• 생명과학회지
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• 제27권11호
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• pp.1233-1244
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• 2017

Calcium-dependent protein kinases (CDPKs)는 칼슘 이온을 매개로 한 신호전달 경로에서 중요한 역할을 한다. 벼(Oryza sativa)에는 29개의 CDPKs가 확인되었지만 그들의 기능은 완벽히 밝혀지지 않았다. 이 연구는 OsCPK11 유전자에 초점을 맞춰 그것의 기능적인 특징을 조사하였다. 벼의 어린 잎, 성장한 잎, 꽃에서 OsCPK11 유전자의 조직-특이적 발현이 확인되었고, 지베렐린이 처리된 벼의 호분층에서도 이 유전자의 발현을 확인할 수 있었다. Tos-17이 삽입된 oscpk11의 표현형에서 돌연변이체 각각의 키는 야생형과 구분되지 않았지만, 영과의 수나 무게는 통계적으로 유의미한 차이가 있었다. 덧붙여 많은 돌연변이체 낟알의 배젖에서 white belly materials이 확인되었다. OsCPK11의 cDNA가 cloning되었고, 약 60.5 kD인 OsCPK11 단백질이 GST affinity chromatography와 SDS-PAGE에 의해 얻어졌다. 아미노산 서열 분석을 통해 OsCPK11이 전형적인 CDPKs의 구조적 특징을 가짐을 알 수 있었다. 이 결과는 OsCPK11유전자의 기능과 식물에서 칼슘 이온을 매개로 한 신호전달 경로에 CDPK 역할의 유용한 정보를 제공해줄 것이다.

• 대한약학회:학술대회논문집
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• 대한약학회 2001년도 Proceedings of International Convention of the Pharmaceutical Society of Korea
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• pp.109-113
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• 2001

Oxidized low-density lipoprotein (oxLDL) has been shown to modulate transactivations by the peroxisome proliferator activated receptor (PPAR)$\gamma$ and nuclear factor-kappa B (NF$\kappa$B). In this study, the oxLDL signaling pathways involved with the NF$\kappa$B transactivation were investigated by utilizing a reporter construct driven by three upstream NF$\kappa$B binding sites, and various pharmacological inhibitors. OxLDL and its constituent lysophophatidylcholine (lysoPC) induced a rapid and transient increase of intracellular calcium and stimulated the NF-KB transactivation in resting RAW264.7 macrophage cells in an oxidation-dependent manner. The NF$\kappa$B activation by oxLDL or lysoPC was inhibited by protein kinase C inhibitors or an intracellular calcium chelator. Tyrosine kinase or PI3 kinase inhibitors did not block the NF$\kappa$B transactivation. Furthermore, the oxLDL-induced NF$\kappa$B activity was abolished by the PPAR$\gamma$ ligands. When the endocytosis of oxLDL was blocked by cytochalasin B, the NF$\kappa$B transactivation by oxLDL was synergistically increased, while PPAR transactivation was blocked. These results suggest that oxLDL activates NF-$\kappa$B in resting macrophages via protein kinase C- and/or calcium-dependent pathways, which does not involve the endocytic processing of oxLDL. The endocytosis-dependent PPAR$\gamma$ activation by oxLDL may function as an inactivation route of the oxLDL induced NF$\kappa$B signal. Short heterodimer partner (SHP), specifically expressed in liver and a limited number of other tissues, is an unusual orphan nuclear receptor that lacks the conventional DNA-binding domain. In this work, we found that SHP expression is abundant in murine macrophage cell line RAW 264.7 but suppressed by oxLDL and its constituent I3-HODE, a ligand for peroxisome proliferator-activated receptor y. Furthermore, SHP acted as a transcription coactivator of nuclear factor-$\kappa$B (NF$\kappa$B) and was essential for the previously described NF$\kappa$B transactivation by lysoPC, one of the oxLDL constituents. Accordingly, NF$\kappa$B, transcriptionally active in the beginning, became progressively inert in oxLDL-treated RAW 264.7 cells, as oxLDL decreased the SHP expression. Thus, SHP appears to be an important modulatory component to regulate the transcriptional activities of NF$\kappa$B in oxLDL-treated, resting macrophage cells.

