• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.357-365
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• 2020

The aim of present study was to investigate regulatory mechanism of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation of porcine oocytes. Basically, immature cumulus-oocyte complexes (COCs) were incubated for 22 h in IVM-I to which hormone was added, and then further incubated for 22 h in IVM-II without hormone. As a result, relative cumulus expansion was increased at 22 h after IVM and it was enhanced by treatment of ALA compared with control group (p < 0.05). During IVM process within 22 h, cAMP level in oocytes was decreased at 6 h (p < 0.05) and it was recovered at 12 h in ALA-treated group, while oocytes in control group recovered cAMP level at 22 h. In cumulus cells, it was reduced in all time point (p < 0.05) and ALA did not affect. Treatment of ALA enhanced metaphase-I (MI) and MII population of oocytes compared with oocytes in control group at 22 and 44 h, respectively (p < 0.05). Intracellular GSH levels in ALA group was increased at 22 and 44 h after IVM (p < 0.05), whereas it was increased in control group at 44 h after IVM (p < 0.05). In particular, the GSH in ALA-treated oocytes during 22 h of IVM was higher than control group at 22 h (p < 0.05). Lipid amount in oocytes from ALA group was higher than control group (p < 0.05). Treatment of ALA did not influence to absorption of glucose from medium. Cleavage and blastocyst formation of ALA-treated oocytes were enhanced compared with control group (p < 0.05). These findings suggest that supplementation of ALA could improve oocyte maturation and development competence through increasing GSH synthesis, lipid storage, and regulation of cAMP accumulation during early 22 h of IVM, and these might be mediated by cumulus expansion.

• Journal of Microbiology and Biotechnology
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• v.1 no.3
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• pp.157-162
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• 1991

Conditions for glutathione production in E. coli cells which possess pGH501 (2 gshI+gshII) were studied. In terms of ATP supply for the glutathione synthesis, two different systems have been constructed and compared. When the acetate kinase reaction of E. coli was used for ATP generation, 20 mM of L-cysteine was completely converted to glutathione by toluene-treated E. coli cells (100 mg/ml) harboring pGH501 within 2 h at $37^{\circ}C$. However, considering the economical aspects, the glycolytic pathway of yeast was chosen as a better system for ATP generation. The optimal concentrations of reactants for glutathione production were determined to be as follows; 80 mM L-glutamate, 20 mM L-cysteine, 20 mM glycine, 20 mM $MgCl_2$, 50 mM potassium phosphate buffer (pH 7.5), 400 mM glucose, polyoxyethylene stearylamine ($5\;\mul/ml$), toluene-treated E. coli HB101/pGH501 (100 mg/ml), and dried yeast cells (400 mg/ml). The conversion ratio of L-cysteine to glutathione was 80% (about 5 mg/ml) under optimal condition within 6 h at $37^{\circ}C$.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.385-393
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• 1998

This study was conducted to investigate the effect of cysteine (CySH) and glutathione (GSH) on in vitro maturation of porcine follicular oocytes. The results obtained were summarized as follows: 1. When the immature oocytes were cultured at 0, 0.04, 0.14, 0.6 and 1.2 mM of cysteine (CySH) for 36h, the germinal vesicle breakdown (GVBD) rates were 90.8, 89.9, 90.5, 92.0 and 91.3%, respectively, and the maturation rates of the oocytes with metaphase-II were 56.1, 50.7, 41.9, 49.0 and 61.5%, respectively. Especially, the maturation rates of 0.14 and 0.6 mM treated groups were significantly lower than those of control (non-treated) group (P＜0.05). After 44h of culture in the same treatments of CySH, the GVBD rates of porcine immature oocytes were 90.0, 91.8, 89.8, 90.5 and 89.6%, respectively, and the maturation rates were 80.2, 76.3, 69.4, 66.7 and 72.6%, respectively. Especially, the maturation rates of 0.14 and 0.6 mM treated groups were significantly lower than those of control group (P＜0.05). 2. When the immature oocytes were cultured at 0, 0.05, 0.1, 0.5 and 1.0 mM of glutathione (GSH) for 36h, the GVBD rates of porcine immature oocytes were 91.0, 90.9, 89.5, 92.0 and 91.1%, respectively, and the maturation rates were 59.0, 48.5, 47.8, 38.6 and 37.5%, respectively. All treated groups of GSH showed lower maturation rates than the control group (P＜0.05). After 44h of culture in the same treatments of GSH, the GVBD rates of porcine immature oocytes were 91.8, 94.1, 89.1, 91.3 and 91.1%, respectively, and the maturation rates were 84.6, 57.1, 69.6, 71.3 and 64.3% respectively. All treated groups of GSH showed lower maturation rates than the control group (P＜0.05).

