• Journal of the Korean Chemical Society
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• v.52 no.5
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• pp.488-494
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• 2008

• Korean journal of applied entomology
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• v.51 no.3
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• pp.299-303
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• 2012

This study aims to investigate whether geographical variation affects the antibacterial component properties of honeybee (Apis mellifera L.) venom in Korea. Honeybee venom samples were collected from May to September, during 2010 and 2011, from 35 different sites, and were analyzed for major components, including melittin, apamin and phospholipase A2 were determined by a liquid chromatography using ammonium formate, acetonitrile, trifluoracetic acid. On average, melittin, apamin and phospholipase A2 were determined $55.2{\pm}2.07%$, $22.57{\pm}0.103%$, and $12.51{\pm}0.37%$, respectively. The ratio of the major components, including melittin, apamin and phospholipase A2 did not differ significantly according to flower or temperature during collections (One way-ANOVA, Duncan's test (${\alpha}$=0.05)).

• Journal of Life Science
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• v.21 no.8
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• pp.1100-1108
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• 2011

The immunomodulating effects of moxifloxacin seem to be effective in downregulating inflammatory reactions. This presumed effect was tested in endotoxin (ETX)-induced acute lung injury (ALI) in rats. After moxifloxacin treatment (10 mg/kg) of ETX-given rats, lung myeloperoxidase (MPO) activity, bronchoalveolar-lavage (BAL) protein, and the number of neutrophils in the BAL cells were measured. Light and electron microscopic structures were also examined. Electron microscopic $CeCl_3$ histochemistry for the detection of hydrogen peroxide in the lungs and immunohistochemistry of cytosolic phospholipase A2 (cPLA2) in the lung tissues and BAL cells were performed. To examine the expression of TNF${\alpha}$ in the lungs, western blotting was carried out with the lung tissues. ETX had accumulated neutrophils in the lungs, which was followed by lung leak. Oxidative stress occurred, and increased expression of cPLA2 in the lung tissues and BAL cells was observed in the ETX-given rats. Simultaneously, the expression of TNF${\alpha}$ was enhanced by ETX. Moxifloxacin, however, decreased all these parameters, indicating that ALI may have been ameliorated. Moxifloxacin appears to ameliorate ETX-induced ALI partially through the suppression of cPLA2 in the lungs of rats.

• Journal of the Korean Chemical Society
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• v.59 no.1
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• pp.22-30
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• 2015

The effect of diphtheria toxin on cell membrane lipids was studied by examining the phospholipase D (PLD) activity and free fatty acids (FFA) release in HepG2 cells. The diphtheria toxin effects on lipid alteration show apparently maximal at pH 5.1, stimulating PLD activity nearly 3.5 fold and enhancing FFA release approximately 5 fold over the control. These results indicate that the membrane is perturbed and its lipid component is rearranged during the diphtheria toxin translocation. Digitonin, a random membrane perturbing detergent, exhibit about four-fold higher perturbation effect over the diphtheria toxin at neutral pH. This observation suggests that the membrane perturbation induced by diphtheria toxin appears to be rather selective. To investigate the cause of the membrane perturbation, Cibacron blue, an inhibitor of membrane pore formation, and hemagglutinin, an influenza virus with fusion peptide, were tested for their effects on diphtheria toxin action. Cibacron blue decreased the diphtheria toxin effect by almost 50%, but the lipid alteration induced by hemagglutinin was similar to the diphtheria toxin effect. These observations imply that the membrane perturbation induced by diphtheria toxin may be caused by a combination of pore formation and insertion of hydrophobic peptide of toxin to the membrane as well. Additionally, we found that the diphtheria toxin increased the HepG2 cells permeability but the cells viability was maintained at high level at the same time. DNA fragmentation which is related to apoptosis was not induced by the toxin. Under these conditions, we could demonstrate that the lipid alteration of HepG2 cells was brought about by diphtheria toxin at acidic pH.

• Journal of the Korean Chemical Society
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• v.50 no.5
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• pp.362-368
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• 2006

SH group modifying chemicals were used to characterize the eight cysteine residues of cabbage PLD. 5,5-dithiobis(2-nitrobenzoate)(DTNB) was used to titrate the SH group of cysteine residues . Based on the optical density at 412nm due to the reduced DTNB, 4 SH groups are found to be present in a native PLD while 8 SH groups in the denatured PLD whose tertiary structure was perturbed by 8M urea. The results imply that among the 8 cysteine residues of PLD, the half(4) are exposed on the surface whereas the other half are present at the interior of the enzyme tertiary structure. The PLD was inactivated by SH modifying reagents such as p-chloromercuribenzoate(PCMB), iodoacetate, iodoacetamide, and N-ethylmaleimide. At the addition of dithiothreitol(DTT) only the PCMB inhibited PLD activity was recovered reversibly. The micro-environment of the exposed SH group of cysteine residues was examined with various disulfide compounds with different functional groups and we found that anionic or neutral disulfides appear to be more effective than the positively charged cystamine for inactivating the PLD activity. The effect of redox state of cysteine residues on the PLD activity was further explored with H2O2. The oxidation of SH groups by H2O2 inhibited the PLD activity more than 70%, which was mostly recovered by DTT. From these results, we could confirm chemically that all the cysteine residues of PLD are present as in their reduced SH forms and the 4 SH groups exposed on the surface of the enzyme may play important roles in the regulation of PLD activity.