• The Korean Journal of Physiology and Pharmacology
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• 제8권1호
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• pp.57-63
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• 2004

Fluoxetine, a widely used anti-depressant compound, has several additional effects, including blockade of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells by using fura-2-based digital calcium imaging and assay for $[^3H]-inositol$ phosphates (IPs). Treatment with ATP $(100\;{\mu}M)$ for 2 min induced $[Ca^{2+}]_i$ increases. The ATP-induced $[Ca^{2+}]_i$ increases were significantly decreased by removal of extracellular $Ca^{2+}$ and treatment with the inhibitor of endoplasmic reticulum $Ca^{2+}$ ATPase thapsigargin $(1\;{\mu}M)$. Treatment with fluoxetine for 5 min blocked the ATP-induced $[Ca^{2+}]_i$ increase concentration-dependently. Treatment with fluoxetine $(30\;{\mu}M)$ for 5 min blocked the ATP-induced $[Ca^{2+}]_i$ increase following removal of extracellular $Ca^{2+}$ and depletion of intracellular $Ca^{2+}$ stores. While treatment with the L-type $Ca^{2+}$ channel antagonist nimodipine for 10 min inhibited the ATP-induced $[Ca^{2+}]_i$ increases significantly, treatment with fluoxetine alone blocked the ATP-induced responses. Treatment with fluoxetine also inhibited the 50 mM $K^+-induced$ $[Ca^{2+}]_i$ increases completely. However, treatment with fluoxetine did not inhibit the ATP-induced $[^3H]-IPs$ formation. Collectively, we conclude that fluoxetine inhibits ATP-indueed $[Ca^{2+}]_i$ increases in PC12 cells by inhibiting both an influx of extracellular $Ca^{2+}$ and a release of $Ca^{2+}$ from intracellular stores without affecting IPs formation.

• Biomolecules & Therapeutics
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• 제23권5호
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• pp.421-427
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• 2015

Imperatorin has been known to exert many biological functions including anti-inflammatory activity. In this study, we investigated the inhibitory effects of imperatorin on the production of inflammatory mediators in mouse bone marrow-derived mast cells (BMMC). Imperatorin inhibited degranulation and the generation of eicosanoids (leukotriene $C_4$ ($LTC_4$) and prostaglandin $D_2$ ($PGD_2$) in IgE/antigen (Ag)-stimulated BMMC. To elucidate the molecular mechanism involved in this process, we investigated the effect of imperatorin on intracellular signaling in BMMC. Biochemical analyses of the IgE/Ag-mediated signaling pathway demonstrated that imperatorin dramatically attenuated degranulation and the production of 5-lipoxygenase-dependent $LTC_4$ and cyclooxygenase-2-dependent $PGD_2$ through the inhibition of intracellular calcium influx/phospholipase $C{\gamma}1$, cytosolic phospholipase $A_2$/mitogen-activated protein kinases and/or nuclear factor-${\kappa}B$ pathways in BMMC. These results suggest that the effects of imperatorin on inhibition of degranulation and eicosanoid generation through the suppression of multiple steps of IgE/Ag-mediated signaling pathways would be beneficial for the prevention of allergic inflammation.

• The Korean Journal of Physiology and Pharmacology
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• 제12권5호
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• pp.267-274
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• 2008

Since first discovered in chick skeletal muscles, stretch-activated channels (SACs) have been proposed as a probable mechano-transducer of the mechanical stimulus at the cellular level. Channel properties have been studied in both the single-channel and the whole-cell level. There is growing evidence to indicate that major stretch-induced changes in electrical activity are mediated by activation of these channels. We aimed to investigate the mechanism of stretch-induced automaticity by exploiting a recent mathematical model of rat atrial myocytes which had been established to reproduce cellular activities such as the action potential, $Ca^{2+}$ transients, and contractile force. The incorporation of SACs into the mathematical model, based on experimental results, successfully reproduced the repetitive firing of spontaneous action potentials by stretch. The induced automaticity was composed of two phases. The early phase was driven by increased background conductance of voltage-gated $Na^+$ channel, whereas the later phase was driven by the reverse-mode operation of $Na^+/Ca^{2+}$ exchange current secondary to the accumulation of $Na^+$ and $Ca^{2+}$ through SACs. These results of simulation successfully demonstrate how the SACs can induce automaticity in a single atrial myocyte which may act as a focus to initiate and maintain atrial fibrillation in concert with other arrhythmogenic changes in the heart.

• Journal of Ginseng Research
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• 제45권4호
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• pp.490-497
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• 2021

Background and objective: Synthetic ginsenoside compounds G-Rp (1,3, and 4) and natural ginsenosides in Panax ginseng 20(S)-Rg3, Rg6, F4 and Ro have inhibitory actions on human platelets. However, the inhibitory mechanism of ginsenoside Rk1 (G-Rk1) is still unclear thus, we initiated investigation of the anti-platelet mechanism by G-Rk1 from Panax ginseng. Methodology: Our study focused to investigate the action of G-Rk1 on agonist-stimulated human platelet aggregation, inhibition of platelet signaling molecules such as fibrinogen binding with integrin α_IIbβ₃ using flow cytometry, intracellular calcium mobilization, fibronectin adhesion, dense granule secretion, and thromboxane B2 secretion. Thrombin-induced clot retraction was also observed in human platelets. Key Results: Collagen, thrombin, and U46619-stimulated human platelet aggregation were dose-dependently inhibited by G-Rk1, while it demonstrated a more effective suppression on collagen-stimulated platelet aggregation using human platelets. Moreover, G-Rk1 suppressed collagen-induced elevation of Ca²⁺ release from endoplasmic reticulum, granule release, and α_IIbβ₃ activity without any cytotoxicity. Conclusions and implications: These results indicate that G-Rk1 possess strong anti-platelet effect, proposing a new drug candidate for treatment and prevention of platelet-mediated thrombosis in cardiovascular disease.

