles cortos en forma de horquilla o fuertemente retorcidos con puntas dilatadas que apuntan hacia la luz del VNO. Estos capilares ralentizan la velocidad de la sangre y pueden conducir a un mayor intercambio de oxígeno, nutrientes y odorizantes, a base de agua en las cámaras intermedias y de feromonas, en el VNO. En este último, están presentes estructuras vasculares que podrían servir como una bomba vascular.

First parasitological study of the African clawed frog (Xenopus laevis, Amphibia) in Chile / Primeiro estudo parasitológico em rã com garras Africano (Xenopus laevis, Anfibia) no Chile

Abstract Introduced species can arrive into new territories with parasites; however, these species are expected to face lower parasite richness than in their original regions. Both introduced hosts and parasites can affect native fauna. Since their release into the wild in Chile following laboratory use, Xenopus laevis Daudin, 1802 has widely spread throughout central Chile. The only pathogen described on the host is the fungus Batrachochytrium dendrobatidis Longcore, Pessier, Nichols, 1999; thus, this is the first parasitological study of this species in Chile. In 10 localities in central Chile, 179 specimens of X. laevis were captured and examined for parasites in the gastrointestinal tube, cavities, lungs, liver, and skin. Only nine specimens of the genus Contracaecum Railliet, Henry, 1912 were found in six specimens of X. laevis from a private dam in La Patagua. It is likely that these parasites originated from species of native birds. This is the first record of Contracaecum sp. in Chilean amphibians.

Resumo Espécies exóticas podem se introduzir em um novo território com seus parasitas, porém nesses casos, a riqueza parasitária seria menor. Contudo, hospedeiros exóticos e seus parasitas associados podem afetar a fauna nativa. Depois de ser dispensado do uso em laboratórios e solto em ambientes naturais, Xenopus laevis Daudin, 1802 tem se espalhado massivamente no Chile central. O único patógeno descrito para este anuro é o fungo Batrachochytrium dendrobatidis Longcore, Pessier, Nichols, 1999. O presente estudo constitui a primeira pesquisa parasitológica realizada nesta espécie de rã introduzida no Chile. Em 10 localidades do Chile central, foram capturados 179 espécimes de X. laevis que foram examinadas em busca de parasitos dentro tubo digestivo, cavidades corporais, pulmões, fígado e pele. Nove espécimes do gênero Contracaecum Railliet, Henry, 1912 foram encontrados em seis espécimes de X. laevis de uma barragem em La Patagua. É provável que a origem destes parasitas sejam espécies de aves nativas. Este é o primeiro relato de Contracaecum sp. em anuros do Chile.

Consumption and expenditure on food prepared away from home among Mexican adults in 2006 / Consumo y gasto en alimentos preparados fuera de casa en adultos mexicanos en 2006

Objective. To describe food expenditure and consumption of foods prepared away from home among Mexican adults. Materials and methods. Data were from 45 241 adult participants in the National Health and Nutrition Survey 2006, a nationally-representative, cross-sectional survey of Mexican households. Descriptive statistics and multivariable linear and logistic regression were used to assess the relationship between location of residence, educational attainment, socioeconomic status and the following: 1) expenditure on all food and at restaurants, and 2) frequency of consumption of comida corrida or restaurant food and street food. Results. Food expenditure and consumption of food prepared away from home were positively associated with socioeconomic status, educational attainment, and urban vs. rural residence (p<0.001 for all relationships in bivariate analyses). Conclusions. Consumption of food prepared outside home may be an important part of the diet among urban Mexican adults and those with high socioeconomic status and educational attainment.

Objetivo. Describir los gastos en alimentos y el consumo de alimentos preparados fuera de casa en población mexicana. Material y métodos. Los datos fueron de 45 241 adultos mexicanos en la Encuesta Nacional de Salud y Nutrición de 2006, representativa al nivel nacional. Se utilizaron estadísticas descriptivas y regresión linear y logística para estimar la relación entre el lugar de residencia, el nivel educativo y el nivel socioeconómico, con el gasto en todos los alimentos y en restaurantes, y con la frecuencia de consumo de comida corrida, en restaurantes y de la calle. Resultados. El gasto en alimentos y el consumo de alimentos preparados se asociaron positivamente con el nivel socioeconómico, el nivel educativo y la residencia rural (p<0,001 para todas las relaciones). Conclusiones. El consumo de alimentos preparados puede ser una parte importante de la dieta de los adultos urbanos y de aquéllos con altos niveles socioeconómicos y educativos.