• Korean Journal of Veterinary Research
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• v.44 no.4
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• pp.497-505
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• 2004

The proliferation of mesangial cells has been associated with the development of diabetic nephropathy. The cell proliferation has been regulated by diverse growth factors. Among them, insulin like growth factors(IGFs) are also involved in the pathogenesis of diabetic nephropathy. However, it is not yet known about the effect of high glucose on IGF-I and IGF-II secretion and the relationship between high glucose-induced secretion of IGFs and PKC or oxidative stress in the mesangial cells. Thus, we examined the mechanisms by which high glucose regulates secretion of IGFs in mesangial cells. High glucose(25 mM) increased IGF-I and IGF-II secretion. High glucose-induced increase of IGF-I and IGF-II secretion were blocked by taurine($2{\times}10^{-3}$ M), N-acetyl cystein(NAC, $10^{-5}M$), or GSH($10^{-5}M$) (antioxidants), suggesting the role of oxidative stress. High glucose-induced secretion of IGF-I and IGF-II were blocked by H-7, staurosporine, and bisindolylmaleimide I(protein kinase C inhibitors). On the other hand, high glucose also increased lipid peroxide (LPO) formation in a dose dependent manner. In addition, high glucoseinduced stimulation of LPO formation was blocked by PKC inhibitors. These results suggest that PKC is responsible for the increase of oxidative stress in the action of high glucose-induced secretion of IGF-I and IGF-II in mesangial cells. In conclusion, high glucose stimulates IGF-I and IGF-II secretion via PKCoxidative stress signal pathways in mesangial cells.

• Journal of Embryo Transfer
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• v.32 no.4
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• pp.287-295
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• 2017

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.39-45
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• 2009

This study was carried out to evaluate the nuclear, cytoplasmic maturation and developmental potential of bovine oocytes selected by brilliant cresyl blue (BCB) as indirect measurement of oocytes growth phase. Cumulus-oocyte complexes (COCs) were collected from 2 to 8 mm follicles from slaughterhouse Hanwoo ovaries. The COCs were divided into stained cytoplasm to blue (BCB+) and unstained (BCB-) according to their ooplasm BCB coloration stained by $26{\mu}m$ of BCB after 90 min. Selected COCs were cultured in a TCM 199 for 18 to 26 h. Nuclear maturation and total cell number was evaluated after in vitro maturation (IVM) or in vitro culture (IVC) using $10{\mu}g/ml$ Hoechst 33342, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay before (0 h) and after (24 h) IVM. The oocyte diameters were not differed significantly between BCB+ ($157.4{\pm}5.8{\mu}m$) and BCB+ ($149.0{\pm}31.0{\mu}m$) groups (p>0.05). However, the proportion of metaphase II oocytes in BCB+ group was significantly higher than BCB- group after IVM (p<0.05). GSH content of BCB+ group oocytes was significantly higher than that of BCB- group just after collection ($7.3{\pm}0.6$ vs. $4.8{\pm}0.6\;pmol/oocyte$, p<0.05), but not varied after IVM($13.1{\pm}0.9$ and $12.6{\pm}2.5\;pmol/oocytes$ for BCB+ and BCB- respectively; p>0.05). The proportion of blastocyst formation and total cell number in BCB+ group (23.5% and $105.5{\pm}28.6$) was significantly higher than that in BCB- (9.8% and $72.4{\pm}26.1$; p<0.05). The results indicate that BCB+ group oocytes may provide a cellular and functional basis for the greater developmental competence in Korean Native Cow (KNC) oocytes.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.2
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• pp.177-183
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• 2008

This study aimed to investigate the protective effect of dandelion water extract (DWE) on liver injury induced by D-galactosamine (GalN) in Sprague-Dawley rats. Fifty rats were divided into 5 groups; normal control (C), DWE-control (DWE-C: saline injection after feeding 3% DWE diet), GalN-control (GalN-C: GalN injection after normal diet), DWE I (GalN injection after feeding 1.5% DWE diet), and DWE II (GalN injection after feeding 3% DWE diet). After 2 weeks, the acute hepatitis was induced by GalN (650 mg/kg, i.p.) and 24 hrs later, all rats were sacrificed. The DWE supplement ameliorated the serum alanine and aspartate aminotransferase (AST, ALT) as well as alkaline phosphatase (ALP) and tumor necrosis $factor-{\alpha}\;(TNF-{\alpha})$. Hepatic antioxidative enzyme activities, such as catalase, GSH peroxidase, GSH reductase, and Mn-superoxide dismutase (SOD) were slightly or significantly elevated by the treatment of DWE. Moreover, the histological examination corresponded with these biochemical observations. According to these findings, dandelion could be used as a potential therapeutic material for treating chemically induced acute hepatitis.

