• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.11a
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• pp.147-172
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• 1994

Aziridinylbenzoquinones such as 3, 6-diaziridinyl-1, 4-benzoquinone (DZQ) and its 2, 5-methyl analog (MeDZQ) require bioreductive activation in order to elicit their anticancer activities. To determine the involvement of DTD in the activation of these drugs, we have used a ligation-mediated polymerase chain reaction to map the intracellular alkylation sites in a sing1e copy gene at the nucleotide level. We have performed this analysis in two human colon carcinoma cells, one proficient (HT-29) and one deficient (BE) in DT-diaphorase (DTD) activity. In the DTD proficient HT-29 cell line, DZQ and MeDZQ were found to alkylate both 5'-(A/T)G(C)-3' and 5'-(A/T)A-3' sequences. This is consistent with the nucleotide preferences observed when DZQ and MeDZQ are activated by purified DTD to reactive metabolites capable of alkylating DNA in vitro [Lee, C. -S., Hartley, J. A., Berardini, M. D., Butler, J., Siegel., D., Ross, D., & Gibson, N. W. (1992) Biochemistry, 31: 3019-3025]. Surprisingly in the DTD-deficient BE cell line a pattern of alkylation induced by DZQ and MeDZQ similar to that observed in the DTD-proficient HT-29 cells was observed. This suggests that reductive enzymes other than DTD can be involved in activating DZQ and MeDZQ to DNA reactive species in vivo.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.156-162
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• 2012

A mannitol dehydrogenase (StMDH) gene was cloned from Salmonella typhimurium LT2 (KCTC 2421) and overexpressed in Escherichia coli. It has a 1,467 bp open reading frame encoding 488 amino acids with deduced molecular mass of 54 kDa, which shares approximately 36% of amino acid identity with known long-chain dehydrogenase/reductatse (LDR) family enzymes. The recombinant StMDH showed the highest activity at $30^{\circ}C$, and pH 5.0 and 10.0 for D-fructose reduction and D-mannitol oxidation, respectively. On the contrary, it has no activity on glucose, galactose, xylose, and arabinose. StMDH can catalyze the oxidative/reductive reactions between D-fructose and D-mannitol only in the presence of $NAD^+$/NADH as coenzymes. These results indicate that StMDH is a typical $NAD^+$/NADH-dependent mannitol dehydrogenase (E.C. 1.1.1.67).

• Korean Journal of Microbiology
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• v.40 no.3
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• pp.183-188
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• 2004

Stenotrophomonas sp. OK-5 capable of degrading TNT has been found to have three nitroreductase fractions designated as NTR fractions I, II, and III. NTR in a previous study. This study was attempted to reveal physiological and molecular characteristics of NTR fractions I, II, and III in strain OK-5. Several chemicals (e.g., EDTA, NaCl, dithiothreitol, $\beta$-mercaptoethanol) were tested for their effect on enzyme activity of NTRs, demonstrating that enzyme activities of NTR fractions I, II, and III from OK-5 were inhibited in the presence of $\beta$-mercaptoethanol. Substrate specificity test showed that NTR fractions I, II, and III all have over 70% enzyme activities for nitrobenzene or RDX as a substrate. N-terminal amino acid sequence of NTR fraction I from Stenotrophomonas sp. OK-5 was $^1MSDLLNADAVVQLFRTARDS^20$ and exhibited 70% sequence homology with that of NTR from Xanthomonas campestris. NTR I gene from Stenotrophomonas sp. OK-5 (SmOK5nrI) shared extensive sequence homology in deduced amino acid sequence of PCR product with NTRs from Xanthomonas campestris (81 %), X. axonopodis (75%), Streptomyces avermitilis(30%), whereas they had low homology with that from P. putida KT2440 (pnrB) (16%).

