• Bulletin of the Korean Chemical Society
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• 제34권3호
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• pp.931-935
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• 2013

Lipid events in liposome-mediated transfection (lipofection) are largely unknown. Here we studied whether phospholipase D (PLD), an important enzyme responsible for phospholipid breakdown, was affected during lipofection of HepG2 cells with a luciferase plasmid. Synthetic cholesterol (Chol) derivatives, including $3{\beta}$[L-ornithinamide-carbamoyl]Chol, [polyamidoamine-carbamoyl]Chol and $3{\beta}$[N-(N',N'-dimethylaminoethane)-carbamoyl]Chol, and a cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride were mixed with a helper lipid dioleoylphosphatidylethanolamine to form respective cationic liposomes. All cationic liposomes were found to stimulate PLD. Although orders of magnitude effects of the cationic liposomes on PLD stimulation did not consistently match those on cytotoxicity and luciferase expression, a causal relationship between PLD activation and cytotoxic effect was remarkable. PLD stimulation by the cationic liposomes was likely due to their amphiphilic characters, leading to membrane perturbation, as supported by similar results obtained with other membrane-perturbing chemicals such as oleate, melittin, and digitonin. Our results suggest that lipofection induces cellular lipid changes such as a PLD-driven phospholipid turnover.

• 대한의생명과학회지
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• 제15권4호
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• pp.287-293
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• 2009

Phospholipase D (PLD) is an enzyme hydrolyzing phosphatidylcholine to phosphatidic acid (PA) and choline. We investigated the involvement of PLD1 in the uptake of norepinephrine (NE) in PC12 cells, pheochromocytoma cells. NE uptake was specific in PC12 cells because nomifensine, a specific blocker of NE transporter, blocked NE uptake. Inhibition of PLD function in PC12 cells by the treatment of butanol suppressed the NE uptake. In contrast, overexpression of PLD1 in PC12 cells increased NE uptake efficiently. These results suggest that PLD activity is involved in NE uptake. We explored the action mechanism of PLD in NE uptake. PA phosphatase inhibitor, propranolol, blocks the formation of PKC activator diacylglycerol from PA. Propranolol treatment to PC12 cells blocked dramatically the uptake of NE. Specific PKC inhibitors, GF109203X and Ro31-8220, blocked NE uptake. Taken together, we suggest for the first time that PLD1 activity is involved in NE uptake via the activation of PKC.

• BMB Reports
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• 제29권1호
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• pp.11-16
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• 1996

The present study showed that receptor-mediated activation of rabbit kidney proximal tubule cells by angiotensin II, the $Ca^{2+}$ ionophore A23187, or the protein kinase C activator phorbol myristate acetate (PMA) all stimulated phospholipase D (PLD). This was demonstrated by the increased formation of phosphatidic acid, and in the presence of 0.5% ethanol, phosphatidylethanol (PEt) accumulation. Angiotensin II leads to a rapid increase in phosphatidic acid and diacylglycerol, and phosphatidic acid formation preceeded the formation of diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine hydrolyzed by Pill. On the other hand, EGTA substantially attenuated angiotensin II and A23187-induced PEt formation, and when the cells were pretreated with verapamil angiotensin II-induced Pill activation was completely abolished. These results provide the evidence that calcium ion influx is essential for the agonist-induced Pill activation. In addition, staurosporine, an inhibitor of protein kinase C, strongly inhibited PMA-induced PEt formation, but was ineffective on angiotensin II-induced PEt accumulation. $GTP{\gamma}S$ also stimulates PEt formation in digitonin-permeabilized cells, but pretreatment of the cells with pertussis toxin failed to suppress angiotensin II-induced PEt formation. From these results, we conclude that in the rabbit kidney proximal tubule cells the mechanisms of angiotensin II- and PMA-induced Pill activation are different from each other and mediated via a pertussis toxin-insensitive trimeric G protein.