• Journal of the Korean Society of Food Science and Nutrition
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• v.36 no.6
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• pp.708-719
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• 2007

Glutathione S-transferase genotypes GSTT1, GSTM1 and GSTP1 were characterized in 104 healthy male and female subjects and compared with parameters of oxidative stress at the level of DNA and lipids, with antioxidant enzymes, and with plasma antioxidants in smokers and non.smokers. Of the 104 subjects studied, 57.4% were GSTT1 present and 47.6% were GSTM1 present. The GSTP1 polymorphisms a and b were represented as follows: a/a, 75.5%; a/b, 21.6%; b/b type, 2.9%. The GSTT1 null genotype was associated with decreased glutathione in erythrocytes and elevated lymphocytes DNA damage. GST-Px was higher in GSTT1 null compared with GSTT1 present type. The homozygous GSTP1 genotype was not associated with any antioxidant status or DNA damage. The difference in plasma ${\alpha}$-carotene and erythrocytes GSH-Px and GST activities between smokers and non-smokers was detected in the GSTT1 null genotype. Plasma ${\gamma}$-tocopherol and ${\beta}$-carotene decreased significantly in smokers having GSTM1 null genotype. When GSTT1 and GSTM1 were combined, plasma lycopene and erythrocyte GST were reduced in smokers in both null types of these genes. As for GSTP1 genotype, plasma ${\alpha}$-carotene and erythrocytes GSH-Px decreased significantly in smokers with GSTP1 b/b, while erythrocytes GSH-Px activities decreased in smokers with GSTP1 a/b. The different ${\beta}$-carotene level between smokers and non-smokers was seen with both GSTP1 a/a and a/b genotype. It seems that polymorphisms in the phase II metabolizing enzyme glutathione S-transferase may be important determinants of commonly measured biomarkers.

• Korean Journal of Food Science and Technology
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• v.35 no.1
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• pp.138-143
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• 2003

Total methanolic extract of Chrysanthemum coronarium L. var. spatiosum (Compositae) was revealed to have anti-hepatotoxic activity against galactosamine-induced toxicity on primary cultured rat hepatocytes. After successive partitioning with chloroform, n-butanol, and water, the chloroform fraction showed a significant inhibition activity of 51% at 50 ppm, compared with that of silybin, 45.9% at $100\;{\mu}M$. The chloroform fraction was subjected to silica gel column chromatography and yielded active CH-II, CH-V and CH-VI subfractions, and the anti-hepatotoxic activity of these subfractions were 47.6, 56.3, and 23.2%, respectively, at 50 ppm. Total glutathione contents of CH-II, CH-V, and CH-VI increased by 49.8, 43.9, and 47.5% of the control, respectively at 50 ppm, whereas that of silymarin was, 59.7% at $100\;{\mu}M$ after challenged with galactosamine. The ratio of (reduced glutathione) / (total glutathione) in CH-II, CH-V and CH-VI subfraction showed similar values of $0.86{\sim}0.87$ at 50 ppm, whereas that of silymarin was, 0.85 at $100\;{\mu}M$. The incorporation of $[^3H]-uridine$ uptake into RNA was not affected by these active subfractions.

• Journal of the Korean Society of Food Science and Nutrition
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• v.42 no.6
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• pp.869-876
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• 2013

To develop functional products based on black garlic, a black garlic extract (BG) of 7 brix, a gaeddongssuk extract (GS) of 0.7 brix and two types of mixtures (MBS-I, black garlic 7 brix : gaeddongssuk 0.7 brix; MBS-II, black garlic 14 brix : gaeddongssuk 1.4 brix, 93:7, v/v) were supplemented to rats training on a treadmill for 4 weeks. Body weight from the training did not decrease during the experimental period. Serum albumin content significantly increased in the groups fed an experimental diet compared to the control. The BUN content significantly decreased in BG and MBS-II groups compared to the control. AST and ALP activities significantly decreased in the groups fed an experimental diet compared to the control. Serum triglyceride and total cholesterol levels in MBS-I and MBS-II groups significantly decreased compared to the control. Lipid levels in the serum and liver tissue were not significantly different between the MBS-I and MBS-II groups. The lipid peroxide content in the serum and liver tissue was significantly reduced in the groups fed all extracts compared to the control; the serum and liver lipid contents was lowest in the MBS-I and MBS-II groups, respectively. Hepatic catalase activity in the GS and MBS groups increased by 1.8~2.3 times compared to the control. SOD and GSH-px activities significantly increased from treatment with the extracts by 1.3~1.5 times and 1.2~1.7 times, respectively. These results indicate that a mixture of BG and GS extracts has higher biological activity than a single supplementation of BG or GS extract. Therefore, the addition of gaeddongssuk to black garlic (MBS-I and MBS-II) is effective as a defense material against oxidative stress. MBS-I may be especially effective for its biological activities.