Effects of Paroxetine on a Human Ether-a-go-go-related Gene (hERG) K⁺ Channel Expressed in Xenopus Oocytes and on Cardiac Action Potential

K⁺ channels are key components of the primary and secondary basolateral Cl- pump systems, which are important for secretion from the salivary glands. Paroxetine is a selective serotonin reuptake inhibitor (SSRI) for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. We studied the effects of paroxetine on a human K⁺ channel, human ether-a-go-go-related gene (hERG), expressed in Xenopus oocytes and on action potential in guinea pig ventricular myocytes. The hERG encodes the pore-forming subunits of the rapidly-activating delayed rectifier K⁺ channel (I(Kr)) in the heart. Mutations in hERG reduce I(Kr) and cause type 2 long QT syndrome (LQT2), a disorder that predisposes individuals to life-threatening arrhythmias. Paroxetine induced concentration-dependent decreases in the current amplitude at the end of the voltage steps and hERG tail currents. The inhibition was concentration-dependent and time-dependent, but voltage-independent during each voltage pulse. In guinea pig ventricular myocytes held at 36℃, treatment with 0.4 µM paroxetine for 5 min decreased the action potential duration at 90% of repolarization (APD₉₀) by 4.3%. Our results suggest that paroxetine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects of clinical administration of paroxetine.

Differential Effects of Quercetin and Quercetin Glycosides on Human α7 Nicotinic Acetylcholine Receptor-Mediated Ion Currents

Quercetin is a flavonoid usually found in fruits and vegetables. Aside from its antioxidative effects, quercetin, like other flavonoids, has a various neuropharmacological actions. Quercetin-3-O-rhamnoside (Rham1), quercetin-3-O-rutinoside (Rutin), and quercetin-3-(2(G)-rhamnosylrutinoside (Rham2) are mono-, di-, and tri-glycosylated forms of quercetin, respectively. In a previous study, we showed that quercetin can enhance α7 nicotinic acetylcholine receptor (α7 nAChR)-mediated ion currents. However, the role of the carbohydrates attached to quercetin in the regulation of α7 nAChR channel activity has not been determined. In the present study, we investigated the effects of quercetin glycosides on the acetylcholine induced peak inward current (I(ACh)) in Xenopus oocytes expressing the α7 nAChR. I(ACh) was measured with a two-electrode voltage clamp technique. In oocytes injected with α7 nAChR copy RNA, quercetin enhanced I(ACh), whereas quercetin glycosides inhibited I(ACh). Quercetin glycosides mediated an inhibition of I(ACh), which increased when they were pre-applied and the inhibitory effects were concentration dependent. The order of I(ACh) inhibition by quercetin glycosides was Rutin≥Rham1>Rham2. Quercetin glycosides-mediated I(ACh) enhancement was not affected by ACh concentration and appeared voltage-independent. Furthermore, quercetin-mediated I(ACh) inhibition can be attenuated when quercetin is co-applied with Rham1 and Rutin, indicating that quercetin glycosides could interfere with quercetin-mediated α7 nAChR regulation and that the number of carbohydrates in the quercetin glycoside plays a key role in the interruption of quercetin action. These results show that quercetin and quercetin glycosides regulate the α7 nAChR in a differential manner.

Depression help-seeking attitudes and behaviors among an Internet-based sample of Spanish-speaking perinatal women / Actitudes y comportamientos de búsqueda de ayuda para la depresión en una muestra basada en internet de mujeres de habla hispana en período perinatal

