• 한국어병학회지
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• 제20권2호
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• pp.119-127
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• 2007

Phosphoinositide-specific phospholipase Cδ (PLCδ) plays an important role in many cellular responses and is involved in the production of second messenger. The present study was conducted to characterize the catalytic and regulatory properties of the PLCδ of Misgurnus mizolepis (ML-PLCδ). The ML-PLCδ gene was cloned and expressed under according to the method of the previous report (Kim et al., 2004), and its recombinant protein was purified by successive chromatography using Ni2+-NTA affinity column. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol (PI). Its activity was absolutely Ca2+-dependence, which was similar to mammalian PLCδ isozymes. The Ca2+ concentration yielding maximal activation of ML-PLCδ was 100 μM. However, the activity was decreased interestingly by a polyamine, such as spermine and spermidine. In vitro assay using cholate-micelle cell, ML-PLCδ activity was inhibited in dose-dependent manner by sphinogosine but increased by phosphocholine . In the lipid-binding assay, ML-PLCδ was strongly bound to LPA, PI(3)P, PI(4)P, PI(5)P, PI(3,5)P2, PI(4,5)P2, PI(3,4,5)P3 and PA, but it showed the low affinity to S1P, PI(3,4)P2 and PS. Taken together our results, it is suggested that the general catalytic and regulatory properties of ML-PLCδ are similar with those of mammalian PLCδ1 isozymes, but the N-terminal extended piscine phospholipase Cδ1 (ML-PLCδ) might reflect some distinctions in regulatory properties and inositol-lipid binding specificity between piscine ML-PLCδ and mammalian PLCδ isozymes.

• 대한의생명과학회지
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• 제12권4호
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• pp.419-425
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• 2006

Phospholipase $C-{\gamma}1\;(PLC-{\gamma}1)$ is an important signaling molecule for cell proliferation and differentiation. $PLC-{\gamma}1$ contains two pleckstrin homology (PH) domains, which are responsible for protein-protein interaction and protein-lipid interaction. $PLC-{\gamma}1$ also has two Src homology (SH)2 domains and a SH3 domain, which are responsible for protein- protein interaction. To identity proteins that specifically binds to PH domain of $PLC-{\gamma}1$, we prepared and incubated the glutathione S-transferase(GST)-fused PH domains of $PLC-{\gamma}1$ with COS7 cell lysate. We found that 90 kDa protein specifically binds to PH domain of $PLC-{\gamma}1$. By matrix-assisted laser desorption ionization time of flight-mass spectrometry, the 90 kDa protein revealed to be heat shock protein (Hsp) $90{\beta}$. Hsp $90{\beta}$ is a molecular chaperone that stabilizes and facilitates the folding of proteins that are involved in cell signaling, including receptors for steroids hormones and a variety of protein kinases. To know whether Hsp $90{\beta}$ affects on $PLC-{\gamma}1$ activity, we performed $PIP_2$ hydrolyzing activity of $PLC-{\gamma}1$ in the presence of purified Hsp $90{\beta}$ in vitro. Our results show that the Hsp $90{\beta}$ dose-dependently inhibits the enzymatic activity of $PLC-{\gamma}1$ and further suggest that Hsp $90{\beta}$ regulates cell growth and differentiation via regulation of $PLC-{\gamma}1$ activity.

• 대한수의학회지
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• 제45권4호
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• pp.507-515
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• 2005

Melatonin, the principal hormone of the vertebral pineal gland, participates in the regulation of cardiovascular system in vitro and in vivo. However, the effects of melatonin on vascular tissues are still vague. The aim of this study was to assess the relationship between phospholipase C (PLC) and nitric oxide synthase (NOS)/cyclic guanosine 3',5'-monophosphate (cGMP) signaling cascade in the relaxatory action of melatonin in isolated rat aorta. Melatonin induced a concentration-dependent relaxation in phenylephrine (PE)- and KCl-precontracted endothelium intact (+E) aortic rings. In KCl-precontracted +E aortic rings, the melatonin-induced vasorelaxation was not inhibited by endothelium removal or by pretreatment with NOS inhibitors, L-$N^G$-nitor-arginine (L-NNA) and L-$N^G$-nitor-arginine methyl ester (L-NAME), guanylate cyclase (GC) inhibitors, methylene blue (MB) and 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (ODQ). In PE-precontracted +E aortic rings, the melatonin-induced vasorelaxation was inhibited by endothelium removal or by pretreatment with L-NNA, L-NAME, MB, ODQ and 2-nitro-4-carboxyphenyl-n,n-diphenylcarbamate (NCDC). Moreover, in without endothelium (-E) aortic rings and in the presence of L-NNA, L-NAME, MB and ODQ in +E aortic rings, the melatonin-induced residual relaxations and residual contractile responses to PE were not affected by NCDC, a PLC inhibitor. It is concluded that melatonin can evoke vasorelaxation due to inhibition of PLC pathway through the protein kinase G activation of endothelial NOS/cGMP signaling cascade.

• Bulletin of the Korean Chemical Society
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• 제35권1호
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• pp.250-252
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• 2014

Lipoprotein-associated phospholipase A2 (Lp-$PLA_2$) is a crucial enzyme in atherosclerosis as a potential drug target. The most remarkable Lp-$PLA_2$ inhibitory drug is Darapladib. We determined the binding pose of Darapladib to Lp-$PLA_2$ through docking study. Darapladib formed two hydrogen bonding interactions with the side chain of Tyr160 and Gln352 and several pi-pi interactions with aromatic and aliphatic hydrophobic residues of Lp-$PLA_2$. It is known that the dietylpropan-amine moiety of Darapladib has influence on the improvement of its oral bioavailability and we supposed this in our docking results.

