• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1244-1251
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• 2020

Phospholipase A₂ (PLA₂) from Streptomyces violaceoruber is a lipolytic enzyme used in a wide range of industrial applications including production of lysolecithins and enzymatic degumming of edible oils. We have therefore investigated expression and secretion of PLA₂ in two workhorse microbes, Pichia pastoris and Escherichia coli. The PLA₂ was produced to an activity of 0.517 ± 0.012 U/ml in the culture broth of the recombinant P. pastoris. On the other hand, recombinant E. coli BL21 star (DE3), overexpressing the authentic PLA₂ (P-PLA₂), showed activity of 17.0 ± 1.3 U/ml in the intracellular fraction and 21.7 ± 0.7 U/ml in the culture broth. The extracellular PLA2 activity obtained with the recombinant E. coli system was 3.2-fold higher than the corresponding value reached in a previous study, which employed recombinant E. coli BL21 (DE3) overexpressing codon-optimized PLA₂. Finally, we observed that the extracellular PLA₂ from the recombinant E. coli P-PLA₂ culture was able to hydrolyze 31.1 g/l of crude soybean lecithin, an industrial substrate, to a conversion yield of approximately 95%. The newly developed E. coli-based PLA₂ expression system led to extracellular production of PLA₂ to a productivity of 678 U/l·h, corresponding to 157-fold higher than that obtained with the P. pastoris-based system. This study will contribute to the extracellular production of a catalytically active PLA₂.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2002년도 Molecular and Cellular Response to Toxic Substances
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• pp.68-88
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• 2002

Phospholipase A$_2$ (s) are esterases that hydrolyze the sn-2 ester bond in phospholipids, releasing a fatty acid and a lysophospholipid. We previously showed that most PLA$_2$ activity in rabbit renal proximal tubule cells (RPTC) was Ca$\^$2+/-independent, localized to the endoplasmic reticulum (ER-iPLA$_2$), and inhibited by the specific Ca$\^$2+/-independent PLA$_2$ inhibitor bromoenol lactone (BEL).(omitted)

• Journal of Photoscience
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• 제2권1호
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• pp.1-5
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• 1995

The activity of phospholipase A$_2$ (PLA$_2$) was identified and characterized from cytosolic and membrane fractions of oat cells, respectively. PLA$_2$ activity was determined fluorometrically in the presence of serum albumin using phospholipids labeled at sn-2-acyl position with 10-pyrenyldecanoic acid. When the cell-free extracts of oat tissues were fractionated by ultracentrifugation at 100,000 x g and the PLA$_2$ activity was assayed, we found that most of the PLA$_2$ activity was revealed from the cytoplasmic fraction rather than from the membrane fraction. The activity of cytosolic PLA$_2$ was dependent on Ca$^{2+}$ concentration and the optimum concentration of Ca$^{2+}$ was found to be 100 $\mu$M. It was also found that PLA$_2$ could be translocated toward the membrane site from the cytosol upon increasing Ca$^{2+}$ concentration. These results might suggest that an increased [Ca$^{2+}$]$_i$ by phytochrome action could promote the translocation of the cytosolic PLA$_2$ toward the membrane site.

• 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.

• The Korean Journal of Physiology and Pharmacology
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• 제14권6호
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• pp.427-433
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• 2010

This study was conducted to investigate the effects of extremely low frequency electromagnetic fields (EMF) on signal pathway in plasma membrane of cultured cells (RAW 264.7 cells and RBL 2H3 cells), by measuring the activity of phospholipase $A_2$ ($PLA_2$), phospholipase C (PLC) and phospholipase D (PLD). The cells were exposed to the EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h. The basal and $0.5\;{\mu}M$ melittin-induced arachidonic acid release was not affected by EMF in both cells. In cell-free $PLA_2$ assay, we failed to observe tbe change of $cPLA_2$ and $sPLA_2$ activity. Also both PLC and PLD activities did not show any change in the two cell lines exposed to EMF. This study suggests that the exposure condition of EMF (60 Hz, 0.1 or 1 mT) which is 2.4 fold higher than the limit of occupational exposure does not induce phospholipases-associated signal pathway in RAW 264.7 cells and RBL 2H3 cells.

