• Proceedings of the Korean Society of Toxicology Conference
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• 1997.05a
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• pp.65-74
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• 1997

By using guinea pig lung mast cells, this study aimed to examine the effects of Aloe component(NY945) on the mediator releases caused by mast cell activation, and also aimed to assess the effects of NY945 on the mechanism of mediator releases in the mast cell activation. We partially purified mast cells from guinea pig lung tissues by using the enzyme digestion, the rough and the discontinuous density percoll gradient method. Mast cells were sensitized with $IgG_1$ (anti-OA) and challenged with ovalbumin. Histamine was assayed by fluorometric analyzer, leukotrienes by radioimmunoassay The phospholipase D activity was assessed more directly by the production of labeled phosphatidylethanol or phosphatidylbutanol which was produced by phospholipase D-mediated transphosphatidylation in the presence of ethanol or butanol. The amount of mass 1,2-diacylglycerol was measured by the [$^3H$]1,2-diacylgycerol produced when prelabeled with [$^3H$]myristic acid. In the mast cells prelabeled with L-[$^3H$]methyl methionine the phospholipid methylation was assessed by measuring the incorporation of the [$^3H$]methyl moiety into phospholipids. Pretreatment of NY945(10$\mu$g) significantly decreased histamine and leukotrienes releases during mast cell activation. The decrease of histamine release was stronger than that of leukotrienes during mast cell activation. The phospholipase D activity increased by the mast cell activation was decreased by the dose-dependent manner in the pretreatment of NY945. The amount of mass 1,2-diacylglycerol produced by activation of mast cells were decreased in the pretreatment of NY945. NY945 pretreatment strongly inhibited the incorporation of the [$^3H$]methyl moiety into phospholipids. The data suggest that NY945 purified from Aloe inhibits in part an increase of 1,2-diacylglycerol which is produced by activating mast cells with antigen-antibody complexes which is mediated via phosphatidylcholine-phospholipise D and phosphatidylinositole-phospholipise C systems, and then followed by the inhibition of histamine release. Furthermore, NY945 reduces the phosphatidylcholine production by inhibiting the methyltransfsrase I and II, which decrease the conversion of phosphatidylcholine into arachidonic acid and inhibits the production of leukotrines.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.4
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• pp.211-215
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• 2003

To examine the localization pattern of phospholipase D2 (PLD2) in the pancreatic islet (the islet of Langerhans) depending on species, we conducted a morphological experiment in the rat and guinea pig. Since individual islets display a typical topography with a central core of B cell mass and a peripheral boundary of A, D, and PP cells, double immunofluorescent staining with a panel of antibodies was performed to identify PLD2-immunoreactive cells in the islets PLD2 immunoreactivity was mainly present in A and PP cells of the rat pancreatic islets. And yet, in the guinea pig, PLD2 immunoreactivity was exclusively localized in A cells, and not in PP cells. These findings suggest a possibility that PLD2 is mainly located in A cells of rodent pancreatic islets, and that the existence of PLD2 in PP cells is not universal in all species. Based on these results, it is suggested that PLD2 may play a significant role in the function of A and/or PP cells via a PLD-mediated signaling pathway.

• Journal of Pharmacopuncture
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• v.5 no.2
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• pp.40-51
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• 2002

Objectives : The purpose of this study was to investigate the effect of Bee Venom on the lipopolysaccharide-induced expression phospholipase $A_2$, cyclooxygenase-2 and inducible nitrogen oxide synthase, and the generation of arachidonic acid, prostaglandin D2 and E2 in RAW 264.7 cells, a murine macrophage cell line. Methods : The expression of phospholipase $A_2$, cyclooxygenase and inducible nitrogen oxide synthase was determined by western blotting with corresponding antibodies, and the generation of arachidonic acid, prostaglandin $D_2$ and $E_2$ was assayed by ELISA method in RAW 264.7 cells. The non-toxic concentrations (0.1 to $5\;{\mu}g/ml$) of bee venom determined by MTT assay, were used in this study. Results : 1. Bee venom inhibited lipopolysaccharide-induced expression of phospholipase $A_2$ in a dose dependent manner after 48 hours treatment. 2. Bee venom inhibited lipopolysaccharide-induced expression of cyclooxygenase-2 in a dose dependent manner after 24 and 48 hours treatment. 3. Bee venom inhibited lipopolysaccharide-induced expression of inducible nitrogen oxidesynthase in a dose dependent manner after 48 hours treatment. 4. The generation of arachidonic acid, prostaglandin $D_2$ and $E_2$ was not much affected by the treatment of bee venom on the lipopolysaccharide-induced generation of arachidonic acid, prostaglandin $D_2$ and $E_2$ in RAW 264.7 cells.

• BMB Reports
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• v.45 no.1
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• pp.7-13
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• 2012

Phospholipase D (PLD), a superfamily of signaling enzymes that most commonly generate the lipid second messenger Phosphatidic Acid (PA), is found in diverse organisms from bacteria to man and functions in multiple cellular pathways. A fascinating member of the family, MitoPLD, is anchored to the mitochondrial surface and has two reported roles. In the first role, MitoPLD-generated PA regulates mitochondrial shape through facilitating mitochondrial fusion. In the second role, MitoPLD performs a critical function in a pathway that creates a specialized form of RNAi required by developing spermatocytes to suppress transposon mobilization during meiosis. This spermatocyte-specific RNAi, known as piRNA, is generated in the nuage, an electron-dense accumulation of RNA templates and processing proteins that localize adjacent to mitochondria in a structure also called intermitochondrial cement. In this review, we summarize recent findings on these roles for MitoPLD functions, highlighting directions that need to be pursued to define the underlying mechanisms.