• International Journal of Oral Biology
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• 제47권3호
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• pp.33-40
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• 2022

Store-operated Ca²⁺ entry (SOCE) represents one of the major Ca²⁺ entry routes in non-excitable cells. It is involved in a variety of fundamental biological processes and the maintenance of Ca²⁺ homeostasis. The Ca²⁺ release-activated Ca²⁺ (CRAC) channel consists of stromal interaction molecule and Orai; however, the role and action of Homer proteins as an adaptor protein to SOCE-mediated Ca²⁺ signaling through the activation of CRAC channels in non-excitable cells still remain unknown. In the present study, we investigated the role of Homer2 in the process of Ca²⁺ signaling induced by the interaction between CRACs and Homer2 proteins in non-excitable cells. The response to Ca²⁺ entry by thapsigargin-mediated Ca²⁺ store depletion remarkably decreased in pancreatic acinar cells of Homer2^-/- mice, as compared to wild-type cells. It also showed critical differences in regulated patterns by the specific blockers of SOCE in pancreatic acinar cells of Homer2^-/- mice. The response to Ca²⁺ entry by the depletion in Ca²⁺ store markedly increased in the cellular overexpression of Orai1 and STIM1 as compared to the overexpression of Homer2 in cells; however, this response was remarkably inhibited by the overexpression of Orai1, STIM1, and Homer2. These results suggest that Homer2 has a critical role in the regulatory action of SOCE activity and the interactions between CRAC channels.

• Parasites, Hosts and Diseases
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• 제48권4호
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• pp.319-324
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• 2010

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4-EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in Echerichia coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.

• BMB Reports
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• 제51권6호
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• pp.296-301
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• 2018

Mitochondrial DNA (mtDNA) mutations are often observed in various cancer types. Although the correlation between mitochondrial dysfunction and cancer malignancy has been demonstrated by several studies, further research is required to elucidate the molecular mechanisms underlying accelerated tumor development and progression due to mitochondrial mutations. We generated an mtDNA-depleted cell line, ${\rho}^0$, via long-term ethidium bromide treatment to define the molecular mechanisms of tumor malignancy induced by mitochondrial dysfunction. Mitochondrial dysfunction in ${\rho}^0$ cells reduced drug-induced cell death and decreased the expression of pro-apoptotic proteins including p53. The p53 expression was reduced by activation of nuclear $factor-{\kappa}B$ that depended on elevated levels of free calcium in $HCT116/{\rho}^0$ cells. Overall, these data provide a novel mechanism for tumor development and drug resistance due to mitochondrial dysfunction.

• Molecules and Cells
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• 제20권3호
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• pp.305-314
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• 2005

Investigation of chemically synthesized inositol 1,4,5-trisphosphate [$Ins(1,4,5)P_3$] analogs has led to the isolation of a novel binding protein with a molecular size of 130 kDa, characterized as a molecule with similar domain organization to phospholipase C-${\delta}1$ (PLC-${\delta}1$) but lacking the enzymatic activity. An isoform of the molecule was subsequently identified, and these molecules have been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and -2. Regarding its ability to bind $Ins(1,4,5)P_3$ via the pleckstrin homology domain, the involvement of PRIP-1 in $Ins(1,4,5)P_3$-mediated $Ca^{2+}$ signaling was examined using COS-1 cells overexpressing PRIP-1 and cultured neurons prepared from PRIP-1 knock-out mice. Yeast two hybrid screening of a brain cDNA library using a unique N-terminus as bait identified GABARAP ($GABA_A$ receptor associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible involvement of PRIP in $GABA_A$ receptor signaling. For this purpose PRIP knock-out mice were analyzed for $GABA_A$ receptor function in relation to the action of benzodiazepines from the electrophysiological and behavioral aspects. During the course of these experiments we found that PRIP also binds to the b-subunit of $GABA_A$ receptors and PP2A (protein phosphtase 2A). Here, we summarize how PRIP is involved in $Ins(1,4,5)P_3$-mediated $Ca^{2+}$ signaling and $GABA_A$ receptor signaling based on the characteristics of binding molecules.