• BMB Reports
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• 제32권2호
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• pp.119-126
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• 1999

The SH3 domain of PLC-${\gamma}1$ has been known to induce DNA synthesis. However, little is known about the putative effector proteins that associate with the domain. In this report, we provide evidence that the SH3 domain of PLC-${\gamma}1$ associates with Shc, which has been implicated in the activation of p21Ras in response to many growth factors. The association between Shc and PLC-${\gamma}1$ is enhanced either by v-Src-induced transformation or EGF-stimulation in vivo and in vitro. Furthermore, from transient expression studies with COS-7 cells, we show that the SH3 domain of PLC-${\gamma}1$ is required for association with Shc in vivo, whereas tyrosyl phosphorylation of PLC-${\gamma}1$ is not. Taken together, we suggest that Shc might be involved in the PLC-${\gamma}1$-mediated signaling pathway.

• Journal of Yeungnam Medical Science
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• 제9권2호
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• pp.288-301
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• 1992

소의 중추신경계의 신경전달인자에 의한 세포막에서의 정보전달 과정에 관여하는 PLC 활성화에 G-단백질의 관여 여부를 관찰하기 위하여 소의 뇌조직의 PLC ${\beta}$, ${\gamma}$ 및 ${\delta}$를 얻어 각 isozyme의 특성을 관찰하였다. 기질용액에 phosphatidyl choline(PC)을 첨가시 PLC 각 isozyme 마다 정도의 차이는 있으나 증가 양상을 보였으며 PLC ${\delta}$는 $100{\mu}M$ $Ca^{2+}$ 농도에서 높은 활성도 증가를 보였다. 세포막 소포체를 형성하기 위하여 $PIP_2$기질과 PC에 detergent로 cholate와 deoxycholate 농도에 따른 PLC 효과 관찰에서 cholate 농도 0.2%에서 1%까지 증가할 때 효소 활성도의 지속적인 상승이 관찰되었고, deoxycholate는 농도가 0.2%에서 높았다가 0.4%에서 낮아졌고 1%까지 증가함에 따라 PLC 효소 활성도는 약간 증가하였다. 기질액에 뇌추출액을 첨가하여 cholic acid 농도에 따른 PLC의 효과를 관찰한 결과 cholic acid 농도 0.2%에서 보다 1%에서 각 isozyme 모두에서 PLC활성도가 증가하였다. 소의 여러 장기에서 PLC isozyme의 분포정도를 방사면역측정방법으로 관찰하였을 때 뇌조직에 가장 많이 분포하고 있으며 특히 PLC ${\beta}$, ${\gamma}$가 많았고, PLC ${\delta}$는 부신에서 가장 많이 분포하였다. 다음으로 PLC ${\beta}$는 부신과 위, PLC${\gamma}$는 부신과 폐순이었다. PLC 효소가 활성화될 때 G-단백질의 관여 여부에 관하여 cholate 0.2%와 0.1%에서 G-단백질과 GTPrS 및 PLC의 결합정도의 관찰은 조직분쇄시료를 소의 뇌 및 부신조직을 이용하여 $^{35}S$-GTPrS 첨가시와 단세포군 항체를 이용한 경우 모두에서 1.49% 이하의 낮은 결합 정도를 관찰하였다. 그래서 정제된 PLC isozyme과 G-단백질 $Go{\alpha}$, $G{\beta}{\gamma}$, Gmix, $Gi{\alpha}$ 및 $Gt{\alpha}$ 각각에 대한 효과 관찰에 서 $Go{\alpha}$와 $G{\beta}{\gamma}$는 PLC ${\beta}$와 ${\delta}$의 활성도를 증가시켰고, PLC ${\gamma}$는 별 영향이 없었으며 Gmix에서는 세효소 모두 증가시켰다. $Gi{\alpha}$는 PLC ${\beta}$와 ${\gamma}$에서만 증가하였다. $Gt{\alpha}$는 PLC ${\beta}$ 및 ${\gamma}$에서 억제하였고 PLC ${\delta}$에서는 증가 양상을 보였다. 그러므로 PLC 활성화에 G-단백질의 관여가 인지되며 PLC isozyme과 G-단백질의 종류에 따라 대개의 경우 증가하는 경향이나 일부는 억제 내지는 별 영향이 없는 것으로 나타났다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.186-186
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• 1994

