• The Korean Journal of Physiology and Pharmacology
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• 제19권6호
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• pp.549-555
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• 2015

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors /PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.

• BMB Reports
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• 제44권3호
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• pp.205-210
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• 2011

Insulin has antiapoptotic activity in various cell types. However, the signaling pathways underlying the antiapoptotic activity of insulin is not yet known. This study was conducted to determine if cAMP affects the antiapoptotic activity of insulin and the activity of PI3K and ERK in CHO cells expressing human insulin receptors (CHO-IR). Insulin-stimulated ERK activity was completely suppressed by cAMP-elevating agents like as pertussis toxin (Ptx) and cholera toxin (Ctx) after 4 h treatment. Insulin-stimulated PKB/Akt activity was not affected at all. Ptx treatment together with insulin increased the number of apoptotic cells and the degree of DNA fragmentation. Ctx or 8-br-cAMP treatment also increased the number of apoptotic cells and stimulated the cleavage of caspase-3 and the hydrolysis of PARP. Taken together, cAMP antagonizes the antiapoptotic activity of insulin and the main target molecule of cAMP in this process is likely ERK, not PI3K-dependent PKB/Akt.

• International Journal of Oral Biology
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• 제33권4호
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• pp.217-221
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• 2008

Bicuculline is one of the most commonly used $GABA_A$ receptor antagonists in electrophysiological research. Because of its poor water solubility, bicuculline quaternary ammonium salts such as bicuculline methiodide (BMI) and bicuculline methbromide are preferred. However, a number of studies have shown that BMI has non-$GABA_A$ receptor-mediated effects. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is implicated in the processing of nociceptive signaling. In this study, we investigated whether BMI has non-GABA receptor-mediated activity in Vc SG neurons using a whole cell patch clamp technique. SG neurons were depolarized by application of BMI ($20{\mu}M$) using a high $Cl^-$ pipette solution. GABA ($30-100{\mu}M$) also induced membrane depolarization of SG neuron. Although BMI is known to be a $GABA_A$ receptor antagonist, GABA-induced membrane depolarization was enhanced by co-application with BMI. However, free base bicuculline (fBIC) and picrotoxin (PTX), a $GABA_A$ and $GABA_C$ receptor antagonist, blocked the GABA-induced response. Furthermore, BMI-induced membrane depolarization persisted in the presence of PTX or an antagonist cocktail consisting of tetrodotoxin ($Na^+$ channel blocker), AP-5 (NMDA receptor antagonist), CNQX (non-NMDA receptor antagonist), and strychnine (glycine receptor antagonist). Thus BMI induces membrane depolarization by directly acting on postsynaptic Vc SG neurons in a manner which is independent of $GABA_A$ receptors. These results suggest that other unknown mechanisms may be involved in BMI-induced membrane depolarization.

• Radiation Oncology Journal
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• 제14권4호
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• pp.281-290
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• 1996

