• YAKHAK HOEJI
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• v.43 no.6
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• pp.743-750
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• 1999

Human and bovine dopamine transporters (DAT) demonstrate discrete functional differences in the dopamine (DA) transport and cocaine binding. The functional analyses on the chimeras of human and bovine DAT have revealed that the region from the $133^{rd}{\;}to{\;}186^{th}$ residue(encompassing the $3^{rd}$ trans-membrane domain (TM) is responsible for the substrate transport and cocaine binding. The present studies have been done to find out the specific amino acid(s) which is essential for the binding of cocaine to DAT by interchanging the amino acids in that region between human and bovine DAT. When isoleucine, the $152^{nd}$ residue of chimera B3 (bovine DAT sequence) was transformed back to valine, the human DAT residue at the identical position, the cocaine binding was remarkably recovered to 98% of the human DAT values. In addition, the cocaine binding of the human DAT was decreased by 57% by substituting isoleucine for valine at position 152. When isoleucine at position 152 of the chimera B3 was converted to the other amino acids to provide an possible molecular basis for the functional role of the $152^{nd}$ residue, only the conversion to alanine among acids tested significantly the cocaine by 34%, but these effect were not as much as those by the conversion to valine. In conclusion, valine at position 152 is a crucial amino acid for the interaction of cocaine to the DAT.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2006.11a
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• pp.43-58
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• 2006

As a bioreactor, bird has proved to be most efficient system for producing useful therapeutic proteins. More than half of the egg white protein content derives from the ovalbumin gene with four other proteins(lysozyme, ovomucoid, ovomucin and conalbumin) present at levels of 50 milligrams or greater. And the naturally sterile egg also contains egg white protein at high concentration allowing for a long shelf life of recombinant protein without loss in activity. In spite of these advantages, transgenic procedures for the bird have lagged far behind because of its complex process of fertilized egg and developmental differences. Recently, a system to transplant mouse testis cells from a fertile donor male to the seminiferous tubules of an infertile recipient male has been developed. Spermatogenesis is generated from transplanted cells, and recipients are capable of transmitting the donor haplotype to progeny. After transplantation, primitive donor spermatogonia migrate to the basement membrane of recipient seminiferous tubules and begin proliferating. Eventually, these cells establish stable colonies with a characteristic appearance, which expands and produces differentiating germ cells, including mature spermatozoa. Thus, the transplanted cells self-renew and produce progeny that differentiate into fully functional spermatozoa. In this study, to develop an alternative system of germline chimera production that operates via the testes rather than through developing embryos, the spermatogonial stem cell techniques were applied. This system consisted of isolation and in vitro-culture of chicken testicular cells, transfer of in vitro-maintained cells into heterologous testes, production of germline chimeras and confirmation of germline transmission for evaluating production of heterologous, functional spermatozoa.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.1031-1037
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• 2004

The interaction of chemoattractant receptors and $G{\alpha}_{16}$ was studied to provide the molecular basis to elucidate the interaction of chemoattractant receptors with $G{\alpha}_{16}$ subunit, thereby possibly contributing to finding novel targets for designing new type of G protein antagonists with anti-inflammatory effects. Experiments were performed to characterize the $G{\alpha}_{16}$ subunit domains responsible for efficient coupling to chemoattractant receptors. Thus, a series of chimeric $G{\alpha}_{11}G{\alpha}_{16}$ and $G{\alpha}_{16}G{\alpha}_{11}$ cDNA constructs were expressed, and the ability of chimeric proteins to mediate C5a, IL-8, and fMLP-induced release of inositol phosphate in transfected Cos-7 cells was tested. The results showed that short stretches of residues 154 to residue 167 and from residue 174 to residue 195 of $G{\alpha}_{16}$ contribute to efficient coupling to the C5a receptor. On the other hand, a stretch of amino acid residues 220-240 of $G{\alpha}_{16}$ that is necessary for interacting with C5a receptor did not play any role in the interaction with IL-8 receptor. However, a stretch from residue 155 to residue 195 of $G{\alpha}_{16}$ was found to be crucial for efficient coupling to IL-8 receptor in concert with C-terminal 30 amino acid residues of this ${\alpha}$ subunit. Coupling profiles of a variety of chimeras, composed of $G{\alpha}_{11}G{\alpha}_{16}$ to fMLP receptor indicate that the C-terminal 30 amino acids are most critical for the coupling of $G{\alpha}_{16}$ to fMLP receptor. Taken together, $G{\alpha}_{16}$ subunit recruits multiple and distinctive coupling regions, depending on the type of receptors, to interact.