OBJECTIVE: To examine attitudes and beliefs related to help-seeking for depression among an international sample of pregnant women, a majority of whom were Spanish-speakers residing in Latin America. METHODS: More than 6 000 (n = 6 672) pregnant women met eligibility criteria and consented to participate between 15 January 2009-12 August 2011. Of these, 1 760 with a Latino/Hispanic background completed a baseline survey as part of a larger study. Group comparisons analyzed attitudes and behaviors related to seeking help for depression, while a logistic regression was conducted to identify demographic characteristics related to help-seeking support. RESULTS: Of the participants, three-fourths reported experiencing depression during or after their current or past pregnancies. The majority of participants did not seek help, and generally reported ambivalence about their depressive symptoms and uncertainty as to the helpfulness of others. However, 44.8% did seek help, mostly by speaking to family or partners and reported feeling fear, shame, and embarrassment about their symptoms. A current major depressive episode and an income less than or equal to US$ 10 000 were significant predictors of help-seeking behaviors. CONCLUSIONS: Data from this study suggest that when feeling sad or depressed, perinatal Latinas tend to seek emotional support first from family and friends and may underutilize mental health services when needed. The Internet is an effective means for reaching perinatal women, especially those in areas of the world where there may be barriers to accessing psychological resources.

OBJETIVO: Analizar las actitudes y las creencias relacionadas con la búsqueda de ayuda para la depresión en una muestra internacional de mujeres embarazadas, la mayor parte de ellas hispanohablantes y residentes en América Latina. MÉTODOS: Más de 6 000 mujeres embarazadas (n = 6 672) cumplieron los criterios de selección y aceptaron participar entre el 15 de enero del 2009 y el 12 de agosto del 2011. De estas, 1 760 de origen latino o hispano completaron una encuesta básica que formaba parte de un estudio más amplio. Mediante comparaciones de grupo, se analizaron las actitudes y los comportamientos relacionados con la búsqueda de ayuda para la depresión, mientras que, mediante regresión logística, se determinaron las características demográficas relacionadas con la búsqueda de ayuda o apoyo. RESULTADOS: De todas las participantes, tres cuartas partes notificaron sentimientos de depresión durante o después de los embarazos actuales o pasados. La mayor parte de ellas no buscaron ayuda, y en general manifestaron ambivalencia acerca de sus síntomas depresivos e incertidumbre en cuanto a la capacidad de ayuda de otras personas. Sin embargo, 44,8% buscaron ayuda, principalmente hablando con familiares o compañeros, y notificaron sentimientos de temor, culpabilidad y vergüenza acerca de sus síntomas. Un episodio depresivo mayor actual y unos ingresos iguales o inferiores a US$ 10 000 fueron factores predictivos significativos de comportamientos de búsqueda de ayuda. CONCLUSIONES: Los datos de este estudio indican que, cuando se sienten tristes o deprimidas, las mujeres latinas en período perinatal tienden a buscar en primer lugar el apoyo emocional de la familia y los amigos, y podrían subutilizar los servicios de salud mental cuando son necesarios. La internet es un medio eficaz para llegar a las mujeres en período perinatal, especialmente a las que viven en zonas del mundo donde pueden existir barreras para el acceso a los recursos psicológicos.

Sox9 regulates development of neural crest and otic placode in a time- and dose-dependent fashion / 동물의과학연구지

Neural crest and placodes share a number of important features, pointing to a possible common evolutionary origin. They both arise from the neural plate border, which is the boundary between the non-neural ectoderm and neural plate. The transcription factor Sox9 has been implicated in neural crest and otic placode induction in several species. To investigate the differential regulation of neural crest and otic placode induction by Sox9, a gain of function assay was performed using a hormone-inducible version of the Sox9 construct at different doses and time periods. Sox9 was expressed in both neural crest and otic placode cell populations in the same stage embryos by in situ hybridization. Using a gain of function approach, increased expression of neural crest marker (Snail2) and otic placode marker (Pax8) in Sox9-overexpressed embryos was observed. Higher dose of Sox9 reduced or eliminated both neural crest and placode cells in the embryos. Interestingly, otic placodes cells were more strongly affected as compared to neural crest cells. So, optimal dosage and timing of Sox9 expression are important for the development of the neural crest and otic placode. The development of the neural crest and otic placode are affected by Sox9 in a time- and dose-dependent manner.

