• Herbal Formula Science
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• v.13 no.2
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• pp.111-123
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• 2005

The purpose of this study was to investigate the anticancer effects of Caesalpiniae Lignum (Somok) on HepG2 cells, a human liver cancer cell line. To study the cytotoxic effect of Caesalpiniae Lignum methanol extract (CL-MeOH) on HepG2 cells, the cells were treated with various concentrations of CL-MeOH and then cell viability was determined by XTT reduction method and trypan blue exclusion assay. CL-MeOH reduced proliferation of HepG2 cells in a dose-dependent manner. To confirm the induction of apoptosis, HepG2 cells were treated with various concentrations of CL-MeOH. The activation of caspase 3 and the cleavage of poly ADP-ribose polymerase (PARP), a substrate for caspase-3 and a typical sign of apoptosis, was examined by western blot analysis. CL-MeOH decreased procaspase 3 level in a dose-dependent manner and induced the clevage of PARP at concentration> $200{\mu}/ml$. Mitogen-activated protein (MAP) kinase signaling cascades are multi-functional signaling networks that influence cell growth, differentiation, apoptosis, and cellular responses to stress. CL-MeOH-induced MAPK activation was examined by Western blot for phosphorylated ERK, p38 and JNK. CL-MeOH significantly increased p38 phosphorylation and JNK phosphorylation in a dose-dependent manner. Inhibition of p38 function using the selective inhibitor SB20358O results in inhibition of apoptosis by CL-MeOH. These results suggest that CL-MeOH-induced apoptosis is MAP kinase-dependent apoptoric pathway. These results suggest that CL-MeOH is potentially useful as a chemotherapeutic agent in human liver cancer.

• Journal of Microbiology and Biotechnology
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• v.19 no.1
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• pp.65-71
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• 2009

Microbial oxidoreductive systems have been widely used in asymmetric syntheses of optically active alcohols. However, when reused in multi-batch reaction, the catalytic efficiency and sustainability of non-growing cells usually decreased because of continuous consumption of required cofactors during the reaction process. A novel method for NADPH regeneration in cells was proposed by using pentose metabolism in microorganisms. Addition of D-xylose, L-arabinose, or D-ribose to the reaction significantly improved the conversion efficiency of deracemization of racemic 1-phenyl-1,2-ethanediol to (S)-isomer by Candida parapsilosis cells already used once, which afforded the product with high optical purity over 97%e.e. in high yield over 85% under an increased substrate concentration of 15 g/l. Compared with reactions without xylose, xylose added to multi-batch reactions had no influence on the activity of the enzyme catalyzing the key step in deracemization, but performed a promoting effect on the recovery of the metabolic activity of the non-growing cells with its consumption in each batch. The detection of activities of xylose reductase and xylitol dehydrogenase from cell-free extract of C. parapsilosis made xylose metabolism feasible in cells, and the depression of the pentose phosphate pathway inhibitor to this reaction further indicated that xylose facilitated the NADPH-required deracemization through the pentose phosphate pathway in C. parapsilosis. moreover, by investigating the cofactor pool, the xylose addition in reaction batches giving more NADPH, compared with those without xylose, suggested that the higher catalytic efficiency and sustainability of C. parapsilosis non-growing cells had resulted from xylose metabolism recycling NADPH for the deracemization.

• Food Science and Biotechnology
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• v.14 no.6
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• pp.732-737
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• 2005

The combinational effect of gamma irradiation and moist heating on the trypsin inhibitor activity (TIA) in soaked and dried soybeans was evaluated by measuring the inhibition using N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. Gamma irradiation significantly decreased the TIA level in soybean at doses above 5 kGy, and the $ID_{50}$ (the gamma irradiation dose required to reach 50% inhibition) value for TIA was 13.53 kGy. Soaking prior to gamma irradiation significantly lowered the $ID_{50}$ to 8.44 kGy, and the soaking process enhanced the efficiency to inactivate TIA by as much as 48%. When soaking prior to gamma irradiation was followed by subsequent mild heating ($60^{\circ}C$) process, the $IT_{50}$ (heating time required to reach the 50% inhibition of TIA) value at even 1 kGy (5.28 min) was greatly reduced by over 50% compared to the level for the no-soaking process. In addition, the activation energy of soaking prior to gamma irradiation at 1 kGy was 2.45 kcal/mole, which was also about 50% lower than the 5.10 kcal/mole of dried soybean gamma-irradiated. Based on these results, soaking prior to gamma irradiation is an effective method for TIA inhibition. Furthermore, a combination of two or more processing methods such as soaking, heating and gamma irradiation is much more effective than any single processing method.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.114-114
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• 1995

