• Biomolecules & Therapeutics
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• v.27 no.2
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• pp.193-200
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• 2019

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z $588.6{\rightarrow}264.4$ for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

• Animal Bioscience
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• v.36 no.11
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• pp.1693-1699
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• 2023

Objective: Cows that are nursing get around 80% of their glucose from liver gluconeogenesis. Propionate, a significant precursor of liver gluconeogenesis, can regulate the key genes involved in hepatic gluconeogenesis expression, but its precise effects on the activity of enzymes have not yet been fully elucidated. Therefore, the aim of this study was to investigate the effects of propionate on the activity, gene expression, and protein abundance of the key enzymes involved in the gluconeogenesis of dairy cow hepatocytes. Methods: The hepatocytes were cultured and treated with various concentrations of sodium propionate (0, 1.25, 2.50, 3.75, and 5.00 mM) for 12 h. Glucose content in the culture media was determined by an enzymatic coloring method. The activities of gluconeogenesis related enzymes were determined by enzyme linked immunosorbent assay kits, and the levels of gene expression and protein abundance of the enzymes were detected by real-time quantitative polymerase chain reaction and Western blot, respectively. Results: Propionate supplementation considerably increased the amount of glucose in the culture medium compared to the control (p<0.05); while there was no discernible difference among the various treatment concentrations (p>0.05). The activities of cytoplasmic phosphoenolpyruvate carboxylase (PEPCK1), mitochondrial phosphoenolpyruvate carboxylase (PEPCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC) were increased with the addition of 2.50 and 3.75 mM propionate; the gene expressions and protein abundances of PEPCK1, PEPCK2, PC, and G6PC were increased by 3.75 mM propionate addition. Conclusion: Propionate encouraged glucose synthesis in bovine hepatocytes, and 3.75 mM propionate directly increased the activities, gene expressions and protein abundances of PC, PEPCK1, PEPCK2, and G6PC in bovine hepatocytes, providing a theoretical basis of propionate-regulating gluconeogenesis in bovine hepatocytes.

• Korean Journal of Food Science and Technology
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• v.31 no.6
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• pp.1661-1666
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• 1999

Yungmijihwgang-Won, Yollyunggobon-Dan and Palmi-Hwan, Korea traditional prescriptions composed of oriental medical herbs, have been used successfully to improve human health and regimen. This study was designed to examine the mechanism of healthful effects of the Korea traditional prescriptions through its antioxidative potentials. Using in vitro antioxidative activity assay system such as DPPH radical quenching assay, superoxide anion radical scavenging assay and inhibition of TBARS production, three Korea traditional prescriptions were observed to have nearly the same antioxidative potentials as ascorbic acid, a well-known strong water-soluble antioxidant. Moreover, we observed reinforced antioxidative effects of these drugs in liver from mouse fed these drugs with 4 weeks. When liver homogenate was incubated with 2.2'-azobis(amidinopropane) dihydrochloride(AAPH), as a free radical initiator, we observed that oxidative damages were decreased and antioxidative potentials were increased in liver homogenate treated these drugs. However, enzymatic antioxidative system as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase was not affected by drug administration.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.278-285
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• 2016

The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained $pheA^{FBR}$ gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about $290{\mu}M$) for prephenate in comparison with that (about $850{\mu}M$) of wild type PheA protein. Kinetic analysis showed that the saturation curve of $PheA^{FBR}$ against phenyalanine is hyperbolic rather than that of $PheA^{WT}$, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type $PheA^{WT}$ was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.

• Journal of the Korean Society of Tobacco Science
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• v.27 no.1 s.53
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• pp.19-30
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• 2005

