• Journal of Microbiology
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• v.44 no.1
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• pp.50-53
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• 2006

Cys-29 and Cys-251 of Streptomyces albus valine dehydrogenase(ValDH) were highly conserved in the corresponding region of $NAD(P)^+$-dependent amino acid dehydroganase sequences. To ascertain the functional role of these cysteine residues in S. albus ValDH, site-directed mutagenesis was performed to change each of the two residues to serine. Kinetic analyses of the enzymes mutated at Cys-29 and Cys-251 revealed that these residues are involved in catalysis. We also constructed mutant ValDH by substituting valine for leucine at 305 by site-directed mutagenesis. This residue was chosen, because it has been proposed to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). Kinetic analysis of the V305L mutant enzyme revealed that it is involved in the substrate binding site. However it displayed less activity than the wild type enzyme toward all aliphatic and aromatic amino acids tested.

• Proceedings of the Korean Society of Toxicology Conference
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• 2001.10a
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• pp.9-9
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• 2001

Several micronutrients (vitamins and minerals) are required as co-factors in DNA synthesis, DNA repair, DNA methylation and apoptosis. Some notable examples include (a) folic acid and vitamin B12 required for maintenance methylation of DNA and the synthesis of dTTP from dUTP, thus prevent the misincorporation of uracil into DNA, a highly mutagenic and chromosome-breaking event, (b) niacin, is essential in the form of the coenzymes NAD and NADP which act as a substrate for polyADPribose polymerase (PARP), an enzyme thought to facilitate efficient DNA repair and telomere length regulation and (c) zinc, apart from its antioxidant role as a co-factor in Cu/Zn SOD, it is required in its stabilizing role of the DNA-binding domain of p53 (residues 102-292) and thus is essential for apoptotic response to DNA damage. (omitted)

• Journal of Ginseng Research
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• v.10 no.1
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• pp.55-65
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• 1986

Effects of various nucleotides including GMP, glnsenoslde $Rb_2$, and redoxidants on the activities of both particulate and soluble guanylate cyclase from rat brain have been studied. At the low concentra狀onto of GMP, AMP, ADP, and ATP the activity of guanylate cyclase is not substantially affected, whereas the inhibitory effects of these nucleotides on the enzyme activities are increased with the increasing concentrations of the nucleotides. Similarly, the activity of the soluble guanylate cyclase is inhibited with the increasing concentrations of the nucleotides. Inhibitory effects of GMP, AMP, ADP, and ATP on the activities of particulate guanylate cyclase and soluble guanylate cyclase is reduced in the presence of ginsenoside $Rb_2$. It is apparent broom this finding that there are seperate binding sites on the guanylate cyclase molecule specific for nucleootides and for ginsenoside $Rb_2$. $NAD^+$ shows no significant effect on the activities of particulate guanylate cyclase, whereas NADH inhibits the activities of the enzyme. The activity of particulate guanylate cyclase is slightly inhibited by iodine, whereas that of soluble gllanylate cyclase is strongly inhibited.

• Journal of Life Science
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• v.25 no.3
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• pp.317-322
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• 2015

Alcohol-induced fatty liver (steatosis) results from excessive generation of reducing equivalents by ethanol metabolism. Generally, chronic ethanol treatment causes hepatosteatosis by regulating sterol regulatory element-binding protein 1c (SREBP-1c), which increases the synthesis of hepatic lipids. The effect of ethanol on SREBP-1c is mediated through mammalian sirtuin-1 (SIRT-1), a NAD⁺-dependent protein deacetylase that regulates hepatic lipid metabolism. Ginseng is a widely used herbal medicine that is used in Asia for its anti-diabetes and anti-obesity effects. The pharmacological and therapeutic effects of ginseng are primarily produced by bioactive constituents known as ginsenosides. Here, we examined the regulatory effects of Korean red ginseng (KRG) extracts on SREBP-1c and SIRT-1 on lipid homeostasis in AML-12 mouse hepatocytes. AML-12 cells were treated with ethanol and/or KRG extracts (0 - 1,000 μg/ml). Lipid droplets were assayed using Oil red O staining, and western blotting was used to measure SIRT-1 and SREBP-1 expression. Treatment with KRG extracts restored SIRT-1 expression and reduced SREBP-1c expression in ethanol-treated cells. We also showed that KRG extract and ginsenosides Rb₂ and Rd significantly decreased SREBP-1 acetylation in ethanol-treated cells. These results show that treatment with KRG extract and its active ginsenoside constituents Rb₂ and Rd protected against alcohol-related hepatosteatosis via regulation of SIRT-1 and downstream acetylation of SREBP-1c, which altered hepatic lipid metabolism.