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.206-212
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• 1999

The gene orf7(oxi III) was expressed using an E. coli system in anticipation that it would encode dTDP-glucose 4,6-dehydratase which is involved in the biosynthesis of the olivose moiety of chlorothricin produced from Streptomyces antibioticus Tu99. The solubility of the expressed protein increased up to 20% under optimal induction conditions. The expressed protein was purified from the E. coli BL 21(DE3) cell lysate by a 28.5-fold purification in two chromatography steps with a 38% recovery to near homogeneity. The molecular weight and N-terminal amino acid sequence of the purified protein correlated with the predicted mass and sequence deduced from the orf7 gene. The purified protein was a homodimer with a subunit relative molecular weight of 38,000 Dalton. The expressed protein was found to exhibit dTDP-glucose 4,6-dehydratase activity and be highly specific for dTDP-glucose as a substrate. The values of K'm and V'max for dTDP-glucose were 28 $\mu$M and 295 nmol $min^{-1} (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of this enzyme.$NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

• Preventive Nutrition and Food Science
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• v.4 no.2
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• pp.122-124
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• 1999

Induction of NAD(P)H : (quinone-acceptor) oxidoreductase (QR) which obligatory two electron reduction of quinones and prevents their participation in oxidative cycling and thereby the depletion of intracellular glutathione, has been used as a marker for chemopreventive agents. We postulated that vitamin E, an antioxidant, which induces QR as the gene of QR was reported to contain antioxidant reponsive element in the 5'-flanking region. Vitamin E resulted in significant induction of QR in both hepalclc7 cells and mouse tissues. QR induction was observed; to be maximal at 25uM vitamin E for hepalclc7 cells while it was maximal in the level of 2.5∼5 μmoles vitamin E/㎏ BW for mouse tissues. Thus the cancer-preventive effect of vitamin E may be exerted by it induction of intracellular detoxifying enzymes.

• Microbiology and Biotechnology Letters
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• v.18 no.3
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• pp.268-272
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• 1990

The structural gene (zadhII) encoding an alcohol dehydrogenase II from Zyrnornonas mobilis was cloned into Escherichia coli in our laboratory (Yoon et al., 1989. Kor. J. Microbiol. Biotechnol.). From E. coli (pADS93) carrying the zadhII gene, the Z mobilis alcochol dehydrogenase II (ZADH-II) was purified by sonication, $(NH_4)_2SO_4$, fractionation, and chromatography. The ZADH-I1 enzyme produced by Z. mobilis cell was also purified to compare to the enzyme produced by E. coli (pADS93). The purified enzyme from cell extract of E. coli (pADS93) was identified to be a tetramer being composed of four identical subunits having molecular weight of 40, 000 dalton like that of Z. mobilis. The pH optimum for the reaction oxidizing ethanol to acetaldehyde was 10.0 while the optimum for the reverse reaction was 7.5-8.5. The apparent $K_m$ values for ethanol and NAD + were $1.2 \times 10^{-1}M$and $5.1\times 10^{-5}M$, respectively. In addition, it was found that the $K_m$ value for acetaldehyde was very lower than that for ethanol.

• Journal of Life Science
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• v.25 no.11
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• pp.1290-1297
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• 2015

Studies of the inactivation of a gene encoding pyruvate decarboxylase, pdc, in an ethanol-producing bacterium, Zymomonas mobilis, identified a mutant strain with 50% reduced PDC activity. To evaluate the possibility of a carbon-flux shift from an ethanol pathway toward higher value fermentation products, including pyruvate, succinate, and lactate, fermentation studies were carried out. Despite attempts to silence pdc expression in the wild-type strain ZM4 using cat-inserted pdc and pdc-deleted homologs by electroporation, the strain isolated showed partial gene activation. Fermentation experiments with the PDC mutant strain showed that the reduced expression level of PDC activity resulted in decreased rates of substrate uptake and ethanol production, together with increased pyruvate accumulation of 2.5 g l^-1 , although lactate and succinate concentrations were not significantly enhanced in these modified strains. Despite numerous attempts, no strains were isolated in which complete pdc inactivation occurred. This result indicates that the ethanol fermentation pathway of this bacterium is totally dependent on the activity of the PDC enzyme. To ensure a redox balance of intracellular NAD and NADH levels, other enzymes, such as lactate dehydrogenase for lactate, and enzymes involved in the production of succinic acid, such as pyruvate dehydrogenase (PDH) and malic enzymes, may be needed for their increased end-product production.

