• KSBB Journal
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• 제29권1호
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• pp.1-8
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• 2014

Tyrosinases catalyze the hydroxylation of monophenolic compounds and the conversion of o-diphenols to oquinones. The enzymes are mainly involved in the modification of tyrosine into L-3,4-dihydroxyphenyl-alanine (L-DOPA) and DOPA/DOPAquinone-drived intermolecular cross-linking, which play the key roles of pigmentation to the cells. It is ubiquitously distributed in microorganisms, plants, and animals all around the nature world. They are classified as copper- containing dioxygen activating enzymes; two copper ions are coordinated with six histidine residues in their active sites and they are distinguished as met-, deoxy-, and oxy-form depending on their oxidative states. Natural extraction and recombinant protein approaches have been tried to obtain practical amounts of the enzymes for industrial application. Tyrosinases have been widely applied to industrial and biomedical usages such as detoxification of waste water containing phenolic compounds, L-DOPA as a drug of Parkinson's disease, biomaterials preparation based on the cross-linking ability and biosensors for the detection of phenolic compounds. Therefore, this review reports the mechanism of tyrosinase, biochemical and structural features and potential applications in industrial field.

• 한국식품영양과학회지
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• 제43권8호
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• pp.1166-1173
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• 2014

본 연구는 굴 가수분해물이 아세트아미노펜에 의한 간독성의 무독화 효과를 HepG-2 세포를 사용하여 조사하였다. 굴 가수분해물은 굴 단백질의 가교연결을 위해 TGase로 전처리하거나(TGPN) 혹은 하지 않고(PN), 1% Protamex와 1% Neutrase 단백질 분해효소로 2단 가수분해하였다. 두 종류의 굴 가수분해물은 아세트아미노펜으로 간 손상을 유도한 세포에 각각 처리하여 세포 생존율을 측정하였으며, 세포 배양 시 배양액으로 유출된 GOT와 GPT 활성을 측정하였다. TGPN 가수분해물의 경우 아세트아미노펜만을 처리한 negative 대조군($60.7{\pm}3.2%$)에 비하여 $100{\mu}g/mL$와 $200{\mu}g/mL$의 농도에서 각각 $136.2{\pm}1.4%$와 $179.6{\pm}3.8%$의 높은 세포 생존율을 보였다. PN 가수분해물은 $100{\mu}g/mL$와 $200{\mu}g/mL$의 농도에서 각각 $107.9{\pm}8.8%$와 $130.6{\pm}7.6%$의 세포 생존율을 보였다. GOT 활성은 negative 대조군의 경우에 $38.3{\pm}0.2$ Karmen/mL이었으며, TGPN($200{\mu}g/mL$)과 PN($200{\mu}g/mL$)에서는 $19.9{\pm}0.5$와 $22.0{\pm}2.4$ Karmen/mL로 농도에 따라 유의적인 활성 감소를 확인할 수 있었다. 그리고 GPT 활성도 GOT와 같은 경향의 활성을 나타내었다. 이 같은 결과에 미루어 굴 유래 가수분해물의 간 보호 건강 기능 식품 혹은 약물 개발의 가능성을 확인하였으며 앞으로 가수분해 펩티드 중의 유효성분에 대한 구조동정과 작용 기전에 관한 연구가 필요할 것으로 보인다.

• Nutrition Research and Practice
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• 제11권3호
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• pp.214-222
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• 2017

BACKGROUND/OBJECTIVES: Glutathione S-transferase (GST) forms a multigene family of phase II detoxification enzymes which are involved in the detoxification of xenobiotics by conjugating substances with glutathione. The aim of this study is to assess the antioxidative status and the degree of DNA damage in the subclinical hypertensive patients in Korea using glutathione S-transferase polymorphisms. SUBJECTS/METHODS: We examined whether DNA damage and antioxidative status show a difference between GSTM1 or GSTT1 genotype in 227 newly diagnosed, untreated (systolic blood pressure $(BP){\geq}130mmHg$ or diastolic $BP{\geq}85mmHg$) subclinical hypertensive patients and 130 normotensive subjects (systolic BP < 120 mmHg and diastolic BP < 80 mmHg). From the blood of the subjects, the degree of the DNA damage in lymphocyte, the activities of erythrocyte superoxide dismutase, the catalase, and the glutathione peroxidase, the level of glutathione, plasma total radical-trapping antioxidant potential (TRAP), anti-oxidative vitamins, as well as plasma lipid profiles and conjugated diene (CD) were analyzed. RESULTS: Of the 227 subjects studied, 68.3% were GSTM1 null genotype and 66.5% were GSTT1 null genotype. GSTM1 null genotype had an increased risk of hypertension (OR: 2.104, CI: 1.38-3.35), but no significant association in GSTT1 null genotype (OR 0.982, CI: 0.62-1.55). No difference in erythrocyte activities of superoxide dismutase, catalase, or glutathione peroxidase, and plasma TRAP, CD, lipid profiles, and GSH levels were observed between GSTM1 or GSTT1 genotype. Plasma levels of ${\alpha}-tocopherol$ increased significantly in GSTT1 wild genotype (P < 0.05); however, plasma level of ${\beta}-carotene$ increased significantly in GSTT1 null genotype (P < 0.01). DNA damage assessed by the Comet assay was significantly higher in GSTM1 null genotype than wild genotype (P < 0.05). CONCLUSIONS: These results confirm the association between GSTM1 null genotype and risk of hypertension as they suggest that GSTM1 null genotype leads to an increased oxidative stress compared with wild genotype.

