• 생명과학회지
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• 제15권3호
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• pp.344-351
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• 2005

산화질소(NO)와 칼슘 결합 단백질은 중추신경계의 다양한 세포들에서 나타나며, 이들은 각각 중요 신호전달 분자와 칼슘 완충 분자이다. 본 연구는 햄스터의 시각피질에서 뇌산화질소 합성효소 (nNOS)와 parvalbumin을 포함하는 뉴런들의 분포와 이들의 co-localization 양상을 면역세포화학적 기법을 이용하여 알아보았다. 햄스터 시각피질에서 parvalbumin에 대한 면역 반응성을 나타내는 뉴런들의 전체 수는 nNOS에 대한 면역 반응성을 보이는 뉴런들의 수보다 17배나 많았다. 가장 큰 차이는 시각피질 제5충에서 발견되었으며, 이곳에서 parvalbumin-면역 반응성 뉴런이 nNOS-면역 반응성 뉴런들의 수보다 54.7배나 높았다. nNOS-또는 parvalbumin-면역 반응성 뉴런들은 크기와 형태, 분포 방식이 시각피질에서 유사하게 나타났다. 그러나 이색 면역형광 기법은 햄스터 시각피질에서 nNOS와 parvalbumin을 모두 발현하는 뉴런은 없음을 보여주었다. 본 연구의 결과는 nNOS와 칼슘 결합 단백질 사이의 co-localization양상이 종간에 차이가 존재함을 나타내며 또한 시각피질에 있는 nNOS-면역 반응성 뉴런들의 다양성과 이질성뿐만 아니라 동물 다양성 이해의 중요성을 함께 제시한다고 볼 수 있다.

• Molecules and Cells
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• 제19권3호
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• pp.375-381
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• 2005

Phospholipase C-${\beta}$ (PLC-${\beta}$) hydrolyses phosphatidylinositol 4,5-bisphosphate and generates inositol 1,4,5-trisphosphate in response to activation of various G protein-coupled receptors (GPCRs). Using glial cells from knock-out mice lacking either PLC-${\beta}1$ [PLC-${\beta}1$ (-/-)] or PLC-${\beta}3$ [PLC-${\beta}3$ (-/-)], we examined which isotype of PLC-${\beta}$ participated in the cellular signaling events triggered by thrombin. Generation of inositol phosphates (IPs) was enhanced by thrombin in PLC-${\beta}1$ (-/-) cells, but was negligible in PLC-${\beta}3$ (-/-) cells. Expression of PLC-${\beta}3$ in PLC-${\beta}3$ (-/-) cells resulted in an increase in pertussis toxin (PTx)-sensitive IPs in response to thrombin as well as to PAR1-specific peptide, while expression of PLC-${\beta}1$ in PLC-${\beta}1$ (-/-) cells did not have any effect on IP generation. The thrombin-induced $[Ca^{2+}]_i$ increase was delayed and attenuated in PLC-${\beta}3$ (-/-) cells, but normal in PLC-${\beta}1$ (-/-) cells. Pertussis toxin evoked a delayed $[Ca^{2+}]_i$ increase in PLC-${\beta}3$ (-/-) cells as well as in PLC-${\beta}1$ (-/-) cells. These results suggest that activation of PLC-${\beta}3$ by pertussis toxin-sensitive G proteins is responsible for the transient $[Ca^{2+}]_i$ increase in response to thrombin, whereas the delayed $[Ca^{2+}]_i$ increase may be due to activation of some other PLC, such as PLC-${\beta}4$, acting via PTx-insensitive G proteins.

• The Korean Journal of Physiology and Pharmacology
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• 제14권1호
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• pp.21-28
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• 2010

Phenolic compounds affect intracellular free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced $Ca^{2+}$ signaling in PC12 cells using fura-2-based digital $Ca^{2+}$ imaging and whole-cell patch clamping. Treatment with ATP ($100\;{\mu}M$) for 90 s induced increases in $[Ca^{2+}]_i$ in PC12 cells. Pretreatment with octyl gallate (100 nM to $20\;{\mu}M$) for 10 min inhibited the ATP-induced $[Ca^{2+}]_i$ response in a concentration-dependent manner ($IC_{50}=2.84\;{\mu}M$). Treatment with octyl gallate ($3\;{\mu}M$) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular $Ca^{2+}$ with nominally $Ca^{2+}$-free HEPES HBSS or depletion of intracellular $Ca^{2+}$ stores with thapsigargin ($1\;{\mu}M$). Treatment for 10 min with the L-type $Ca^{2+}$ channel antagonist nimodipine ($1\;{\mu}M$) significantly inhibited the ATP-induced $[Ca^{2+}]_i$ increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the $[Ca^{2+}]_i$ increase induced by 50 mM KCI. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein ($50\;{\mu}M$) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced $[Ca^{2+}]_i$ increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of $Ca^{2+}$ from extracellular space and P2Y receptor-induced release of $Ca^{2+}$ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced $Ca^{2+}$ responses by inhibiting the secondary activation of voltage-gated $Ca^{2+}$ channels.