이 연구에서는 혈전증 치료에 사용되는 혈전 응해제를 국내 독사독으로부터 개발하기 위한 실험을 실시하였다. 그 내용을 요약하면 다음과 같다. 국내에 서식하는 독사인 Agkistrodon blomoffi brevicaudus, Agkistrodon caliginosus와 Agkistrodon saxatilis에서 각각 사독을 채취하여 fibrin plate 방법으로 fibrin 분해능을 조사하여 Agkistrodon blomoffi brevicaudus의 독이 분해능이 가장 우수함을 밝혔다. 이와 같은 사실에 기초하여 A. blomoffi brevicaudus의 독으로부터 p-Aminobenzamidine affinity chromatography와 DEAE ion-exchange chromatography를 이용하여 분자량이 50.8 kDa인 황성 단백질을 정제하였다. 위에서와 같은 방법으로 정제한 단백질은 fibrin 분해능이 우수하고 fibrinogen의 ${\gamma}$ chain은 분해하지 않으나 B$\beta$ chain을 $A\alpha$ chain에 비하여 보다 선택적으로 분해하는 단백분해 효소임을 증명하였다. 이 정제 효소의 Fibrin에 대한 분해능은 266$\mu\textrm{g}$/${\mu}\ell$의 농도에서 Plasmin 1.0 unit (=3.0 WHO unit)보다 높게 나타났다. 정제된 효소는 chromogenic substrate인 N-Benzoyl-Phe-Val-Arg-pNA와 N-p-Tosyl- Gly-Pro-Arg-pNA의 arginine carboxyl side를 분해하고 pH 7.5에서 최대 활성을 보이며, Vmax는 5.46 umo1/1ㆍmin이고, Km 값은 0.20mM이며, 그리고 Cu$^{2+}$, $Zn^{2+}$, soybean trypsin inhibtor에 의해 25～50% 정도, serine proteinase inhibitor인 phenylmethylsulfonyl floride에 의해 80%정도 활성이 억제되는 특성이 있음을 규명하였다.

• Analytical Science and Technology
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• v.12 no.3
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• pp.256-259
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• 1999

Tyrosinases are related to the enzymatic browning of plants and attract the major scientific interest for the prevention of it. Three tyrosinase isozymes ($P_1$, $P_2$ and $P_3$) from pine needles were purified to homogeneity and characterized the factors that affect their activities. The L-ascorbic acid and ${\beta}$-mercaptoethanol notably inhibited the enzymatic activities of the three isozymes. The sodium diethyldithiocarbamate was a competitive inhibitor of isozymes with the $K_i$ values of $P_1$(0.030 mM), $P_2$(0.015 mM) and $P_3$(0.019 mM), respectively. Their enzyme activities were however, increased by the addition of most metal ions. The optimum pH for the three isozymes was 9.0~9.5 and the optimum temperatures ranged from 55 to $60^{\circ}C$ using L-DOPA as substrate.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.36 no.3
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• pp.199-205
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• 2010

The structure-activity relationships (SARs) between a series of phenyl-2,2'-methylenebis(cyclohexane-1,3-dione) analogues (1-22) as substrate molecules and their inhibitory activity against tyrosinase were studied quantitatively using molecular hologram quantitative structure-activity relationships (HQSARs). Based on the results, new compounds with the increased tyrosinase inhibitory activity were designed. In addition, statistically favored E-2 model ($q_2$ = 0.564 & $r_2$ = 0.929) was derived. It is predicted that the activity of the most potent one (P1) of compounds newly designed by the optimized HQSAR E-2 model was $Pred.pI_{50}$ = 5.48 and the predicted inhibitory activity was about 13.4 fold higher than that of the kojic acid used as standard compound.

• Archives of Pharmacal Research
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• v.28 no.3
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• pp.311-318
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• 2005

Oxidative stress has been reported to elevate ceramide level during cell death. The purpose of the present study was to modulate cell death in relation to cellular glutathione (GSH) level and GST (glutathione S-transferase) expression by regulating the sphingolipid metabolism. LLC­PK1 cells were treated with H$_2$O$_2$ in the absence of serum to induce cell death. Subsequent to exposure to H$_2$O$_2$, LLC-PK1 cells were treated with desipramine, sphingomyelinase inhibitor, and N-acetylcysteine (NAC), GSH substrate. Based on comparative visual observation with H202-treated control cells, it was observed that 0.5 $\mu$M of desipramine and 25 $\mu$M of NAC exhibited about 90 and $95\%$ of cytoprotection, respectively, against H$_2$O$_2$-induced cell death. Desipramine and NAC lowered the release of LDH activity by 36 and $3\%$ respectively, when compared to $71\%$ in H$_2$O$_2$-exposed cells. Cellular glutathione level in 500 $\mu$M H202-treated cells was reduced to 890 pmol as compared to control level of 1198 pmol per mg protein. GST P1-1 expression was decreased in H$_2$O$_2$-treated cells compared to healthy normal cells. In conclusion, it has been inferred that H$_2$O$_2$-induced cell death is closely related to cellular GSH level and GST P1-1 expression in LLC-PK1 cells and occurs via ceramide elevation by sphingomyelinase activation.