• Bulletin of the Korean Chemical Society
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• 제34권11호
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• pp.3223-3226
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• 2013

Spherical phospholipid-bilayers, vesicles, were prepared using the layer-by-layer double emulsion technique, which allows the bilayer to be formed asymmetrically. On the outer layer of the vesicles, the phospholipase D (PLD) reacted to convert phosphatidylcholine (PC) to phosphatidic acid (PA). The reaction induced the curvature change of the vesicles, which eventually led to rupture. The response time from the time of PLD injection to the time of rupture was measured against different vesicle curvatures and the outer layer phase, using the fluorescence intensity change of a pH-sensitive dye encapsulated within the vesicles. The effect of the vesicle curvature on the response was observed to be more significantly dramatic at the solid phase, compared to the liquid phase. Furthermore, in the solid phase, the response time was faster for 80 and 155 nm vesicles and, slower for 605 nm vesicles than similarly sized vesicles in the liquid phase vesicles. This difference in the response time was thought to result from the configuration determined by the phase difference and the PLD behavior.

• BMB Reports
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• 제29권6호
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• pp.535-539
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• 1996

The activation of phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and choline in plants as well as animals. To determine the presence of PLD in oat cells, we prepared inside-out plasma membrane and cytosolic fractions from oat tissues. PLD activities in both cytosol and plasma membrane were detected by ion chromatography method. The activity of PLD in plasma membrane was dependent upon $Ca^{2+}$ concentration and was heat stable. To investigate whether G-protein couples to PLD, the effects of $GTP{\gamma}S$ and $GDP{\beta}S$ on the PLD activity were measured. PLD activity was dramatically increased 300~400% in the presence of 50 ${\mu}M$ $GTP{\gamma}S$ but not in the presence of 50 ${\mu}M$ $GDP{\beta}S$. These results indicate that G-protein may be involved in regulation of PLD activity. To identify whether PLD is regulated by red light receptor, phytochrome, we irradiated red, far-red, or red/far-red/red light on oat protoplasts. PLD activity has increased 5-fold and 3-fold by treatment with red light and red/far-red/red light, respectively. In contrast, irradiation with far-red light had little or no effect on PLD activity. These results suggest that phytochrome regulates PLD activity through activation of G-protein in oat cells.

• 한국수산과학회지
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• 제34권4호
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• pp.370-377
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• 2001

본 연구에서는 넙치, Pafalichthys olivaceus 뇌 조직에서 인지질가수분해효소 D (phospholipase D, PLD) 활성의 특성 규명 및 이 활성을 억제하는 단백질을 분리 정제하여 그 특성을 규명하였다. 넙치 뇌 조직에서 PLD 활성이 관찰되었으며, 이 활성은 포스파티딜 이노시톨 비스인산염 (phosphatidyl-inositol 4,5-bisphos-phate, $PIP_2$)에 대해서 의존성을 나타내었으나, ADP-rebosylation factor (ARF)에 의해서는 영향을 받지 않았다. PLD 억제물은 넙치 뇌 조직의 세포질 분획물을 사용하여 여러 종류의 칼럼을 통하여 분리 정제하였고, 그 억제물의 분자크기 및 기작의 특성을 규명하였다. 마지막 chromatography을 통하여 여섯 개의 억제를 나타내는 분획물을 얻었으며, 이 중 두 개의 분획물인 IIA IIB는 $PIP_2$-phosphatase activities를 나타내었다. 이 중 IIA 분획물은 면역화학적 분석을 통하여 inositolpolyphosphate 5-phosphatase family로 알려져 있는 신경말단 단백질인 synaptojanin으로 동정되었다. 그리고 IIB fraction은 Superose 12 gel filtration chromatography을 통하여 158-kDa의 크기로 확인되었으며, 이것은 면역화학적 분석을 통하여 synaptojanin과는 별개의 단백질로 판명되었다. 또한, IIB 분획물은 $PIP_2$ phosphatase activity 확인 실험에서 대사산물로서 phosphatidylinositol phosphate (PIP)만을 생성하였다. 이 결과는 IIB 분획물이 $PIP_2$의 4혹은 5 위치의 인산 (phosphate) 중 어느 하나만을 선택적으로 가수분해시킨다는 것을 암시한다. 이상의 연구 결과들을 종합하여 보면, 넙치 뇌 조직에는 다양한 형태의 $PIP_2$-phosphatases가 존재하며, 이들은 $PIP_2$-의존적인 PLD 활성의 억제조절과정에서 중요한 역할을 할 것으로 사료된다.