• Journal of Animal Science and Technology
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• v.50 no.1
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• pp.9-18
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• 2008

Species-specific polymorphisms in chicken, pheasant, turkey, and quail were identified by cloning and sequencing of the immunoglobulin constant domain (IgLC). A set of species-specific primers were then designed on the basis of polymorphisms in the IgLC between species, as well as two additional sets of primers for the cytochrome b and tapasin genes, for the purpose of species identification. Together, the primers successfully distinguished specific species from chicken by species-specific PCR. This simple but unambiguous method may be used to screen avian inter-species germline chimeras, which are valuable models for the conservation of endangered species.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.4
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• pp.211-216
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• 2008

TREK (TWIK-RElated $K^+$ channels) and TRAAK (TWIK-Related Arachidonic acid Activated $K^+$ channels) were expressed in COS-7 cells, and the channel activities were recorded from inside-out membrane patches using holding potential of - 40 mV in symmetrical 150 mM $K^+$ solution. Intracellular application of an oxidizing agent, 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB), markedly decreased the activity of the TREK2, and the activity was partially reversed by the reducing agent, dithiothreitol (DTT). In order to examine the possibility that the target sites for the oxidizing agents might be located in the C-terminus of TREK2, two chimeras were constructed: TREK2 (1-383)/TASK3C and TREK2 (1-353)/TASK3C. The channel activity in the TREK2 (1-383)/TASK3C chimera was still inhibited by DTNB, but not in the TREK2 (1-353)/TASK3C chimera. These results indicate that TREK2 is inhibited by oxidation, and that the target site for oxidation is located between the amino acid residues 353 and 383 in the C-terminus of the TREK2 protein.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1271-1278
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• 2012

Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.

• Korean Journal of Ichthyology
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• v.21 no.sup1
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• pp.73-73
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• 2009

The modern cartilaginous fishes, class Chondrichthyes, are small group of Pisces, and comprisingmore 1,000 described valid species, and including approximately 190 genera, 55 families, and 15 orders. The Chondrichthyes consists of two subclasses, the Holocephali (chimeras) and the Elasmobranchii(sharks and rays). The dominant subclass Elasmobranchii includes sharks (Selachii) and rays (Batoidea), the former is more diverse than the latter in higher taxa (order, family, genus) but has fewer species. The Batoidea, batoids or rays, has long been recognized as monophyletic, however, the interrelationships of the groups are problematic and uncertain. In order to review biodiversity and systematics of batoids, valid species name and supraspecific taxa from the published materials are studied. Batoids (including sawfishes, Pristiformes; electric rays, Torpediniformes; stingrays, Myliobatiformes; skates, Rajiformes; wedgefishes, Rhiniformes; and guitarfishes, Rhinobatiformes) is speciose and worldwide with about 580 species in 4 to 6 orders, 17 to 19 families, and about 75 genera. In East Asian waters, the previous records of the Batoidea reveal about 170 species belonging to 41 genera, 18 families, 6 orders. The largest group of batoid fauna in the area is order Myliobatiformes comprising about 80 species, 16 genera of 7 families. The predominant families are Dasyatidae (whiptail stingrays: 46 spp. of 6 genera), Arhynchobatidae (softnose skates: 26 spp. of 3 genera), and Rajidae (skates: 21 spp. of 6 genera). The predominant genera are Bathyraja (softnoseskates: 20 spp.), Himantura (whiprays:19spp.), and Dasyatis (fantailstingrays: 18 spp.). There are 23 species in the Yellow Sea, 70 species in the East China Sea, 82 species in the South China Sea, and 58 species in the North Pacific coast of Japan. Batoid fauna from the East Sea (Sea of Japan) is the poorest of the five regions, consisting of 14 species. Korean waters include only 28 species, 16 genera, 10 families and 5 orders of batoids. Two species is torpediniforms, 4 rhinobatiforms, arhynchobatiforms, 11 rajiforms and 10 myliobatiforms. By the earlier works, the systematics and a check list of the valid species of batoids will be presented.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2001.11a
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• pp.40-46
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• 2001

