Our previous results showed that FGF signaling, which is important for the mesoderm and neuroectoderm induction, should be blocked for the endoderm formation in Xenopus. Here, Xenopus embryos were collected according to the two time points of MBT or stage 10.5. FGF signal was blocked with SU5402, chemical inhibitor of FGF signal, in the stage-specific embryos, to understand the role of FGF signal during the endoderm formation in the stage-specific embryos. Embryos subjected with the blocking of FGF signal before stage 10.5 showed the expanded abdominal volume in which endodermal mass was increased about 2 times but abdominal organs were not found. The tissue recombinant experiment showed that mesodermal tissue was necessary for the differentiation of endoderm. Embryos subjected with the blocking of FGF signal after stage 10.5 showed that abdomen was not expanded, the neural tube was opened instead. Our data indicate that blocking of FGF signal before stage 10.5 may be necessary for the endoderm induction and signals from neighboring endoderm tissue and mesoderm are required for the endoderm differentiation.
Abdomen ; Embryonic Development* ; Embryonic Structures ; Endoderm* ; Female ; Mesoderm ; Neural Plate ; Neural Tube ; Pregnancy ; Xenopus laevis* ; Xenopus*

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

The effects of dimethyl sulfoxide (DMSO) were studied in two groups of Xenopus oocytes, one expressing ATP sensitive K+ (KATP) channel comprised of sulfonylurea receptor SUR1 and inwardly rectifying K+ channel subunit Kir6.2, and the other expressing renal KATP channel ROMK2. At concentrations of 0.3~10% (vol/vol) DMSO inhibited whole cell Kir6.2/SUR1 currents elicited by bath application of sodium azide (3 mM) in a concentration-dependent manner. The inhibition constant and Hill coefficient were 2.93% and 1.62, respectively. ROMK2 currents, however, was not affected significantly by DMSO. The results support the idea that DMSO inhibits KATP channel expressed in Xenopus oocyte through a protein-specific mechanism(s) that remains to be further elucidated.
Adenosine Triphosphate ; Baths ; Dimethyl Sulfoxide* ; Oocytes* ; Sodium Azide ; Xenopus*

The pancreas is a mixed exocrine and endocrine gland involved in the control of many homeostatic functions.During embryogenesis,the pancreas arises from dorsal and ventral evaginations of the foregut,which subse- quently fuse into a single organ.The characterization of early genes expressed in the developing pancreas is critical to understand its specification and differentiation.Pdx1 is one of the earliest markers of pancreatic development and a key molecule in its development.Sox proteins form a large class of transcriptional regulators implicated in the control of a variety of developmental processes.One member of this family,Sox9,is expressed in the developing pancreas, but little is known about the function of Sox9 in the developing pancreas.We further investigated Sox9 function during pancreatic development in Xenopus .Using a hormone-inducible inhibitory mutant of Sox9 ,we found that Pdx1 expres- sion was reduced in the ventral pancreatic buds in Sox9-depleted embryos.We suggest that Sox9 gene expression may be involved in pancreatic development in Xenopus.
Animals ; Endocrine Glands ; Gene Expression ; Hoof and Claw* ; Pancreas ; Xenopus

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.
Anesthesia, General ; Anesthetics ; Animals ; Ginsenosides ; Incidence ; Isoflurane ; Mice ; Nausea ; Negotiating ; Oocytes ; Postoperative Nausea and Vomiting ; Serotonin ; Vomiting ; Xenopus ; Xenopus laevis

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.
Animals ; Cell Membrane ; Ectoderm ; Embryonic Development ; Epithelium ; Female ; Gastrulation ; Gills ; Humans ; In Situ Hybridization ; Ion Pumps ; Larva ; Pregnancy ; Protein Isoforms ; Retina* ; Retinal Rod Photoreceptor Cells ; Retinaldehyde ; Xenopus laevis ; Xenopus*

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.
Animals ; Calcium ; Dental Papilla ; Enamel Organ ; Extracellular Matrix ; Humans ; In Situ Hybridization ; Mice ; Odontoblasts ; Odontogenesis ; Osteocalcin* ; Osteogenesis ; Rats ; RNA, Messenger ; Tooth Germ ; Tooth* ; Xenopus ; Xenopus laevis*

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

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.
Acetylcholine* ; Carbohydrates ; Flavonoids ; Fruit ; Glycosides* ; Humans* ; Oocytes ; Quercetin* ; Receptors, Nicotinic ; RNA ; Rutin ; Vegetables ; Xenopus

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.
Ectoderm ; Embryonic Structures ; In Situ Hybridization ; Neural Crest* ; Neural Plate ; Transcription Factors ; Xenopus