Ko, Kyong-Cheol;Choi, Mi Hee;Park, Sang Hyun;Cho, Kyung-Hyun;Lee, Ki-Teak
Journal of Radiation Industry
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v.3
no.1
/
pp.25-29
/
2009
Microarrays can be used to screen thousands of binding events in a parallel and high throughput fashion and are of major importance in disease diagnosis and drug discovery. The use of radioisotope is conventionally regarded as one of the most sensitive detection methods. Atherosclerosis is a common disorder affecting arterial blood vessels. It happens when fat, cholesterol, and other substances made in the arterial blood vessels form a hard substances called plaque. Lipoprotein-associated phospholipase $A_2$ ($Lp-PLA_2$), a phospholipase $A_2$ enzyme, is used as a marker for cardiac disease. The detection of $Lp-PLA_2$ was accomplished by using radioactive [$^3H-acetyl$] PAF as a substrate and a feasibility study on RI biochip application to detection of $Lp-PLA_2$, a risk factors of atherosclerosis was performed. Inhibitive activity of a native plant extract was also determined by using the RI biochip. It was found to be applicable to a high-throughput screening of inhibitors for developing atherosclerosis therapeutic agents.
Fu, Zhicheng;Yun, So Yoon;Won, Jong Hoon;Back, Moon Jung;Jang, Ji Min;Ha, Hae Chan;Lee, Hae Kyung;Shin, In Chul;Kim, Ju Yeun;Kim, Hee Soo;Kim, Dae Kyong
Biomolecules & Therapeutics
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v.27
no.2
/
pp.193-200
/
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.
PURPOSE: This study was to investigate the Glycolysis mediated sarcoplasmic reticulum (SR) $Ca^{2+}$ signal regulates mitochondria $Ca^{2+}$ during skeletal muscle contraction by using glycolysis inhibitor. METHODS: To examine the effect of Glycolysis inhibitor on SR and mitochondria $Ca^{2+}$ content, we used skeletal muscle fiber from gastrocnemius muscle. 2-deoxy glucose and 3-bromo pyruvate used as glycolysis inhibitor, it applied to electrically stimulated muscle contraction experiment. Intracellular $Ca^{2+}$ content, SR, mitochondria $Ca^{2+}$ level and mitochondria membrane potential (MMP) was detected by confocal microscope. Mitochondrial energy metabolism related enzyme, citric acid synthase activity also examined for mitochondrial function during the muscle contraction. RESULTS: Treatment of 2-DG and 3BP decreased the muscle contraction induced SR $Ca^{2+}$ increase however the mitochondria $Ca^{2+}$ level was increased by treatment of inhibitors and showed and overloading as compared with the control group. Glycolysis inhibitor and thapsigargin treatment showed a significant decrease in MPP of skeletal muscle cells compared to the control group. CS activity significantly decreased after pretreatment of glycolysis inhibitor during skeletal muscle contraction. These results suggest that regulation of mitochondrial $Ca^{2+}$ levels by glycolysis is an important factor in mitochondrial energy production during skeletal muscle contraction CONCLUSIONS: These results suggest that mitochondria $Ca^{2+}$ level can be regulated by SR $Ca^{2+}$ level and glycolytic regulation of intraocular $Ca^{2+}$ signal play pivotal role in regulation of mitochondria energy metabolism during the muscle contraction.
