• Proceedings of the PSK Conference
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• 2003.04a
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• pp.161.1-161.1
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• 2003

NAD(P)H:quinone oxidoreductase (quinone reductase: QR: EC1.6.99.2), a cytosolic FAD-containing flavoprotein, form one of the important component of the phase II drug-metabolizing enzyme systems. It is found in all mammalian species tested and is expressed in many organs including the liver. QR catalyses two-electron reduction of qui nones to hydroquinones thereby suppresses the formation of superoxide anion radical. (omitted)

• Microbiology and Biotechnology Letters
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• v.25 no.4
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• pp.374-378
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• 1997

Glutathione S-transferase (GST) was purified from Pseudomonas sp. DJ77, and its N-terminal sequence was determined to be MKLFISPGACSL. A specific tyrosyl residue in the vicinity of the N terminus is conserved in all the known cytosolic GSTs and has been shown to function as a catalytic residue in $\alpha$, $\mu$, $\pi$ class GSTs from mammals. However, Pseudomonas sp. DJ77 GST has the Phe-4 and Ile-5 instead of Tyr in N-terminus. Its replacement with tyrosine did not significantly affect the enzyme activity. Results from in vitro biochemical analyses were confirmed by the in vivo activity-based CDNB growth inhibition analyses. Our results clearly indicate that GST of Pseudomonas sp. DJ77 has a novel reaction mechanism different from that of mammalian GSTs.

• Food Science and Biotechnology
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• v.15 no.6
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• pp.975-979
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• 2006

The extracts of Oenanthe javanica were evaluated for their effects on the expression of cyclooxygenase-2 (COX-2), which is mediated by the translocation of the p65 subunit into the nucleus. Fractions of ethyl acetate and chloroform from 80% ethanol extracts of O. javanica exhibited inhibitory effects on the secretion of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) from lipopolysaccharide (LPS)-stimulated peritoneal macrophages; however, the aqueous- and hexane-fractions showed no significant effect. The ethyl acetate- and chloroform-fractions also reduced the COX-2 enzyme levels after 24-hr treatment. RT-PCR showed that the mRNA levels of COX-2 decreased following treatment with these fractions, suggesting that COX-2 expression is transcriptionally regulated by these extracts. We examined the effects of the chloroform- and ethyl acetate-fractions on the cytosolic activation of nuclear factor-${\kappa}B$ ($NF-{\kappa}B$, p65 subunit) and on the degradation of inhibitor-${\kappa}B{\alpha}$ ($I-{\kappa}B{\alpha}$) in order to determine the mechanism of COX-2 regulation. The LPS-stimulated activation of the p65 subunit was significantly blocked upon the addition of $50\;{\mu}g/mL$ of these fractions, and the cytosolic $I-{\kappa}B{\alpha}$ degradation process was simultaneously inhibited. These findings suggest that the inhibition of COX-2 expression by the ethyl acetate-and chloroform-fractions may result from the inhibition of p65 translocation by blocking the degradation of $I-{\kappa}B{\alpha}$; this may be the mechanistic basis for the anti-inflammatory effects of O. javanica.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.224-224
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• 2017

In plants, cysteine(Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur containing secondary products. Cys formation is involved in the consecutive two reactions using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast and mitochondria. OASTL is able to produce mimosine with 3-hydroxy-4-pyridone (3H4P) in lieu of $H_2S$ for Cys. In this report, we describe the first time cloning, purification and characterization of cytosolic(cy)OASTL from M. pudica and its expression in Escherichia coli and try to find out the cross link between this OASTL and the mimosine formation and to elucidate the metabolic role of cy-OASTL in M. pudica. The purified recombinant protein was 34.7 KDa. The optimum reaction pH and temperature was 6.5 and $50^{\circ}C$, respectively. The Michaelis constant (Km) and the Vmax value of the enzyme was $252{\pm}25{\mu}M$ and $57{\pm}3{\mu}M\;cysteine\;min^{-1}\;{\mu}g\;protein^{-1}$ for sulfide and $159{\pm}21{\mu}M$ and $58{\pm}2.4{\mu}M\;cysteine\;min^{-1}\;{\mu}g\;protein^{-1}$ for OAS subsequently. After cleaving the His-tag, we tried to observe cy-OASTL to form mimosine with appropriate substrate but it was not successful. It may be concluded that cy-OASTL of the present study is only Cys specific, not mimosine.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.3
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• pp.342-347
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• 2003