Many agonists have been known to activate the hydrolysis of membrane phospholipids through the bindings with corresponding receptors on the various cells. Diacylglycerol and inositol 1,4,5-trisphosphate(IP3) generated by the action of phosphoinositide-specific phospholipase C (PI-PLC) are well known second messengers for the activation of protein kinase C and the mobilization of Ca2+ in many cells. Three types of PI-PLC isozyme (${\alpha}$,${\gamma}$, and $\delta$) and several subtrpes for each type have been identified from mammalian sources by purification of enzymes and cloning of their cDNAs. Each type PI-PLC isozyme is coupled to different receptors and mediators, for example, ${\beta}$-types are coupled to the seven-transmembrane-receptors via Gq family of G-proteins and ${\beta}$-types directly to the receptor tyrosine kinases. Specific modulators for the signaling pathway through each type of PI-PLC should be very useful as potential potential candidates for lend substances in developing novel drugs. To establish the sensitive and convenient screening systems for searching modulators on PI-PLC mediated signaling, two kinds of approaches have been tried. (1) Establishment of in vitro assay condition for each type of PI-PLC isozyme: Overexpression by using vaccinia virus and purification of each isozyme was carried out for the preparation of large amounts of enaymes. Optimum and sensitive assay condition for the measurements of PI-ELC activities were established. (2) Development of the cell lines in which each type of PI-PLC is permanently overexpressed: A fibroblast cell line (3T3${\gamma}$1-7) in which PI-PLC-${\gamma}$1 was overexpressed by using pZip-neo expression vector was developed and used for the measurement of PDGF-induced IP3 formation. The responses for IP3 formed in 3T3${\gamma}$1-7 cells by the treatment of PDGF is 8 times more sensitive than those in control cells. 3T3${\gamma}$l-7 cell is useful for the screening of the inhibitors on the PDGF-induced cellular responses from large number of samples in a small volume(50 ${\mu}$l) and short time(5-15 min). Using these systems, we screened hundreds of herb-extracts for the inhibition of PDGF-induced IP3 formation and selected several extracts that showed the inhibition as the candidates for isolation and characterization of active substances. The determination of the acting point of selected extracts or fractions in the PDGF signaling pathway has been analyzing.

• Radiation Oncology Journal
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• 제12권3호
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• pp.271-284
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• 1994

Purpose : Phospholipase C(PLC) isozymes play significant roles in transmembrane signal transduction. PLC-${\gamma}1$ acts as the intracellular effector in signal transduction for cellular proliferation and differentiation. Ras oncoprotein is also involved in cell growth. We determined the biological significance of PLC and ras oncoprotein in regeneration following radiation and the effect of different modes of administration of 5-FU. Materials and Methods : To determine the effect of the administration mode of 5-FU on the regeneration of intestinal mucosa of rats following radiation, we compared the expression of PLC and ras oncoprotein in six groups. Group I had no treatment. Group II received radiation(8 Gy) only. Group III received radiation(8 Gy) and 5-FU(150mg/kg) continuous intravenous (iv) infusion for 12 hours. Group IV received radiation(8 Gy) and 5-FU(750mg/kg) iv bolus injection. Group V received only 5-FU(150mg/kg) continuous iv infusion for 12 hours, Group VI received only 5-FU (150mg/kg) iv bolus injection. Through immunoblotting and immunohistochemistry, we examined the expression of PLC and ras oncoprotein in rat jejunum at 96 hours after radiation or 5-FU administration and at 120 hours after radiation and 5-FU adminstration. We also investigated the histological findings using hematoxylin and eosin stain. Results : In the immunohistochemistry study, PLC-${\gamma}1$ expression was the highest in group III followed by groups II and VI in that order and was weakly positive in groups V and VI. PLC-${\gamma}1$ was hardly detected in the control group. The expression of ras oncoprotein was the same as the PLC-${\gamma}1$ expression for all groups. These results were confirmed by the histological findings regarding the mucosal regeneration. In the immunoblotting analysis, PLC-${\gamma}1$ expression was the highest in group III followed by group IV and II in that order. This difference between the immunoblotting and immunohistochemistry study was due to the high expression of PLC-${\gamma}1$ on the damaged surface epithelium rather than to its expression in the regeneration region as observed in the immunohistochemistry study for group IV. The expression of PLC-${\delta}1$ was positive only in group V and VI, which received both radiation and 5-FU, and the expression of PLC-${\beta}1$ was negligible for all groups. Conclusion : These results suggest that PLC-${\gamma}1$ mediated signal transduetion and ras oncoprotein may have a significant role in mucosal regeneration after radiation, and that continuous iv infusion of 5-FU may induce active regeneration in intestinal mucosa following radiation. In addition, the expression of PLC-${\delta}1$ in combined group of radiation and 5-FU implies that PLC-${\delta}1$ may be involved in signal transduction mediated by concerted action between radiation and 5-FU.

