• 한국자기공명학회논문지
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• 제9권2호
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• pp.74-92
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• 2005

The high resolution solution structure of MCP-3 was determined using multinuclear, multidimensional NMR spectroscopy with an expressed and $^{13}C-\;and\;^{15}N-labeled$ protein. The MCP-3 has a typical chemokine fold including 3 anti-parallel $\beta-sheets$, and a C-terminal helix, but it exists as a monomer in solution under the conditions where the structure was determined (2 mM, pH 5.1 at $30^{\circ}C$). Based on the structure and the amino acid sequence compared to other chemokines we propose that Ile20 and Leu25 in MCP-3 play key roles in the formation of N-loop (residues between the $2^{nd}$ cysteine and the I sheet) which has been implicated as a determinant of chemokine specificity. Additional receptor binding surface is supplied by the 40s loop (residues between the 2 and the 3 sheet) and the binding interface of the acidic N-terminal region of chemokine receptor to MCP-3 would resemble the dimerization interface of CC type dimer.

• Journal of Ginseng Research
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• 제23권2호
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• pp.105-114
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• 1999

본 실험은 백서의 간세포막에서 transferrin receptor(TfR)의 발현에 미치는 인삼 및 3'-methyl-4-dimethylaminobenzene(3'-Me-DAB)의 영향을 연구하기 위하여, 인삼, 3'-Me-DAB 또는 3'-Me-DAB처치후 인삼을 투여한 백서를 부분 간절제한 후 간조직에서 $[^{3}H]thymidine$및 transferrin수용체의 유전자 발현 실험, 그리고 간세포막에서$[^{3}H]thymidine$ 결합 실험을 시행하여 다음과 같은 결과를 얻었다. 1. 8주간 3'-Me-DAB를 투여한 백서에서 간조직의 조직학적 소견은 림프구의 침윤, 담관세포의 증식,교량성괴사 및 이상세포의 증식 소견을 보였다. 그러나 정상 또는 3'-Me-DAB를 투여한 백서에 인삼을 투여하였을 때 간조직의 조직학적 소견은 각각의 대조군에 비해 크게 변화되지 않았다. 2. 인삼 또는 3'-Me-DAB를 투여한 백서에서의 $[^{3}H]thymidine$ uptake는 대조군에서와 비슷하였으나, 부분간절제 수술 후 1일째 간에서는 수술 전보다 현저하게 증가되었다. 특히 3'-Me-DAB투여군의 간조직에서 더 증가되었으며, 이는 인삼 투여에 의해 억제되는 경향을 보였다. 3. 간세포막에서의 transferrin 결합량(Bmax) 및 Kd는 정상 백서에서 각각 6.87pmole/mg protein 및 10.64nM이었으며, 인삼 투여에 의해 크게 변동되지 않았다 그러나 3'-Me-DAB를 투여한 백서에서는 Bmax 및 Kd가 증가되었고, 이 증가는 인삼투여에 의해 감소되었다. 부분 간절제 수술 후 간세포막에서의 Bmax 및 Kd는 모든 그룹에서 수술 후 3일째에는 증가, 수술 후 5일째에는 감소되는 양상을 나타냈다. 그러나 3'-Me-DAB투여 후 간절제 수술군에서의 증가가 가장 컸으며, 이 증가효과는 인삼 투여에 의하여 억제되었다. 4. 부문 간절제 수술 후 간조직에서의 TfR mRrfA 발현은 수술 후 24시간에 최대로 증가되었으며, 이는 3'-Me-DAB를 투여한 백서에서 더 현저하였다. 그리고 인삼은 3'-Me-DAB투여 후 간절제 수술군에서 증가된 발현양을 약간 감소시켰다. 이상의 결과로 백서에서 부분 간절제 수술 및 발암 물질에 의해 간세포막 transffirin수용체는 친화력이 저하된 TfR이 증가될 수 있으며, 이는 일부 이 수용체의유전자 수준에서 조절될 수 있는 것으로 생각된다. 그리고 인삼은 발암물질 투여등으로 간세포의 증식이 촉진되었을 때, 간세포내 핵산 합성 및 TfR mRNA 발현 조절을 통하여 세포막의 TfR출현 및 세포증식을 억제시킬 수 있을 것으로 추측된다.