• Journal of Conservation Science
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• v.27 no.2
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• pp.135-144
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• 2011

When metal artifacts have to undergo conservation treatment, the person in charge of the treatment selects and uses various coating mixtures based on his judgment regarding their condition, material, or environment. Since the kinds of coating mixtures or solvents make a difference in the set time of cyanoacrylate adhesives, they have something to do with the efficiency of the conservation treatment. This study examines the effects and causes that affect the set time of cyanoacrylate adhesives according to the kinds of coating mixtures and solvents and suggests ways to increase the set time. As a result, it is thought that as the surface roughness gets flatter, the wettability of adhesive is improved further, which increases the set time. Moreover, the C-F binding of V-Flon, C-O-C absorption peak, molecular weight of the coating mixtures, and glass transition temperature (Tg) were the factors that significantly affected the set time. According to the result of measuring the set time based on the result of superficial and chemical analysis, relative difference was shown according to the kind and viscosity of adhesive, but all the adhesives indicated the following order of the set time: V-Flon > Paraloid B-72 (in xylene) > Paraloid NAD-10 > Paraloid B-72 (in acetone).

• Journal of Life Science
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• v.26 no.2
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• pp.174-180
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• 2016

Dicumarol is a coumarin derivative isolated from sweet clover (Melilotus alba), and has anti-coagulant activity with the inhibitory activity of NAD(P)H quinone oxidoreductase1 (NQO1). NQO1 catalyzes the two-electron reduction of quinones to hydroquinones. Dicumarol competes with NAD(P)H for binding to NQO1, resulting in the inhibition of NQO1 enzymatic activity. The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. The expression of MMPs is regulated by cytokines and signal transduction pathways, including those activated by phorbol myristate acetate (PMA). However, the effects of dicumarol on metalloproteinase (MMP)-9 expression and activity are not investigated here. This study investigated whether dicumarol inhibits MMP-9 expression and activity in PMA-treated human renal carcinoma Caki cells. Dicumarol markedly inhibited the PMA-induced MMP-9 mRNA expression and MMP-9 activity. NF-κB and AP1 promoter activity, which is important in MMP-9 expression, also decreased in dicumarol-treated cells. Furthermore, dicumarol markedly suppressed the ability of PMA-mediated migration in Caki cells. When the relevance of NQO1 in the dicumarol-mediated inhibitory effect on PMA-induced MMP9 activity was elucidated, knock-down of NQO1 with siRNA was found to have no effect on PMA-induced MMP9 activity, suggesting that the stimulating effect of dicumarol on PMA-induced MMP9 activity is independent of NQO1 activity. Taken together, the present studies suggested that dicumarol may inhibit PMA-induced migration via down-regulation of MMP-9 expression and activity.