The enzyme $20{\alpha}$-hydroxysteroid dehydrogenase ($20{\alpha}$-HSD) catabolizes progesterone to $20{\alpha}$-dihydroprogesterone ($20{\alpha}$-OHP), and is appeared in rat corpora luteal and placenta. A polled samples of 10-15 placental and ovarian tissues collected from each individual rat were subjected to measurement of $20{\alpha}$-HSD activity. A $20{\alpha}$-HSD activity in the cytosol fraction was based on the generation of NADPH. In this study, it is designed to study cytosolic $20{\alpha}$-HSD activity in rat ovarian and placenta during pregnancy, and its relationship to embryonic mortality. It was found that from days 5 to 18 of pregnancy the $20{\alpha}$-HSD activities steady by decreased but at parturition time rapidly increased in ovary. On the other hand, placental cytosolic $20{\alpha}$-HSD activities were high detected from days 8 to 10 of pregnancy, not detectable from days 11 to 20 of pregnancy, but again very high at the time of parturition. Analysis of DEAE column chromatography revealed that two different types of $20{\alpha}$-HSD (HSD-1 and HSD-2) were found with similar activity in the placental cytosol on day 10 of pregnancy. The number of fetuses on day 10 of pregnancy was 15.4 and decreased significantly to 12.9 on day 12. The results suggested that expression of $20{\alpha}$-HSD in the placental tissues seems to be related the number of fetal survived in the specific time (days 11 and 12) which spontaneous fetal loss occurs.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.55-64
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• 2012

Peroxiredoxin II (Prdx II; a typical 2-Cys Prdx) has been originally isolated from erythrocytes, and its structure and peroxidase activity have been adequately studied. Prdx II has been reported to protect a wide range of cellular environments as antioxidant enzyme, and its dysfunctions may be implicated in a variety of disease states associated with oxidative stress, including cancer and aging-associated pathologies. But, the precise mechanism is still obscure in various aspects of aging containing ovarian aging. Identification and relative quantification of the increased proteins affected by Prdx II deficiency may help identify novel signaling mechanisms that are important for oxidative stress-related diseases. To identify the increased proteins in Prdx $II^{-/-}$ mice, we performed RBC comparative proteome analysis in membrane fraction and cytosolic fractions by nano-UPLC-$MS^E$ shotgun proteomics. We found the increased 86 proteins in membrane (32 proteins) and cytosolic (54 proteins) fractions, and analyzed comparative expression pattern in healthy RBCs of Prdx $II^{+/+}$ mice, healthy RBCs of Prdx $II^{-/-}$ mice, and abnormal RBCs of Prdx $II^{-/-}$ mice. These proteins belonged to cellular functions related with RBC lifespan maintain, such as cellular morphology and assembly, cell-cell interaction, metabolism, and stress-induced signaling. Moreover, protein networks among the increased proteins were analyzed to associate with various diseases. Taken together, RBC proteome may provide clues to understand the clue about redox-imbalanced diseases.