The objective of this study was to characterize the enzymatic hydrolysis of lipopolysaccharide (LPS) by wheat phytase and to investigate the effects of wheat phytase-treated LPS on in vitro toxicity, cell viability and release of a pro-inflammatory cytokine, interleukin (IL)-8 by target cells compared with the intact LPS. The phosphatase activity of wheat phytase towards LPS was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine. In vitro toxicity of LPS hydrolyzed with wheat phytase in comparison to intact LPS was assessed. Cell viability in human aortic endothelial (HAE) cells exposed to LPS treated with wheat phytase in comparison to intact LPS was measured. The release of IL-8 in human intestinal epithelial cell line, HT-29 cells applied to LPS treated with wheat phytase in comparison to intact LPS was assayed. Wheat phytase hydrolyzed LPS, resulting in a significant release of inorganic phosphate for 1 h (p < 0.05). Furthermore, the degradation of LPS by wheat phytase was nearly unaffected by the addition of L-phenylalanine, the inhibitor of tissue-specific alkaline phosphatase or L-homoarginine, the inhibitor of tissue-non-specific alkaline phosphatase. Wheat phytase effectively reduced the in vitro toxicity of LPS, resulting in a retention of 63% and 54% of its initial toxicity after 1-3 h of the enzyme reaction, respectively (p < 0.05). Intact LPS decreased the cell viability of HAE cells. However, LPS dephosphorylated by wheat phytase counteracted the inhibitory effect on cell viability. LPS treated with wheat phytase decreased IL-8 secretion from intestinal epithelial cell line, HT-29 cell to 14% (p < 0.05) when compared with intact LPS. In conclusion, wheat phytase is a potential therapeutic candidate and prophylactic agent for control of infections induced by pathogenic Gram-negative bacteria and associated LPS-mediated inflammatory diseases in animal husbandry.
Background: Abnormalities of myelin, which increases the efficiency of action potential conduction, are found in neurological disorders. Korean Red Ginseng (KRG) demonstrates therapeutic efficacy against some of these conditions, however effects on oligodendrocyte (OL)s are not well known. Here, we examined the effects of KRG-derived components on development and protection of OL-lineage cells. Methods: Primary OL precursor cell (OPC) cultures were prepared from neonatal mouse cortex. The protective efficacies of the KRG components were examined against inhibitors of mitochondrial respiratory chain activity. For in vivo function of Rb1 on myelination, after 10 days of oral gavage into adult male mice, forebrains were collected. OPC proliferation were assessed by BrdU incorporation, and differentiation and myelination were examined by qPCR, western blot and immunocytochemistry. Results: The non-saponin promoted OPC proliferation, while the saponin promoted differentiation. Both processes were mediated by AKT and extracellular regulated kinase (ERK) signaling. KRG extract, the saponin and non-saponin protected OPCs against oxidative stress, and both KRG extract and the saponin significantly increased the expression of the antioxidant enzyme. Among 11 major ginsenosides tested, Rb1 significantly increased OL membrane size in vitro. Moreover, Rb1 significantly increased myelin formation in adult mouse brain. Conclusion: All KRG components prevented OPC deaths under oxidative stress. While non-saponin promoted proliferation, saponin fraction increased differentiation and OL membrane size. Furthermore, among all the tested ginsenosides, Rb1 showed the biggest increase in the membrane size and significantly enhanced myelination in vivo. These results imply therapeutic potentials of KRG and Rb1 for myelin-related disorders.
Jaewon Shin;Harim Lee;Seunghyun Ahn;Won Seok Jeong;CheongTaek Kim;Seyeon Park
Journal of Applied Biological Chemistry
/
v.65
no.4
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pp.299-306
/
2022
In this study, derivatives of trimethoxybenzene were investigated as inhibitors of melanogenesis. We examined the effects of methyl 3,4,5-trimethoxybenzoate (MTB), ethyl 3,4,5-trimethoxybenzoate (ETB), methyl 3,4,5-trimethoxycinnamate (MTC), and ethyl 3,4,5-trimethoxycinnamate (ETC). First, the inhibitory effects of these agents on melanin production were evaluated using α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells. We found that all derivatives decreased α-MSH-induced melanin production in B16F10 melanoma cells; ETC showed a strong inhibitory effect at half of the concentration of the other derivatives. As tyrosinase is considered a key enzyme of melanogenesis, we also examined whether the derivatives inhibited tyrosinase activity. MTC and ETC reduced mushroom tyrosinase activity and expression levels of α-MSH-induced B16F10 cellular tyrosinase protein. Inhibitory effects of all derivatives on α-MSH-induced B16F10 cellular tyrosinase activity were shown in a dose-dependent manner. Additionally, the derivatives were exposed to diphenylpicrylhydrazyl free radical to examine their antioxidant characteristics. All derivatives showed considerable antioxidant activity, which was 2-fold higher than that of arbutin. In conclusion, the trimethoxybenzene derivatives, including MTB, ETB, MTC, and ETC exerted anti-melanogenic and antioxidant effects on α-MSH-stimulated melanogenesis, demonstrating their potential for use as novel hypopigmenting agents and antioxidants.