• Animal Bioscience
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• v.34 no.6
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• pp.1006-1013
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• 2021

Objective: Cells have increased susceptibility to activation of apoptosis when suffering heat stress (HS). An effective supplementation strategy to mimic heat-induced apoptosis of bovine mammary epithelial cells (MECs) is necessary to maintain optimal milk production. This study aimed to investigate possible protective effects of the anti-apoptotic activity of 5-aminolevulinic acid (5-ALA) against HS-induced damage of bovine MECs. Methods: Bovine MECs were pretreated with or without 5-ALA at concentrations of 10, 100, and 500 µM for 24 h followed by HS (42.5℃ for 24 h and 48 h). Cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Real-time quantitative polymerase chain reaction and Western blotting were used to explore the regulation of genes associated with apoptosis, oxidative stress, and endoplasmic reticulum (ER) stress genes. Results: We found that 5-ALA induces cytoprotection via inhibition of apoptosis markers after HS-induced damage. Pretreatment of bovine MECs with 5-ALA resulted in dramatic upregulation of mRNA for nuclear factor erythroid-derived 2-like factor 2, heme oxygenase-1, and NAD(P)H quinone oxidoreductase 1, all of which are antioxidant stress genes. Moreover, 5-ALA pretreatment significantly suppressed HS-induced ER stress-associated markers, glucose-regulated protein 78, and C/EBP homologous protein expression levels. Conclusion: 5-ALA can ameliorate the ER stress in heat stressed bovine MEC via enhancing the expression of antioxidant gene.

• BMB Reports
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• v.36 no.5
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• pp.442-449
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• 2003

NAD(P)H quinone oxidoreductase is a ubiquitous enzyme that is known to directly reduce quinone substrates to hydroquinones by a two-electron reaction. We report the identification of NADPH quinone oxidoreductase from Kluyveromyces marxianus (KmQOR), which reduces quinone substrates directly to hydroquinones. The KmQOR gene was sequenced, expressed in Escherichia coli, purified, and characterized. The open-reading frame of the KmQOR gene consists of 1143 nucleotides, encoding a 380 amino acid polypeptide. The nucleotide sequence of the KmQOR gene was assigned to EMBL under accession number AY040868. The $M_r$ that was determined by SDS-PAGE for the protein subunit was about 42 kDa, and the molecular mass of the native KmQOR was 84 kDa, as determined by column calibration, indicating that the native protein is a homodimer. The KmQOR protein efficiently reduced 1,4-benzoquinone, whereas no activities were found for menadiones and methoxyquinones. These observations, and the result of an extended sequence analysis of known NADPH quinone oxidoreductase, suggest that KmQOR possesses a different action mechanism.

• Journal of the Korean Society of Food Science and Nutrition
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• v.23 no.6
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• pp.964-972
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• 1994

The 4.3-kb gene coding for L-lactate dehydrogenase of Bacillus stearothermophilus has been subcloned and expressed in E. coli cells. The enzyme was purified 200-fold with 25% yield by heat treatment , DEAE-Sephadex, and NAD++ -Sepharose CL-4B affinity chromatography followed by gel filtration through Sephadex G-200 . The molecular weight of the purfied enzyme was estimated to be about 35, 000 and 140, 000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively. indicating that the enzyme is composed of four identical subunits. THe enzyme for pyruvate reduction and lactate oxdiation was stable at 60 and 75$^{\circ}C$ for 30 min, and the optimal temperatures for both reactions were 60 and 7$0^{\circ}C$, respectively. The enzyme had an optimal pH at 5.5 and 8.5 in pyruvate reduction and lactate oxidation, respectively. The pH stability of enzyme of pyruvate reduction was table between pH 5 and 7. more than 90% of enzyme activity was lost at 1mM FeSO4 and p-chloromercuribonzoate. The maximal activation of the enzyme was obtained with 0.8mM fructose 1, 6-bisphosphate.