• Journal of Ginseng Research
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• 제43권2호
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• pp.236-241
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• 2019

Background: Thromboxane A2 ($TXA_2$) induces platelet aggregation and promotes thrombus formation. Although ginsenoside Ro (G-Ro) from Panax ginseng is known to exhibit a $Ca^{2+}-antagonistic$ antiplatelet effect, whether it inhibits $Ca^{2+}-dependent$ cytosolic phospholipase $A_2$ ($cPLA_{2{\alpha}}$) activity to prevent the release of arachidonic acid (AA), a $TXA_2$ precursor, is unknown. In this study, we attempted to identify the mechanism underlying G-Ro-mediated $TXA_2$ inhibition. Methods: We investigated whether G-Ro attenuates $TXA_2$ production and its associated molecules, such as cyclooxygenase-1 (COX-1), $TXA_2$ synthase (TXAS), $cPLA_{2{\alpha}}$, mitogen-activated protein kinases, and AA. To assay COX-1 and TXAS, we used microsomal fraction of platelets. Results: G-Ro reduced $TXA_2$ production by inhibiting AA release. It acted by decreasing the phosphorylation of $cPLA_{2{\alpha}}$, p38-mitogen-activated protein kinase, and c-Jun N-terminal kinase1, rather than by inhibiting COX-1 and TXAS in thrombin-activated human platelets. Conclusion: G-Ro inhibits AA release to attenuate $TXA_2$ production, which may counteract $TXA_2-associated$ thrombosis.

• 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.

• 대한의생명과학회지
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• 제27권4호
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• pp.223-230
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• 2021

Tumor necrosis factor alpha (TNF-α) is a principal regulator of inflammation and immunity. The proinflammatory properties of TNF-α can be attributed to its ability to activate the enzyme cytosolic phospholipase A₂ (cPLA₂), which generates potent inflammatory lipid mediators, eicosanoids. L-glutamine (Gln) plays physiologically important roles in various metabolic processes. We have reported that Gln has a potent anti-inflammatory activity via rapid upregulation of mitogen-activated protein kinases (MAPKs) phosphatase (MKP)-1, which preferentially dephosphorylates the key proinflammatory enzymes, p38 MAPK and cytosolic phospholipase A₂ (cPLA₂). In this study, we have investigated whether Gln could inhibit TNF-α-induced cPLA₂ activation. Gln inhibited TNF-α-induced increases in cPLA₂ phosphorylation in the lungs and blood levels of the cPLA₂ metabolites, leukotrine B4 (LTB4) (lipoxygenase metabolite) and prostaglandin E2 (PGE2) (cyclooxygenase metabolite). TNF-α increased p38 and cPLA₂ phosphorylation and blood levels of LTB4 and PGE2, which were blocked by the p38 inhibitor SB202190. Gln inhibited TNF-α-induced p38 and cPLA₂ phosphorylation and production of the cPLA₂ metabolites. Such inhibitory activity of Gln was no longer observed in MKP-1 small interfering RNA-pretreated animals. Our data indicate that Gln inhibited TNF-α-induced cPLA₂ phosphorylation through MKP-1 induction/p38 inhibition, and suggest that the utility of Gln in inflammatory diseases in which TNF-α plays a major role in their pathogenesis.

• Journal of Chest Surgery
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• 제35권5호
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• pp.343-349
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• 2002