• Journal of Yeungnam Medical Science
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• 제12권1호
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• pp.32-47
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• 1995

생쥐의 백혈병세포 L1210과 항암제에 대하여 내성이 유도된 L1210AdR, L1210VcR과 L1210Cis에서 glutathione의 농도와 glutathione의 합성 조절에 관여하는 ${\gamma}$-glutamylcysteine synthetase(GCS)와 ${\gamma}$-glutamyl transpeptidase (GGT), 세포 이물질을 축합하는데 촉매하는 glutathione S-transferase(GST)의 효소 활성도와 유전자의 발현 여부를 관찰하였다. 세포내 glutathione농도(${\mu}M/mg$ protein)는 L1210이 $0.41{\pm}0.003$, L1210AdR가 $0.73{\pm}0.006$, L1210VcR은 $1.16{\pm}0.060$, L1210Cis가 $2.19{\pm}0.282$으로 모세포에 비하여 내성세포에서 통계적으로 유의한 증가를 관찰하였다. Buthionine sulfoxamine(BSO)를 1 ${\mu}M$농도로 첨가하여 12시간 배양한 세포들에서의 glutathione농도는 L1210이 88%, L1210AdR가 85%, L1210VcR이 89%, 그리고 L1210Cis는 79%의 감소를 보였다. GCS의 활성도(nM/mg protein/min)는 L1210이 104인데 비하여 L1210AdR가 128, L1210VcR는 227, 및 L1210Cis는 212로 증가하였다. GGT의 활성도(nM/mg protein/min)는 L1210이 $2.15{\pm}0.531$이었고, L1210AdR은 $2.80{\pm}0.498$, L1210VcR은 $2.42{\pm}0.389$, 그리고 L1210Cis는 $2.98{\pm}0.623$으로 내성인 세포들에서 증가하였으며 L1210AdR과 L1210Cis에서 유의하였다. GST활성도(nM/mg protein/min)는 L1210이 $16.70{\pm}4.798$이었고, L1210AdR은 $14.51{\pm}3.402$, L1210VcR은 $19.52{\pm}4.255$, L1210Cis $17.77{\pm}4.495$로 L1210VcR과 L1210Cis가 약간의 증가를 보였으며, L1210AdR은 오히려 감소를 보였다. DNA의 slot blot에서 GCS, GGT, GST 유전자의 모세포와 내성세포간에 별다른 차이를 보이지 않았다. Northern hybridization에서 GCS는 약 4.5kb 크기의 band, GST-${\pi}$는 약 1.05kb 크기의 band를 보였으며 내성세포 모두에서 발현 증가가 관찰되었다. GGT의 경우 크기가 다른 6개의 band가 보였으며 특히 11.5 kb크기의 band에서 L1210AdR과 L1 210VcR의 발현이 증가하였으며, L1210VcR에서는 L1210과 다른 내성세포에서 보이는 1.95kb크기의 band가 보이지 않고 2.2kb 크기의 다른 band가 관찰되었다. 이상에서 L1210AdR과 L1210VcR의 내성에는 mdr1 유전자가 관여하고, L1210Cis의 내성에는 특히 glutathione이 중요하다. GCS, GGT 및 GST등의 활성도 및 유전자의 발현도 내성세포들에서 증가하였으며 이중 GCS는 내성세포내의 glutathione 합성에 가장 중요한 조절인자라 할 수 있다.