목적 : 흉부방사선조사시 심장이 포함됨으로써 다양한 정도의 심장손상이 발생되고 이로써 방사선치료의 효과를 감소시키게 된다. 따라서 이들의 발생빈도를 감소시키기 위해 여러방법이 시도되었으며 이중 혈류장애 개선제로 알려진 pentoxifylline (3,7-dimethyl-1(5-oxyhexyl-xanthine)) (이하 PTX로 약함)을 방사선조사와 동시에 투여하여 심장에서의 방사선손상을 감소시키고자 하였으며 이의 효과는 병리조직학적 검사를 통해 관찰하였다. 대상 및 방법 : ICR마우스 수컷 180마리를 각각 대조군, 방사선조사단독군, 방사선조사와 PTX투여군으로 구분하고 각 군은 다시 방사선조사후의 기간에 따라 1, 3, 6, 10일과 2, 3, 4, 6, 8, 12, 16, 20주의 12개 소군으로 나누었다. 방사선조사는 4 MV 선형가속기를 이용하여 15Gy를 일회로 심장을 포함한 흉부에 시행하였고 PTX투여는 1일 50mg/kg을 방사선조사 다음날부터 실험완료시까지 매일 배부의 좌우측에 번갈아 피하주사 하였다. 일정한 관찰기간후 심장을 절제 하여 병리학적 검사를 시행하였고 이들의 변화를 급성과 만성으로 나누어 병변의 정도에 따라 각각 4등급으로 분류한 후 합산하여 점수화하였다. 이들 결과는 Student's t-test를 이용하여 각군의 관찰기간에 따른 변화와 각 군간의 병리소견상 변화의 차이점에 대해 통계적 유의성이 검증되었다. 결과 : 방사선조사가 시행되지 않은 대조군은 비특이성심근염외에는 별 특이한 소견을 보여주지 않았다. 방사선조사후의 급성변화는 이염성백혈구성 심낭염과 혈관내피세포의 공포화 및 풍선화가 있었고 만성기 심장손상의 변화로는 단핵세포성 심낭염, 심낭섬유화, 혈관내피세포변화, 심근의 퇴행성 변화와 심근섬유화등이 관찰되었다. 심장손상의 병리학적 소견을 각 군에 따라 점수화하여 비교한 결과 급성변화에서는 방사선조사단독군이 1일-6주에 2-4점을, PTX투여군이 1-3점의 분포를 보여 주었다. 이들 변화는 각 군 모두 3주까지 유사하였으며 4주이후부터 PTX투여군의 변화소견이 방사선조사단독군에 비해 빨리 회복됨을 보여 주었고 이는 통계학적으로 의미있는 소견임이 확인 되었다. 만성심장손상소견은 방사선조사단독군이 4-8주에 5-6점의 변화소견을 보였고 이후 12주에 심근의 섬유화현상시작으로 7-9점의 병리소견을 보였으며 PTX투여군은 4-12주에 이르기까지 1-4점을 보인후 16주의 심근섬유화현상으로 5-6점의 소견을 보여주었고 이들 군간의 변화정도의 차이는 통계학적으로 유의성이 있음이 확인 되었다. 결론 : PTX투여 결과 급성 방사선심장손상의 병리학적 변화정도는 유사하였으나 빨리 회복되는 추세가 관찰되었고 만성 방사선심장손상에서는 방사선조사단독군에 비해 심근의 퇴행성 변화 및 섬유화정도의 의미 있는 억제 및 감소효과를 보여주었다.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.227.2-228
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• 2002

ZD1839 is a new anticancer agent which selectively inhibits EGFR tyrosine kinase. Oxaliplatin (LOHP), 5-FU (FU), and paclitaxel (PTX) have shown to be highly active against the gastric carcinomas. and ZD1839 is considered as a good candidate for the treatment of gastric cancers when combined with cytotoxic agents. In this study, we evaluated the antitumor effects of these agents in SNU-l human gastric cancer cells either alone or when given as a doublet. (omitted)

• 한국식품과학회지
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• 제41권4호
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• pp.458-463
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• 2009

설사성패류독의 신속정밀 분석조건 확립을 위하여 LC-MS/MS를 사용하여 이동상, 분석용 column 및 collision energy 등을 변화시키면서 시험하였다. 50 mM formic acid와 2 mM ammonium formate가 함유된 acetonitrile 수용액을 이동상으로 사용하였을 때 OA와 DTX1이 검출되었다. Collision energy는 독소 성분에 따라 달리하는 것이 다성분 동시분석에 적합하였으며 OA와 DTX1 고유의 fragment ion들은 48 V 정도에서 최적의 intensity로 확인되었다. Column의 종류에 따라서는 $C_8$ column의 경우 OA, DTX1, DTX3, PTX2 및 YTX 모두 검출 가능하였으나 실제 검출 대상이 OA와 DTX1인 경우에는 일반적으로 사용되는 $C_{18}$ column도 적합한 것으로 확인되었다. 본 연구에서 확립한 LC-MS/MS 분석 조건의 검출한계는 OA와 DTX1 모두 1 ng/g, 정량한계는 각각 3 ng/g이었고, 표준독 성분을 첨가한 시료에서 process efficiency는 굴의 경우 91-118%, 진주담치에서는 96-117%이었고, matrix의 영향은 거의 없었다. 마우스 시험에서 양성을 나타낸 시료를 LCMS/MS법으로 분석한 결과, 일부 시료에서만 OA 및 DTX1이 검출되어 두 시험법의 독성은 일치하지 않았으며 LC-MS/MS법은 마우스 시험법보다 하루 이상 분석시간을 단축할 수 있었다.

