• 생약학회지
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• 제38권3호통권150호
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• pp.277-280
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• 2007

In our continuing effort to investigate compounds having anti-inflammatory activity from natural products, Ailanthus altissima was examined. Among six compounds isolated from Ailanthus altissima, Luteolin-7-O-glucoside (L7G) along with ethanol extract of Ailnathus altissima (EAa) were chosen to determine their inhibitory activity on secretory recombinant phospholipase $A_2s$ enzyme activity in vitro. As a results, EAa inhibited human recombinant $sPLA_2-V$ ($IC_{50}$ of about 100 ${\mu}g/ml$) and $cPLA_2$, ($IC_{50}$ of about 59 ${\mu}g/ml$), while L7G showed strong inhibitory effect on $sPLA_2-A$, V and $cPLA_2$ with an $IC_{50}$ value of approximately 40 ${\mu}M$, respectively.

• Biomolecules & Therapeutics
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• 제17권4호
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• pp.379-387
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• 2009

We have previously reported that N-ethylmaleimide (NEM) induces apoptosis through activation of $K^+$, $Cl^-$-cotransport (KCC) in HepG2 human hepatoblastoma cells. In this study we investigated the possible role of phospholipase $A_2$ ($PLA_2$)-arachidonic acid (AA) signals in the mechanism of the NEM-induced apoptosis. In these experiments we used arachidonyl trifluoromethylketone ($AACOCF_3$), bromoenol lactone (BEL) and p-bromophenacyl bromide (BPB) as inhibitors of the calcium-dependent cytosolic $PLA_2$ ($cPLA_2$), the calcium-independent $PLA_2$ ($iPLA_2$) and the secretory $PLA_2$ ($sPLA_2$), respectively. BEL significantly inhibited the NEM-induced apoptosis, whereas $AACOCF_3$ and BPB did not. NEM increased AA liberation in a dose-dependent manner, which was markedly prevented only by BEL. In addition AA by itself induced $K^+$ efflux, a hallmark of KCC activation, which was comparable to that of NEM. The NEM-induced apoptosis was not significantly altered by treatment with indomethacin (Indo) and nordihydroguaiaretic acid (NDGA), selective inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Treatment with AA or 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, significantly induced apoptosis. Collectively, these results suggest that AA liberated through activation of $iPLA_2$ may mediate the NEMinduced apoptosis in HepG2 cells.

• Journal of Microbiology and Biotechnology
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• 제28권12호
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• pp.2046-2056
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• 2018

Phospholipase D has great commercial value due to its transphosphatidylation products that can be used in the food and medicine industries. In order to construct a strain for use in the production of PLD, we employed a series of combinatorial strategies to increase PLD expression in Bacillus subtilis WB600. These strategies included screening of signal peptides, selection of different plasmids, and optimization of the sequences of the ribosome-binding site (RBS) and the spacer region. We found that using the signal peptide amyE results in the highest extracellular PLD activity (11.3 U/ml) and in a PLD expression level 5.27-fold higher than when the endogenous signal peptide is used. Furthermore, the strain harboring the recombinant expression plasmid pMA0911-PLD-amyE-his produced PLD with activity enhanced by 69.03% (19.1 U/ml). We then used the online tool \RBS Calculator v2.0 to optimize the sequences of the RBS and the spacer. Using the optimized sequences resulted in an increase in the enzyme activity by about 26.7% (24.2 U/ml). In addition, we found through a transfer experiment that the retention rate of the recombinant plasmid after 5 generations was still 100%. The final product, phosphatidylserine (PS), was successfully detected, with transphosphatidylation selectivity at 74.6%. This is similar to the values for the original producer.