• Molecules and Cells
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• 제20권2호
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• pp.263-270
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• 2005

Transactivation of EGF-receptor (EGFR) by G-protein coupled receptors (GPCRs) is emerging as an important pathway in cell proliferation, which plays a crucial role in the development of atherosclerotic lesion. Angiotensin II (Ang II) has been identified to have a major role in the formation of atherosclerotic lesions, although the underlying mechanisms remain largely unclear. We hypothesize that Ang II promotes the proliferation and migration of smooth muscle cells through the release of heparin-binding epidermal growth factor like growth factor (HB-EGF), transactivation of EGFR and activation of Akt and Erk 1/2, with matrix metalloproteases (MMPs) playing a dispensable role. Primary rat aortic smooth muscle cells were used in this study. Smooth muscle cells rendered quiescent by serum deprivation for 12 h were treated with Ang II (100 nM) in the presence of either GM6001 ($20{\mu}M$), a specific inhibitor of MMPs or AG1478 ($10{\mu}M$), an inhibitor of EGFR. The levels of phosphorylation of EGFR, Akt and Erk 1/2 were assessed in the cell lysates. Inhibition of MMPs by GM6001 significantly attenuated Ang II-stimulated phosphorylation of EGFR, suggesting that MMPs may be involved in the transactivation of EGFR by Ang II receptor. Furthermore Ang II-stimulated proliferation and migration of smooth muscle cells were significantly blunted by inhibiting MMPs and EGFR and applying HB-EGF neutralization antibody, indicating that MMPs, HB-EGF and EGFR activation is necessary for Ang-II stimulated migration and proliferation of smooth muscle cells. Our results suggest that inhibition of MMPs may represent one of the strategies to counter the mitogenic and motogenic effects of Ang II on smooth muscle cells and thereby prevent the formation and development of atherosclerotic lesions.

• 대한의생명과학회지
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• 제13권3호
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• pp.197-206
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• 2007

P2X receptors are membrane-bound ion channels that conduct $Na^+,\;K^+$, and $Ca^{2+}$ in response to ATP and its analogs. There are seven subunits identified so far ($P2X_1-P2X_7$). $P2X_2$ receptors are known to be expressed in a wide range of organs including brains and adrenal grands. PC12 cells are originated from adrenal grand and differentiated by nerve growth factor or pituitary adenylate cyclase activating poly peptide (PACAP). Previous studies indicate that $P2X_2$ receptor activation in PC12 cells couples to $Ca^{2+}-dependent$ release of catecholamine and ATP. It is known that acidic pH potentiates ATP currents at $P2X_2$ receptors. This leads to a hypothesis that $P2X_2$ receptors may play an important role in PC12 cell differentiation, one of the characteristics of which is neurite outgrowth, induced by the hormones under lower pH. In the present study, we isolated several clones which potentiate neurite outgrowth by PACAP in acidic pH (6.8), but not in alkaline pH (7.6). RT-PCR and electrophysiology data indicate that these clones express only functional $P2X_2$ receptors in the absence or presence of PACAP for 3 days. Potentiation of neurite outgrowth resulted from PACAP (100 nM) in acidic pH is inhibited by the two P2X receptor antagonists, suramin and PPADS ($100\;{\mu}M)$ each), and exogenous exprerssion of ATP-binding mutant $P2X_2$ receptor subunit ($P2X_2[K69A]$). However, acid sensing ion channels (ASICs) are not involved in PACAP-induced neurite outgrowth potentiation in lower pH since treatments of an inhibitor of ASICs, amyloride ($10\;{\mu}M$), did not give any effects to neurite extension. The vesicular proton pump ($H^+-ATPase$) inhibitor, bafilomycin (100 nM), reduced neurite extension indicating that ATP release resulted from $P2X_2$ receptor activation in PC12 cells is needed for neurite outgrowth. These were confirmed by activation of mitogen activated protein kinases, such as ERKs and p38. These results suggest roles of ATP and $P2X_2$ receptors in hormone-induced cell differentiation or neuronal synaptogenesis in local acidic environments.

• 약학회지
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• 제35권4호
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• pp.295-300
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• 1991

Ralationship between noradrenergic nervous system activity and luteinizing hormone releasing hormone(LHRH) content mediated by testosterone in hypothalamus was tested. Three groups of adult male animals were prepared; (1) Intact; (2) Castration+Vehicle (Cast+V); (3) Castration+Testosterone (Cast+T). Silastic capsule containing vehicle or testosterone was implanted into neck region of animals two weeks following castration. Norepinephrine content, alpha-adrenergic receptor binding characteristics using H$^{3}$-WB4101, and content of LHRH by LHRH RIA procedure were determined. Testosterone replacement to castrated male rats augmented the content of norepinephrine and LHRH. Testosterone replacement increased the alpha-adrenergic receptor density but did not change alpha-receptor affinity. The data from the present study suggest that increase in LHRH content by testosterone may be positively coupled to the activity of central noradrenergic nervous system.

