• Journal of Life Science
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• v.29 no.1
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• pp.118-122
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• 2019

Oxytocin (Oxt) and vasopressin (Avp) are mainly synthesized in neuronal cells of the hypothalamus and are released from the posterior pituitary. The structure and sequences of Oxt and Avp genes imply that they are closely related and that they are the result of a duplication event during evolution. A previous study suggested that a small regulatory microRNA (miRNA), miR-24, regulated Oxt after binding. However, it is not clear whether this miRNA can modulate Avp simultaneously. The aim of the present study was to investigate putative targeting miRNAs of Avp, including miR-24. Targeted candidate miRNA oligonucleotides were transfected into COS-7 cells to elucidate the binding activity of miRNAs and Avp using dual-luciferase assays. The luciferase assay showed that only miR-24 displayed elevated binding activity with Avp as compared to a control and other candidate miRNAs. Transfection with seed mutants of Avp and miR-24 inhibitors clearly showed that miR-24 can directly bind to the Avp gene. These results provide new insight into the regulatory mechanism of neurohypophysial hormones by a single miRNA.

• Parasites, Hosts and Diseases
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• v.49 no.1
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• pp.85-90
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• 2011

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8$^+$ T-cells and CD4$^+$ T-cells. However, in mice immunized using a gene gun, significantly higher (P<0.05) proportions of CD8$^+$ cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique.

• 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 Korean Neurosurgical Society
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• v.29 no.10
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• pp.1283-1288
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• 2000

Objectives : The subcellular locations of Bad, Bid, Bax and Bcl-XL change during apoptosis and this change is important for the regulation of cell death. The purpose this study was to elucidate binding of Bad with Bcl-XL in vivo Methods : We mads Bad with Green Fluorescent Protein(GFP) using PCR method. We transfected and overexpressed GFP-Bad with or without Bcl-XL cotransfection in living COS-7 cell. Results : Bad and Bcl- XL bind one another in healthy living cells and this association controled mitochondrial docking. In the absence of Bad-XL, Bad was mainly cytosolic and partially bound to mitochondria. Upon coexpression of Bad and Bcl-XL, most of Bad translocated to mitochondria. These should suggest that Bad binds to the mitochondrial and cytoplasmic forms of Bcl-XL and Bad bound to cytoplasmic Bcl-XL translocates to mitochondria. These in vivo findings confirm that Bad make a complexes with Bcl- XL and cause mitochondrial translocation of Bad-Bcl-XL complex.

• 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.26 no.12
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• pp.2192-2198
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• 2016

The myeloid-specific IgA Fc receptor ($Fc{\alpha}R$) is a cell surface molecule on immunocytes that provides a fundamental connection between humoral and cellular immunity. In this study, the full-length cDNA sequence of swine $Fc{\alpha}RI$ ($swFc{\alpha}RI$) was isolated and characterized and found to contain a 792-base-pair open reading frame, encoding a 264-amino-acid transmembrane glycoprotein with a predicted molecular mass of 29.4 kDa. The $swFc{\alpha}RI$ shares high amino acid sequence homology (>50%) with its counterparts from cattle, seal, and horse. Rosetting analysis confirmed that COS-7 cells transfected with an $swFc{\alpha}RI$ expression plasmid was able to combine with chicken erythrocytes sensitized with porcine IgA, but not IgG.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.43-43
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• 2003

Large-conductance $Ca^{2+}$-actived $K^{+}$ channels ($BK_{Ca}$ channels) play a key role in setting the pace of contractile activity in muscle and are involved in the regulation of neurotransmitter release in neuron. $BK_{Ca}$ channels are activated by depolarizing membrane potential and the elevated level of intracellular calcium. Using yeast-two hybrid assay, we have identified a novel protein interacting with the cytosolic carboxyl terminus of rSlo, the brain isoform of rat large-conductance $Ca^{2+}$-activated $K^{+}$ channel $\alpha$-subunit. The novel gene encodes 51 kDa protein and is named as SIRK(rSlo-interacting RGS-like protein). SIRK is expressed in various tissues and localized in the cytosolic and the membrane fraction. Biochemical and immunological studies indicated that SIRK physically interacted with the cytosolic region of rSlo. To investigate whether SIRK can modulate the activity of rSlo, GFP-fused SIRK and rSlo were transiently transfected into COS-7 cells and the effects of SIRK was studied using electrophysiological means. We concluded that the overexpression of SIRK alters the surface expression of rSlo channel with only a limited effect on the biophysical characteristics of the channel.the channel.