• Journal of Yeungnam Medical Science
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• 제21권2호
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• pp.177-190
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• 2004

Background: Secretory phospholipase $A_2$ ($sPLA_2$) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA $sPLA_2$ ($sPLA_2$-IIA) has been detected in the inflammatory fluids, and its plasma level increases in the inflammatory disease. This study examined the effect of $sPLA_2$-IIA on mouse macropahges in order to investigate the potential mechanism of $sPLA_2$-induced inflammation. Materials and Methods: Wild type $PLA_2$ and mutant H48Q $PLA_2$ were purified from HEK293 cells transfected with the corresponding plasmids, and the $PLA_2$ activities were measured using 1-palmitoyl-2-[1-$^{14}C$]linoleoyl-3-phosphatidylethanolamine as substrates. The TNF-${\alpha}$ and IL-6 released in the supernatants were determined by ELISA. In addition, the TNF-${\alpha}$ and IL-6 mRNA were analyzed by RT-PCR. Results: $sPLA_2$-IIA stimulated the production of TNF-${\alpha}$ and IL-6 in a dose- and time-dependent manner. In addition, the effect of $sPLA_2$-IIA on cytokine production from the macrophage was found to be associated with the accumulation of their specific mRNA. The mRNA levels of TNF-${\alpha}$ and IL-6 peaked at 2 and 6 hours in a time-dependent manner, respectively. Conclusion: In conclusion, the production of proinflammatory cytokine might be mediated by the binding of $sPLA_2$-IIA to the receptors.

• Archives of Pharmacal Research
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• 제24권6호
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• pp.552-556
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• 2001

Histamine and arachidonic acid (AA) release was measured using the P2-purinoceptor antaongists, phospholipase $A_2{\;}(PLA_2)$ and cyclooxygenase (COX)/lipoxygenase (LOX) inhibitors to determine whether or not ATP-induced histamine release is associated with arachidonic acid (AA) release in rat peritoneal mast cells. ATP increased histamine release in a dose dependent manner, whereas adenosine did not. PPADS (a selective P2X-purinoceptor antagonist) and suramin (a nonselective P2X,2Y-purinoceptor antagonist) inhibited ATP-induced histamine release in a dose dependent manner. However, RB-2 (a P2Y-purinoceptor antagonist) did not block ATP-induced histamine release. Manoalide and oleyloxyethyl phosphorylcholine (OPC), secretory PLA$_2$ inhibitors, also inhibited ATP-induced histamine release dose-dependently. Both COX inhibitors (ibuprofen and indomethacin) and LOX inhibitors (baicalein and caffeic acid) inhibited ATP-induced histamine in a dose dependent manner. ATP significantly increased [$^3H$]AA release by 54%. PPADS and suramin significantly inhibited ATP-induced [3H]Ph release by 81% and 39%, respectively. ATP-induced histamine release was significantly inhibited by a variety of protein kinase inhibitors, such as bisindolmaleimide, genistein, methyl 2,5-dihydroxycinnamate, W-7 and trifluoperazine. Overall, the results suggest that ATP-induced histamine release is in part related to the PLA2-mediated AA metabolism and P2X-purinoceptors.

• The Korean Journal of Physiology and Pharmacology
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• 제13권5호
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• pp.401-408
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• 2009

$K^+$-$Cl^-$-cotransport (KCC) has been reported to have various cellular functions, including proliferation and apoptosis of human cancer cells. However, the signal transduction pathways that control the activity of KCC are currently not well understood. In this study we investigated the possible role of phospholipase $A_2$ ($PLA_2$)-arachidonic acid (AA) signal in the regulatory mechanism of KCC activity. Exogenous application of AA significantly induced $K^+$ efflux in a dose-dependent manner, which was completely blocked by R-(+)-[2-n-butyl-6,7 -dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1Hinden-5-yl]oxy]acetic acid (DIOA), a specific KCC inhibitor. N-Ethylmaleimide (NEM), a KCC activatorinduced $K^+$ efflux was significantly suppressed by bromoenol lactone (BEL), an inhibitor of the calciumindependent $PLA_2$ ($iPLA_2$), whereas it was not significantly altered by arachidonyl trifluoromethylketone ($AACOCF_3$) and p-bromophenacyl bromide (BPB), inhibitors of the calcium-dependent cytosolic $PLA_2$ ($cPLA_2$) and the secretory $PLA_2$ ($sPLA_2$), respectively. NEM increased AA liberation in a doseand time-dependent manner, which was markedly prevented only by BEL. In addition, the NEM-induced ROS generation was significantly reduced by DPI and BEL, whereas $AACOCF_3$ and BPB did not have an influence. The NEM-induced KCC activation and ROS production was not significantly affected by treatment with indomethacin (Indo) and nordihydroguaiaretic acid (NDGA), selective inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Treatment with 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, markedly produced ROS and activated the KCC. Collectively, these results suggest that $iPLA_2$-AA signal may be essentially involved in the mechanism of ROS-mediated KCC activation in HepG2 cells.