• Biomolecules & Therapeutics
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• 제23권5호
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• pp.421-427
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• 2015

Imperatorin has been known to exert many biological functions including anti-inflammatory activity. In this study, we investigated the inhibitory effects of imperatorin on the production of inflammatory mediators in mouse bone marrow-derived mast cells (BMMC). Imperatorin inhibited degranulation and the generation of eicosanoids (leukotriene $C_4$ ($LTC_4$) and prostaglandin $D_2$ ($PGD_2$) in IgE/antigen (Ag)-stimulated BMMC. To elucidate the molecular mechanism involved in this process, we investigated the effect of imperatorin on intracellular signaling in BMMC. Biochemical analyses of the IgE/Ag-mediated signaling pathway demonstrated that imperatorin dramatically attenuated degranulation and the production of 5-lipoxygenase-dependent $LTC_4$ and cyclooxygenase-2-dependent $PGD_2$ through the inhibition of intracellular calcium influx/phospholipase $C{\gamma}1$, cytosolic phospholipase $A_2$/mitogen-activated protein kinases and/or nuclear factor-${\kappa}B$ pathways in BMMC. These results suggest that the effects of imperatorin on inhibition of degranulation and eicosanoid generation through the suppression of multiple steps of IgE/Ag-mediated signaling pathways would be beneficial for the prevention of allergic inflammation.

• 미생물학회지
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• 제24권1호
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• pp.38-45
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• 1986

Rhizopus oryzae 균사체 내의 인지질 구성 성분은 6종으로 나타났고 sodium deoxycholate와 sodium dodecyl sulfate를 각각 0.48mM, O.18mM의 낮은 농도로 영양배지에 첨가했을때 성장군략의 형태변화와 아울러 미량 존재하던 cerdiolipin과 phospha tidylinositol 그러고 Iy sophospholipid의 함량이 증가하였다. 또한 phospholipase는 A와 C, 그리고 D가 존재하였으며 A와 D는 sodium deoxycholate에 의하여. C는 sodium dodecyl sulfate에 의하여 활성도 증가를 나타냈다.

• 생명과학회지
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• 제13권6호
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• pp.924-932
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• 2003

녹차의 주성분인 에피갈로 갈레이트 (EGCG)는 건강에 이로운 효과로 나타내고 있는것으로 알려져 있어 많은 주목을 받고 있는 실정이다 본 연구에서는 처음으로 EGCG가, U87사람의 교세포에서 포스포리파제 D의 효소활성을 증가시킨다는 사실을 규명하였다. EGCG에 의한 포스포리파제 D의 활성화는 포스포리파제 C효소의 특이적인 억제제와 지질분해효소 활성이 억제제 포스포리파제 \gama1$의 변이체를 이용하여 실험한 결과 세포내 칼슘에 의존적인 양상을 보였주었다. 이러한 포스포리파제 D의 활성화는 아마도 칼슘 의존성 단백질 키나아제 II (CaM kinase II)를 통하여 일어나는것으로 여겨진다. 흥미롭게도, EGCG는 포스포리파제 \gama1$를 세포질에서 세포막으로의 이동을 유도하였으며, 포스포리파제 \gama1$와 PLD1의 결합을 유도하였음을 관찰하였다. 이상의 결과들을 종합하여 볼 때, EGCG가 사람의 교세포에서 포스포리파제 \gama1$-칼슘-CaM kinase II의 신호전달 경로를 통하여 포스포리파제 D의 활성을 유도하는 것으로 사료된다.

• 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.