Bisphenol A and 4-tert-Octylphenol Inhibit Cx46 Hemichannel Currents

Connexins (Cx) are membrane proteins and monomers for forming gap junction (GJ) channels. Cx46 and Cx50 are also known to function as conductive hemichannels. As part of an ongoing effort to find GJ-specific blocker(s), endocrine disruptors were used to examine their effect on Cx46 hemichannels expressed in Xenopus oocytes. Voltage-dependent gating of Cx46 hemichannels was characterized by slowly activating outward currents and relatively fast inward tail currents. Bisphenol A (BPA, 10 nM) reduced outward currents of Cx46 hemichannels up to ~18% of control, and its effect was reversible (n=5). 4-tert-Octylphenol (OP, 1 microM) reversibly reduced outward hemichannel currents up to ~28% (n=4). However, overall shapes of Cx46 hemichannel current traces (outward and inward currents) were not changed by these drugs. These results suggest that BPA and OP are likely to occupy the pore of Cx46 hemichannels and thus obstruct the ionic fluxes. This finding provides that BPA and OP are potential candidates for GJ channel blockers.

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.

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.

As causas das internações no Hospital Adauto Botelho (Cariacica, ES) na segunda metade do século XX / The motives for hospitalization at Adauto Botelho Hospital (Cariacica, ES) in the second half of the twentieth century

Discutem-se aqui as formas de encaminhamento de pacientes ao Hospital Adauto Botelho, localizado em Cariacica, Espírito Santo. A pesquisa se deu por meio de prontuários médicos datados desde a inauguração em 1954 e de depoimentos de pessoas que trabalharam lá durante a segunda metade do século XX. Foram analisados 102 prontuários e entrevistadas quatro pessoas. A pesquisa dos prontuários mostra forte inserção da Chefatura de Polícia no processo de internação. As falas dos entrevistados reiteram esse ponto, mostrando também a longa duração das internações. São as histórias de vida dos internos que dão o tom deste trabalho. Conclui-se, a partir delas, que o Hospital Adauto Botelho, mais que uma instituição de tratamento, era um espaço de confinamento.

This paper discusses the procedures for referring patients to Adauto Botelho Hospital, in Cariacica, Espírito Santo state, Brazil. The research is based on the medical records since its inauguration in 1954 and statements by people who worked there in the second half of the twentieth century. One hundred and two records were analyzed and four people were interviewed. The records revealed the active involvement of the Chief of Police in hospitalizations. The interviews corroborate this, while also showing the long duration of the hospitalizations. The tone of the paper is set by the life stories of the people hospitalized there. The conclusion is that this hospital served not so much for treatment as for confinement.

A Novel Long-Acting Glucagon-Like Peptide-1 Agonist with Improved Efficacy in Insulin Secretion and beta-Cell Growth

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by cleavage of proglucagon in intestinal L-cells. In the pancreas, GLP-1 stimulates post-prandial insulin secretion, promotes insulin biosynthesis, and improves insulin sensitivity. Because of its insulinotropic activity, GLP-1 has been considered a good candidate drug for treatment of diabetes mellitus. However, clinical use of GLP-1 has been limited by its short half-life, as a result of rapid degradation by dipeptidyl peptidase-IV (DPP-IV). METHODS: We designed a novel GLP-1 analog, Xenopus GLP-1 (xGLP)-E4. The Ala residue in the second position of xGLP was replaced with a Ser residue to increase the half-life in the body. The C-terminal tail of exendin-4 was added to enhance the binding affinity for the GLP-1 receptor (GLP1R). The potency of GLP-1 and its analogs was determined by luciferase assay. The stability of GLP1R agonists was evaluated by determining the activity of agonists that had been preincubated in the presence of fetal bovine serum, which contains innate DPP-IV activity. The effects of xGLP-E4 on insulin secretion and beta-cell growth were investigated using insulin enzyme-linked immunosorbent assay and cell counting. RESULTS: xGLP-E4 exhibited improved stability against DPP-IV activity and increased potency to GLP1R, compared with GLP-1. An increase in glucose-dependent insulin secretion was observed in xGLP-E4-treated pancreatic beta-cells. The effect of xGLP-E4 on beta-cell growth was greater than that of GLP-1. CONCLUSION: We developed a novel GLP-1 analog, xGLP-E4, that shows prolonged longevity and improved efficacy. This analog is a potential candidate for treatment of type 2 diabetes.