• BMB Reports
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• 제41권8호
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• pp.560-567
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• 2008

The importance of lipids in cell signaling and tissue physiology is demonstrated by the many CNS pathologies involving deregulated lipid metabolism. One such critical metabolic event is the activation of phospholipase $A_2$ ($PLA_2$), which results in the hydrolysis of membrane phospholipids and the release of free fatty acids, including arachidonic acid, a precursor for essential cell-signaling eicosanoids. Reactive oxygen species (ROS, a product of arachidonic acid metabolism) react with cellular lipids to generate lipid peroxides, which are degraded to reactive aldehydes (oxidized phospholipid, 4-hydroxynonenal, and acrolein) that bind covalently to proteins, thereby altering their function and inducing cellular damage. Dissecting the contribution of $PLA_2$ to lipid peroxidation in CNS injury and disorders is a challenging proposition due to the multiple forms of $PLA_2$, the diverse sources of ROS, and the lack of specific $PLA_2$ inhibitors. In this review, we summarize the role of $PLA_2$ in CNS pathologies, including stroke, spinal cord injury, Alzheimer's, Parkinson's, Multiple sclerosis-Experimental autoimmune encephalomyelitis and Wallerian degeneration.

• BMB Reports
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• 제43권9호
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• pp.604-608
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• 2010

Tumor necrosis factor-$\alpha$ (TNF-$\alpha$) mediates proinflammatory responses in primary human umbilical vein endothelial cells (HUVECs), and it upregulates the expression of secretory group IIA phospholipase $A_2$ ($sPLA_2$-IIA). $sPLA_2$-IIA plays a pivotal role in inflammation, and antithrombin (AT) possesses properties that are beneficial to endothelial cells. Therefore, we investigated the effects of AT on the expression of $sPLA_2$-IIA in TNF-$\alpha$-stimulated HUVECs. TNF-$\alpha$ potently upregulated the expression of $sPLA_2$-IIA, and prior treatment of cells with AT inhibited the expression of $sPLA_2$-IIA in HUVECs. Also, antibodies or siRNA for syndecan-4 blocked the protective effect of AT. Furthermore, PI3-kinase and the AKT pathway are significantly involved in the AT-mediated inhibition of the expression of $sPLA_2$-IIA. These results show that AT effectively suppresses the upregulated $sPLA_2$-IIA expression, which might contribute to the cytoprotective effects of AT in the treatment of severe inflammatory diseases.

• Toxicological Research
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• 제31권1호
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• pp.33-40
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• 2015

Bee venom (BV) has long been used in traditional Eastern and Western medicine for chronic inflammation, pain and skin therapy. Human exposure to BV, however, often causes unwanted adverse effects and is even fatal in some cases. Phospholipase $A_2$ ($PLA_2$) of BV is now suspected to play a key role in these adverse effects. We investigated the potential use of $PLA_2$-free bee venom (PBV) as a replacement for BV in cosmetic products. PBV prepared by molecular weight cut-off ultrafiltration exhibits a superior profile in comparison with regular BV, by inhibiting elastase activity and suppressing the induction of nitric oxide (NO) and metalloproteinase-9 (MMP-9), while retaining the effects of cell proliferation and protection against ultraviolet B (UVB)-induced damage in human dermal fibroblast cells. PBV thus appears to be more promising than BV as a cosmetic ingredient with a reduced potential for adverse reactions in the recipient.

• Bulletin of the Korean Chemical Society
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• 제10권6호
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• pp.595-599
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• 1989

The effects of phosphatidic acid(PA) on the activity of phospholipase D were examined in detail. The enzyme activity was examined in the liposome system containing phosphatidylcholine and PA, which was suspended in a desired buffer solution by ultrasonication. The substrate of large unilamella vesicle (LUV) state by ultrasonication was more effective on the enzyme activity than that of multilamella vesicle(MLV) by water-bath type sonication. The most effective molar ratio of PC-PA liposome for enzyme activity was found to be 1:0.7. The other optimum conditions were found 5 mM $Ca^{2+}$ ion, pH 6.6, and incubation temperature of $27^{\circ}C. K_m \;and \;V_{max}$ values were estimated to be 1.43 mM and 0.8 $nmole/min/{\mu}g$ protein respectively. These properties in a PC-PA liposome system were compared with those in a PC-SDS mixed micelle system. The effects of other phospholipids and organic phosphates on the enzyme activity were also examined.

• 생약학회지
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• 제34권1호통권132호
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• pp.100-108
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• 2003

The regulation of plasma lipid level, particularly LDL cholesterol, represents the focus of current therapy for atherosclerosis. And $Lp-PLA_2$ is able to hydrolyse oxidized phosphatidylcholine within LDL into lyso-PC and oxidized fatty acids. $Lp-PLA_2$ is a potential biomarker of coronary heart disease and plays an important proinflammatory role in the progression of atherosclerosis. We investigated the inhibitory effects of methanol extracts of 224 natural plants on $Lp-PLA_2$ activity. Seven kinds of methanol extracts of tested plants showed above 50% inhibitory effect with the concentration of $100\;{\mu}g/ml$. The concentrated aqueous suspensions of each methanol extract were partitioned with n-hexane, $CHCl_3$, and EtOAc. Among them, EtOAc extracts of Astilbe chinensis var. davidii (root) and Pourthiaea villosa var. brunnea (leaf) significantly inhibited $Lp-PLA_2$ activity at the same concentration.

• 대한약학회:학술대회논문집
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• 대한약학회 2001년도 Proceedings of International Convention of the Pharmaceutical Society of Korea
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• pp.178.3-179
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• 2001