• Toxicological Research
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• v.25 no.1
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• pp.23-28
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• 2009

Some of the edible plants like apricot kernel, flaxseed, and cassava generate hydrogen cyanide (HCN) when cyanogenic glycosides are hydrolyzed. Rhodanese (thiosulfate: cyanide sulfurtransferases of TSTs; EC: 2.8.1.1) is a sulfide-detoxifying enzymes that converts cyanides into thiocyanate and sulfite. This enzyme exists in a liver and kidneys in abundance. The present study is to evaluate the conversion of apricot cyanogenic glycosides into thiocyanate by human hepatic (HepG2) and colonal (HT-29) cells, and the induction of the enzymes in the rat. The effects of short term exposure of amygdalin to rats have also been investigated. Cytosolic, mitochondrial, and microsomal fractions from HepG2 and HT-29 cells and normal male Spraque-Dawley rats were used. When apricot kernel extract was used as substrate, the rhodanese activity in liver cells was higher than the activity in colon cells, both from established human cell line or animal tissue. The cytosolic fractions showed the highest rhodanese activity in all of the cells, exhibiting two to three times that of microsomal fractions. Moreover, the cell homogenates could metabolize apricot extract to thiocyanate suggesting cellular hydrolysis of cyanogenic glycoside to cyanide ion, followed by a sulfur transfer to thiocyanate. After the consumption of amygdalin for 14 days, growth of rats began to decrease relative to that of the control group though a significant change in thyroid has not been observed. The resulting data support the conversion to thiocyanate, which relate to the thyroid dysfunction caused by the chronic dietary intake of cyanide. Because Korean eats a lot of Brassicaceae vegetables such as Chinese cabbage and radish, the results of this study might indicate the involvement of rhodanese in prolonged exposure of cyanogenic glycosides.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1635-1643
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• 2009

The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.

• Nutritional Sciences
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• v.3 no.1
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• pp.11-17
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• 2000

This study was intended to examine whether dehydroepiandrosterone (DHEA) and dietary fat level or source could modulate glutathione utilizing detoxifying system activity and the cytosolic NADPH generation in rat liver. Male Sprague-Dawley rats were fed semipurifed diet containing either 2%(w/w) corn oil (low level of corn oil diet: 5 ca% of fat) 15% corn oil (high level of corn oil diet: 31 cal% of fat) or 13% sardine oil plus 2% corn oil(high level of fish oil diet: 31 cal% of fat) for 9 weeks. Half of the rats in each diet group were fed a diet supplemented with 0.2% DHEA (w/w). DHEA administration increased plasma total cholesterol level in low corn oil diet-fed rats. The high fish oil diet significantly decreased plasma total cholesterol level compared to the high corn oil diet. Plasma triglyceride level was not significantly changed by DHEA administration and dietary fat level and source. Fasting plasma glucose level was increased by DHEA administration and fish oil diet. Glucose 6-phosphate dehydrogenase activity in liver tissue was significantly increased by DHEA administration and high fat diet, especially fish oil diet. Malic enzyme activity in liver tissue was significantly increased by DHEA administration and high fat diet, especially fish oil diet. Malic enzyme activity in liver tissue was significantly increased by DHEA administration. DHEA suppressed the glutathione peroxidase, glutathione-dependent enzymes compared to the low corn oil diet, while fish oil diet elevated the activity of glutathione peroxidase and glutathione reductase compared to corn oil diet. These results suggest that DHEA administration and high level of corn oil diet may suppress the cellular detoxifying system activity through reduction of glutathione utilization, while the fish oil diet did not show these effects.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.259-265
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• 2006

This study is investigated the effect on mechanism of nephrotoxicity formation of methotrexate(MTX) by hyperglycemic by streptozotocin(STZ). MTX was injected daily at two doses of 3, 6 mg/kg for 1 week in STZ-induced hyperglycemic rats. Activities of BUN, creatinine and LDH were significantly increased by treatment with MTX in STZ-induced diabetic group when compared to MTX treatment group in normal rats' Renal lipid peroxide content and activities of cytosolic enzyme were significantly increased in the treatment of MTX in diabetic group. The concentration of glutathione and glutathione biosynthesis enzymes were decreased by treatment with MTX in STZ-induced diabetic group. These results suggest that nephrotoxicity of MTX in STZ-induced hyperglycemic rat was caused by activation of renal metabolizing enzymes in cytosol and decrease of glutathione concentration.