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.

Expression and localization of Rdd proteins in Xenopus embryo / 대한해부학회지

The previous study has shown that repeated D domain-like (Rdd) proteins, a group of novel secretory proteins consisting of repeated domains of a cysteine-rich sequence, are involved in the process of blood vessel formation in Xenopus embryo. We performed further experiments to examine the localization of Rdd proteins in embryogenesis. Detection of tagged Rdd proteins expressed in blastomeres showed that Rdd proteins formed a high molecular weight complex and existed in the extracellular space. A rabbit antibody against the Rdd synthetic peptide identified a single band of 28 kD in embryonic tissue extract. By whole-mount immunostaining analysis, signal was detected in the regions of inter-somites, vitelline veins, and branchial arches at the tailbud stage. Staining of Rdd was remarkably reduced in the embryos injected with vascular endothelial growth factor Morpholino. We suggest that Rdd proteins interact with a molecule(s) associated with vascular precursor cells.

Camphor modulates TRPV3 cation channels activity by interacting with critical pore-region cysteine residues

TRPV3 ion channels mediate thermo-transduction, nociception, inflammation and dermatitis in mammals. TRPV1-4 proteins have been shown to have conserved cysteine-residues in the pore-forming regions. These residues participate in channel activation via S-nitrosylation of channel proteins. Camphor is a commonly used ligand for TRPV3 channels. Thus the knowledge about the potential binding/interacting site[s] for camphor will help to design effective and potent analgesic compounds. In an overlap-extension PCR method, following primer-pairs were used to mutate conserved cysteine-residues in the pore-region of TRPV3 channels; GATTGAGAATcCTCCAAGGACAAAAAGGAC, TRPV3-C612S-Fw and GTCCTTGGAGgACTTCTCAATCAGTCAGTGAGG, TRPV3-C612S-Rv primers pair. And for TRPV3-C619S: GGACTCcAGTTCCTATGGCCAGC, TRPV3-C619S-Fw and GCTGGCCATAgGAACTGGAGTCC, TRPV3-C619S-Rv respectively. All cDNA constructs were confirmed by DNA-sequencing and used to make cRNAs. Oocytes expressing mTRPV3-C619S and mTRPV3-C612S mutant channels were challenged with 2-APB [1 mM], camphor [10 mM] and dihydrocarveol [10 mM] either at -40 mV or +40 mV holding potentials in voltage-clamp experiments. Responses of both mutants to 2-APB were similar to wild-type mTRPV3. Interestingly, responses to camphor were totally lost in mTRPV3-C619S mutant, while responses to dihydrocarveol remained intact. In contrast mTRPV3-C612S displayed slightly altered [16 +/- 2% reduction] phenotype with respect to camphor sensitivity. It is concluded that pore-region cysteines play critical role in camphor sensitivity of TRPV3 ion channels

Activation of Lysophosphatidic Acid Receptor Is Coupled to Enhancement of Ca(2+)-Activated Potassium Channel Currents

The calcium-activated K+ (BKCa) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. Ca2+ is the main regulator of BKCa channel activation. The BKCa channel contains two high affinity Ca2+ binding sites, namely, regulators of K+ conductance, RCK1 and the Ca2+ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular Ca2+ levels through diverse G proteins such as Galphaq/11, Galphai, Galpha12/13, and Galphas and the related signal transduction pathway. In the present study, we examined LPA effects on BKCa channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated BKCa channel activation was also attenuated by the PLC inhibitor U-73122, IP3 inhibitor 2-APB, Ca2+ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated BKCa channel activation. The present study indicates that LPA-mediated activation of the BKCa channel is achieved through the PLC, IP3, Ca2+, and PKC pathway and that LPA-mediated activation of the BKCa channel could be one of the biological effects of LPA in the nervous and vascular systems.