• Biomolecules & Therapeutics
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• 제26권2호
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• pp.109-114
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• 2018

Liquiritigenin (LQ) is a flavonoid that can be isolated from Glycyrrhiza radix. It is frequently used as a tranditional oriental medicine herbal treatment for swelling and injury and for detoxification. However, the effects of LQ on cognitive function have not been fully explored. In this study, we evaluated the memory-enhancing effects of LQ and the underlying mechanisms with a focus on the N-methyl-D-aspartic acid receptor (NMDAR) in mice. Learning and memory ability were evaluated with the Y-maze and passive avoidance tests following administration of LQ. In addition, the expression of NMDAR subunits 1, 2A, and 2B; postsynaptic density-95 (PSD-95); phosphorylation of $Ca^{2+}$/calmodulin-dependent protein kinase II (CaMKII); phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2); and phosphorylation of cAMP response element binding (CREB) proteins were examined by Western blot. In vivo, we found that treatment with LQ significantly improved memory performance in both behavioral tests. In vitro, LQ significantly increased NMDARs in the hippocampus. Furthermore, LQ significantly increased PSD-95 expression as well as CaMKII, ERK, and CREB phosphorylation in the hippocampus. Taken together, our results suggest that LQ has cognition enhancing activities and that these effects are mediated, in part, by activation of the NMDAR and CREB signaling pathways.

• Archives of Pharmacal Research
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• 제24권2호
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• pp.114-118
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• 2001

The present study was undertaken to investigate whether or not the hepatoprotective activity of acetylbergenin was superior to bergenin in carbon tetrachloride ($CCl_4$)-intoxicated rat. Acetylbergenin was synthesized by acetylating bergenin, which was isolated from Mallotus japonicus. The hepatoprotective effects of acetylbergenin were examined against $CCl_4$-induced liver damage in rats by means of serum and liver biochemical Indices. Acetylbergenin was administered orally once daily for 7 successive days, then a 0.5 ${m/kg}$ mixture of $CCl_4$in olive oil (1:1) was intraperitoneally injected at 12 h and 36 h after the final administration of acetylbergenin. Pretreatment with acetylbergenin reduced the elevated serum enzymatic activities of alanine/aspartate aminotransferase, sorbitol dehydrogenase and $\gamma$-glutamyltransferase in a dose dependent fashion. Acetylbergenin also prevented the elevation of hepatic malondialdehyde formation and depletion of glutathione content dose dependently in $CCl_4$-intoxicates rats. In addition, the decreased activities of glutathione S-transferase and glutathione reductase were restored to almost normal levels. The results of this study strongly suggest that acetylbergenin n has potent hepatoprotective activity against $CCl_4$-induced hepatic damage in rats by glutathione-mediated detoxification as well as having free radical scavenging activity. In addition, acetylbergenin doses of 50 ${mg/kg}$showed almost the same levels of hepatoprotection activity as 100 ${mg/kg}$ of bergenin, indicating that lipophilic acetylbergenin is more active against the antihepatotoxic effects of $CCl_4$ than those of the much less lipophilic bergenin.

• Archives of Pharmacal Research
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• 제20권5호
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• pp.454-458
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• 1997

Two human liver UDP-glucuronosyltransferase cDNA clones, HLUG25 and UDPGTh2 were previously shown to encode isozymes active in the glucuronidation of hyodeoxycholic acid (HDCA) and certain estrogen derivatives (e.g., estriol and 3,4-catechol estrogens), respectively. in this study we have found that the UDPGTh2-encoded isoform (UDPGTh2) and HLUG25-encoded isoform (UDPGThl) have parallel aglycone specificities. When expressed in COS 1 cells, each isoform metabolized three types of dihydroxy- or trihydroxy-substituted ring structures, including the 3,4-catechol estrogen (4-hydroxyestrone), estriol, 17-epiestriol, and HDCA, but the UDPGTh2 isozyme was 100-fold more efficient than UDPGTh1. UDPGTh1 and UDPGTh2 were 86% identical overall (76 differences out of 528 amino acids), including 55 differences in the first 300 amino acids of the amino terminus, a domain which conferred the substrate specificity. The data indicated that a high level of conservation in the amino terminus was not required for the preservation of substrate selectivity. Analysis of glucuronidation activity encoded by UDPGTh1/UDPGTh2 chimeric cDNA constructed at their common restriction sites, Sac I (codon 297), Nco I (codon 385), and Hha I (codon 469), showed that nine amino acids between residues 385 and 469 were important for catalytic efficiency, suggesting that this region represented a domain which was critical for the catalysis but distinct from that responsible for aglycone-selection. These data indicate that UDPGTh2 is a primary isoform responsible for the detoxification of the bile salt intermediate as well as the active estrogen intermediates.