• The Korean Journal of Physiology and Pharmacology
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• 제1권4호
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• pp.425-434
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• 1997

Many reports represent that angiotensin II (ANG II) caused a dose dependent biphasic effects on fluid transport in the proximal tubule. However, respective roles of different signaling pathways in mediating these effects remain unsettled. The aim of the present study was to examine signaling pathways at high doses of ANG II on the $Na^+$ uptake of primary cultured rabbit renal proximal tubule cells(PTCs) in hormonally defined serum-free medium. High concentrations of ANG II $(>10^{-9}\;M)$ inhibited $Na^+$ uptake and increased $[Ca^{2+}]_i\;level$ in the PTCs. However, low concentrations of $(<10^{-11}\;ANG\;II)$ stimulated $Na^+$ uptake and did not affect $[Ca^{2+}]_i\;level$. 8-(N, N-diethylamino)-octyl-3,3,5- trimethoxybenzoate (TMB-8), ethylene glycol-bis$({/beta}-amino\;ethyl ether)-N,N,N'$, N'-tetra acetic acid (EGTA), and nifedifine partially blocked the inhibitory effects of ANG II on $Na^+$ uptake. When ANG II and bradykinin (BK) were treated together, $Na^+$ uptake was further reduced $(88.47{\pm}1.98%\;of\;that\;of\;ANG\;II,\;81.85{\pm}1.84%\;of\;that\;of\;BK)$. In addition, W-7 and KN-62 blocked the ANG II-induced inhibition of $Na^+$ uptake. Arachidonic acid reduced $Na^+$ uptake in a dose-dependent manner. When ANG II and arachidonic acid were treated together, inhibitory effects on $Na^+$ uptake significantly exhibited greater reduction than that of each group, respectively. When PTCs were treated by mepacrine $(10^{-6}\;M)$ and AACOCF3 $(10^{-5}\;M)$ for 1 hr before the addition of $(<10^{-9}\;ANG\;II)$, the inhibitory effect of ANG II was reversed. In addition, econazole $(>10^{-6}\;M)$ blocked ANG II-induced inhibition of $Na^+$ uptake. In conclusion, the $[Ca^{2+}]_i$ (calcium-calmodulin-dependent kinase) and phospholipase $A_2\;(PLA_2)$ metabolites are involved in the inhibitory effects of ANG II on $Na^+$ uptake in the PTCs.

• Asian-Australasian Journal of Animal Sciences
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• 제32권12호
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• pp.1942-1949
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• 2019

Objective: Leukocyte cell-derived chemotaxin 2 (LECT2) is associated with several physiological processes including inflammation, tumorigenesis, and natural killer T cell generation. Chicken LECT2 (chLECT2) gene was originally identified as one of the differentially expressed genes in chicken kidney tissue, where the chickens were fed with different calcium doses. In this study, the molecular characteristics and gene expression of chLECT2 were analyzed under the stimulation of toll-like receptor 3 (TLR3) ligand to understand the involvement of chLECT2 expression in chicken metabolic disorders. Methods: Amino acid sequence of LECT2 proteins from various species including fowl, fish, and mammal were retrieved from the Ensembl database and subjected to Insilco analyses. In addition, the time- and dose-dependent expression of chLECT2 was examined in DF-1 cells which were stimulated with polyinosinic:polycytidylic acid (poly [I:C]), a TLR3 ligand. Further, to explore the transcription factors required for the transcription of chLECT2, DF-1 cells were treated with poly (I:C) in the presence or absence of the nuclear factor ${\kappa}B$ ($NF{\kappa}B$) and activated protein 1 (AP-1) inhibitors. Results: The amino acid sequence prediction of chLECT2 protein revealed that along with duck LECT2 (duLECT2), it has unique signal peptide different from other vertebrate orthologs, and only chLECT2 and duLECT2 have an additional 157 and 161 amino acids on their carboxyl terminus, respectively. Phylogenetic analysis suggested that chLECT2 is evolved from a common ancestor along with the actinopterygii hence, more closely related than to the mammals. Our quantitative polymerase chain reaction results showed that, the expression of chLECT2 was up-regulated significantly in DF-1 cells under the stimulation of poly (I:C) (p<0.05). However, in the presence of $NF{\kappa}B$ or AP-1 inhibitors, the expression of chLECT2 is suppressed suggesting that both $NF{\kappa}B$ and AP-1 transcription factors are required for the induction of chLECT2 expression. Conclusion: The present results suggest that chLECT2 gene might be a target gene of TLR3 signaling. For the future, the expression pattern or molecular mechanism of chLECT2 under stimulation of other innate immune receptors shall be studied. The protein function of chLECT2 will be more clearly understood if further investigation about the mechanism of LECT2 in TLR pathways is conducted.