배경: 흰쥐의 기도에 과산화수소를 분무함으로서 급성 염증성 반응을 일으켜 phospholipase $A_2$(PLA$_2$)와 endogenous oxidative stress에 산소기가 어떤 영항을 미치는지에 대해 알아보고자 하였다. 대상 및 방법: 호중구에 의해 유리된 산소기가 phospholipase $A_2$를 다시 활성화시킨다는 가설을 증명하기위해 phospholipase $A_2$의 활성도와 lysoplatelet activating factor acetyltransferase(lysoPAF AT)를 수산화수소 분무 5시간 후에 측정하였다. 또한, PLA$_2$활성화에 따른 나쁜 영향에 대해 알아보기 위하여 폐 질량/체중의 비, bronchoalveolar lavage(BAL)내의 단백 함량을 측정하였다. 폐장 내의 염증반응은 호중구의 폐장내 침윤에 따른 respiratory burst에 의한 oxidative stress가 그 원인일 것으로 추정되므로 BAL 내의 호중구 수와 myeloperoxidase(MPO)수치를 측정하였다. 또한 oxidative stress에 의한 영향을 화인하기 위하여 형태학적, 조직화학적 검사도 시행하였다. 결과: 과산화수소를 투여하고 5시간 뒤에 폐장은 심한 호중구의 침윤 및 폐 질량의 증가를 볼 수 있었다. BAL 내의 단백 함량은 정상폐의 경우에서보다 훨씬 증가된 것을 볼 수 있었다. PLA$_2$의 활성도 또한 증가되어 있었다. 흥미롭게도, 과산화수소를 투여한 폐에서 lysoPAF AT 활성도의 증가를 측정함으로서 platelet activating factor(PAF) 생성의 증가를 확인할 수 있었다. 형태학적으로, 광학현미경상 폐장의 무기폐 및 염증세포의 침윤을 관찰할 수 있었다. 이것은 PLA$_2$의 활성화에 따라 생성된 염증성 지질 분자에 의해 초래된 결과라고 생각되어진다. Cerium chloride 세포화학 전자 현미경상 많은 양치 cerrous perhydroxide의 침윤을 관찰할 수 있었다. 반면 정상 폐장에서는 전혀 관찰되어지지 않았다. 결론: 이상의 모든 결과들은 호중구에 의한 oxidative stress에 의한 폐장의 손상과 일치하므로 산소기에 의해 생성된 PLA$_2$가 내인성 oxidative stess에 관여한다고 볼 수 있다.

• 한국응용곤충학회지
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• 제55권2호
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• pp.129-138
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• 2016

아이코사노이드는 탄소수 20 개의 다가불포화지방산 산화물로 구성된다. 이들 다가불포화지방산의 생합성 전구물질을 탐지하기 위해 파밤나방(Spodoptera exigua)의 서로 다른 조직으로부터 지방산을 분리하여 GC/MS로 조성을 분석하였다. 파밤나방 5령 유충에서 소화관, 지방체, 혈구 및 체벽을 분리하고, 각 조직에서 지질을 추출하여 각각 중성지질, 당지질 및 인지질로 분리하였다. 대부분의 조직은 palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) 그리고 linolenic acid (18:3)를 주요 지방산으로 함유하였다. 그러나 이들 지방산의 조성은 조직과 지질 종류에 따라 상이하였다. 지방체와 혈구세포는 이들 주요 지방산 이외에 myristic acid (14:0)와 3 종류의 미동정 지방산들이 추가로 검출되었다. 서로 다른 지질 종류 가운데 인지질은 중성지질이나 당지질에 비해 상대적으로 높은 linolenic acid를 지닌 반면 포화지방산의 함유량은 낮았다. 전체 불포화지방산의 조성도 조직과 지질 종류에 따라 상이하였다. 인지질은 지방체, 혈구 및 소화관에서 높은 불포화지방산 함유량을 나타냈다. 세포성 인지질분해효소인 calcium-independent phospholipase $A_2$ ($iPLA_2$)는 지방산 조성을 조절하는 데 역할을 담당하였다. 이 유전자의 RNA 간섭은 중성지질과 인지질에서 지방산 조성의 변화를 유발하였다. 본 연구는 아이코사노이드 생합성의 전구물질로 여겨지는 아라키도닉산을 검출하지 못했다. 이는 곤충에 있어서 아이코사노이드는 포유동물과는 다른 새로운 생합성 과정을 통해 형성되는 것으로 추정된다.

