• 대한약리학회지
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• 제29권1호
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• pp.121-130
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• 1993

흰쥐 말초 T림프구에 분열유발 물질인 PMA와 Con A를 투여하여 인산화되는 단백을 확인하고, PKC 억제제인 H-7, CaM kinase 억제제인 W-7을 전처치한 후의 인산화 변동과 시간 경과에 따른 인산화 변동을 관찰하였다. 그 결과 흰쥐 T림프구를 PMA로 자극하면 5개의 인산화 단백이 새로이 나타나고 7개 단백의 인산화가 증가 되었으며, Con A자극으로는 1개의 단백이 새로이 인산화 되고 7개 단백의 인산화가 증가되었다. PMA 및 Con A자극으로 인산화 되는 13개 단백은 kinase억제제 전처치에 의하여 3군으로 각각 구분되며, H-7 전처치로 24 kDa/pI 7.1, 24/7.2, 26/6.1, 74/6.2 단백의, W-7 전처치로 14 kDa/pI5.9, 28/6.8, 29/6.9, 28/7.0, 44/6.8, 58/6.2 단백의 인산화가 현저히 감소 되었으며, 18 kDa/p1 5.4, 25/7.3 및 54/5.2단백은 두 억제제에 의해 영향을 받지 않았다. 이들 인산화 단백은 대부분 세포의 soluble fraction에서 확인되며 자극후 반응 초기에 인산화 된 후 인산화가 감소하나, 침전물에서 관찰되는 소수의 인산화 단백은 지속적인 인산화를 보였다. 한편 Kinase 억제제 처리에 의하여 구분된 3군에 속하는 단백들의 시간에 따른 인산화 양상을 관찰한 결과 각 군에 따른 인산화 양상에 상호 연관성이 없었다. 이상의 실험결과로 보아 림프구 활성의 초기 단계에서 인산화 되는 단백에는 PKC, CaM kinase 및 다른 kinase에 의해 인산화 되는 3종류의 단백이 존재하며, 3종류의 kinase의 활성은 단계적인 활성이 아니라 독립적 또는 상호 협동적으로 작용하여 림프구 활성을 유발시키는 것으로 생각된다.

• Archives of Pharmacal Research
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• 제29권5호
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• pp.375-383
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• 2006

The anti platelet effects of a novel guanidine derivative, KR-32570 ([5-(2-methoxy-5-chlorophenyl) furan-2-ylcarbonyl]guanidine), were investigated with an emphasis on the mechanisms underlying its inhibition of collagen-induced platelet aggregation. KR-32570 significantly inhibited the aggregation of washed rabbit platelets induced by collagen $(10{\mu}g/mL)$, thrombin (0.05 U/mL), arachidonic acid $(100{\mu}M)$, a thromboxane (TX) $A_2$ mimetic agent U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin $F_2,\;1{\mu}M$) and a $Ca^{2+}$ ATPase inhibitor thapsigargin $(0.5{\mu}M)$ ($IC_{50}$ values: $13.8{\pm}1.8,\;26.3{\pm}1.2,\;8.5{\pm}0.9,\;4.3{\pm}1.7\;and\;49.8{\pm}1.4{\mu}M$, respectively). KR-32570 inhibited the collagen-induced liberation of $[^3H]$arachidonic acid from the platelets in a concentration dependent manner with complete inhibition being observed at $50{\mu}M$. The $TXA_2$ synthase assay showed that KR-32570 also inhibited the conversion of the substrate $PGH_2$ to $TXB_2$ at all concentrations. Furthermore, KR-32570 significantly inhibited the $[Ca^{2+}]_i$ mobilization induced by collagen at $50{\mu}M$, which is the concentration that completely inhibits platelet aggregation. KR-32570 also decreased the level of collagen $(10{\mu}g/mL)$induced secretion of serotonin from the dense-granule contents of platelets, and inhibited the NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. These results suggest that the antiplatelet activity of KR-32570 against collagen-induced platelet aggregation is mediated mainly by inhibiting the release of arachidonic acid, $TXA_2$ synthase, the mobilization of cytosolic $Ca^{2+}$ and NHE-1.