Journal of the Korean Society of Food Science and Nutrition
/
v.34
no.4
/
pp.446-450
/
2005
The biological activity of water and ethanol extracts from Chamomaile for functional food source were examined. Total phenol contents in the $60\%$ ethanol extracts $(24.98\pm0.20\;mg/g)$ from Chamomaile leaf was higher than those of water extracts $(23.64\pm0.35\;mg/g)$ The major phenolic compound by HPLC were rosemarinic acid and Quercetin. $60\%$ ethanol extracts had higher content of these phenolics than water extacts. Electron donating ability showed $91.05\%$ in the water extracts and $95.49\%$ in the $60\%$ ethanol extracts. Antioxidant protection factor (PF) showed $0.71\pm0.02 $ in the water extracts and $1.48\pm0.03 $ in the $60\%$ ethanol extracts. The water extracts of Chamomaile leaves did not have antimicrobial activity against H. pylori, but the $60\%$ ethanol extracts revealed the slight antimicrobial activity as 9.42 mm of clear zone. Angiotensin converting enzyme inhibition was $57.98\%$ in water extracts and $91.36\%$ in $60\%$ ethanol extracts. Xanthine oxidase activity was $73.48\%$ in water extracts and $81.96\%$ in $60\%$ ethanol extracts. The results suggest that Chamomailes extract may be useful as potential source as antioxidant, angiotensin converting enzyme and xanthine oxidase inhibitors.
Lee, Chea Yeon;Park, Hyo Sung;Kong, Deok-Hoon;Kim, Young Kwan;Cho, Whajung
Journal of Nutrition and Health
/
v.53
no.5
/
pp.452-463
/
2020
Purpose: Aster tataricus (AT) is one of the Asteraceae perennial herbs used in traditional Chinese medicine. The herb contains various bioactive substances, such as flavonoids, isoflavonoids, and phenolic compounds in the roots, and exhibits a range of effects including anti-bacterial, anti-oxidant, and anti-inflammatory activities. This study compared the immunomodulatory effects of ethanol and water extracts of whole AT, except the roots, and analyzed the molecular mechanisms for the regulatory effects on cytokine secretion from THP-1 cells. Methods: The effects of AT extract on the cell viability and proliferation of THP-1 cells were analyzed using the Cell Counting Kit-8 method. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell culture supernatant of the AT-treated THP-1 cells were measured using an enzyme-linked immunosorbent assay. The protein levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor kappa B (IκBα), and mitogen-activated protein kinase (MAPK) phosphorylation in the cell lysates were determined by western blotting. Results: The water extract and the ethanol extract of AT did not affect the cell viability, and increased the proliferation of THP-1 cells significantly compared to the vehicle. The water extract increased the secretion of IL-1β from THP-1 cells in a dose-dependent manner, but the ethanol extract had no effect. The expression of COX-2 and iNOS protein and the phosphorylation of MAPK and Akt were induced in AT-treated cells. In addition, IκBα was degraded by AT in a concentration-dependent manner. IL-1β secretion by AT was reduced by extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors, while TNF-α secretion was decreased by inhibitors of ERK, p38 MAPK, and JNK. Interestingly, the p38 MAPK inhibitor increased the production of IL-1β by AT further. Conclusion: The water extract of the above-ground parts of AT contains immunomodulatory bioactive substances that stimulate immune cells through the MAPK signaling pathway.