• BMB Reports
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• 제33권6호
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• pp.525-530
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• 2000

The nerve growth factor (NGF) induces neuronal differentiation and neurite outgrowth of PC12 cells, whereas epidermal growth factors (EGF) stimulate growth and proliferation of the cells. In spite of this difference, NGF-or EGF-treated PC12 cells share various properties in cellular-signaling pathways. These include the activation of the phosphoinositide (PI)-3 kinase, 70 kDa S6 kinase, and in the mitogen-activated protein (MAP) kinase pathway, following the binding of these growth factors to intrinsic receptor tyrosine kinases (RTKs). Therefore, many studies have been attempted to access the critical signaling events in determining the differentiation and proliferation of PC12 cells. In this study, we investigated the cytosolic phospholipase $A_2$ ($cPLA_2$) in neurite behavior in order to identify the differences of signaling pathways between the NGF-induced differentiation and the EGF-induced proliferation of PC12 cells. We have showed here that the $cPLA_2$ was translocated from cytosol to membrane only in NGF-treated cells. We also demonstrated that this translocation is associated with NGF-induced activation of phospholipase $C-{\gamma}(PLC-{\gamma})$, which elevates intracellular $Ca^{2+}$ concentration. These results reveal that the translocation of $cPLA_2$ may be a requisite event in the neuronal differentiation of PC12 cells. Various phospholipase inhibitors were used to confirm the importance of these enzymes in the differentiation of PC12 cells. Neomycin B, a PLC inhibitor, dramatically inhibited the neurite outgrowth, and two distinct $PLA_2$ inhibitors, 4-bromophenacyl bromide (BPB) and arachidonyltrifluoro-methyl ketone ($AACOCF_3$) also suppressed the neurite outgrowth of the cells, as well Taken together, these data indicated that $cPLA_2$ is involved in NGF-induced neuronal differentiation and neurite outgrowth of PC12 cells.

• 동의생리병리학회지
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• 제18권5호
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• pp.1322-1330
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• 2004

This experimental studies were performed in order to prove the effect of Phospholipase A₂(PLA₂) Herbal-acupuncture by using rats that had neuronal damage due to the Middle Cerebral Artery Occulsion(MCAO). We observed the change of extracellular concentrations(μM) Of dopamine, DOPAC, HVA, HIAA, glutamate, aspartate, GABA, glysine, taurine, alanine, and tyrosine as extracted by vivo microdialysis, in the PLA₂ Herbal-acupuncture administrated rats(240-260g, Sprague-Dawley) subjected to the MCAO. The dialysates were extracted three times before the MCAO and six times after the MCAO every 20 minutes, and ana lysed by highperformance liquid chromatography(HPLC). PLA₂ Herbal-acupuncture significantly inhibited glutamate and tyrosine which are stimulant neurotransmitters at brain ischemia, and it significantly decreased glycine, GABA, taurine, and alanine which are inhibitory neurotransmitters at brain ischemia. PLA₂ Herbal-acupuncture may prevent delayed neuronal death(DND) in selectively vulnerable focal areas of the brain effectively.

• The Korean Journal of Physiology and Pharmacology
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• 제2권2호
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• pp.251-260
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• 1998

Cromakalim (BRL 34915), known as an airway smooth muscle relaxant, inhibited the releases of mediators in the antigen-induced mast cell activation. It has been suggested that cromakalim, in part, inhibited mediator releases by inhibiting the initial increase of 1,2-diacylglycerol (DAG) produced by the activation of the other phospholipase system which is different from phosphatidylcholine-phospholipase D pathway. The aim of this study is to further examine the inhibitory mechanism of cromakalim on the mediator release in the mast cell activation. Guinea pig lung mast cells were purified by using enzyme digestion and percoll density gradient. In purified mast cells prelabeled with $[^3H]PIP_2$, phospholipase C (PLC) activity was assessed by the production of $[^3H]$insitol phosphates. Protein kinase C (PKC) activity was assessed by measuring the protein phosphorylated from mast cells prelabeled with $[{\gamma}-32P]ATP$, and Phospholipase $A_2\;(PLA_2)$ activity by measuring the lyso-phosphatidylcholine produced from mast cell prelabeled with 1-palmitoyl-2-arachidonyl $phosphatidyl-[^{14}C]choline$. Histamine was assayed by fluorometric analyzer, and leukotrienes by radioimmunoassay. The PLC activity was increased by activation of the passively sensitized mast cells. This increased PLC activity was decreased by cromakalim pretreatment. The PKC activity increased by the activation of the passively sensitized mast cells was decreased by calphostin C, staurosporine and cromakalim, respectively. The $PLA_2$ activity was increased in the activated mast cells. The pretreatment of cromakalim did not significantly decrease $PLA_2$ activity. These data show that cromakalim inhibits histamine release by continuously inhibiting signal transduction processes which is mediated via PLC pathway during mast cell activation, but that cromakalim does not affect $PLA_2$ activity related to leukotriene release.

• 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.

• Parasites, Hosts and Diseases
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• 제49권1호
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• pp.1-8
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• 2011

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phospholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase $A_2$ (PLA$_2$). and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA$_2$. Acanthamoeba exhibited optimal phospholipase activities at $37^{\circ}C$ and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA$_2$-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.