• 생명과학회지
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• 제26권3호
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• pp.331-337
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• 2016

섬유모세포성장인자-23(fibroblast growth factor 23, FGF23)은 뼈를 형성하는 세포에서 주로 생성되지만 그 작용은 신장에서 이루어진다. FGF23은 신장의 나트륨-인산염 공동수송체(Na-phosphate cotransporter)를 억제하여 인산염 재흡수를 감소시킨다. 이렇게 함으로써 인산염 항상성을 조절하는 작용과는 별개로 이것은 in vivo에서 뼈 형성을 억제하는 것으로 알려져 있다. 두개골 조골세포를 이용한 연구에서도 FGF23은 조골세포의 발달, 즉 분화 및 기질의 광화(mineralization)에 악영향을 미쳤다. 본 연구는 FGF23이 골수 유래 간엽줄기세포에서 조골세포로의 발달에 있어서도 유사한 영향을 줄 것인지를 조사한 것이다. 간엽줄기세포주인 D1 세포를 β-glycerophosphate, ascorbic acid, dexamethazone이 포함된 조골배(osteogenic medium)에 배양하여 alkaline phosphatase (Alp) 염색으로 분화를, Alizarin red 염색과 기질의 칼슘 함량의 분석을 통해 광화를 평가하였다. 분화 촉진 유전자인 Runx2, osteocalcin, Alp와 광화 억제 유전자인 Enpp1, Ank의 발현은 RT-PCR로 분석하였다. D1 세포의 증식과 조골세포로의 분화는 생리학적 농도를 훨씬 초과하는 FGF23의 농도에 의해서도 달라지지 않았다. FGF23 처치 1주, 2주, 3주 후 Alizarin red 염색에 의한 광화 정도의 평가에서도 대조군과 실험군의 차이는 발견되지 않았다. 그러나 두 군 모두 시간이 경과함에 따라 광화는 증가되었다. 기질에 침착된 칼슘의 양 또한 차이가 없었다. 분화 촉진 유전자와 광화 억제 유전자의 발현도 양 군 간에 다르지 않았다. 이러한 부정적인(negative) 결과는 FGF23에 의한 세포 내 신호전달의 장애가 아님이 Erk 인산화로 확인되었다. 이상의 결과로 미루어 두개골의 조골세포와 달리 FGF23은 간엽줄기세포에서 조골세포로의 분화와 광화에는 영향을 미치지 않을 것으로 사료된다.

• Molecular & Cellular Toxicology
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• 제2권4호
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• pp.236-243
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• 2006

Microarray analysis of gene expression has become a powerful approach for exploring the biological effects of drugs, particularly at the stage of toxicology and safety assessment. Acetaminophen (APAP) has been known to induce necrosis in liver, but the molecular mechanism involved has not been fully understood. In this study, we investigated gene expression changes of APAP using microarray technology. APAP was orally administered with a single dose of 50 mg/kg or 500 mg/kg into ICR mice and the animals were sacrificed at 6, 24 and 72 h of APAP administration. Serum biochemical markers for liver toxicity were measured to estimate the maximal toxic time and hepatic gene expression was assessed using high-density oligonucleotide microarrays capable of determining the expression profile of >30,000 well-substantiated mouse genes. Significant alterations in gene expression were noted in the liver of APAP-administered mice. The most notable changes in APAP-administered mice were the expression of genes involved in apoptosis, cell cycle, and calcium signaling pathway, cystein metabolism, glutatione metabolism, and MAPK pathway. The majority of the genes upregulated included insulin-like growth factor binding protein 1, heme oxygenase 1, metallothionein 1, S100 calcium binding protein, caspase 4, and P21. The upregulation of apoptosis and cell cycle-related genes were paralleled to response to APAP. Most of the affected gene expressions were returned to control levels after 72 hr. In conclusion, we identified potential hepatotoxicity makers, and these expressions profiling lead to a better understanding of the molecular basis of APAP-induced hapatotoxicity.