• 한국수정란이식학회지
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• 제31권3호
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• pp.293-297
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• 2016

For evaluating the boar semen quality, sperm motility is an important parameter because the movement of sperm indicates active metabolism, membrane integrity and fertilizing capacity. Phospholipase C zeta (PLCz) is important enzyme in spermatogenesis, but the effect has not been confirmed in pigs yet. Therefore, this study was aimed to analyze their association with sperm motility and kinematic characteristics. DNA samples from 124 Duroc pigs with records of sperm motility and kinematic characteristics [total motile spermatozoa (MOT), curvilinear velocity (VCL), straight-line velocity (VSL), the ratio between VSL and VCL (LIN), amplitude of lateral head displacement (ALH)] were subjected. A SNP in non-coding region of PLCz g.158 A > C was associated with MOT (p < 0.05), VCL (p < 0.01), LIN (p < 0.01) and ALH (p < 0.05) in Duroc population. Therefore, we suggest that the intron region of the porcine PLCz gene may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not defined yet. Whether the association is due to the candidate gene or not require further verification. Thus, it will be of interest to continue association studies in the regions surrounding those genes.

• The Korean Journal of Physiology and Pharmacology
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• 제5권4호
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• pp.287-297
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• 2001

Contraction of smooth muscle is initiated by an increase in cytosolic $Ca^{2+}$ leading to activation of $Ca^{2+}$/ calmodulin-dependnet myosin light chain (MLC) kinase and phosphorylation of MLC. The types of contraction and signaling mechanisms mediating contraction differ depending on the region. The involvement of these different mechanisms varies depending on the source of $Ca^{2+}$ and the kinetic of $Ca^{2+}$ mobilization. $Ca^{2+}$ mobilizing agonists stimulate different phospholipases $(PLC-{\beta},\;PLD\;and\;PLA_2)$ to generate one or more $Ca^{2+}$ mobilizing messengers $(IP_3\;and\;AA),$ and diacylglycerol (DAG), an activator of protein kinase C (PKC). The relative contributions of $PLC-{\beta},\;PLA_2$ and PLD to generate second messengers vary greatly between cells and types of contraction. In smooth muscle cell derived form the circular muscle layer of the intestine, preferential hydrolysis of $PIP_2$ and generation of $IP_3$ and $IP_3-dependent\;Ca^{2+}$ release initiate the contraction. In smooth muscle cells derived from longitudinal muscle layer of the intestine, preferential hydrolysis of PC by PLA2, generation of AA and AA-mediated $Ca^{2+}$ influx, cADP ribose formation and $Ca^{2+}-induced\;Ca^{2+}$ release initiate the contraction. Sustained contraction, however, in both cell types is mediated by $Ca^{2+}-independent$ mechanism involving activation of $PKC-{\varepsilon}$ by DAG derived form PLD. A functional linkage between $G_{13},$ RhoA, ROCK, $PKC-{\varepsilon},$ CPI-17 and MLC phosphorylation in sustained contraction has been implicated. Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to $M_2$ muscarinic receptors activating at least three intracellular phospholipases, i.e. phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD) and the high molecular weight (85 kDa) cytosolic phospholipase $A_2\;(cPLA_2)$ to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic $M_3$ receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the $G_{q/11}$ type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate $(PIP_2),$ producing inositol 1, 4, 5-trisphosphate $(IP_3)$ and DAG. $IP_3$ causes release of intracellular $Ca^{2+}$ and formation of a $Ca^{2+}$-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway.

• 대한후두음성언어의학회지
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• 제12권2호
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• pp.126-132
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• 2001

