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*

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

Animals ; Oocytes ; metabolism ; physiology ; Receptors, G-Protein-Coupled ; physiology ; Receptors, Purinergic P2X ; physiology ; Xenopus laevis

Homeodomain transcription factors functioning downstream of BMP ventral pathway have been reported to share similar domain of roles in mesoderm patterning along the dorsal-ventral axis. To elucidate the differential role of PV.1 in the aspect of relationship between dorsal and ventral region, we tried to screen PV.1- interacting proteins. Twenty-four PV.1-interacting proteins were identified by yeast two-hybrid screening. Xvent-2 and Xclaudin-6 among these, went under domain study. The C-terminus of PV.1, more specifically 197-241 region was found to interact with Xclaudin-6. Meanwhile Xvent-2 has mild affinity to overall C-terminal region of PV.1. At the same time it was found that Xvent-2 homodimerizes and also binds to Xclaudin-6.
Axis, Cervical Vertebra ; Mass Screening* ; Mesoderm ; Transcription Factors ; Xenopus laevis ; Yeasts

The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 Å resolution cryo-electron microscopic (cryo-EM) structure of the outer rings containing nuclear ring (NR) and cytoplasmic ring (CR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 Å. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the outer rings, including the Y complexes and flanking components. In this most comprehensive and accurate model of outer rings to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. In addition to two copies of Y complexes, each asymmetric subunit in CR contains five copies of Nup358, two copies of the Nup214 complex, two copies of Nup205 and one copy of newly identified Nup93, while that in NR contains one copy of Nup205, one copy of ELYS and one copy of Nup93. These in-depth structural features represent a great advance in understanding the assembly of NPCs.
Animals ; Artificial Intelligence ; Cryoelectron Microscopy ; Nuclear Pore/ultrastructure* ; Oocytes/metabolism* ; 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.
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 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*

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

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.
Anesthetics ; Electrodes ; Electrophysiology ; Halothane ; Ligand-Gated Ion Channels ; Oocytes ; Patch-Clamp Techniques ; Receptors, Serotonin, 5-HT3 ; Serotonin ; Xenopus laevis

AIMTo explore a method of the stable and persistent expression of HERG(human ether-a-go-go-related gene) channels in Xenopus oocytes, and investigate the alteration of rest membrane potential of oocytes and electrophysiological properties of expressed channel in different culture duration.

METHODSHERG mRNA for injection was prepared with in intro transcription using vector plasmid pSP64 containing HERG cDNA fragment. Expressed HERG current was recorded using standard two-microelectrode voltage-clamp technique.

RESULTS(1) Functional channels, with electrophysiological properties consistent with those of HERG channels were persistently expressed in oocytes membrane with this method. Furthermore, channel current could be recorded stably in 10-15 days. (2) The negative value of rest membrane potential increased gradually in the 3, 6, and 9 days of culture, and then decreased in the 12 days. The potential of peak value of inward rectification shifted gradually to the positive direction in 3, 6 and 9 days, and recovered in 12 days. Half-maximal activation potential (V1/2) of heterological expressed current shifted gradually to the negative direction in 3, 6 and 9 days of culture and then recovered in 12 days, the tendency of change was coincident with that of membrane rest potential.

CONCLUSIONThe investigation provides a method of persistent expression of HERG channel in Xenopus oocytes and offers evidences for the difference of electrophysiological experimental data of studies of molecular site and drugs effect of HERG channel in different experimental conditions.

Animals ; Ether-A-Go-Go Potassium Channels ; genetics ; metabolism ; Humans ; Membrane Potentials ; Oocytes ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Xenopus laevis