• 생명과학회지
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• 제20권10호
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• pp.1451-1457
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• 2010

Serotonin (5-hydroxytryptamine (5-HT))는 신경계의 세포-세포 간의 신호전달의 주요한 신경전달물질이다. 세포막에 존재하는 serotonin transporter (SERT)는 연접간격에 존재하는 5-HT를 세포 내로 재흡수 하여 세포외부의 5-HT 농도를 조절하지만 그 기전은 아직 밝혀지지 않았다. 본 연구에서는 yeast two-hybrid system을 사용하여 SERT의 C-말단이 protein kinase C-$\varepsilon$ (PKC-$\varepsilon$)과 특이적으로 결합함을 알았다. PKC-$\varepsilon$는 PKC의 isotype으로 calcium 비의존적이며 phorbol ester/diacylglycerol 민감성 serine/threonine kinase이다. $Na^+/Cl^-$ 의존성 SLC6 gene family의 다른 수송체는 PKC-$\varepsilon$과 결합하지 않았다. Deletion mutant들을 사용하여 SERT는 PKC-$\varepsilon$의 C-말단부위와 결합함을 알았으며, 또한 이 단백질간의 결합을 GST pull-down assay로 확인하였다. PKC-$\varepsilon$는 in vitro에서 SERT의 N-말단의 펩티드를 인산화시켰다. 이러한 결과들은 PKC-$\varepsilon$에 의한 SERT의 인산화가 세포막에 존재하는 SERT의 활성을 조절하는 역할을 할 가능성을 시사한다.

• Biomolecules & Therapeutics
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• 제15권3호
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• pp.150-155
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• 2007

In the present study, we have tested the effect of dioleoyl phosphatidic acid (PA) on intracellular $Ca_{2+}$ concentration ($[Ca^{2+}]_{i}$) in two human colon cancer cell lines (HCT116 and HT29). PA and lysophosphatidic acid (LPA), a bioactive lysolipid, increased $[Ca^{2+}]_{i}$ in both HCT116 and HT29 cell lines. Increases of $[Ca^{2+}]_{i}$ by PA and LPA were more robust in HCT116 cells than in HT29 cells. A specific inhibitor of phospholipase C (U73122), however, was not inhibitory to the cell responses. Pertussis toxin, a specific inhibitor of $G_{i/o}$ type G proteins, however, had an inhibitory effect on the responses except for an LPA-induced one in HT29 cells. Ruthenium red, an inhibitor of the ryanodine receptor, was not inhibitory on the responses, however, 2-APB, a specific inhibitor of inositol 1,4,5-trisphosphate receptor, completely inhibited both lipid-induced $Ca^{2+}$ increases in both cell types. Furthermore, by using Ki16425 and VPC32183, two structurally dissimilar specific antagonists for $LPA_{1}/LPA_{3}$ receptors, an involvement of endogenous LPA receptors in the $Ca^{2+}$ responses was observed. Ki16425 completely inhibited the responses but the susceptibility to VPC32183 was different to PA and LPA in the two cell types. Expression levels of five LPA receptors in the HCT116 and HT29 cells were also assessed. Our data support the notion that PA could increase $[Ca^{2+}]_{i}$ in human colon cancer cells, probably via endogenous LPA receptors, G proteins and $IP_{3}$ receptors, thereby suggesting a role of PA as an intercellular lipid mediator.

• 생명과학회지
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• 제33권2호
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• pp.176-182
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• 2023

석곡의 줄기는 전통 동양의학에서 위를 보하고, 진액을 보충하며, 열을 내리는 것에 사용되어 왔다. 본 연구에서는 RBL-2H3 세포에서 비만세포 탈과립과 TNF-α, IL-4, histidine decarboxylase (HDC) 발현에 미치는 석곡 열수추출물(DME)의 효과를 조사하였다. DME는 PMA와 Calcium ionophore 병행처리(PMACI)에 의해 유도되는 β-hexosaminidase 분비와 TNF-α, IL-4, HDC 발현을 현저히 억제하였다. 또한 PMACI에 의해 유도되는 NF-κB, AP-1 활성과 p38 kinase, extracellular signal-regulated kinase 1/2 (ERK1/2)과 c-Jun N-terminal kinase (JNK)의 인산화가 DME 전처리에 의해 저해되었다. 이러한 결과들은DME가 비만세포 탈과립을 억제하고, MAPKs/NF-κB/AP-1 신호전달 경로를 통해 TNF-α, IL-4, HDC 발현을 억제한다는 것을 시사한다. 본 연구결과들로 보아 DME는 과민반응과 염증성 질환을 치료하는 약물로 개발될 가능성을 가지는 것으로 사료된다.