Osteocalcin Expression and Mineralization in Developing Tooth of Xenopus laevis

Osteocalcin (OC) is the most abundant noncollagenous protein of extracellular matrix in the bone. In an OC deficient mouse, bone formation rates are increased in cancellous and cortical bones. OC is known as a negative regulator of mineral apposition. OC is also expressed in the tooth of the rat, bovine, and human. However, little is known about OC during tooth development in Xenopus. The purpose of this study is to compare the expression of OC with mineralization in the developing tooth of Xenopus, by using von Kossa staining and in situ hybridization. At stage 56, the developmental stage of tooth germ corresponds to the cap stage, and an acellular zone was apparent between the dental papilla and the enamel organ. From stage 57, calcium deposition was revealed by von Kossa staining prior to OC expression, and the differentiated odontoblasts forming predentin were located at adjoining predentin. At stage 58, OC transcripts were detected in the differentiated odontoblasts. At stage 66, OC mRNA was expressed in the odontoblasts, which was aligned in a single layer at the periphery of the pulp. These findings suggest that OC may play a role in mineralization and odontogenesis of tooth development in Xenopus.

Expression analysis of ciliary rootlet coiled coil protein mRNA during Xenopus development / 대한수의학회지

Ciliary rootlet coiled coil protein (CROCC), the structural component that originates from the basal body at the proximal end of the ciliary rootlet, plays a crucial role in maintaining the cellular integrity of ciliated cells. In the current study, we cloned Xenopus CROCC and performed the expression analysis. The amino acid sequence of Xenopus laevis was related to those of Drosophila, cow, goat, horse, chicken, mouse and human. Reverse transcription polymerase chain reaction analysis revealed that CROCC mRNA encoding a coiled coil protein was present maternally, as well as throughout early development. In situ hybridization indicated that CROCC mRNA occurred in the animal pole of embryo during gastrulation and subsequently in the presumptive neuroectoderm at the end of gastrulation. At tailbud stages, CROCC mRNA expression was localized in the anterior roof plate of the developing brain, pharyngeal epithelium connected to gills, esophagus, olfactory placode, intestine and nephrostomes of the pronephric kidney. Our study suggests that CROCC may be responsible for control of the development of various ciliated organs.

Na, K-ATPase beta2 isoform (atp1b2) expressed in the retina of Xenopus / 동물의과학연구지

The ubiquitous Na, K-ATPase is a membrane-bound ion pump located in the plasma membrane in all animal cells and plays an essential role in a variety of cellular functions. Studies in several organisms have shown that this protein regulates different aspects of embryonic development and is responsible for the pathogenesis of several human diseases. Na, K-ATPase is an important factor for retinal development, and combinations of the isoforms of each of its subunits are expressed in different cell types and determine its functional properties. In this study, we performed RT-PCR assay to determine temporal expression and in situ hybridization to determine spatial expression of Na, K-ATPase beta2 isoform (atp1b2) in Xenopus laevis. Focusing on retinal expression to distinguish the specific expression domain, we used retinal marker genes sox4, sox11, vsx1, and . Xenopus atp1b2 was expressed from late gastrulation to the tadpole stage. Using whole mount in situ hybridization, we showed that Xenopus atp1b2 was expressed broadly in the eye, the whole surface ectoderm, and gills. In situ hybridization on sections revealed detailed and specific expression in the outer nuclear layer of the retina, which consists of two major classes of photoreceptors, rods and cones, surface ectoderm, pharyngeal epithelium, and gills. These findings indicate that atp1b2 may play an important role for the development of Xenopus retina.