Inhibitory Effects of Ginsenoside Metabolites, Compound K and Protopanaxatriol, on GABAC Receptor-Mediated Ion Currents

Ginsenosides, one of the active ingredients of Panax ginseng, show various pharmacological and physiological effects, and they are converted into compound K (CK) or protopanaxatriol (M4) by intestinal microorganisms. CK is a metabolite derived from protopanaxadiol (PD) ginsenosides, whereas M4 is a metabolite derived from protopanaxatriol (PT) ginsenosides. The gamma-aminobutyric acid receptorC (GABAC) is primarily expressed in retinal bipolar cells and several regions of the brain. However, little is known of the effects of ginsenoside metabolites on GABAC receptor channel activity. In the present study, we examined the effects of CK and M4 on the activity of human recombinant GABAC receptor (rho1) channels expressed in Xenopus oocytes by using a 2-electrode voltage clamp technique. In oocytes expressing GABAC receptor cRNA, we found that CK or M4 alone had no effect in oocytes. However, co-application of either CK or M4 with GABA inhibited the GABA-induced inward peak current (IGABA). Interestingly, pre-application of M4 inhibited IGABA more potently than CK in a dose-dependent and reversible manner. The half-inhibitory concentration (IC50) values of CK and M4 were 52.1+/-2.3 and 45.7+/-3.9 microM, respectively. Inhibition of IGABA by CK and M4 was voltage-independent and non-competitive. This study implies that ginsenoside metabolites may regulate GABAC receptor channel activity in the brain, including in the eyes.

Resveratrol Inhibits GABAC rho Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

Resveratrol is a phytoalexin found in grapes, red wine, and berries. Resveratrol has been known to have many beneficial health effects, such as anti-cancer, neuroprotective, anti-inflammatory, and life-prolonging effects. However, relatively little is known about the effects of resveratrol on the regulation of ligand-gated ion channels. We have previously reported that resveratrol regulates subsets of homomeric ligand-gated ion channels such as those of 5-HT3A receptors. The gamma-aminobutyric acidC (GABAC) receptor is mainly expressed in retinal bipolar cells and plays an important role in visual processing. In the present study, we examined the effects of resveratrol on the channel activity of homomeric GABAC receptor expressed in Xenopus oocytes injected with cRNA encoding human GABAC rho subunits. Our data show that the application of GABA elicits an inward peak current (IGABA) in oocytes that express the GABAC receptor. Resveratrol treatment had no effect on oocytes injected with H2O or with GABAC receptor cRNA. Co-treatment with resveratrol and GABA inhibited IGABA in oocytes with GABAC receptors. The inhibition of IGABA by resveratrol was in a reversible and concentration-dependent manner. The IC50 of resveratrol was 28.9+/-2.8 microM in oocytes expressing GABAC receptor. The inhibition of IGABA by resveratrol was in voltage-independent and non-competitive manner. These results indicate that resveratrol might regulate GABAC receptor expression and that this regulation might be one of the pharmacological actions of resveratrol on the nervous system.

Cloning and bioinformatic analysis of TAGLN2 cDNA of Bufo japonicus formosus / 药学学报