• 대한핵의학회지
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• 제38권4호
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• pp.294-299
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• 2004

목적 : 현재 생체 내로 이식된 세포를 추적하는데 여러 가지 리포터 유전자들이 이용되고 있다. 이 연구에서는 사람 나트륨 옥소 공동 수송체 (hNIS)와 도파민 2 수용체($D_2R$)를 이중 리포터 유전자로 사용하여 각각을 비교하였다. 대상 및 방법: hNIS와 $D_2R$의 발현이 동시에 이루어지도록 하기 위해서 IRES (Internal ribosome entry site)로 연결된 재조합 플라스미드(pIRES-hNIS/D_2R)를 제조하였다. $pIRES-hNIS/D_2R$를 사람의 간암세포주인 SK-Hep1에 lipofactamine을 이용하여 형질을 도입시킨 후, 항생제(G418)를 농도별로 처리하여 2주간 선별하였다(HEP-ND). hNIS와 $D_2R$발현 유무와 발현 정도를 알아보기 위하여 각 유전자에 특이적인 프라이머를 이용하여 RT-PCR을 수행하였다. 각 형질 도입세포군에서, hNIS의 활성은 $^{125}I$ 섭취율을 이용하여 측정하고 $D_2R$의 활성은 $[^3H]spiperone$을 리간드로 이용하여 수용체 결합 정도를 측정하였다. 결과: 선별된 HEP-ND세포에서 hNIS와 $D_2R$의 발현을 RT-PCR로 확인하였을 때 IRES로 연결된 hNIS와 $D_2R$의 발현 정도는 서로 비슷하였다. HEP-ND세포의 $^{125}I$ 섭취율은 대조군인 SK-Hep1세포에 배해 30-40배 증가되었고, $KClO_4$에 의해 $^{125}I$ 섭취가 저해되었다. $D_2R$의 발현 정도를 측정할 수 있는 수용체 결합 분석법을 통해G418 농도별로 나눈 두 종류의 세포주에서, $[^3H]spiperone$을 이용한 해리상수 ($K_d$)와 최대결합 부위농도 ($B_{max}$)는 각각 2.92 nM, 745.25 fmol/mg protein과 8.91nM, 1323 fmole/mg protein이었다. hNIS와 $D_2R$발현의 상관관계에서는 높은 상관관계를 나타내었다. 결론: 이 연구에서 hNIS와 $D_2R$가 이입된 세포주에서 이중 유전자, 감마 영상 리포터 시스템을 개발하였으며, $D_2R$와 HNIS 유전자를 이중 핵 영상 시스템으로서 서로 상호보완적으로 이용할 수 있을 것으로 기대하고 있다.

• Molecules and Cells
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• 제41권9호
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• pp.842-852
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• 2018

Notch signaling is an evolutionarily conserved pathway and involves in the regulation of various cellular and developmental processes. Ligand binding releases the intracellular domain of Notch receptor (NICD), which interacts with DNA-bound CSL [CBF1/Su(H)/Lag-1] to activate transcription of target genes. In the absence of NICD binding, CSL down-regulates target gene expression through the recruitment of various corepressor proteins including SMRT/NCoR (silencing mediator of retinoid and thyroid receptors/nuclear receptor corepressor), SHARP (SMRT/HDAC1-associated repressor protein), and KyoT2. Structural and functional studies revealed the molecular basis of these interactions, in which NICD coactivator and corepressor proteins competitively bind to ${\beta}-trefoil$ domain (BTD) of CSL using a conserved ${\varphi}W{\varphi}P$ motif (${\varphi}$ denotes any hydrophobic residues). To date, there are conflicting ideas regarding the molecular mechanism of SMRT-mediated repression of CSL as to whether CSL-SMRT interaction is direct or indirect (via the bridge factor SHARP). To solve this issue, we mapped the CSL-binding region of SMRT and employed a 'one- plus two-hybrid system' to obtain CSL interaction-defective mutants for this region. We identified the CSL-interaction module of SMRT (CIMS; amino acid 1816-1846) as the molecular determinant of its direct interaction with CSL. Notably, CIMS contains a canonical ${\varphi}W{\varphi}P$ sequence (APIWRP, amino acids 1832-1837) and directly interacts with CSL-BTD in a mode similar to other BTD-binding corepressors. Finally, we showed that CSL-interaction motif, rather than SHARP-interaction motif, of SMRT is involved in transcriptional repression of NICD in a cell-based assay. These results strongly suggest that SMRT participates in CSL-mediated repression via direct binding to CSL.