This study was to evaluate the biological activity of flavor components extracted from E. ciliata and E. splendens in order to survey the possibility applicable to tobacco and food industry. Flavor components were extracted with dividing into three parts; essential oil, absolute, oleoresin. In the nonenzymatic lipid peroxidation system, the inhibition rate($\%$) of essential oil were $67.3\;\pm\;20.7\%,\;58.1\;\pm\;19.3\%$ at the concentration of 50 ${\mu}g/mL$ of E. ciliata and E. splendens, respectively. The inhibition rate($\%$) of the oleoresin in E. ciliata was higher than one in E. splendens. In the enzymatic lipid peroxidation system, the inhibition rate($\%$) of essential oil and oleoresin was$14.28\;\pm\;2.38\%,\;and\;65.93\;\pm\;0.01\%,\;and\;was\;22.58\;\pm\;2.84\%\;and\;40.73\;pm\;6.04\%$. The oleoresin of two species were showed above $90\%$ of the inhibition rate($90\%$) against autooxidative lipid peroxidation system. $EC_{50}$ values in neutral red uptake assays 24 h of exposure times were $23.3\;{\mu}g/mL,\;341.0\;{\mu}g/mL\;and\;17.2\;{\mu}g/mL$ in essential oil, absolute and oleoresin from E. ciliata respectively, and were $46.4\;{\mu}g/mL,\;681.7\;{\mu}g/mL\;17.6\;{\mu}g/mL$ in three extractions of E. splendens. Oleoresin of two species showed high rate in the cytotoxic effect by neutral red uptake assay. Absolute and oleoresin did not show antibiotic and mutagenic activity. On the contrary, essential oil with over 500 ug/plate showed antibiotic and mutagenic activity in Ames test. Essential oil and oleoresin have a prolongating effect the ciliostasis of rat trachea. This results indicate that flavor components extracted from E. ciliata and E. splendens can be considered to be toxicological safe and to be the possibility applicable the cigarette, food and drug industry as a flavor for expectoration.

• YAKHAK HOEJI
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• v.56 no.3
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• pp.191-197
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• 2012

We previously reported that the novel isoflavone-free peptide mixture (black soybean peptide, BSP) had several beneficial effects like antiobesity and hypotriglyceridemic effect. However, there are no reports for BSP on anti-hypertensive activity. BSP induced heme oxygenase-1 (HO-1) in HUVECs, thus investigated the HO-1-induced activity in HUVECs and the anti-hypertensive effects in SHR animal model. BSP significantly induced HO-1 expression both at transcriptional and protein levels in a time- and dose-dependent manner as measured by RT-PCR and Western blot analysis, respectively. These inductions were abolished by pretreatment of N-acetyl-cystein (NAC, 1~10 mM), but not by employing Tempol, a superoxide dismutase (SOD) mimetic (1~5 mM). As expected, enzymatic activity (~2 fold) determined by bilirubin formation assay and cGMP concentration (~6 fold) were significantly increased in BSP-treated cells. Based on the numerous evidences on the beneficial effects of HO-1 and our results, we investigated in vivo effects of BSP on the antihypertensive activity. The administration of BSP (1% in drinking water) significantly decreased mean blood pressure (BP) (from $218.6{\pm}6.99$ to $190.0{\pm}3.42$ mm Hg, p<0.01). This result indicates that BSP is strong inducer of HO-1 expression, which may be triggered by oxidative stress, and has anti-hypertensive activity.

• Journal of Nutrition and Health
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• v.54 no.6
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• pp.618-630
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• 2021

Purpose: The ginger rhizome (Zingiber officinale) is widely cultivated as a spice for its aromatic and pungent components. One of its constituents, 6-hydroxydopamine (6-OHDA) is usually thought to cross the cell membrane through dopamine uptake transporters, and induce inhibition of mitochondrial respiration and the generation of intracellular reactive oxygen species (ROS). This study examines the neuroprotective effect and acetylcholinesterase (AChE) inhibitory activity of fermented ginger extracts (FGEs) on 6-OHDA induced toxicity in SH-SY5Y human neuroblastoma cells. Methods: Ginger was fermented using 2 species of Bacillus subtilis, with or without enzyme pretreatment. Each sample was extracted with 70% ethanol. Neurotoxicity was assessed by applying the EZ-Cytox cell viability assay and by measuring lactic dehydrogenase (LDH) release. Morphological changes of apoptotic cell nuclei were observed by Hoechst staining. Cell growth and apoptosis of SH-SY5Y cells were determined by Western blotting and enzyme activity analysis of caspase-3, and AChE enzymatic activity was determined by the colorimetric assay. Results: In terms of cell viability and LDH release, exposure to FGE showed neuroprotective activities against 6-OHDA stimulated stress in SH-SY5Y cells. Furthermore, FGE reduced the 6-OHDA-induced apoptosis, as determined by Hoechst staining. The occurrence of apoptosis in 6-OHDA treated cells was confirmed by determining the caspase-3 activity. Exposure to 6-OHDA resulted in increased caspase-3 activity of SH-SY5Y cells, as compared to the unexposed group. However, pre-treatment with FGE inhibited the activity of caspase-3. The neuroprotective effects of FGE were also found to be caspase-dependent, based on reduction of caspase-3 activity. Exposure to FGE also inhibited the activity of AChE induced by 6-OHDA, in a dose-dependent manner. Conclusion: Taken together, our results show that FGE exhibits a neuroprotective effect in 6-OHDA treated SH-SY5Y cells, thereby making it a potential novel agent for the prevention or treatment of neurodegenerative disease.