• YAKHAK HOEJI
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• v.44 no.1
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• pp.47-51
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• 2000

To examine whether DGRF inhibits $cPLA_2$ activity in vitro, we purified a 100 kDa $cPLA_2$enzyme from porcine spleen and performed an inhibition study at two concentrations of 5.0 and 50.0 $\mu$M 1-stearoyl-2-[1-$^{l4C}$ ]arachidonoyl-sn -glycero-3-phosphocholine as a substrate to rule out an apparent inhibition due to "substrate depletion". Here we reported that DGRF inhibited $cPLA_2$activity with $ID_{50}$ of 3.2 $\mu$M and virtually complete inactivation of the enzyme occurred at 60 $\mu$M. Interaction experiment between enzyme protein and inhibitor by ultrafiltration method indicated that 1-desgalloylrugosin-F inactivates $cPLA_2$enzyme by an irreversible mechanism.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.79-79
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• 1997

자연계에 존재하는 수은중 유기수은은 생태계 먹이사슬을 통하여 체내의 여러장기에 축적되어 조직손상을 일으키는 것으로 잘 알려져 있다. 그러나 이러한 세포독성에 대한 정확한 생화학적 기전에 대해서는 자세히 알려진 바가 없다. 포스포리파아제 $A_2$(PLA$_2$)는 세포막의 인지질로부터 Arachidonic acid (AA)와 Lysophospholipid를 유리시키는 효소로 최근 세포손상과 관련하여 그 역할이 주목되고 있으며, 극히 최근, 일차배양 소뇌신경세포를 이용한 연구에서 메칠수은처리에 의해 세포독성의 지표인 Lactate dehydrogenase (LDH)의 유리와 함께 AA 유리가 증가되는 것이 관찰되었으나 여러형태의 PLA$_2$중 어느형태의 효소가 관련되어 있는지, 또한, 그 자세한 기전에 대해서는 불분명한 점이 많다. 본 연구에서는 신장세포의 일종인 MDCK세포를 이용하여 메칠수은의 처리에 의한 PLA$_2$의 활성화 및 그 생화학적인 기전을 구명하고자 하였다. [$^3$H]AA를 MDCK세포의 배양액에 첨가하여 라벨링한 후 메칠수은을 처리하였을때 [$^3$H]AA가 대조군에 비해 농도의존적 및 경시적으로 현저하게 증가하였으며 동시에 LDH의 유리도 함께 관찰되었다. 이러한 [$^3$H]AA의 유리 증가는 세포질 PLA$_2$에 특이적인 저해제로 알려진 AACOCF$_3$의 전처리에 의해 거의 완전히 억제되었으나 LDH의 유리는 오히려 증가하였다. 또한, 글루타치온(GSH)의 전구체인 NAC (N-Acetyl Cysteine)에 의해 [$^3$H]AA의 유리는 부분적으로 감소하였으나, LDH의 유리는 변함이 없었다. 돼지비장이나 MDCK 세포에서 얻어진 세포질 PLA$_2$에 메칠수은을 직접 처리하였을때는 오히려 PLA$_2$의 활성은 감소되었다. 위의 결과들로부터 메칠수은에 의한 [$^3$H]AA의 유리 증가는 세포질 PLA$_2$효소에 대한 직접적인 작용이 아니라 세포내 -SH기의 차단이나 Oxidative Stress에 의해 간접적으로 활성화되는 것으로 예상되며, 세포질 PLA$_2$에 의해 유리된 AA의 세포독설과 관련된 세포내의 역할에 대해 의문이 제기되었다.

• Journal of fish pathology
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• v.18 no.1
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• pp.67-80
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• 2005

Phosphoinositide-specific phospholipase $C\delta$ $PLC\delta$) plays an important role in many cellular responses and is involved in the production of second messenger. The present study was conducted to obtain the biochemical characteristics of the expressed recombinant $PLC\delta$ in E. coli cloned from Misgurnus mizolepis and partially purified $PLC\delta$ enzymes from liver tissues of M. mizolepis (wild ML-$PLC\delta$). The ML $PLC\delta$ gene was cloned and expressed under the previous report (Kim et al., 2004), and purified the recombinant protein by successive chromatography using $Ni^{2+}$-NTA affinity column and gel iltration FPLC column. The wild ML-$PLC\delta$ protein was solublized with 2 M KCI and purified by successive chromatography on open heparin-Sephagel and analytical TSKgel heparin-5PW. Both the recombinant and wild ML-$PLC\delta$ form of protein showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP$_2$) or phosphatidylinositol (PI). Its activity was absolutely $Ca^{2+}$- dependant, which was similar to mammalian $PLC\delta$ isozymes. Maximal PI-hydrolytic activations of recombinant and wild ML- TEX>$PLC\delta$ was at pH 7.0 and pH 7.5, respectively. In addition, the enzymatic activities of recombinant and wild ML-$PLC\delta$ were increased in concentration-dependent manner by detergent, such as sodium deoxycholate SDC), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). The activities decreased in contrast by a polyamine, such as spermine. Western blotting showed that several types of $PLC\delta$ isozymes exist in various organs. Taken together our results, it suggested that the biochemical characteristics of ML-$PLC\delta$ are similar with those of mammalian $PLC\delta1$ and ${\delta}3$ isozymes.