The use of pluripotent stem cells has tremendous advantages for various purposes but these cell lines with proven germ-line transmission have been completely established only in the mouse. Embryonic germ (EG) cell lines are also pluripotent and undifferentiated stem cells established from primordial germ cells (PGCs). This study was conducted to establish and characterize the chicken EG cells derived from gonadal primordial germ cells. We isolated gonadal PGCs from 5.5-day-old (stage 28) White leghorn (WL) embryos and established chicken EG cells lines with EG culture medium supplemented with human stem cell factor (hSCF), murine leukemia inhibitory factor (mLIF), bovine basic fibroblast growth factor (bFGF), human interleukin-11 (hIL-11), and human insulin-like growth factor-I (hIGF-I). These cells grew continuously for 4 months (10 passages) on a feeder layer of mitotically active chicken embryonic fibroblasts. These cells were characterized by screening with the Periodic acid-Shiff＇s reaction, anti-SSEA-1 antibody, and a proliferation assay after several passages. As the results, the chicken EG cells maintained characteristics of undifferentiated stem cells as well as that of gonadal PGCs. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types when re-seeded onto culture dish. The chicken EG cells were injected into blastodermal layer at stage X and dorsal aorta of recipient embryo at stage 14 (incubation of 53hrs) and produced chimeric chickens with various differentiated tissues derived from the EG cells. The germline chimeras were also successfully induced by using EG cells. Thus, Chicken EG cells will be useful for the production of transgenic chickena and for studies of germ cell differentiation and genomic imprinting.

• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.133-137
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• 2009

Pluripotent embryonic stem (ES) cells isolated from inner cell mass (ICM) of blastocyst-stage embryos are capable of differentiating into various cell lineages and demonstrate germ-line transmission in experimentally produced chimeras. These cells have a great potential as tools for transgenic animal production, screening of newly-developed drugs, and cell therapy. Miniature pigs, selectively bred pigs for small size, offer several advantages over large breed pigs in biomedical research including human disease model and xenotransplantation. In the present study, factors affecting primary culture of somatic cell nuclear transfer blastocysts from miniature pigs for isolation of ES cells were investigated. Formation of primary colonies occurred only on STO cells in human ES medium. In contrast, no ICM outgrowth was observed on mouse embryonic fibroblasts (MEF) in porcine ES medium. Plating intact blastocysts and isolated ICM resulted in comparable attachment on feeder layer and primary colony formation. After subculture of ES-like colonies, two putative ES cell lines were isolated. Colonies of putative ES cells morphologically resembled murine ES cells. These cells were maintained in culture up to three passages, but lost by spontaneous differentiation. The present study demonstrates factors involved in the early stage of nuclear transfer ES cell isolation in miniature pigs. However, long-term maintenance and characterization of nuclear transfer ES cells in miniature pigs are remained to be done in further studies.

• Journal of Microbiology and Biotechnology
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• v.32 no.10
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• pp.1245-1252
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• 2022

Induced pluripotent stem cells (iPSCs) can be generated from somatic cells using Oct4, Sox2, Klf4, and c-Myc (OSKM). Small molecules can enhance reprogramming. Licochalcone D (LCD), a flavonoid compound present mainly in the roots of Glycyrrhiza inflata, acts on known signaling pathways involved in transcriptional activity and signal transduction, including the PGC1-α and MAPK families. In this study, we demonstrated that LCD improved reprogramming efficiency. LCD-treated iPSCs (LCD-iPSCs) expressed pluripotency-related genes Oct4, Sox2, Nanog, and Prdm14. Moreover, LCD-iPSCs differentiated into all three germ layers in vitro and formed chimeras. The mesenchymal-to-epithelial transition (MET) is critical for somatic cell reprogramming. We found that the expression levels of mesenchymal genes (Snail2 and Twist) decreased and those of epithelial genes (DSP, Cldn3, Crb3, and Ocln) dramatically increased in OR-MEF (OG2^+/+/ROSA26^+/+) cells treated with LCD for 3 days, indicating that MET effectively occurred in LCD-treated OR-MEF cells. Thus, LCD enhanced the generation of iPSCs from somatic cells by promoting MET at the early stages of reprogramming.