• Bulletin of the Korean Chemical Society
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• v.17 no.10
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• pp.905-908
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• 1996

A substrate specificity of cabbage phospholipase D (PLD) was studied using the synthetic phospholipids having different head groups. The phospholipids were synthesized from phosphatidylcholine and appropriate bases by transphosphatidylation of PLD. The bases used were ethanolamine, serine, ethanol and γ-hydroxybutyric acid. The phosphatidic acid, the product of PLD, was separated in TLC and measured densitometrically. The kinetic parameters were estimated for each substrate and the effects of pH, SDS, Ca2+ and other metal ions were examined. Vmax values found were 3.75, 2.36, 5.59, 1.63, 2.30 nmol/min/μg protein for phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylethanol, and phosphatidylburytic acid, respectively. These results indicate a broad specificity of cabbage PLD toward phospholipids with different head groups. Particularly phosphatidylserine was most easily hydrolyzed by PLD and its activity did not depend on Ca2+.

• Bulletin of the Korean Chemical Society
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• v.15 no.11
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• pp.1001-1003
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• 1994

A series of phosphodiesters of p-nitrophenyl phosphoryl derivatives were synthesized and used as a model substrate for phospholipase D (PLD). The phosphodiester substrates were synthesized from p-nitrophenyl phosphorodichloridate and corresponding alcohols with different chain lengths and polar groups. To measure the activity of PLD, either spectroscopic method for p-nitrophenol or pH-stat titration method was employed. For each substrate, effects of substrate concentration, pH, and $Ca^{2+}$ ion were examined. The kinetic parameters $V_{max}$ for the different substrates were varied depending on the chain lengths or charge of the alcohols. No calcium effect was observed in the hydrolysis of neutral and negatively charged alcohol derivatives, while positively charged choline derivative showed a strong $Ca^{2+}$ ion dependence.

• Bulletin of the Korean Chemical Society
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• v.13 no.4
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• pp.381-384
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• 1992

The hydrolytic susceptibility of large unilamellar vesicle (LUV) toward cabbage phospholipase D (PLD) was studied. The activity of PLD was determined by pH stat titration method. Using phosphatidylcholine LUV as substrate a pH optimum of 6.96 was observed. For maximal activity the optimal temperature of $31^{\circ}C$ and 10 mM of Ca2+ were required. The apparent Km value estimated was 2.5 mM. The hydrolytic activity of PLD toward PC LUV was somewhat high despite the absence of activator in assay system and this high susceptibility of PC LUV may be attributed to the structural properties of LUV. The effect of amphiphatic substances such as dicetyl phosphate and phosphatidic acid on the enzyme activity were also examined in mixed LUVs.

• Bulletin of the Korean Chemical Society
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• v.10 no.6
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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.

• Journal of Life Science
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• v.13 no.5
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• pp.551-558
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• 2003

Hepatitis C Virus (HCV) is associated with a severe liver disease and increased frequency in the development of hepatocellular carcinoma. Overexpression of HCV core protein is known to transform fibroblast cells. Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals, and PLD has been also reported to be overexpressed and hyperactivated in some human cancer. The aim of this study was to understand how PLD can be regulated in HCV core protein-transformed NIH3T3 mouse fibroblast cells. We observed that in unstimulated state, basal PLD activity was higher in NIH3T3 cells overexpressing HCV core protein than in vector-transfected cells. Although expression of PLD and protein kinase C (PKC) in core protein-transformed cells was similar with that of control cells, phorbol 12-myristate 13-acetate (PMA), which is known to activate PKC, stimulated significantly PLD activity in core protein-transformed cells, compared with that of the control cells. PLD activity assay using PKC isozyme-specific inhibitor, and PKC translocation experiment showed that PKC-$\delta$ was mainly involved in the PMA-induced PLD activation in the core-transformed cells. Taken together, these results suggest that PLD might be implicated in core protein-induced transformation.

• Journal of Life Science
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• v.13 no.6
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• pp.903-910
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• 2003

Phospholipase D (PLD) plays an important role as a signaling molecule in the activation of neutrophils. In this study, effect of nitric oxide (NO) and cGMP on the activation of PLD in human neutrophils was investigated. Sodium nitroprusside (SNP), an agent to produce NO spontaneously in cells, alone increased PLD activity and the maximal activation was obtained with 0.5 mM SNP. Dibutyryl-cAMP, an agent to increase an intracellular cAMP concentration inhibited formyl-Met-Leu-Phe (fMLP)-stimulated PLD activity but 8-bromo-cGMP (300 $\mu$M), an agent to increase an intracellular cGMP concentration did not affect basal and fMLP-stimulated PLD activity. NO-induced activation of PLD was not blocked by KT 5823, an inhibitor of cGMP-dependent protein kinase (PKG), suggesting that NO-induced PLD activation is not mediated by cGMP. NO also stimulated p38 mitogen activated protein kinase (MAPK) in human neutrophils, indicated by increased phosphorylation of p38 MAPK in Western blotting. NO-induced phosphorylation of p38 MAPK was not inhibited by KT 5823 or n-butanol. RhoA, an regulatory factor of PLD activation was trans-located from cytosolic fraction to plasma membranes by fMLP or phorbol ester, and fMLP-stimulated but not phorbol ester-stimulated translocation of RhoA was inhibited by cGMP. These results suggest that NO stimulates PLD activity through other unidentified facto.(s) than cGMP even though cGMP inhibits the artivation of RhoA.