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- 저자 : Dawon Kang,Changyong Choe,Chang-Woon Kim,Ae Jin Goo,Jaehee Han (검색결과 88 건)
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TASK-2 Expression Levels are Increased in Mouse Cryopreserved Ovaries
Kang, Dawon,Choe, Changyong,Kim, Chang-Woon,Goo, Ae Jin,Han, Jaehee The Korean Society of Embryo Transfer 2015 한국동물생명공학회지 Vol.30 No.4
Cryopreservation affects osmotic tolerance and intracellular ion concentration through changes in expression levels of water and ion channels. Control of these changes is important for cell survival after cryopreservation. Relatively little is known about changes in $K^+$ channel expression compared to water channel expression. This study was performed to investigate changes in TASK-2 channel (KCNK5: potassium channel, subfamily K, member 5), a member of two-pore domain $K^+$ channel family, in cryopreserved mouse ovaries. Cryopreservation increased TASK-2 mRNA expression in mouse ovaries. In addition, TASK-2 protein expression was upregulated in vitrified and slowly frozen ovaries. TASK-2 protein was expressed in all area of granulosa cells that surround the oocyte within the follicle, except nucleus. Viability of cells overexpressed with TASK-2 was higher than that of vector-transfected cells. Our results found that TASK-2 expression was increased by cryopreservation and overexpression of TASK-2 decreased cryopreservation-induced cell death. These results suggest that TASK-2 upregulation might reduce cryodamage.
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Dawon Kang,Gyu-Tae Kim,Jaehee Han 한국동물생명공학회(구 한국동물번식학회) 2007 Reproductive & developmental biology Vol.31 No.1
Aging causes thymus involution, and genes in thymus play an important role in the development of the immune system. In this study, we compared genes expressed in thymus of neonatal and peripubertal rats using annealing control primers (ACPs)-based GeneFishing polymerase chain reaction (PCR) and semiquantitative reverse transcription (RT)-PCR. We identified 10 differentially expressed genes (DEGs) with 20 ACPs. Of 10 DEGs, bystin-like, collagen type V alpha 1 (COL5A1), and T-cell receptor beta-chain segment 2 (TCRB2) that are related to immune-function were detected in rat thymus. Bystin-like and TCRB2 were up-regulated, while COL5A1 was down-regulated in peripubertal thymus. Semiquantitative RT-PCR confirmed postnatal changes in expression of bystin-like, COL5A1, and TCRB2. These results suggest that bystin-like, COL5A1, and TCRB2 could regulate immune function controlled in thymus as age increases.
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Kang, Dawon,Kim, Donghee American Physiological Society 2006 American journal of physiology. Cell physiology Vol.291 No.1
<P>Dorsal root ganglion (DRG) neurons express mRNAs for many two-pore domain K(+) (K(2P)) channels that behave as background K(+) channels. To identify functional background K(+) channels in DRG neurons, we examined the properties of single-channel openings from cell-attached and inside-out patches from the cell bodies of DRG neurons. We found seven types of K(+) channels, with single-channel conductance ranging from 14 to 120 pS in 150 mM KCl bath solution. Four of these K(+) channels showed biophysical and pharmacological properties similar to TRESK (14 pS), TREK-1 (112 pS), TREK-2 (50 pS), and TRAAK (73 pS), which are members of the K(2P) channel family. The molecular identity of the three other K(+) channels could not be determined, as they showed low channel activity and were observed infrequently. Of the four K(2P) channels, the TRESK-like (14 pS) K(+) channel was most active at 24 degrees C. At 37 degrees C, the 50-pS (TREK-2 like) channel was the most active and contributed the most (69%) to the resting K(+) current, followed by the TRESK-like 14-pS (16%), TREK-1-like 112-pS (12%), and TRAAK-like 73-pS (3%) channels. In DRG neurons, mRNAs of all four K(2P) channels, as well as those of TASK-1 and TASK-3, were expressed, as judged by RT-PCR analysis. Our results show that TREKs and TRESK together contribute >95% of the background K(+) conductance of DRG neurons at 37 degrees C. As TREKs and TRESK are targets of modulation by receptor agonists, they are likely to play an active role in the regulation of excitability in DRG neurons.</P>
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Choe, Changyong,Kang, Dawon 韓國受精卵移植學會 2014 한국동물생명공학회지 Vol.29 No.2
K+ channels are involved in the regulation of a variety of physiological functions, including proliferation, apoptosis and differentiation, in mammalian cells. Our previous study demonstrated that the blockage of K+ channels inhibits mouse early embryonic development. This study was designed to identify the effect of K+ channels during bovine embryonic development. K+ channel blockers (tetraethylammonium (TEA), BaCl2, quinine, ruthenium red and fluoxetine) were added to the culture medium during in vitro fertilization (IVF) for 6 h to first identify the short-term effect of these chemicals. Among K+ channel blockers, fluoxetine, which is used as a selective serotonin reuptake inhibitor, significantly increased the blastocyst formation rate by approximately 6% when compared to control. During the in vitro maturation (IVM) of immature oocytes and the in vitro culture (IVC) of embryos, the oocytes and embryos were exposed to fluoxetine for either a short-term (6 h) or a long-term (24 h) to compare the embryonic development in response to exposure time. The 6 h exposure to fluoxetine during IVM did not affect the blastocyst formation rate, but the rate of blastocyst formation was reduced after the 24 h exposure. On the other hand, embryonic development increased approximately 10% in both groups of embryos exposed to fluoxetine for 6 and 24 h during IVC. Taken together, fluoxetine treatment during IVF and IVC, but not IVM, enhances bovine embryonic development. These results suggest that fluoxetine-modulated signals in oocytes and embryos could be an important factor towards enhancing bovine embryonic development.