• BMB Reports
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• v.33 no.4
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• pp.317-320
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• 2000

The succinic semialdehyde dehydrogenase from bovine brain was inactivated by treatment with phenylglyoxal, a reagent that specifically modifies arginine residues. The inhibition at various phenylglyoxal concentrations shows pseudo-first-order kinetics with an apparent secondorder rate constant of 30 $M^{-1}min^{-1}$ for inactivation. Partial protection against inactivation was provided by the coenzyme $NAD^+$, but not by the substrate succinic semialdehyde. Spectrophotometric studies indicated that complete inactivation of the enzyme resulted from the binding of 2 mol phenylglyoxal per mol of enzyme. These results suggest that essential arginine residues, located at or near the coenzyme-binding site, are connected with the catalytic activity of brain succinic semialdehyde dehydrogenase.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1636-1643
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• 2014

3-Hydroxybutyryl-CoA dehydrogenase is an enzyme that catalyzes the second step in the biosynthesis of n-butanol from acetyl-CoA, in which acetoacetyl-CoA is reduced to 3-hydroxybutyryl-CoA. To understand the molecular mechanisms of n-butanol biosynthesis, we determined the crystal structure of 3-hydroxybutyryl-CoA dehydrogenase from Clostridium butyricum (CbHBD). The monomer structure of CbHBD exhibits a two-domain topology, with N- and C-terminal domains, and the dimerization of the enzyme was mostly constituted at the C-terminal domain. The mode of cofactor binding to CbHBD was elucidated by determining the crystal structure of the enzyme in complex with $NAD^+$. We also determined the enzyme's structure in complex with its acetoacetyl-CoA substrate, revealing that the adenosine diphosphate moiety was not highly stabilized compared with the remainder of the acetoacetyl-CoA molecule. Using this structural information, we performed a series of site-directed mutagenesis experiments on the enzyme, such as changing residues located near the substrate-binding site, and finally developed a highly efficient CbHBD K50A/K54A/L232Y triple mutant enzyme that exhibited approximately 5-fold higher enzyme activity than did the wild type. The increased enzyme activity of the mutant was confirmed by enzyme kinetic measurements. The highly efficient mutant enzyme should be useful for increasing the production rate of n-butanol.

• Biomolecules & Therapeutics
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• v.24 no.1
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• pp.33-39
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• 2016

Oxidative stress activates several intracellular signaling cascades that may have deleterious effects on neuronal cell survival. Thus, controlling oxidative stress has been suggested as an important strategy for prevention and/or treatment of neurodegenerative diseases. In this study, we found that ginsenoside Rh1 inhibited hydrogen peroxide-induced reactive oxygen species generation and subsequent cell death in rat primary astrocytes. Rh1 increased the expression of phase II antioxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1, superoxide dismutase-2, and catalase, that are under the control of Nrf2/ARE signaling pathways. Further mechanistic studies showed that Rh1 increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to the antioxidant response element (ARE), and increased the ARE-mediated transcription activities in rat primary astrocytes. Analysis of signaling pathways revealed that MAP kinases are important in HO-1 expression, and act by modulating ARE-mediated transcriptional activity. Therefore, the upregulation of antioxidant enzymes by Rh1 may provide preventive therapeutic potential for various neurodegenerative diseases that are associated with oxidative stress.

• Archives of Pharmacal Research
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• v.18 no.4
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• pp.262-266
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• 1995

Bacterial lipopolysaccharide (LPS) is one of the most potent inducers of various cytokines nad other proinflammatory mediators in macrophages. Although pathophysiological consequences of LPS-induced responses are well established, the mechanisms through which LPS-generated singals are transduced remain unclear. In the present study, we attempted to determine early intracellular events after LPS binding which transduced the signal for the induction of arachidonic acid metabolism in rat alveolar macrophages. While H-7, a protein kinase C(PKC) inhibitor, did not affect LPS-stimulated prostaglandin synthesis, staurosporine enhanced archidonic acid etabolism in macropahages treated with LPS. Phorbol-12-myristate-13 acetate snesitive to LPS compare with control group. PMA and H-7 did not alter the effect of flucose. Pertussis toxin did not show nay effect, thus pertussis toxin snesitive G-protein pathway appears not to play a role in this experimental system. Genistein and tyrphostin 25, protein tyrosine kinase 9PTK) inhibitors, markedly inhibited prostaglandin synthesis in macrophages nal transduction events leading to icnreased macrophage arachidonic acid metabolism.