• Toxicological Research
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• 제11권2호
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• pp.199-203
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• 1995

The focus of this study was to investigate that cellular parameters and glucose uptake might be altered by extracellular calcium and starvation. Addition of 1 mM $Ca^{++}$ to hepatocytes (equalling to the free calcium concentration of blood) significantly increased intracellular $Na^+$ and decreased $Na^+$ & LDH leakage. This pertains to the hepatocytes of control rats as well as those of rats fasted for 24 and 48. hr. These effects might be come from the membrane-stabilizing effects of calcium. But calcium had no effects on cell volumes, superoxide-formation and glucose uptake. Actually hepatocytes of starved rats showed changes in several cellular parameters. Starvation increased LDH leakage, glucose uptake and the total concentration of $Na^+$ and $Na^+$ whereas it markedly decreased cell volumes. Since total tonicity remained unchanged, intracellular $Na^+$ and $Na^+$ could contribute to a higher share of total osmolarity in starvation. Starvation increased the cytoplasmic pH because $R-NH^{3+}$ions and their corresponding counterions disappeared. This increase may be related to suppress the protonization of amino groups in proteins. Starvation decreased hepatic glycogen, a major compound that affects cytosolic volume of hepatocytes. The data indicate that starvation increases the glucose transport activity. The possible molecular basis will be discussed.

• Journal of Nutrition and Health
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• 제25권5호
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• pp.360-378
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• 1992

This study was performed to investigate effect of dietary protein and calcium levels on cad-mium intoxication in rats. Adult Sprague-Dawley male rate(245$\pm$21g) were blocked into 18 groups of 7 animals according to body weight Nine experimental diets different with protein(40%, 15%, 7%) and calcium (1.3%, 0.6%, 0.1%) levels were prepared. Nine groups of animals were fed each diet with 50ppm cadmium in drinking water and the other 9 groups without cadmium for 30days. Results were summarized as follows: 1) Body weight gain F. E. R(Food Efficiency Ratio) and weights of liver kidney and femur were higher in high protein groups among cadmium exposed groups. 2) Cadmium contents in liver and intestine were higher in rats fed high protein diet or low calcium diet among cadmium exposed groups. Fecal cadmium excretion was highest in high protein-high calcium diet group among cadmium exposed animals. Metallothionein contents in liver kidney and intestine were higher in animals exposed to cadmium and fed high protein diets. 3) Gel filtration chromatography of cytosolic solution showed that the higher dietary protein and calcium levels were the more cadmium was found in metallothionein fractions. 4) No gross histopathological change was seen in liver kidney and intestine of cadmium exposed rats. However a significant increase of smooth endoplasmic reticulum which was alleveated by high protein-high calcium diet was observed. Results obtained indicated that not only high protein diet but also high calcium diet showed preventive effect on cadmium intoxication by increasing the induction of metallothionein syn-thesis and decreasing the cadmium absorption.

• 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.

• BMB Reports
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• 제47권7호
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• pp.393-398
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• 2014

The role of Bax inhibitor-1 (BI-1) in the protective mechanism against apoptotic stimuli has been studied; however, as little is known about its role in death receptor-mediated cell death, this study was designed to investigate the effect of BI-1 on Fas-induced cell death, and the underlying mechanisms. HT1080 adenocarcinoma cells were cultured in high concentration of glucose media and transfected with vector alone (Neo cells) or BI-1-vector (BI-1 cells), and treated with Fas. In cell viability, apoptosis, and caspase-3 analyses, the BI-1 cells showed enhanced sensitivity to Fas. Fas significantly decreased cytosolic pH in BI-1 cells, compared with Neo cells, and this decrease correlated with BI-1 oligomerization, mitochondrial $Ca^{2+}$ accumulation, and significant inhibition of sodium-hydrogen exchanger (NHE) activity. Compared with Neo cells, a single treatment of BI-1 cells with the NHE inhibitor EIPA or siRNA against NHE significantly increased cell death, which suggests that the viability of BI-1 cells is affected by the maintenance of intracellular pH homeostasis through NHE.

• Journal of Applied Biological Chemistry
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• 제66권
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• pp.236-243
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• 2023