Optimization of coding sequences and expression of antimicrobial peptide magainin II in Escherichia coli and Pichia pastoris / 生物工程学报

The antimicrobial peptide magainin II is expressed in the skin of the African clawed frog, Xenopus laevis, and exhibits a broad spectrum of antimicrobial activity as well as tumoricidal properties at low concentrations. In addition, magaininII plays a synergistic role during antimicrobial and tumoricidal processes with another antimicrobial peptide PGLa that is also expressed in Xenopus laevis. The optimized cDNA sequence of magainin II and magainin II-PGLa hybrid peptide according to E. coli or Pichia pastoris codon usage frequency were synthesized and sub-cloned into prokaryotic expression vector pGEX and Pichia pastoris secreted expression vector pPIC9k. The resulting recombinant plasmids were named as pGEX-magainin II and pPIC9k-magainin II-PGLa. The GST-magainin II fusion protein was highly expressed in E. coli. Furthermore, magainin II was successfully purified by digestion with PreScission Protease to cleave the GST tag. Additionally, our data obtained from the ELISA revealed that magainin II -PGLa hybrid peptide was successfully expressed in Pichia pastoris. These experiments establish a useful system for further studies of these antimicrobial peptides.

Aparición de melanina como pigmento protector en el encéfalo de xenopus laevis para protegerlo de los efectos de la radiación ultravioleta / Emerging melanin protective pigment in the brain of xenopus laevis to protect from the effects of uv radiation

Con el fin de proteger al organismo de condiciones estresantes, tales como cambios de osmolaridad y de temperatura, además de actuar como pantalla protectora en contra de los rayos ultravioleta (UV). Se ha observado que ciertos anfibios han desarrollado pigmentación en su encéfalo como una posible protección ante el aumento de la radiación UV, causada por el daño en la capa de ozono, la cual estaría alterando al ecosistema. En este trabajo se describe la presencia de pigmentación en el encèfalo de X. laevis durante el desarrollo larvario y su posible función protectora frente a la radiación UV. Para ello, se recolectaron individuos de diferentes estados larvarios, los que fueron obtenidos de distintas localidades de la región de Valparaíso (V región, Chile), para ser procesados con el método corriente H-E y el método de Lillie. En los análisis se pudo evidenciar que la pigmentación correspondía a melanina, que se encontraría en la membrana denominada leptomeninge, la cual recubre al encéfalo y estaría actuando como un filtro protector para evitar daños a nivel del desarrollo en el sistema nervioso de estos anuros. En suma, los rayos UV como agentes deletéreos estarían estimulando la producción de eumelanina en la leptomeninge de estos anfibios, para proteger parte del SNC (encéfalo), como al individuo en sí de posibles alteraciones teratogénicas y/o mutagénicas.

It has been observed that certain amphibians have developed pigmentation in brain as a possible increased protection against UV radiation, caused by damage to the ozone layer, which would alter the ecosystem. In this paper we describe the presence of pigment in the brain of X. laevis during larval development and possible protective function against UV radiation. To do this, we collected individuals at various larval stages, which were obtained from different locations in Valparaiso (V Region, Chile), to be processed with HE and the method of Lillie. In the analysis it was evident that pigmentation corresponded to melanin, which would be in the membrane called leptomeninges, which covers the brain and would be acting as a protective filter to prevent damage to the level of development in the nervous system of these frogs. In addition, UV rays would be deleterious agents stimulating production of eumelanin in the leptomeninges of these amphibians, to protect the CNS (brain), and the individual itself of potential teratogenic or mutagenic alterations.

In vivo and in vitro anti-natriuretic activity of twigs fraction from Dorstenia picta: a possible mechanism

The present study examine the in vivo effects of Dorstenia Picta [D. picta] on urinary volume and sodium excretion in streptozotocin-induced diabetic rats, and determine a possible mechanism by which the extract increased sodium transport in A6 cells monolayers. Administration of the plant extract at the dose of 150 mg/kg during two weeks decreased urinary volume and sodium excretion. In vitro study showed that, apical application of the plant extract at the dose of 100 micro g/mL does not significantly increase sodium transport, whereas basolateral administration provoked a significant [P<0.05] increase of sodium transport in a concentration-dependent manner. The plant extract increases the sodium transport by 69.93% versus 55.41% for insulin and 78.44% for adenosine after 30 min. Preincubation of A6 cells monolayers with inhibitor of all adenosine receptors completely suppressed adenosine and plant extract stimulated sodium transport. Interesting is that, the A1 inhibitor receptor [DPCPX], at 100 nM completely abolished the effect of plant extract. The plant extract increased sodium transport by increase PI3-kinase activity and this effect is strongly inhibited by LY-294002. These data also suggest that, the twigs methanol fraction from Dorstenia picta increase sodium transport via PI 3-kinase pathway and requires A1 adenosine receptor

