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

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

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

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

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.
Amino Acid Sequence ; Animals ; Base Sequence ; Bufonidae ; genetics ; metabolism ; Cloning, Molecular ; Gene Library ; Microfilament Proteins ; chemistry ; genetics ; metabolism ; Muscle Proteins ; chemistry ; genetics ; metabolism ; Open Reading Frames ; Phosphorylation ; Phylogeny ; Plasmids ; genetics ; Sequence Homology, Amino Acid ; Skin ; metabolism ; Xenopus ; genetics

The heterodimeric c-Jun/c-Fos, an activator protein-1 (AP-1) has been implicated in mesoderm induction (Dong et al., 1996; Kim et al., 1998) whereas the homodimer of c-Jun was reported to be involved in neural inhibition during the early development of Xenopus embryos. During the early vertebrate development AP-1 involvement in the neural induction is still not clearly understood. We report here that AP-1 has a role in Zic3 expression, a critical proneural gene and a primary regulator of neural and neural crest development (Nakata et al., 1997; Nakata et al., 1998). AP-1 was able to induce the Zic3 gene in a dose dependent manner but other homo- or hetero-dimeric proteins, such as c-Jun/c-Jun, JunD/FosB or JunD/Fra-1 were not. The inhibition of AP-1 activity using morpholino antisenses of c-jun mRNAs blocked the Zic3 expression induced by activin. In addition, co-injection of c-jun mRNA rescued the down-regulated Zic3 expression. The promoter region of isolated Zic3 genomic DNA was found to possess several consensus-binding site of AP-1. Thus, in the functional assays, AP-1 could increase promoter activity of Zic3 gene. These findings suggest that proneural gene, Zic3 may be regulated by heterodimeric AP-1(c-Jun/c-Fos) and it may have a role in activin signaling for the regulation of neural specific gene, Zic3.
Activins/pharmacology/physiology ; Animals ; Base Sequence ; Binding Sites/genetics ; Consensus Sequence/genetics ; Dimerization ; Embryo, Nonmammalian/metabolism ; *Gene Expression Regulation, Developmental ; Homeodomain Proteins/*genetics ; Molecular Sequence Data ; Promoter Regions (Genetics)/genetics ; Proto-Oncogene Proteins c-fos/genetics/physiology ; Proto-Oncogene Proteins c-jun/genetics/physiology ; RNA, Antisense/genetics ; Research Support, Non-U.S. Gov't ; Transcription Factor AP-1/genetics/*physiology ; Transcription Factors/*genetics ; *Transcription, Genetic ; Up-Regulation ; Xenopus Proteins/*genetics ; Xenopus laevis/*embryology/*genetics

Spliceosomal RNAs are a family of small nuclear RNAs (snRNAs) that are essential for pre-mRNA splicing. All vertebrate spliceosomal snRNAs are extensively pseudouridylated after transcription. Pseudouridines in spliceosomal snRNAs are generally clustered in regions that are functionally important during splicing. Many of these modified nucleotides are conserved across species lines. Recent studies have demonstrated that spliceosomal snRNA pseudouridylation is catalyzed by two different mechanisms: an RNA-dependent mechanism and an RNA-independent mechanism. The functions of the pseudouridines in spliceosomal snRNAs (U2 snRNA in particular) have also been extensively studied. Experimental data indicate that virtually all pseudouridines in U2 snRNA are functionally important. Besides the currently known pseudouridines (constitutive modifications), recent work has also indicated that pseudouridylation can be induced at novel positions under stress conditions, thus strongly suggesting that pseudouridylation is also a regulatory modification.
Animals ; Base Sequence ; Molecular Sequence Data ; Nucleic Acid Conformation ; Nucleotides ; metabolism ; Oocytes ; cytology ; metabolism ; Pseudouridine ; metabolism ; RNA Precursors ; metabolism ; RNA Splice Sites ; RNA Splicing ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Nuclear ; genetics ; metabolism ; Ribonucleoproteins, Small Nuclear ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Spliceosomes ; genetics ; metabolism ; Uridine ; analogs & derivatives ; metabolism ; Xenopus ; genetics ; metabolism

We have previously shown that the inhibition of fibroblast growth factor (FGF) signaling induced endodermal gene expression in the animal cap and caused the expansion of the endodermal mass in Xenopus embryos. However, we still do not know whether or not the alteration of FGF signaling controls embryonic cell fate, or when FGF signal blocking is required for endoderm formation in Xenopus. Here, we show that FGF signal blocking in embryonic cells causes their descendants to move into the endodermal region and to express endodermal genes. It is also interesting that blocking FGF signaling between fertilization and embryonic stage 10.5 promotes endoderm formation, but persistent FGF signaling blocking after stage 10.5 restricts endoderm formation and differentiation.
Animals ; Endoderm/drug effects/embryology/*metabolism ; Fibroblast Growth Factors/antagonists & inhibitors/genetics/*physiology ; Gene Expression Regulation, Developmental/drug effects ; In Situ Hybridization ; Pyrroles/administration & dosage/pharmacology ; Receptors, Fibroblast Growth Factor/genetics/physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction/drug effects ; Xenopus Proteins/antagonists & inhibitors/genetics/*physiology ; Xenopus laevis/embryology/genetics/*physiology

Animals ; Animals, Newborn ; Electrophysiology ; Female ; Membrane Potentials ; physiology ; NAV1.3 Voltage-Gated Sodium Channel ; Nerve Tissue Proteins ; genetics ; physiology ; Oocytes ; metabolism ; physiology ; Protein Subunits ; genetics ; physiology ; Rats ; Rats, Sprague-Dawley ; Sodium Channels ; genetics ; physiology ; Xenopus

OBJECTIVETo investigate the molecular mechanism of human ether-a-go-go-related gene (HERG) potassium channels regulated by protein kinase A (PKA) in a human cell line.

METHODSHERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody.

RESULTSElevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated.

CONCLUSIONSPKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.

1-Methyl-3-isobutylxanthine ; pharmacology ; 8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; Adenylyl Cyclases ; metabolism ; Animals ; Anti-Arrhythmia Agents ; pharmacology ; Cation Transport Proteins ; Cell Line ; Colforsin ; pharmacology ; Cyclic AMP ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; DNA-Binding Proteins ; ERG1 Potassium Channel ; Enzyme Activation ; drug effects ; Ether-A-Go-Go Potassium Channels ; Female ; Humans ; Membrane Potentials ; drug effects ; Microinjections ; Oocytes ; Patch-Clamp Techniques ; Phenethylamines ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; Phosphoric Diester Hydrolases ; drug effects ; metabolism ; Phosphorylation ; Potassium Channels ; genetics ; metabolism ; physiology ; Potassium Channels, Voltage-Gated ; RNA, Complementary ; administration & dosage ; genetics ; Sulfonamides ; pharmacology ; Trans-Activators ; Transcriptional Regulator ERG ; Xenopus laevis