혈소판은 1차 및 2차 지혈에서 근본적인 역할을 하는 세포지만 혈소판의 과도한 활성화는 혈전증을 유발할 수 있다. 따라서 혈소판 응집의 적절한 조절은 혈전증 매개 질환을 예방하는 데 중요하다. 최근 곤충소재의 개발이 주목을 받고 있다. 다양한 곤충 자원 중 고영양 기능성 식품원으로는 쌍별귀뚜라미(Gryllus bimaculatus)와 같은 곤충류가 있다. 쌍별귀뚜라미 는 고단백 및 불포화지방산을 함유하고 있으며 2015년 9월 식품의약품안전처로부터 식품원료로 등록되었다. 본 연구에서는 쌍별귀뚜라미 에탄올 추출물(G. bimaculatus extract)이 혈소판 응집, 세포 내 Ca²⁺ 조절, thromboxane A₂ 생산 및 glycoprotein IIb/IIIa (integrin αIIb/β3) 활성화를 억제하는지 여부를 확인하고. 1, 4, 5-triphosphate receptor type I, extracellular signal-regulated kinase, cytosolic phospholipase A₂, mitogen-activated protein kinases p38, vasodilator-stimulated phosphoprotein, phosphatidylinositol-3 kinase, Akt, glycogen synthase kinase-3α/β 및 SYK 같은 신호 분자를 조절할 수 있는지 여부를 조사했다. 우리는 쌍별귀뚜라미 추출물이 혈소판 관련 혈전증 및 심혈관 질환을 예방할 수 있는 잠재적인 치료 약물로 가치가 있음을 규명하였다.

• Journal of Plant Biotechnology
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• 제38권2호
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• pp.169-175
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• 2011

칼슘은 동물과 마찬가지로 식물에서도 다양한 신호를 전달하는 2차 매개체로 잘 알려져 있다. 특히, 세포사멸 현상을 유도하는 신호전달에 칼슘의 관여는 잘 보고되어 있다. 최근 발표된 논문에서 액포막에 존재하는 $Ca^{2+}$-ATPases(ACA4와 ACA11)가 이중으로 돌연변이된 식물체의 잎에서 세포사멸 표현형을 관찰했으며, 이러한 세포사멸 현상이 salicylic acid (SA)에 의존적이라고 보고했다. 또한 앞선 연구들에서 vacuolar processing enzymes (VPEs)이 생물학적 또는 비생물학적 스트레스에 의해서 유발되는 SA에 의해서 활성화 된다고 보고하였다. 본 연구에서는 액포막 $Ca^{2+}$-ATPases (ACA4와 ACA11)가 이중으로 돌연변이 된 식물체에서 VPEs의 유전자 발현이 상당히 증가되어 있고 효소 활성도 증가됨을 확인했다. 이 결과를 바탕으로, aca4/aca11/avpe와 aca4/aca11/${\gamma}$vpe 그리고, aca4/aca11/avpe/${\gamma}$vpe의 삼중과 사중 돌연변이체를 구축했다. 이들과 aca4/aca11의 이중 돌연변이체의 세포사멸 표현형을 비교 관찰한 결과, 삼중과 사중 돌연변이에서 세포사멸 현상이 일정기간 억제되는 것을 관찰했다. 또한, 삼중과 사중의 돌연변이체에서 VPEs의 효소 활성이 많이 감소되는 현상으로 나타났다. 결론적으로, $Ca^{2+}$-ATPases (ACA4와 ACA11)가 이중으로 돌연변이된 식물체에서는 SA가 유발되며, 이러한 SA에 의해서 VPEs의 단백질이 활성화되어 세포사멸 현상이 발생할 것으로 사료된다.

• The Korean Journal of Physiology and Pharmacology
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• 제20권2호
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• pp.213-220
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• 2016

Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular $Ca^{2+}$, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with $0.5{\mu}g/ml$ BG, $100{\mu}g/ml$ peptidoglycan (PGN), or $10{\mu}M$ A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial $Ca^{2+}$ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial $Ca^{2+}$ uniporter has an important regulatory role in BG-induced mast cell degranulation.

• 약학회지
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• 제41권2호
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• pp.212-219
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• 1997

Multiple forms of extracellular phospholipase $A_2$ have been detected in human amniotic fluid (HAF). When HAF was subjected to heparin-Sepharose column chromatography, phospholipase $A_2$ activity was detected in both heparin-non binding and binding fraction. The activity of heparin-non binding fraction was further purified by sequential uses of column chromatographies on butyl-Toy-opearl 650M and DEAE-Sephacel. DEAE-Sephacel fraction contained three different phospholipase $A_2$ activities (Peak I, II, III). The molecular weight of DEAE-Sephacel fraction phospholipase $A_2$ determined by SDS-PAGE were about 52KDa (Peak I). Peak II, III required micromolar $Ca^{2+}$ ion for its maximum activity, but Peak I enzyme showed calcium independent phospholipase $A_2$ activity and showed broad range of pH (6.0~10.0) optimum. All these enzymes were not recognized by a monoclonal antibody raised against phospholipase $A_2$ from human synovial fluid. These results suggest that HAF might contain multiple forms of extracellular phospholipase $A_2$, which may neither belong to the 14KDa group II phospholipase $A_2$ family nor cytosolic phospholipase $A_2$.