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Kang, Dawon,Kim, Sung-Hee,Hwang, Eun-Mi,Kwon, Oh-Sang,Yang, Hae-Young,Kim, Eun-Sook,Choi, Tae Hyun,Park, Jae-Yong,Hong, Seong-Geun,Han, Jaehee Blackwell Publishing Ltd 2007 Experimental dermatology Vol.16 No.12
<P>Abstract: </P><P>Recent studies have shown that keratinocytes can sense temperature via thermo-transient receptor potential (TRP) channels. It is not known whether other thermosensitive ion channels such as TREK-1, TREK-2 and TRAAK (TREKs/TRAAK) that are members of the two-pore domain K<SUP>+</SUP> (K<SUB>2P</SUB>) channel family are expressed in human keratinocytes. Here, we identified the expression of TREKs/TRAAK in human keratinocytes-derived cell line HaCaT cells using RT-PCR, immunocytochemistry, Western blot analysis and patch-clamp technique. RT-PCR showed that all six K<SUB>2P</SUB> channels tested (TASK-1, TASK-3, TREK-1, TREK-2, TRAAK and TASK-2) were expressed in HaCaT cells, as well as in skin and dorsal root ganglion (DRG) of rat. The expression of TREKs/TRAAK mRNA identified by RT-PCR was further studied at the protein level. Using anti-TREK-1, -TREK-2 and -TRAAK, bands of ∼46, ∼60 and ∼43 kDa, respectively, were observed at plasma membrane of HaCaT cells. Immunostaining also showed that TREK-1, TREK-2 and TRAAK were expressed in all area of cells including plasma membrane. Whole-cell K<SUP>+</SUP> currents recorded from HaCaT cells were activated by arachidonic acid and heat. These results suggest that TREKs/TRAAK channels could act as thermosensors in human keratinocytes.</P>
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Mechanism of inhibition of TREK-2 (K2P10.1) by the Gq-coupled M3 muscarinic receptor.
Kang, Dawon,Han, Jaehee,Kim, Donghee American Physiological Society 2006 American journal of physiology. Cell physiology Vol.291 No.4
<P>TREK-2 is a member of the two-pore domain K(+) channel family and provides part of the background K(+) current in many types of cells. Neurotransmitters that act on receptors coupled to G(q) strongly inhibit TREK-2 and thus enhance cell excitability. The molecular basis for the inhibition of TREK-2 was studied. In COS-7 cells expressing TREK-2 and M(3) receptor, acetylcholine (ACh) applied to the bath solution strongly inhibited the whole cell current, and this was markedly reduced in the presence of U-73122, an inhibitor of PLC. The inhibition was also observed in cell-attached patches when ACh was applied to the bath solution. In inside-out patches, direct application of guanosine 5'-O-(3-thiotriphosphate) (10 microM), Ca(2+) (5 microM), or diacylglycerol (DAG; 10 microM) produced no inhibition of TREK-2 in >75% of patches tested. Phosphatidic acid, a product of DAG kinase, had no effect on TREK-2. Pretreatment of cells with 20 microM wortmannin, an inhibitor of phosphatidylinositol kinases, did not affect the inhibition or the recovery from inhibition of TREK-2, suggesting that phosphatidylinositol 4,5-bisphosphate depletion did not mediate the inhibition. Pretreatment of cells with a protein kinase C inhibitor (bisindolylmaleimide, 10 microM) markedly inhibited ACh-induced inhibition of TREK-2. Mutation of two putative PKC sites (S326A, S359C) abolished inhibition by ACh. Mutation of these amino acids to aspartate to mimic the phosphorylated state resulted in diminished TREK-2 current and no inhibition by ACh. These results suggest that the agonist-induced inhibition of TREK-2 via M(3) receptor occurs primarily via PKC-mediated phosphorylation.</P>
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Expression and identification of CatSper3 channel in mouse brain and testis
Dawon Kang,Jaehee Han 한국발생생물학회 2010 한국발생생물학회 학술발표대회 Vol.29 No.-
Sperm specific non selective cation (CatSper) channels belong to the CatSper family of genes and are expressed only in sperm and testis. In general, gene expression profiles in the brains of humans and mice share the highest similarity with those in testis. Therefore, to identify whether CatSper genes are expressed in the mouse brain, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. RT-PCR detected all four CatSper mRNAs in both the testis and the brain, with CatSper3 being the most highly expressed. Consistent with RT-PCR data, Western blot analysis showed that CatSper3 was expressed in the brain. We cloned CatSper3 variant 2 from eight-week mouse brain. We named the gene as CatSper3 variant 1 (V1) because the mouse CatSper3 is orthologous to human CatSper3, and another mouse CatSper3 variant (variant 2, V2) with truncated second transmembrane helix was identified. The open reading frame of mCatSper3 V1 consists of 1185 nucleotides and encodes a putative 395-amino acid protein. At the amino acid level, CatSper3 isolated from brain is 100 and 64.8% identical to that isolated from mouse testis and human CatSper3, respectively. Based on comparison between the mCatSper3 V1 ORF and mCatSper3 V2 using TopPred software, the alignment of amino acid sequences shows that the differences exist mainly in segment 2. The CatSper3 transcript consists of eight exons and seven introns, and alternative splice is present within the third exon. In HT22 cell, a mouse hippocampal neuronal cell line, H2O2-induced changes in CatSper3 expression were studied. H2O2 dramatically increased CatSper3 expression in HT22 cells in a dose-and time-dependent manner. The H2O2-induced increase in CatSper3 expression was offset by the addition of N-acetylcysteine (NAC), which is an antioxidant. Taken together, these data strongly indicate that CatSper3 is expressed in mouse brain as variant 1 and suggest that CatSper3 could be a potential target for the modulation of ROS.
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