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

Characteristic and effect of cadmium on ATP-activated currents mediated by P2X4 receptors / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the characteristic and effect of cadmium on ATP-activated currents (I(ATP)) mediated by P2X4 purinoceptors.</p><p><b>METHODS</b>Transcribe cDNA coding for the rat P2X4 receptor to cRNA in vitro. Inject the cRNA to oocytes of an xenopus laevis using the microinjection technique. Reveal the effect of cadmium on I(ATP) mediated by P2X4 receptor using the two-electrode whole-cell voltage clamp technique.</p><p><b>RESULTS</b>(1) Within a certain concentration range, cadmium was found to reversibly magnify I(ATP) mediated by P2X4 receptors expressed in oocytes of an xenopus. When the concentration of cadmium reached 30 micromol/L, the increase of I(ATP) was the most significant. I(ATP) turned to decrease when the concentration of cadmium was more than 30 micromol/L; (2) The concentration-response curve was shifted to left by applying cadmium at 10 micromol/L; the EC50 was reduced from (17.1 +/- 1.5) micromol/L to (9.8 +/- 1.8) micromol/L (n = 6, P < 0.01) and the Hill coefficient was increased from 1.14 +/- 0.13 to 1.57 +/- 0.36; (3) The effect of cadmium on I(ATP) showed no dependence on membrane voltage; (4) The magnifying effect on I(ATP) reached maximum when preincubating cadmium for 120 seconds.</p><p><b>CONCLUSION</b>The increase I(ATP) by cadmium is reversible, concentration-dependent, time-dependent, and voltage-independent. One reason of this augment effect could be the allosteric modulation on P2X4 receptors.</p>

Mg(2+) inhibits ATP-activated current mediated by rat P2X4 receptors expressed in Xenopus oocytes / 生理学报

To investigate the modulation of Mg(2+) on rat P2X4 receptors and its underlying mechanism, we transcribed cDNA coding for wild-type and mutant P2X4 receptors to cRNA in vitro, injected the cRNA to oocytes of Xenopus laevis using the microinjection technique and revealed the effect of Mg(2+) on ATP-activated currents (I(ATP)) mediated by P2X4 receptors using the two-electrode whole-cell voltage clamp technique. The effects of extracellular Mg(2+) on I(ATP) were as follows: (1) In oocytes expressing P2X4 receptors, Mg(2+) with concentration ranging from 0.5-10 mmol/L inhibited the amplitude of I(ATP) in a concentration-dependent and reversible manner, with a 50% inhibitory concentration value (IC(50)) of (1.24 ± 0.07) mmol/L for current activated by 100 μmol/L ATP. (2) Mg(2+) (1 mmol/L) shifted the dose-response curve for I(ATP) right-downward without changing the EC(50), but reduced the maximal current (E(max)) by (42.0 ± 2.1)%. (3) After being preincubated with Mg(2+) for 80 s, the inhibitory effect of the Mg(2+) on I(ATP) reached the maximum. (4) The inhibition of Mg(2+) on I(ATP) was independent of membrane potential from -120 mV to +60 mV. (5) Compared with the current activated by 100 μmol/L ATP in the wild-type P2X4 receptors, mutant P2X4 D280Q responded to the application of 100 μmol/L ATP with a smaller current. The peak current was only (4.12 ± 0.15)% of that seen in wild-type receptors. Mutant P2X4 D280E responded to ATP stimulation with a current similar to that observed in cells expressing wild-type receptors. (6) When Asp280 was removed from P2X4, the current amplitude of I(ATP) was increased almost one-fold, and Mg(2+) with concentration ranging from 0.5-10 mmol/L did not affect the I(ATP) significantly. The results suggest that Mg(2+) inhibits I(ATP) mediated by P2X4 receptors non-competitively, reversibly, concentration-dependently, time-dependently and voltage-independently. The inhibitory effect of Mg(2+) might be realized by acting on the site Asp280 of the P2X4 receptors.

Isolation and Expression Profile of the Ca(2+)-Activated Chloride Channel-like Membrane Protein 6 Gene in Xenopus laevis / 한국실험동물학회지

To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA.