• YAKHAK HOEJI
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• v.41 no.1
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• pp.126-131
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• 1997

To overcome the problems of the resistance to clavulanic acid, many researchers are developing novel inhibitors that are not sensitive to new mutant ${\beta}$-lactamases. In order to evaluate newly synthesized compound CH2150 (Sodium (3S.5R)-6(Z)-[1-{1-(2-{2-benzoxazoly}thioethyl)-l.2,3-txiazol-4-yl}methylene] penicillanate-1,1-dioxide) as a ${\beta}$-lactamase inhibitor, we examined inhibitory activity of CH2150 against ${\beta}$-lactamases of clinical isolates resistant to ampicillin/sulbactam(12 strains of Escherichia coli and 13 strains of Staphylococcus aureus), and compared with that of sulbactam. Nitrocefin was used as substrate for ${\beta}$-lactamases, and the increase of absorbance was measured spectrophotometerically at 482 nm. ${\beta}$-Lactarnase inhibition of CH2150 against ${\beta}$-lactamases was 73 ~ 96% in E. coli and 76 ~ 79% in S. aureus. Comparatively, that of sulbactam was 96 ~ 100% and 96 ~ 100%, respectively. The inhibitory activity of CH2150 was slightly lower than that of sulbactam. The MIC values of ampicillin combined with CH2150 (2:1) for the clinical isolates were 4~512 ${\mu}$g/ml for E. coli and 1.0 ~ 64 ${\mu}$g/ml for S. aureus, whereas 0.5~16 ${\mu}$g/ml for E. coli and 0.25~8 ${\mu}$g/ml for S. aureus when combined with sulbactam (2:1).

• Applied Biological Chemistry
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• v.41 no.5
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• pp.355-362
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• 1998

The minor form of phosphofructokinase (EC 2.7.1.11; PFK), which was suggested to be of plastid origin from the host fraction of chickpea nodules, was isolated as a small protein with apparent molecular mass near 220 kDa and purified to a high degree. SDS-PAGE and western blot indicated that the enzyme was made up of a homotetrameric structure (55 kDa). The enzyme had sharp pH profiles with maximal activities at pH 8 and displayed Michaelis-Menten kinetics with respect to Fru-6-P and nucleoside triphosphate substrate at the pH optimum (pH 8) and at pH 7. MgATP was the most effective phosphoryl donor. Phosphoenolpyruvate was a potent inhibitor of minor PFK activity, and the enzyme was also strongly inhibited by 3-phosphoglycerate, 2-phosphoglycerate, and to a lesser extent, PPi. Minor PFK was weakly activated by KCl, NaCl and Pi, and was inhibitory at high concentration of KCl and Pi.

• Biomedical Science Letters
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• v.15 no.1
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• pp.55-60
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• 2009

Insect cells such as Spodoptera frugiperda Clone 9 (Sf9) cells are widely chosen as the host for heterologous expression of a mammalian sugar transport protein using the baculovirus expression system. Characterization of the expressed protein is expected to include assay of its function, including its ability to transport sugars and to bind inhibitory ligands such as cytochalasin B. It is therefore very important first to establish the transport characteristics and other properties of the endogenous sugar transport proteins of the host insect cells. However, very little is known of the transport characteristics of Sf9 cells, although their ability to grow on TC-100 medium strongly suggested the presence of endogenous glucose transport system. In order to investigate the substrate and inhibitor recognition properties of the Sf9 cell transporter, the ability of pentoses to inhibit 2-deoxy-D-glucose (2dGlc) transport was investigated by measuring inhibition constants $(K_i)$. To determine the time period over which of sugar into the Sf cells was linear, the uptake of 2dGlc 0.1mM extracellular concentration was measured over periods ranging from 30 seconds to 30 minutes. The uptake was linear for at least 2 minutes at the concentration, implying that uptake made over a 1 minute time course would reflect initial rates of the sugar uptake. The data have also revealed the existence of a saturable transport system for pentose uptake by the insect cells. The transport was inhibited by D-xylose and D-ribose, although not as effective as hexoses. However, L-xylose had a little effect on 2dGlc transport in the Sf9 cells, indicating that the transport is stereoselective. Unlike the human erythrocyte-type glucose transport system, D-ribose had a somewhat greater apparent affinity for the Sf9 cell transporter than D-xylose. It is therefore concluded that Sf9 cells contain an endogenous sugar transport activity that in some aspects resembled the human erythrocyte-type counterpart, although the Sf9 and human transport systems do differ in their affinity for cytochalasin B.