Background and Objectives : Signal traduction through phospholipase C(PLC) participate in the regulation of cell growth and differentiation. Growth factors bind to their receptors and thereby induce tyrosine phophorylation of the phospholipase C-${\gamma}$1(PLC-${\gamma}$1). PLC-${\gamma}$1 is a substrate for several receptor tyrosine kinases and its catalytic activity is increased by tyrosine phosphorylation. Tyrosine kinase phosphorylation of PLC-${\gamma}$1 stimulates PLC activation and cell proliferation. However the signal transduction pathway and the significance of PLC in injured recurrent laryngeal nerve regeneration is unknown. Therefore after we obtained fuctionally recovered rats using PEMF in this study, we attempt to provide some evidence that PLC plays a role in nerve regeneration itself and regeneration related to PEMF through the analysis of the difference between fucntional recovery group and non-recovery group in the recurrent laryngeal nerve. Materials and Method : Using 32 healthy male Sprague-Dawley rats, transections and primary anastomosis were performed on their left recurrent laryngeal nerves. Rats were then randomly assigned to 2 groups. The experimental group(n=16) received PEMS by placing them in custom cages equipped with Helm-holz coils(3hr/day, 5days/wk, for 12wk). The control group(n=16) were handled the same way as the experimental group, except that they did not receive PEMS. Laryngo-videoendoscopy was performed before and after surgery and followed up weekly. Laryngeal EMG was obtained in both PCA and TA muscles. Immunohistochemisty staining and Western blotting analysis using monoclonal antibody was performed to detect PLC-${\gamma}$1 in recurrent laryngeal nerve and nodose ganglion. Results : 10 rats(71%) in experimental group and 4 rats(38%) in the control group showed recovery of vocal fold motion. Functionally-recoverd rats show PLC-${\gamma}$1 positive cells in neuron and ganglion cells after 12 weeks from nerve injury. Conclusion : This study shows that PLC1-${\gamma}$ involved in singnal trasduction pathway in functinal recovery of injured recurrent laryngeal nerve and PEMF enhance the functional recovery by effect on this molecule.

• 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.

• Journal of Acupuncture Research
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• 제21권5호
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• pp.179-190
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• 2004

Objectives : The purpose of this study was to investigate the effect of Bee Venom and Melittin acupuncture solution on the lipopolysaccharide and sodium nitroprusside- induced expression of cytosolic phospholipase $A_2$, tumor necrosis factor-${\alpha}$ and calcium concentration in RAW 264.7 cells, a murine macrophage cell line. Methods : The expression of cytosolic phospholipase $A_2$ and tumor necrosis factor-${\alpha}$ was determined by western blotting with corresponding antibodies, and the generation of intracellular calcium concentration was investigated by delta scan system in RAW 264.7 cells. Results : 1. Compared with control, expressions of lipopolysaccharide-induced cytosolic phospholipase $A_2$ were decreased significantly by $5{\mu}g/mL$ of bee venom and 5, $10{\mu}g/mL$ of melittin acupuncture solution and decreased by 0.5, $1{\mu}g/mL$ of bee venom. 2. Compared with control, expressions of sodium nitroprusside-induced phospholipase $A_2$ were decreased significantly by 0.5, 1, $5{\mu}g/mL$ of bee venom and by 5, $10{\mu}g/mL$ of melittin acupuncture solution. 3. Compared with control, expressions of lipopolysaccharide-induced tumor necrosis factor-${\alpha}$ were decreased significantly by $10{\mu}g/mL$ of melittin acupuncture solution and were not changed significantly by 0.5, 1, $5{\mu}g/mL$ of bee venom and $5{\mu}g/mL$ of melittin acupuncture solution. 4. Compared with control, expressions of sodium nitroprusside-induced tumor necrosis factor-${\alpha}$ were decreased significantly by 1, $5{\mu}g/mL$ of bee venom and 5, $10{\mu}g/mL$ of melittin acupuncture solution and decreased by $0.5{\mu}g/mL$ of bee venom 5. Compared with control, lipopolysaccharide-induced intracellular calcium concentrations were decreased by 0.5, 1, $5{\mu}g/mL$ of bee venom and $10{\mu}g/mL$ of melittin acupuncture solution and increased by $5{\mu}g/mL$ of melittin acupuncture solution. 6. Compared with control, sodium nitroprusside-induced intracellular calcium concentrations were decreased by 0.5, 1, $5{\mu}g/mL$ of bee venom and 5, $10{\mu}g/mL$ of melittin acupuncture solution.