Calcium regulation of nucleocytoplasmic transport

Bidirectional trafficking of macromolecules between the cytoplasm and the nucleus is mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) of eukaryotic cell. The NPC functions as the sole pathway to allow for the passive diffusion of small molecules and the facilitated translocation of larger molecules. Evidence shows that these two transport modes and the conformation of NPC can be regulated by calcium stored in the lumen of nuclear envelope and endoplasmic reticulum. However, the mechanism of calcium regulation remains poorly understood. In this review, we integrate data on the observations of calciumregulated structure and function of the NPC over the past years. Furthermore, we highlight challenges in the measurements of dynamic conformational changes and transient transport kinetics in the NPC. Finally, an innovative imaging approach, single-molecule superresolution fluorescence microscopy, is introduced and expected to provide more insights into the mechanism of calcium-regulated nucleocytoplasmic transport.

Inhibition of Jingzhaotoxin-V on Kv4.3 channel / 生理学报

Kv4.3 channel is present in many mammalian tissues, predominantly in the heart and central nervous system. Its currents are transient, characterized by rapid activation and inactivation. In the hearts of most mammals, it is responsible for repolarization of the action potential of ventricular myocytes and is important in the regulation of the heart rate. Because of its central role in this important physiological process, Kv4.3 channel is a promising target for anti-arrhythmic drug development. Jingzhaotoxin-V (JZTX-V) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Whole-cell patch clamp recording showed that it partly blocked the transient outward potassium channels in dorsal root ganglion neurons of adult rats with an IC(50) value of 52.3 nmol/L. To investigate the effect of JZTX-V on Kv4.3 channel, JZTX-V was synthesized using the solid-phase chemical synthesis and separated by reverse phase high performance liquid chromatography (HPLC). The purity was tested by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MOLDI-TOF mass spectrometry). Two-electrode voltage-clamp technique was used to characterize the action of JZTX-V on Kv4.3 channels expressed in Xenopus laevis oocytes. As a result, JZTX-V displayed fast kinetics of inhibition and recovery from inactivation. Furthermore, it could inhibit Kv4.3 channel current in a time- and concentration-dependent manner with an IC(50) value of 425.1 nmol/L. The application of JZTX-V affected the activation and inactivation characteristics of Kv4.3 channel and caused a shift of the current-voltage relationship curve and the steady-state inactivation curve to depolarizing direction by approximately 29 mV and 10 mV, respectively. So we deduced that JZTX-V is a gating modifier toxin of Kv4.3 channel. Present findings should be helpful to develop JZTX-V into a molecular probe and drug candidate targeting to Kv4.3 channel in the myocardium.

Effect of ginsenosides on the desflurane modulation in the recombinant serotonin type 3A receptor expressed in Xenopus laevis oocytes / 대한마취과학회지

BACKGROUND: Postoperative nausea and vomiting (PONV) is the most frequent and discomforting side effect following general anesthesia. Most volatile anesthetics have a potent effect on serotonin (5-hydroxydtryptamine, 5-HT) type 3 receptor mediating PONV, and their antagonists have been currently used effectively to prevent and/or reduce the incidence and severity of PONV. The authors reported previously that ginsenosides have inhibitory effect on 5-HT3A receptor. In this study we intended to elucidate the inhibitory effect of ginsenosides on the potentiated 5-HT3A receptor by desflurane. METHODS: After in vitro transcription of the recombinant mouse 5-HT3A receptor in the Xenopus laevis oocyte, we examined the effects of ginsenosides (g-Rb1, g-Rg1, g-Rd, g-Rg2) as well as ginsenoside metabolite, compound K on the modulation of desflurane by measuring currents flowing through 5-HT3A receptor using two-electrode voltage clamp technique. RESULTS: Although normalized inhibitory responses of ginsenosides were same regardless of desflurane, some ginsenosides such as g-Rd, g-Rg2, and g-Rg1 showed potential inhibition to the enhanced 5-HT induced current of 5-HT3A receptor by desflurane. CONCLUSIONS: Although ginsenosides have substantial inhibitory effect on 5-HT3A receptor, the effects of ginsenoside on potentiation by desflurane of 5-HT induced current via recombinant 5HT3A receptor may depend on the types of ginsenoside, which suggesting that ginsenoside might have an antagonistic action to nausea and vomiting associated with volatile anesthetics.