• Biomolecules & Therapeutics
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• 제26권6호
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• pp.546-552
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• 2018

A comprehensive collection of proteins senses local changes in intracellular $Ca^{2+}$ concentrations ($[Ca^{2+}]_i$) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular $Ca^{2+}$ concentrations in cat esophageal smooth muscle cells. To measure $[Ca^{2+}]_i$ levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in $[Ca^{2+}]_i$ in the cells. Pretreatment with EGTA, an extracellular $Ca^{2+}$ chelator, decreased the S1P-induced increase in $[Ca^{2+}]_i$, and an L-type $Ca^{2+}$-channel blocker, nimodipine, decreased the effect of S1P. This indicates that $Ca^{2+}$ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an $InsP_3$ receptor blocker, the S1P-evoked increase in $[Ca^{2+}]_i$ was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of $G_i$-protein, suppressed the increase in $[Ca^{2+}]_i$ evoked by S1P. These results suggest that the S1P-induced increase in $[Ca^{2+}]_i$ in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of $Ca^{2+}$ from the $InsP_3$-sensitive $Ca^{2+}$ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular $Ca^{2+}$ via the L type $Ca^{2+}$ channel, which was dependent on activation of the $S1P_4$ receptor coupled to PTX-sensitive $G_i$ protein, via phospholipase C-mediated $Ca^{2+}$ release from the $InsP_3$-sensitive $Ca^{2+}$ pool in cat esophageal smooth muscle cells.

• The Korean Journal of Physiology and Pharmacology
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• 제7권6호
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• pp.349-355
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• 2003

We previously shown that LES contraction depends on $M_3$ receptors linked to PTX insensitive $G_q$ protein and activation of PLC. This results in production of $IP_3$, which mediates calcium release, and contraction through a CaM dependent pathway. In the esophagus ACh activates $M_2$ receptors linked to PTX sensitive $G_{i3}$ protein, resulting in activation of PLD, presumably, production of DAG. We investigated the role of PLC isozymes which can be activated by $G_q$ or $G{\beta}$ protein on ACh-induced contraction in LES and esophagus. Immunoblot analysis showed the presence of 3 types of PLC isozymes, $PLC-{\beta}1$, $PLC-{\beta}3$, and $PLC-{\gamma}1$, but not $PLC-{\beta}2$, $PLC-{\beta}4$, $PLC-{\gamma}2$, $PLC-{\delta}1$, and $PLC-{\delta}2$ from both LES and esophageal muscle. ACh produced contraction in a dose dependent manner in LES and esophageal muscle cells obtained by enzymatic digestion with collagenase. $PLC-{\beta}1$ or $PLC-{\beta}3$ antibody incubation reduced contraction in response to ACh in LES but not in esophageal permeabilized cells, but $PLC-{\gamma}1$ antibody incubation did not have an inhibitory effect. The inhibition by $PLC-{\beta}1$ or $PLC-{\beta}3$ antibody on Ach-induced contraction was antibody concentration dependent. The combination with $PLC-{\beta}_1$ and $PLC-{\beta}_3$ antibody completely abolished the contraction, suggesting that $PLC-{\beta}1$ and $PLC-{\beta}3$ have a synergism to inhibit the contraction in LES. $PLC-{\beta}1$, -${\beta}3$ or -${\gamma}1$ antibody did not reduce the contraction of LES cells in response to DAG ($10^{-6}$ M), suggesting that this isozyme of PLC may not activate PKC. When $G_{q/11}$ antibody was incubated, the inhibitory effect of the incubation of PLC ${\beta}3$, but not of PLC ${\beta}_1$ was additive (Fig. 6). In contrast, when $G_{\beta}$ antibody was incubated, the inhibitory effect of the incubation of PLC ${\beta}_1$, but not of PLC ${\beta}_3$ was additive. This data suggest that $G_{q/11}$/11 or $G{\beta}$ may activate cooperatively different PLC isozyme, $PLC{\beta}_1$ or $PLC{\beta}_3$ respectively.

• 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.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2000년도 제18회 학술발표회 논문개요집
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• pp.164-164
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• 2000

Since Au-Pt alloys have various atomic structures depending upon composition and annealing temperature, it is very interesting to investigate the electronic structures of alloys. We studied the changes of the electronic structure I the Au-Pt alloys by x-ray absorption near edge spectroscopy (XANES). Two kinds of Au-Pt alloy samples were prepared by arc melting methods and ion-beam-mixing technique. The Pt L2, 3-edge and Au L2, 3-edge X-ray absorption spectra (XPS) were measured with the electron yield mode detector at the 3C1 beam line of the Pohang Light Source (PLS). It was found that there was a substantial decrease in the area of the Pt L2, 3 white lines compared with that of pure Pt. The observed decrease in white line area was attributed to an increase in the number of pure Pt. The observed decrease in white line area was attributed to an increase in the number of 5d-electrons at the Pt site upon alloy formation. However, the Au L2, 3 edge spectra for Au-Pt alloys are all similar to that of pure Au. This implies that the 5d hole count of Au is not changed by alloy formation with Pt.