To study the bioactive polypeptides included in Bufo skin and its secretions the plasmid skin cDNA library of adult Japanese toad Bufo japonicus formosus was prepared. The pSD64TR has been used as the vector and the cloning sites are Xho I and EcoR I. To screen cDNAs encoding bioactive components, the plasmid cDNA library was transformed into E. coli DH5 competent cells, and positive colonies were screened by colony PCR (polymerase chain reaction). The suspension of a single colony in LB medium was used as the template, SP6 (the upstream primer of the plasmid cDNA library) and a primer with Xho I site and polyT were used as the primers. As the result, 465 positive colonies out of 1 344 were obtained and their plasmid were collected and sequenced. By homologous analysis, it was found that one of the cDNAs encoding a peptide with high homolog with transgelin-2, which was registered in GenBank (accession number: JX197456), and it was indicated as a partial cDNA sequence with a deletion at the 5' end. The transcript is 997 bp consisting of 31 bp 5', 618 bp 3' untranslated region (UTR) and an open reading frame (ORF) of 348 bp encoding a polypeptide of 115 amino acids. In the putative protein product, there is a calponin homology domain, two cysteine residues for a disulfide bond and three a-helix domains, and five potential phosphorylation sites. The homologous analysis indicates 90% similarity with Xenopus (Silurana) tropicalis and 89% with Xenopus laevis, and 71%-85% with other species.

Effects of clonidine on the activity of the rat glutamate transporter EAAT3 expressed in Xenopus oocytes / 대한마취과학회지

BACKGROUND: Clonidine has been shown to be a potent neuroprotectant by acting at alpha2 receptors on glutamatergic neurons to inhibit the release of glutamate. The aim of this study is to investigate the effects of clonidine on the activity of EAAT3 that can regulate extracellular glutamate. METHODS: EAAT3 was expressed in the Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 microM L-glutamate both in the presence and absence of various concentrations of clonidine. To determine the effects of clonidine on the Km and Vmax of EAAT3 and the reversibility of clonidine effects, membrane currents were recorded after the application of various concentrations of L-glutamate both in the presence and absence of 1.50 x 10(-7) M clonidine. RESULTS: Clonidine reduced the EAAT3 responses to L-glutamate in a concentration-dependent manner. This inhibition was statistically significant at higher concentrations than at the clinically relevant range. Clonidine at 1.50 x 10(-7) M reduced the Vmax, but did not affect the Km of EAAT3 for L-glutamate. CONCLUSIONS: These results suggest that the direct inhibition of EAAT3 activity is not related to the sedation effect of clonidine and that the clonidine-induced reduction of EAAT3 activity provides additional data for the possible involvement of glutamatergic hyperactivity in the proconvulsant effect of clonidine.

Schistosoma mansoni polo-like kinases and their function in control of mitosis and parasite reproduction

Polo-like kinases are important regulators of cell cycle progression and mitosis. They constitute a family of conserved serine/threonine kinases which are highly related in their catalytic domains and contain polo boxes involved in protein-protein interactions and subcellular localization. In mammals, five Plks (Plk 1-5) encompass diverse roles in centrosome dynamics, spindle formation, intra S-phase and G2/M checkpoints and DNA damage response. Plk1 is a key positive regulator of mitosis and is overexpressed in various types of cancers. Plk4 is a divergent member of the Plk family, with essential functions in centriole duplication. Homozygous disruption of Plk1 or Plk4 in mice is lethal in embryos. Two Plk members SmPlk1 and SmSak, homologous to Plk1 and Plk4 respectively, are present in the parasitic platyhelminth Schistosoma mansoni. Structural and functional analyses of SmPlk1 have demonstrated its conserved function in the regulation of cell cycle G2/M transition in Xenopus oocytes. The anti-cancer drug BI 2536 (the most potent and selective Plk1 inhibitor) inhibits specifically the catalytic activity of SmPlk1 and induced profound alterations in schistosome gonads, indicating a role of SmPlk1 in parasite gametogenesis and its potential as a novel chemotherapeutic target against schistosomiasis. Functions of SmSak in cell cycle regulation and schistosome gonad development are currently investigated.

