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.
Connexins ; Endocrine Disruptors ; Gap Junctions ; Membrane Proteins ; Oocytes ; Xenopus

Carrier Proteins ; Humans ; Nutrition Surveys ; Nutritional Status ; Population Surveillance ; Xenopus Proteins

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.
Animals ; Escherichia coli ; metabolism ; Genetic Vectors ; Magainins ; biosynthesis ; genetics ; Peptides ; genetics ; metabolism ; Pichia ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Xenopus Proteins ; biosynthesis ; genetics ; Xenopus laevis

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.
Animals ; Bone Morphogenetic Proteins ; Ectoderm ; Embryonic Structures ; Eye ; Larva ; Mesoderm ; Neurogenesis ; RNA ; Sequence Analysis, RNA ; Xenopus

BMP-4 signaling is mediated through Smad proteins which may translocate to the nucleus to activate transcription. Little is known about how BMP-4 signaling regulates the transcription of its target genes, e.g., Xvent genes. Therefore, we isolated the genomic clone of a BMP-4 responsive homeobox gene, Xbr-1a/Xvent-2. This clone contains a promoter and three exons for the entire coding region. Using the primer extension, we identified the transcription initiation site corresponding to position -64 bp upstream to the ATG codon of the Xvent-2 gene. The promoter was linked to the luciferase reporter gene, and promoter activity determined by luciferase assay. The temporal promoter activity peaked between embryonic stages 13~17, in agreement with its temporal mRNA expression in the whole embryo. Through the serial deletion mutation, the upstream -235 bp of the promoter retains the full transcriptional activity, and is regulated by BMP-4 signaling. The present results suggest that the BMP-4 responsive element is located on the upstream 235 bp of the promoter.
Clinical Coding ; Clone Cells ; Codon ; Embryonic Development* ; Embryonic Structures ; Exons ; Female ; Genes, Homeobox ; Genes, Reporter ; Luciferases ; Pregnancy ; RNA, Messenger ; Sequence Deletion ; Smad Proteins ; Transcription Initiation Site ; Xenopus laevis ; Xenopus*

AIMTo determine the feasibility of establishing the heterologous expression model of human- serotonin transporter(hSERT or 5-HTT).

METHODScRNA of SERT was transcribed from cDNA, which was cloned in the pOTV vector. Each oocyte of mature xenopus laevis was injected with transcribed cRNA in vivo and incubated at room temperature for 4-9 days. Recording the current induced by 5-HT with voltage clamp technique tested the function of the expressed 5-HT transporter.

RESULTSThe transporter current could be observed in Ringer's solution containing 5-HT, and the 5-HT induced current were concentration-dependent. Norepinephrine and dopamine could not induce the transporter current while the 5-HT induced current could be specifically inhibited by 5-HTT blocker, desipramine.

CONCLUSIONThe results demonstrate that the heterologous expression product in xenopus laevis oocytes is human 5-HT transporter.

Animals ; Carrier Proteins ; genetics ; DNA, Complementary ; genetics ; Female ; Gene Expression ; Models, Animal ; Oocytes ; metabolism ; RNA, Messenger ; genetics ; Serotonin ; metabolism ; Serotonin Plasma Membrane Transport Proteins ; biosynthesis ; genetics ; Xenopus laevis