• Journal of Life Science
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• v.22 no.6
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• pp.751-759
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• 2012

Microbulbifer sp. was used to acquire the degrading products from Ecklonia cava (DPEC) and the products were investigated to determine the physiological activities. Firstly, 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity and superoxide dismutase (SOD) assay were about 84.1% and 89.6% at 2.5 mg/ml, respectively. In addition, nitrite scavenging ability was shown to be 56.3% at 0.5 mg/ml on pH 1.2. ${\alpha}$-Glucosidase inhibitory activity was increased in a dose-dependent manner and was about 58.7% at 2.5 mg/ml. To determine the influence of DPEC on alcohol metabolism, the generating activity of reduced-nicotinamide adenine dinucleotide (NADH) by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) were measured. Facilitating rates of ADH and ALDH activities by DPEC were 123.3% and 215.2% at 2.5 mg/ml, respectively. For analyses of anti-wrinkling and whitening effects, its elastase and tyrosinase inhibitory activities were measured and were about 73.1% and 42.2% at 2.5 mg/ml, respectively. These results indicated that DPEC has valuable biological attributes owing to its antioxidant, nitrite scavenging, and alcohol metabolizing activities and ${\alpha}$-glucosidase, elastase, and tyrosinase inhibitory activities.

• Korean Journal of Food Science and Technology
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• v.44 no.4
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• pp.488-492
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• 2012

Two strains, traditionally referred to as rock flower (Bawhi-kot) and buckwheat flower (Memil-kot or Chile-Kot), were isolated from stored traditional soy sauce and were identified by using the 18S ITS1/4 region sequences. The rock flower strain showed 99% of similarity with Cladosporium sp. and buckwheat flower strain was 99% identical with yeast Sterigmatomyces halophilus. Both strains were tentatively named Cladosporium sp. NK1 and Sterigmatomyces halophilus NK2, respectively. The optimal growth pHs and temperatures of both strains in a YPD broth medium were in the range of pH 5.0 to 7.0 and 22 to $27^{\circ}C$. Both strains were able to grow in more than 20% of NaCl. In the enzyme activity assay, high protease activity of Cladosporium sp. NK1 and S. halophilus NK2 were obtained in YPD containing 10% of NaCl. High amylase activities of both stains were in 15% and 5% of NaCl, respectively. Lipase activity was, however, not detected in both strains.

• Journal of Life Science
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• v.26 no.7
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• pp.796-804
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• 2016

In the present study, we screened suitable yeasts for wine fermentation and evaluated the fermentative characteristics of Saccharomyces sp. BCNU 6006 and its anti-oxidant activities. Firstly, various yeasts were isolated from Makgeolli, fruits, and fermented foods. Then, the preliminary selections of suitable yeasts were made using an enzymatic activity assay of glucosidase, glycosidase, protease and tolerance to ethanol and SO₂. In addition, the production of biogenic amines and hydrogen sulfide was also monitored. The 9 yeast strains initially selected were determined to belong to the genera Saccharomyces and Kazachtania phylogenetically. We investigated the optimal conditions for wine fermented with black garlic juice (BGJ). The optimal conditions of alcohol fermentation using BGJ were 26 brix, 28℃, and 10 days. Finally, the fermentation products of black garlic wine (BGW) fermented with Saccharomyces sp. BCNU 6006 exhibited 15.03% ethanol, 12 brix of sugar, and pH 4.01. The contents of total polyphenol, total flavonoid, tannin, and 5-HMF compound of BGW were 3.85 mg/ml, 0.51mg/ml, 5.90 mg/ml, and 0.07 mg/ml respectively, lower than that of BGJ. DPPH radical scavenging activity, ABTS radical scavenging activity, and reducing power of BGW were 90.77%, 95.20% and 1.261 respectively, lower than that of BGJ. Superoxide anion (O²-) radical scavenging activity was 94.42%, higher than that of BGJ. Based on the above results, the industrial potential of Saccharomyces sp. BCNU 6006 as a wine-making yeast was confirmed in the present study.