• Molecules and Cells
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• 제37권9호
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• pp.656-663
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• 2014

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits $[Ca^{2+}]_i$ transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier $K^+$ ($I_{Ks}$) channel is a cardiac $K^+$ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating $I_{Ks}$ channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human $I_{Ks}$ channel activity by expressing human $I_{Ks}$ channels in Xenopus oocytes. We found that gintonin enhances $I_{Ks}$ channel currents in concentration- and voltage-dependent manners. The $EC_{50}$ for the $I_{Ks}$ channel was $0.05{\pm}0.01{\mu}g/ml$. Gintonin-mediated activation 1 of the $I_{Ks}$ channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an $IP_3$ receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the $I_{Ks}$ channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 $[Ca^{2+}]_i$/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on $I_{Ks}$ channel. However, gintonin had no effect on hERG $K^+$ channel activity. These results show that gintonin-mediated enhancement of $I_{Ks}$ channel currents is achieved through binding of the $[Ca^{2+}]_i$/CaM complex to the C terminus of KCNQ1 subunit.

• BMB Reports
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• 제42권11호
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• pp.764-768
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• 2009

The tandem ubiquitin-interacting motif (UIM) domain located at the N-terminus of Receptor Associated Protein 80 (RAP80) plays a crucial role in ionizing radiation (IR)-induced DNA damage response. RAP80 translocates to sites of IR-induced DNA damage through interaction of its UIM domain with ubiquitinated H2A and Lys63-linked polyubiquitin chains. The exact mechanism, however, through which RAP80 associates with Lys63-linked polyubiquitin chains is not clear. Here, we show by in vitro GST-pull down assays that modifying the linker region between the tandem ubiquitin binding domains of RAP80 changes the binding affinity for Lys63-linked polyubiquitin chains and affects translocation to sites of DNA breaks. Based on these findings, we suggest that the length of the linker region between the tandem ubiquitin binding domains of RAP80 may be a key factor in the binding of RAP80 with Lys63-linked polyubiquitin chains as well as in the translocation of RAP80 to DNA break sites.

• Journal of the Korean Academy of Child and Adolescent Psychiatry
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• 제5권1호
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• pp.93-101
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• 1994

반복적으로 공격적인 행동 양상을 보이는 사람들의 세로토닌 반응(serotonergic responsivity)이 정상인과 비교하여 차이가 있을 것인지를 알아보고자 연구를 실시하였다. 나이를 대조하여 공격성군(소년원 재소자 16명)과 대조군(의과대학생 17명)으로 구분한 연구대상에게, 여러 심리검사 척도를 이용하여 공격성의 심한 정도를 정의하고, 두뇌의 세로토닌 기능과 일치하는 것으로 알려진 혈소판의 이미프라민 결합을 측정하여 다음과 같은 결과를 얻었다. 1) 공격성군은 대조군에 비하여 공격척도상 신체적 공격성(physical aggression)의 평균이 모두 유의하게 높은 값을 나타내었다. 이외에도 공격성군은 대조군에 비해 충동성(impulsivity), 적대감(hostility), 정신증(psychoticism)등이 통계적으로 유의하게 높은 평균값을 보였다. 2) 공격성군은 대조군에 비하여 혈소판 이미프라민 최대결합부위 밀도(Bmax)가 낮은 경향을 보였다. 3) 공격성군과 대조군의 혈소판 이미프라민 결합의 결합상수(Kd) 값은 통계적으로 유의한 차이를 보이지 않았다. 4) 연구대상군을 전체로 하였을 때, 공격성척도(PFAV)와 갈등해결 척도(CTS)의 신체적 공격성의 심한 정도는 혈소판 이미프라민 최대결합부위 밀도(Bmax)와 통계적으로 유의한 정도의 역비례 상관관계를 보였다.