• The Korean Journal of Physiology and Pharmacology
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• 제4권3호
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• pp.219-226
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• 2000

In order to elucidate the role of platelet activating factor (PAF) in the acute lung injury induced by endotoxin (ETX), activities of phospholipase A2, lyso PAF acetyltransferase and oxidative stress by neutrophilic respiratory burst were probed in the present study. To induce acute lung injury, $100\;{\mu}g$ of E.coli ETX (type 0127; B8) was instilled directly into the tracheae of Sprague-Dawley rats. Five hours after the ETX instillation, induction of acute lung injury was confirmed by lung leak index and protein contents in the bronchoalveolar lavage (BAL) fluid. At the same time, lung phospholipase A2 (PLA2) activity and expression of group I and II secretory type PLA2 were examined. In these acutely injured rats, ketotifen fumarate, known as lyso PAF acetyltransferase inhibitor and mepacrine were administered to examine the role of PAF in the pathogenesis of the acute lung injury. To know the effect of the ETX in the synthesis of the PAF in the lungs, lyso PAF acetyltransferase activity and PAF content in the lungs were measured after treatments of ETX, ketotifen fumarate and mepacrine. In addition, the role of neutrophils causing the oxidative stress after ETX was examined by measuring lung myeloperoxidase (MPO) and enumerating neutrophils in the BAL fluid. To confirm the oxidative stress in the lungs, pulmonary contents of malondialdehyde (MDA) were measured. After instillation of the ETX in the lungs, lung leak index increased dramatically (p＜0.001), whereas mepacrine and ketotifen decreased the lung leak index significantly (p＜0.001). Lung PLA2 activity also increased (p＜0.001) after ETX treatment compared with control, which was reversed by mepacrine and ketotifen (p＜0.001). In the examination of expression of group I and II secretory PLA2, mRNA synthesis of the group II PLA2 was enhanced by ETX treatment, whereas ketotifen and WEB 2086, the PAF receptor antagonist, decreased the expression. The activity of the lysoPAF acetyltransferase increased (p＜0.001) after treatment of ETX, which implies the increased synthesis of PAF by the remodelling of lysoPAF in the lungs. Consequently, the contents of the PAF in the lungs were increased by ETX compared with control (p＜0.001), while mepacrine (p＜0.001) and ketotifen (p＜0.01) decreased the synthesis of the PAF in the lungs of ETX treated rats. The infiltration of the neutrophils was confirmed by measuring and enumerating lung MPO and the neutrophils in the BAL fluid respectively. Compared with control, ETX increased lung MPO and number of neutrophils in BAL significantly (p＜0.001) whereas mepacrine and ketotifen decrerased number of neutrophils (p＜0.001) and MPO (p＜0.05, p＜0.001, respectively). The lung MDA contents were also increased (p＜0.001) by ETX treatment, but treatment with mepacrine (p＜0.001) and ketotifen (p＜0.01) decreased the lung MDA contents. Collectively, we conclude that ETX increases PLA2 activity, and that the subsequently increased production of PAF was ensued by the remodelling of the lyso PAF resulting in tissue injury by means of oxidative stress in the lungs.