Quinases do tipo Polo ("polo-like") são importantes reguladores da progressão do ciclo celular e da mitose. Elas constituem uma família de serina/treonina quinases que são altamente relacionadas entre si no seu domínio catalítico e contêm blocos "polo" envolvidos com interações proteína-proteína e com localização subcelular. Em mamíferos, cinco Plks (Plk 1-5) englobam diversos papéis na dinâmica do centrossomo, formação do fuso, "checkpoints" dentro da fase S e da transição G2/M, e na resposta aos danos do DNA. Plk1 é um regulador positivo chave da mitose, e é superexpresso em vários tipos de câncer. Plk4 é um membro divergente da família Plk, com funções essenciais na duplicação do centríolo. Deleção homozigótica de Plk1 ou Plk4 em camundongos é letal em embriões. Dois membros da família Plk, SmPlk1 e SmSak, homólogos a Plk1 e Plk4, respectivamente, estão presentes no parasita platelmíntico Schistosoma mansoni. Análises estruturais e funcionais de SmPlk1 demonstraram uma função conservada na regulação da transição G2/M do ciclo celular em ovócitos de Xenopus. A droga anticâncer BI2536 (o inibidor mais potente e seletivo de Plk1) inibe específicamente a atividade catalítica de SmPlk1 e induz alterações profundas nas gonadas de esquistossomos, indicando um papel de SmPlk1 na gametogênese do parasita e seu potencial como um alvo terapêutico novo contra a esquistossomose. As funções de SmSak na regulação do ciclo celular e no desenvolvimento das gônadas de esquistossomos estão sendo investigadas no momento.

Effect of propofol on oxidative stress-attenuated glutamate transporter EAAT4 activity

BACKGROUND: Propofol (2, 6-diisopropylphenol) has been known to have neuroprotective effects. Excitatory amino acid transporter 4 (EAAT4) is a glutamate transporter predominantly expressed in the cerebellar Purkinje cells, which is vulnerable to ischemic injury. Thus, we hypothesized that propofol reverses reduced EAAT4 activity which was induced by oxidative stress and investigated the effects of propofol on EAAT4 under oxidative stress induced by tert-butyl hydroperoside (t-BHP). METHODS: EAAT4 was expressed in Xenopus oocytes by injection of its mRNA. By using two-electrode voltage clamping, membrane currents were recorded before, during, and after application of L-aspartate (3 microM) in the presence or absence of t-BHP and propofol. RESULTS: L-aspartate induced an inward current in EAAT4 expressing oocytes. Exposure of these oocytes to t-BHP (1-20 mM) for 10 min dose-dependently decreased EAAT4 activity (1 +/- 0.01 microC for control; 0.88 +/- 0.05 microC for 1 mM; 0.83 +/- 0.03 microC for 2mM; 0.65 +/- 0.04 microC for 3 mM; 0.51 +/- 0.07 microC for 5 mM; 0.45 +/- 0.03 f microC for 10 mM and 0.24 +/- 0.06 microC for 20 mM). IC50 for t-BTH was 6.05 mM and further study was performed with 10 mM t-BTH. Propofol (3-10 microM) dose-dependently reversed this t-BHP-attenuated EAAT4 activity. CONCLUSIONS: Oxidative stress by t-BHP decreased EAAT4 activity and 3-10 microM propofol restored oxidative stress-reduced EAAT4 activity.

xCITED2 Induces Neural Genes in Animal Cap Explants of Xenopus Embryos

Neural tissue is arisen from presumptive ectoderm via inhibition of bone morphogenetic protein (BMP) signaling during Xenopus early development. Previous studies demonstrate that ectopic expression of dominant negative BMP4 receptor (DNBR) produces neural tissue in animal cap explants (AC) and also increases the expression level of various genes involved in neurogenesis. To investigate detail mechanism of neurogenesis in transcriptional level, we analyzed RNAs increased by DNBR using total RNA sequencing analysis and identified several candidate genes. Among them, xCITED2 (Xenopus CBP/p300-interacting transcription activator) was induced 4.6 fold by DNBR and preferentially expressed in neural tissues at tadpole stage. Ectopic expression of xCITED2 induced anterior neural genes without mesoderm induction and reduced BMP downstream genes, an eye specific marker and posterior neural marker. Taken together, these results suggest that xCITED2 may have a role in the differentiation of anterior neural tissue during Xenopus early development.