• BMB Reports
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• 제41권6호
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• pp.415-434
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• 2008

Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-$\beta$, -$\gamma$, -$\delta$, -$\varepsilon$, -$\zeta$ and -$\eta$. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.

• International Journal of Oral Biology
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• 제45권1호
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• pp.15-24
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• 2020

The fruit of Chaenomeles sinensis (Thouin) Koehne (Chaenomelis Fructus) known as "Mo-Gua" in Korea has been commonly used in traditional medicine to treat inflammatory diseases, such as sore throat. However, its effect on bone metabolism has not been elucidated yet. Here, we examined the effect of Chaenomelis Fructus ethanol extract (CF-E) on receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation and formation. CF-E considerably inhibited osteoclast differentiation and tartrate-resistant acid phosphatase-positive multinuclear cell formation from bone marrow-derived macrophages and osteoclast precursor cells in a dose-dependent manner. In addition, the formation of actin rings and resorption pits were significantly suppressed in CF-E-treated osteoclasts as compared with the findings in non-treated control cells. Consistent with these phenotypic inhibitory results, the expressions of osteoclast differentiation marker genes (Acp5, Atp6v0d2, Oscar, CtsK, and Tm7sf4) and Nfatc1, a pivotal transcription factor for osteoclastogenesis, were markedly decreased by CF-E treatment. The inhibitory effect of CF-E on RANKL-induced osteoclastogenesis was associated with the suppression of NFATc1 expression, not by regulation of mitogen-activated protein kinases and NF-κB activation but by the inactivation of phospholipase C gamma 1 and 2. These results indicate that CF-E has an inhibitory effect on osteoclast differentiation and formation, and they suggest the possibility of CF-E as a traditional therapeutic agent against bone-resorptive diseases, such as osteoporosis, rheumatoid arthritis, and periodontitis.

• BMB Reports
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• 제51권5호
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• pp.230-235
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• 2018

Bone resorption by multinucleated osteoclasts is a multistep process involving adhesion to the bone matrix, migration to resorption sites, and formation of sealing zones and ruffled borders. Macrophage colony-stimulating factor (M-CSF) and osteopontin (OPN) have been shown to be involved in the bone resorption process by respective activation of integrin ${\alpha}v{\beta}3$ via "inside-out" and "outside-in" signaling. In this study, we investigated the link between signal modulators known to M-CSF- and OPN-induced osteoclast adhesion and spreading. M-CSF- and OPN-induced osteoclast adhesion was achieved via activation of stepwise signals, including integrin ${\alpha}v{\beta}3$, $PLC{\gamma}$, $PKC{\delta}$, and Rac1. Osteoclast spreading induced by M-CSF and OPN was shown to be controlled via sequential activation, consistent with the osteoclast adhesion processes. In contrast to osteoclast adhesion, osteoclast spreading induced by M-CSF and OPN was blocked via activation of $PLC{\gamma}/PKC{\alpha}/RhoA$ signaling. The combined results indicate that osteoclast adhesion and spreading are selectively regulated via $PLC{\gamma}/PKC{\alpha}-PKC{\delta}/RhoA-Rac1$ signaling.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 제4회 추계심포지움
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• pp.75-84
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• 1996