• 대한약침학회지
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• 제25권1호
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• pp.52-62
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• 2022

Objectives: Snake venom is a complex mixture of various pharmacologically active substances, such as small proteins, peptides, and organic and mineral components. This paper aims to identify and analyse the proteins in common venomous snakes, such as Gloydius blomhoffii (G. blomhoffii) and Agkistrodon acutus (A. acutus), in Korea. Methods: We used mass spectrometry, electrophoresis, N-terminal sequencing and in-gel digestion to analyse the proteins in these two snake venoms. Results: We identified eight proteins in G. blomhoffii venom and four proteins in A. acutus venom. The proteins detected in G. blomhoffii and A. acutus venoms were phospholipase A₂, snake venom metalloproteinase and cysteine-rich secretory protein. Snake C-type lectin (snaclec) was unique to A. acutus venom. Conclusion: These data will contribute to the current knowledge of proteins present in the venoms of viper snakes and provide useful information for investigating their therapeutic potential.

• Journal of Chest Surgery
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• 제42권1호
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• pp.1-8
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• 2009

배경: 패혈증에 동반된 급성호흡곤란증후군의 병태생리는 내독소에 의해 유발된 급성폐손상이다. 이 연구에서 우리는 내독소로 인해 유발된 급성 폐손상에서 moxifloxacin의 영향을 알아보고자 하였다. 대상 및 방법: 백서를 대조군(C), 내독소처리군(L), 내독소-moxifloxacin처리군(L-M)으로 나누었다. 대장균의 내독소를 기관 내 분무하여 급성폐손상을 유발하였고 L-M군에서는 내독소 분무 30분 후 moxifloxacin을 투여하였다. 내독소 투여 5시간 후 폐질량/체중비, 폐세척액 내의 단백함량, 폐세척액 내의 호중구 수, 폐장 내 myeloperoxidase (MPO) 활성도, 폐장 내 malondialdehyde (MDA) 함량을 측정하였고, 폐장 내 세포질형 $phospholipaseA_2$ ($cPLA_2$)와 분비형 $phospholipaseA_2$ ($sPLA_2$)의 발현을 관찰하고 광학현미경을 이용하여 형태학적인 분석을 시행하였다. 결과: 폐질량/체중비, 폐세척액 내의 단백함량, 폐세척액 내의 호중구 수, 폐장 내 myeloperoxidase (MPO) 활성도, 폐장 내 malondialdehyde (MDA) 함량은 L군에서 대조군 보다 유의하게 증가되어 있었고, L-M군은 L군보다 의미있게 낮은 것으로 나타났다. L군에서 $cPLA_2$와 $sPLA_2$의 발현은 증가되어 있었고 L-M군에서는 $cPLA_2$의 발현이 감소되어 있었다. 그러나 L-M군에서 $sPLA_2$의 발현은 L군과 차이가 없었다. 형태학적 분석으로 L군에서 많은 염증성 소견들을 관찰할 수 있었으나 L-M군에서는 관찰할 수 없었다. 결론: 내독소에 의해 유발된 급성폐손상의 많은 염증성 변화들이 moxifloxacin처리에 의해 소멸됨을 관찰할 수 있었다.

• 한국식품저장유통학회지
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• 제19권2호
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• pp.287-293
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• 2012

본 연구에서는 HMGB1 의해 증가되는 각종 염증관련물질에 대해 라이코펜이 가지는 저해 역할을 규명하고 하였다. 라이코펜은 HMGB1에 의해 증가되는 MCP-1, IL-8, sPLA2-IIA, PGE2의 발현을 NF-${\kappa}B$ 그리고 TNF-${\alpha}$의 활성를 저해함으로써 감소시켰다. 특히, 1 mM에서 그 효능이 통계적으로 유효하였다. 결론적으로 HMGB1에 의해서 발생하는 각종 혈관염증질환에서 라이코펜은 증가하는 각종 염증관련물질을 저해하였고, 결국 라이코펜이 패혈증을 포함하는 염증질환을 효과적으로 치료할 수 있는 방법에 있어 많은 방향성을 제시할 것으로 기대한다.