Regulation of Antiarrhythmic Drug Propafenone Effects on the C-type KV1.4 Potassium Channel by PHo and K+

The effects of the antiarrhythmic drug propafenone at c-type kv1.4 channels in Xenopus laevis oocytes were studied with the two-electrode voltage-clamp techinique. Defolliculated oocytes (stage V-VI) were injected with transcribed cRNAs of ferret Kv1.4 delta N channels. During recording, oocytes were continuously perfused with control solution or propafenone. Propafenone decreased the currents during voltage steps. The block was voltage-, use-, and concentration- dependent manners. The block was increased with positive going potentials. The voltage dependence of block could be fitted with the sum of monoexponential and a linear function. Propafenone accelerated the inactivate of current during the voltage step. The concentration of half-maximal block (IC(50)) was 121 micrometer/L. With high, normal, and low extracellular potassium concentrations, the changes of IC(50) value had no significant statistical differences. The block of propafenone was PH- dependent in high-, normal- and low- extracellular potassium concentrations. Acidification of the extracellular solution to PH 6.0 increased the IC50 values to 463 micrometer/L, alkalization to PH 8.0 reduced it to 58 micrometer/L. The results suggest that propafenone blocks the kv1.4 delta N channel in the open state and give some hints for an intracellular site of action.

Amino acid residues involved in agonist binding and its linking to channel gating, proximal to transmembrane domain of 5-HT3A receptor for halothane modulation / 대한마취과학회지

BACKGROUND: The 5-hydroxytryptamine type 3 (5-HT3) receptor is a member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) and modulated by pharmacologic relevant concentrations of volatile anesthetics or n-alcohols like most receptors of LGICs. The goal of this study was to reveal whether the site-directed single mutations of E-106, F-107 and R-222 in 5-HT3 receptor may affect the anesthetic modulation of halothane known as positive modulator. METHODS: The wild-type and mutant receptors, E106D, F107Y, R222F, R222V, were expressed in Xenopus Laevis oocytes and receptor function was assessed using two electrode voltage clamp techniques. RESULTS: E106D, F107Y, R222F, R222V mutant 5-HT3A receptors were functionally expressed. F107Y mutant 5-HT3A receptors displayed decreased sensitivity to 5-HT compared to the wild type 5-HT3A receptor (P < 0.05). Halothane showed positive modulation in both wild and F107Y mutant 5-HT3A receptors but F107Y mutant 5-HT3 receptor showed greater enhancing modulation comparing to wild-type receptor. Meanwhile, R222F and R222V mutant 5-HT3 receptor lost positive modulation with 1 and 2 MAC of halothane. Most interestingly, positive modulation by halothane was converted into negative modulation in E106D mutant 5-HT3A receptor. CONCLUSIONS: The present study implicate the amino acid residues known for agonist binding and linking agonist binding to channel gating might also have important role for anesthetic modulation in 5-HT3A receptor.

Inhibition of the Human Ether-a-go-go-related Gene (HERG) K+ Channels by Lindera erythrocarpa

Lindera erythrocarpa Makino (Lauraceae) is used as a traditional medicine for analgesic, antidote, and antibacterial purposes and shows anti-tumor activity. We studied the effects of Lindera erythrocarpa on the human ether-a-go-go-related gene (HERG) channel, which appears of importance in favoring cancer progression in vivo and determining cardiac action potential duration. Application of MeOH extract of Lindera erythrocarpa showed a dose-dependent decrease in the amplitudes of the outward currents measured at the end of the pulse (I(HERG)) and the tail currents of HERG (I(tail)). When the BuOH fraction and H2O fraction of Lindera erythrocarpa were added to the perfusate, both I(HERG) and I(tail) were suppressed, while the hexane fraction, CHCl3 fraction, and EtOAc fraction did not inhibit either I(HERG) or I(tail). The potential required for half-maximal activation caused by EtOAc fraction, BuOH fraction, and H2O fraction shifted significantly. The BuOH fraction and H2O fraction (100 microgram/mL) decreased gmax by 59.6% and 52.9%, respectively. The H2O fraction- and BuOH fraction-induced blockades of I(tail) progressively decreased with increasing depolarization, showing the voltage-dependent block. Our findings suggest that Lindera erythrocarpa, a traditional medicine, blocks HERG channel, which could contribute to its anticancer and cardiac arrhythmogenic effect.