• Biomolecules & Therapeutics
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• 제26권2호
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• pp.218-223
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• 2018

The liver is an essential organ for the detoxification of exogenous xenobiotics, drugs and toxic substances. The incidence rate of non-alcoholic liver injury increases due to dietary habit change and drug use increase. Our previous study demonstrated that Ecklonia stolonifera (ES) formulation has hepatoprotective effect against alcohol-induced liver injury in rat and tacrine-induced hepatotoxicity in HepG2 cells. This present study was designated to elucidate hepatoprotective effects of ES formulation against carbon tetrachloride ($CCl_4$)-induced liver injury in Sprague Dawley rat. Sixty rats were randomly divided into six groups. The rats were treated orally with ES formulation and silymarin (served as positive control, only 100 mg/kg/day) at a dose of 50, 100, or 200 mg/kg/day for 21 days. Seven days after treatment, liver injury was induced by intraperitoneal injection of $CCl_4$ (1.5 ml/kg, twice a week for 14 days). The administration of $CCl_4$ exhibited significant elevation of hepatic enzymes (like AST and ALT), and decrease of antioxidant related enzymes (superoxide dismutase, glutathione peroxidase and catalase) and glutathione. Then, it leaded to DNA damages (8-oxo-2'-deoxyguanosine) and lipid peroxidation (malondialdehyde). Administration of ES formulation inhibited imbalance of above factors compared to $CCl_4$ induced rat in a dose dependent manner. Real time PCR analysis indicates that CYP2E1 was upregulated in $CCl_4$ induced rat. However, increased gene expression was compromised by ES formulation treatment. These findings suggests that ES formulation could protect hepatotoxicity caused by $CCl_4$ via two pathways: elevation of antioxidant enzymes and normalization of CYP2E1 enzyme.

• Archives of Pharmacal Research
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• 제27권5호
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• pp.547-553
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• 2004

The pyrrolizidine alkaloids (PAs), contained in a number of traditional remedies in Africa and Asia, show wide variations in metabolism between animal species but little work has been done to investigate differences between animal strains. The metabolism of the PA senecionine (SN) in Fischer 344 (F344) rats has been studied in order to compare to that found in the previously investigated Sprague-Dawley (SO) rats (Drug Metab. Dispos. 17: 387, 1989). There was no difference in the formation of ($\pm$) 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP, bioactivation) by hepatic microsomes from either sex of SO and F344 rats. However, hepatic microsomes from male and female F344 rats had greater activity in the Noxidation (detoxication) of SN by 88% and 180%, respectively, when compared to that of male and female SD rats. Experiments conducted at various pH showed an optimum pH of 8.5, the optimal pH for flavin-containing monooxygenase (FMO), for SN N-oxidation by hepatic microsomes from F344 females. In F344 males, however, a bimodal pattern was obtained with activity peaks at pH 7.6 and 8.5 reflecting the possible involvement of both cytochrome P450 (CYP) and FMO. Use of specific inhibitors (SKF525A, 1-benzylimidazole and methimazole) showed that the N-oxide of SN was primarily produced by FMO in both sexes of F344 rats. In contrast, SN N-oxide formation is known to be catalyzed mainly by CYP2C11 rather than FMO in SD rats. This study, therefore, demonstrated that there were substantial differences in the formation of SN N-oxide by hepatic microsomes from F344 and SD rats and that this detoxification is catalyzed primarily by two different enzymes in the two rat strains. These findings suggest that significant variations in PA biotransformation can exist between different animal strains.

• 대한임상독성학회지
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• 제1권1호
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• pp.6-11
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• 2003

Various forms of dialytic techniques are available for detoxification. Hemodialysis, hemoperfusion and hemofiltration (hemodialfiltration) are the main treatment modalities. Because these modalities are rather invasive and expensive, it must be decided in balance of the risk and benefit to the patient. The prime consideration in the decision is based on the clinical features of poisoning; hemodialysis or hemoperfusion should be considered in general if the patient's condition progressively deteriorates despite intensive supportive therapy. The hemodialysis technique relies on passage of the toxic agent through a semipermeable membrane so that it can equilibrate with the dialysate and subsequently removed. It needs a blood pump to pass blood next to a dialysis membrane, which allows agents permeable to the membrane to pass through and reach equilibrium. Solute (or drug) removal by dialysis has numerous determinants such as solute size, its lipid solubility, the degree to which it is protein bound, its volume of distribution etc. The technique of hemoperfusion is similar to hemodialysis except there is no dialysis membrane or dialysate involved in the procedure. The patient's blood is pumped through a perfusion cartridge, where it is in direct contact with adsorptive material (usually activated charcoal) that has a coating material such as cellulose. This method can be used successfully with lipid-soluble compounds and with higher-molecular-weight compounds than for hemodialysis. Protein binding does not significantly interfere with removal by hemoperfusion. In conclusion, hemodialysis, hemoperfusion and hemofiltration can be used effectively as adjuncts to the management of severely intoxicated patients.