Wnt signaling is known to be important for diverse embryonic and post-natal cellular events and be regulated by the proteins Dishevelled and Axin. Although Dishevelled is activated by Wnt and involved in signal transduction, it is not clear how Dishevelled-mediated signaling is turned off. We report that guanine nucleotide binding protein beta 2 (Gnb2; Gbeta2) bound to Axin and Gbeta2 inhibited Wnt mediated reporter activity. The inhibition involved reduction of the level of Dishevelled, and the Gbeta2gamma2 mediated reduction of Dishevelled was countered by increased expression of Axin. Consistent with these effects in HEK293T cells, injection of Gbeta2gamma2 into Xenopus embryos inhibited the formation of secondary axes induced either by XWnt8 or Dishevelled, but not by beta-catenin. The DEP domain of Dishevelled is necessary for both interaction with Gbeta2gamma2 and subsequent degradation of Dishevelled via the lysosomal pathway. Signaling induced by Gbeta2gamma2 is required because a mutant of Gbeta2, Gbeta2 (W332A) with lower signaling activity, had reduced ability to downregulate the level of Dishevelled. Activation of Wnt signaling by either of two methods, increased Frizzled signaling or transient transfection of Wnt, also led to increased degradation of Dishevelled and the induced Dishevelled loss is dependent on Gbeta1 and Gbeta2. Other studies with agents that interfere with PLC action and calcium signaling suggested that loss of Dishevelled is mediated through the following pathway: Wnt/Frizzled-->Gbetagamma-->PLC-->Ca+2/PKC signaling. Together the evidence suggests a novel negative feedback mechanism in which Gbeta2gamma2 inhibits Wnt signaling by degradation of Dishevelled.
Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Animals ; Blastomeres/cytology/*metabolism ; Cell Line ; Embryonic Development/genetics ; *Feedback, Physiological ; Frizzled Receptors/genetics/metabolism ; GTP-Binding Proteins/genetics/*metabolism ; Gene Expression Regulation, Developmental ; Humans ; Mutation ; Phosphoproteins/genetics/*metabolism ; Protein Binding ; RNA, Small Interfering/genetics ; Repressor Proteins/genetics/metabolism ; Transfection ; Wnt Proteins/*genetics/metabolism ; Xenopus ; Xenopus Proteins/*genetics/metabolism

Neurogenesis is the process that develops neuroectoderm from ectoderm. Bone morphogenetic protein (BMP) inhibition in ectodermal cells is necessary and sufficient for neurogenesis in Xenopus embryos. To isolate genes involved in early neurogenesis, Xenous Affymetrix gene chips representing 14,400 genes were analyzed in early stage of neuroectodermal cells that were produced by inhibition of BMP signaling with overexpression of a dominant-negative receptor. We identified 265 candidate genes including 107 ESTs which were newly expressed during the early neurogenesis by blocking BMP signaling. The candidates of 10 ESTs were selected and examined for upregulation in neuroectoderm. Five EST genes were confirmed to be upregulated in neuroectoderm and examined for time-dependent expression patterns in intact embryos. Two EST genes were cloned and identified as a homology of CYP26c (Xl.1946.1.A1_at) and Kielin containing VWC domain (Xl.15853.1.A1_at). One of them, CYP26c, was further characterized for its transcriptional regulation and role of anterior-posterior patterning during neurogenesis. Taken together, we analyzed and characterized genes expressed in early neurogenesis. The results suggest that neurogenesis by inhibition of BMP provides useful system to isolate genes involved in early events of neurogenesis during early vertebrate embryogenesis.
Bone Morphogenetic Proteins ; Clone Cells ; DNA, Complementary ; Ectoderm ; Embryonic Development ; Embryonic Structures ; Expressed Sequence Tags ; Female ; Neural Plate ; Neurogenesis ; Oligonucleotide Array Sequence Analysis ; Pregnancy ; Up-Regulation ; Vertebrates ; Xenopus

Aberrant chromosomal fusion of the Ewing's sarcoma oncogene (EWS) to several different cellular partners produces the Ewing's family of oncoproteins (EWS-fusion-proteins, EFPs) and associated tumors (EFTs). EFPs are potent transcriptional activators, dependent on the N-terminal region of EWS (the EWS-activation-domain, EAD) and this function is thought to be central to EFT oncogenesis and maintenance. Thus EFPs are promising therapeutic targets, but detailed molecular studies will be pivotal for exploring this potential. Such studies have so far largely been restricted to intact mammalian cells while recent evidence has indicated that a mammalian cell-free transcription system may not support bona fide EAD function. Therefore, the lack of manipulatable assays for the EAD presents a significant barrier to progress. Using Xenopus laevis oocytes we describe a plasmid-based micro-injection assay that supports efficient, bona fide EAD transcriptional activity and hence provides a new vehicle for molecular dissection of the EAD.
Animals ; Biological Assay ; Female ; Oncogene Proteins ; genetics ; Oncogenes ; genetics ; Oocytes ; metabolism ; pathology ; RNA-Binding Protein EWS ; genetics ; metabolism ; Sarcoma, Ewing ; genetics ; pathology ; Xenopus

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.
Binding Sites ; Egtazic Acid ; Estrenes ; GTP-Binding Proteins ; Ion Channels ; Isoxazoles ; Lysophospholipids ; Naphthalenes ; Oocytes ; Potassium ; Potassium Channels ; Propionates ; Pyrrolidinones ; Receptors, Lysophosphatidic Acid ; Signal Transduction ; Xenopus