Platelets from a patient with a mild inherited bleeding disorder and abnormal platelet aggregation and secretion show reduced generation of inositol 1,4,5-trisphosphate (IP$_3$), mobilization of intracellular Ca$\^$2+/, and phosphorylation of pleckstrin in response to several G protein mediated agonists, suggesting a possible defect at the level of phospholipase C (PLC) activation. A procedure was developed that allows quantitation of platelet PLC isozymes. After fractionation of platelet extracts by high-performance liquid chromatography, seven, out often known PLC isoforms were detected by immunoblot analysis. The amount of these isoforms in normal platelets decreased in the order PLC-${\gamma}$2 > PLC-${\beta}$2 > PLC-${\beta}$3 > PLC-${\beta}$l > PLC-${\gamma}$ > PLC-$\delta$1 > PLC-${\beta}$4. Compared with normal platelets, platelets from the patient contained approximately one-third the amount of PLC-${\beta}$2, whereas PLC-${\beta}$4 was increased threefold. These results suggest that the impaired platelet function in the patient in response to multiple G protein mediated agonists is attributable to a deficiency of PLC-${\beta}$2. They document for the first time a specific PLC isozyme deficiency in human platelets and provide an unique opportunity to understand the role of different PLC isozymes in normal platelet function.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.93.3-94
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• 2003

The signaling components of high affinity IgE receptor (Fc RI) were searched by yeast-hybrid screening of the cDNA library constructed from RBL-2H3 cells. The cytoplasmic part of the Fc RI- chain was found to specifically interact with PLC 2, and further comparatives studies were conducted focusing on the differential regulation of two PLC- isoforms through Fc RI. The inhibitors of Src, Syk, and protein kinase C similarly affected the tyrosine phosporylations of PLC 1 and PLC 2 but the inhibitors of PI3-kinase and p42/44 ERK effectively inhibited the activation of PLC 1 but not PLC 2. (omitted)

• 한국산학기술학회논문지
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• 제20권12호
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• pp.298-305
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• 2019

꾸지뽕나무 뿌리 추출물은 항염, 항암, 항산화와 같은 효과를 갖는 많은 생리활성 물질을 포함하고 있다고 알려져 있다. 그러나 꾸지뽕나무 뿌리 추출물(이하 CTE)의 사람 혈소판 응집 억제 기전에 관하여는 알려진 바 없다. 본 연구에서는 CTE가 혈소판에 미치는 영향을 확인하고자 하였다. CTE는 collagen으로 유도한 혈소판 응집 반응에서 cyclooxygenase-1 활성을 억제하고, 세포 내 칼슘 농도를 낮추는 방식으로 thromboxane A₂ 생성을 억제하였다. 또한, phospholipase Cγ2와 syk의 인산화 반응을 억제하였으며, guanosine monophosphate (cGMP)에 의존적인 방식으로 vasodilator-stimulated phosphoprotein(VASP)의 Ser²³⁹ 위치를 인산화하여 항혈소판 효과를 나타내었다. 또한, 고지방 식이로 혈소판 활성화를 유도한 랫드에서 CTE를 경구 투여 하였을 때, 간독성 없이 콜라겐으로 유도한 혈소판 응집반응을 thromboxane A₂ 생성을 억제함으로써 항혈소판 효과를 보였다. 이는 in vivo와 in vitro에서 같은 결과를 제시하고 있다. 결론적으로, CTE는 항혈소판 작용 및 심혈관계 질환 예방을 위한 천연물 유래의 안전하고, 새로운 물질임을 제시하는 바이다.