• Archives of Pharmacal Research
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• v.21 no.4
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• pp.423-428
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• 1998

The ligand binding signals to a wide variety of seven transmembrane cell surface receptors are transduced into intracellular signals through heterotrimeric G-proteins. Recently, there have been reports which show diverse coupling patterns of ligand-activated receptors to the members of Gq family $\alpha$ subunits. In order to shed some light on these complex signal processing networks, interactions between G$\alpha$q family of G protein and neurokinin-2 receptor as well as muscarinic M$_{1}$ receptor, which are considered to be new thearpeutic targets in asthma, were studied. Using washed membranes from Cos-7 cells co-transfected with different G.alpha.q and receptor cDNAs, the receptors were stimulated with various concentrations of carbachol and neurokinin A and the agonist-dependent release of [$^3H$]inositol phosphates through phospholipase C beta-1 activation was measured. Differential coupling of Gaq family of G-protein to muscarinic M$_{1}$ receptor and neurokinin-2 receptor was observed. The neurokinin-2 receptor shows a ligand-mediated response in membranes co-transfected with G$\alpha$q, G$\alpha$11 and G$\alpha$14 but not G$\alpha$16 and the ability of the muscarinic $M_1$ receptor to activate phospholipase C through G$\alpha$/11 but not G$\alpha$14 and G$\alpha$16 was demonstrated. Clearly G$\alpha$/11 can couple $\M_1$ and neurokinin-2 receptor to activate phospholipase C. But, there are differences in the relative coupling of the G$\alpha$14 and G$\alpha$16 subunits to these receptors.

• IMMUNE NETWORK
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• v.11 no.2
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• pp.114-122
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• 2011

Background: The leukocyte common antigen (CD45) is a transmembrane-type protein tyrosine phosphatase that has five isoforms. Methods: We generated seven murine mAbs against human CD45 by injecting cells from different origins, such as human thymocytes, PBMCs, and leukemic cell lines. By using various immunological methods including flow cytometry, immunohistochemistry, and immunoprecipitation, we evaluated the reactivity of those mAbs to CD45 of thymus as well as tonsil lysates. Furthermore, we transiently transfected COS-7 cells with each of gene constructs that express five human CD45 isoforms respectively, and examined the specificities of the mAbs against the transfected isoforms. Results: In case of thymocytes, lymphocytes, and monocytes, all the seven mAbs demonstrated positive reactivities whereas none was reactive to erythrocytes and platelets. The majority of immune cells in formalin-fixed paraffin-embedded thymus and tonsil tissues displayed strong membranous immunoreactivity, and the main antigen was detected near 220 kDa in all cases. Among the mAbs, four mAbs (AP4, DN11, SHL-1, and P6) recognized a region commonly present in all the five isoforms. One mAb, YG27, recognized four isoforms (ABC, AB, BC, and O). Two mAbs, P1 and P14, recognized the isoforms that contain exon A encoded regions (ABC and AB). Conclusion: In this study, we confirmed that AP4, DN11, SHL-1, YG27 and P6, are mAbs reactive with the CD45 antigen whereas P1 and P14 are reactive with the CD45RA antigen.

• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1670-1674
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• 2007

Duffy binding protein (DBP) plays a critical role in Plasmodium vivax invasion of human red blood cells. We previously reported a single-chain antibody fragment (scFv) that was specific to P. vivax DBP (PvDBP). However, the stabilization and the half-life of scFvs have not been studied. Here, we investigated the effect of PEGylated scFvs on their biological activity and stability in vitro. SDS-PAGE analysis showed that three clones (SFDBII-12, -58, and -92) were formed as monomers (about 70 kDa) with PEGylation. Clone SFDBII-58 gave the highest yield of PEGylated scFv. Binding analysis using BIAcore between DBP and scFv showed that both SFDBII-12 and -58 were decreased approximately by two folds at the level of binding affinity to DBP after PEGylation. However, the SFDBII-92 clone still showed a relatively high level of binding affinity ($K_D=1.02{\times}10^{-7}\;M$). Binding inhibition assay showed that PEGylated scFv was still able to competitively bind the PvDBP and playa critical role in inhibiting the interactions between PvDBP protein expressed on the surface of Cos-7 cells and Duffy receptor on the surface of erythrocytes. When both scFvs and their PEGylated counterparts were exposed to trypsin, scFv was completely degraded only after 24 h, whereas 35% of PEGylated scFvs remained intact, maintaining their stability against the proteolytic attack of trypsin until 72 h. Taken together, these results suggest that the PEGylated scFvs retain their stability against proteolytic enzymes in vivo, with no significant loss in their binding affinity to target antigen, DBP.