Lee, Eun Hee;Yim, Hyung Eun;Jang, Gi Young;Yoo, Kee Hwan;Son, Chang Sung;Hong, Young Sook;Lee, Joo Won
Clinical and Experimental Pediatrics
/
v.51
no.9
/
pp.992-997
/
2008
Purpose : Hypertension (HTN) is no longer an exclusively adult disease; the prevalence of pediatric HTN is increasing. To understand the evaluation and treatment of childhood HTN in Korea, we investigated, via a questionnaire, how hypertensive children are currently assessed and managed by pediatric cardiologists (CA) and nephrologists (NE). Methods : We surveyed 82 pediatric CA and 77 NE, regarding how they manage hypertensive children in Korea. Results : A total 75 replies were received to our questionnaire request (response rate: 47.15%). Routine blood pressure (BP) checks were more frequently performed by NE (CA: 5.7%, NE: 25%, P=0.03), but most respondents (86%) did not check BP routinely. Mercury sphygmomanometers were the most commonly used devices and ambulatory blood pressure monitoring was not frequently used. The goal BP in treated patients was set at the 95th percentile by 61% of respondents. NE used a lower BP goal in hypertensive children with renal disease (CA: 24%, NE: 64%, P=0.004) or DM (CA: 12.5%, NE: 50%, P=0.003). Angiotensin converting enzyme (ACE) inhibitors were the most commonly used agents (mean: 59%); following ACE inhibitors, CA preferred diuretics for hypertensive children with renal disease or cardiovascular disease, and NE preferred calcium channel blockers, regardless of underlying disease. Self-monitoring was the most frequent method for BP monitoring at home. Conclusion : In Korea, BP measurement is not yet a routine examination in treating hypertension. There are some differences in management strategies vis-$\grave{a}$-vis hypertension, between pediatric cardiologists and nephrologists. For the appropriate management and prevention of secondary disease in long-term studies, standard guidelines and education are needed for pediatricians.
Lee, Eun Kyeong;Kim, Ju Hyun;Moon, Kyoung Mi;Ha, Sugyeong;Noh, Sang-Gyun;Kim, Dae Hyun;Lee, Bonggi;Kim, Do Hyun;Kim, Su Jeong;Ullah, Sultan;Moon, Hyung Ryong;Chung, Hae Young
Journal of Life Science
/
v.27
no.2
/
pp.139-148
/
2017
The inhibition of tyrosinase, a key enzyme in mammalian melanin synthesis, plays an important role in preventing skin pigmentation and melanoma. Therefore, tyrosinase inhibitors are very important in the fields of medicine and cosmetics. However, only a few tyrosinase inhibitors are currently available because of their toxic effects on skin or lack of selectivity and stability. Therefore, we synthesized a novel series of (E)-2-(substituted benzylidene)-2,3-dihydro-1H-cyclopenta[a]naphthalen-1-one derivatives and evaluated their inhibitory effects on mushroom tyrosinase, with the aim of discovering a novel tyrosinase inhibitor. Among 19 derivatives, MHY3655 ($IC_{50}=0.1456{\mu}M$) showed the strongest inhibitory effect on tyrosinase activity compared to kojic acid ($IC_{50}=17.2{\mu}M$), a well-known tyrosinase inhibitor. In addition, MHY3655 showed competitive inhibition on Lineweaver-Burk plots. We confirmed that MHY3655 strongly interacts with mushroom tyrosinase residues through the docking simulation. Substitutions with a hydroxy group at both R2 and R4 in the phenyl ring indicated that these groups play a major role in the high binding affinity to tyrosinase. Further, MHY3655 did not show cytotoxicity at the concentrations tested in B16F10 melanoma cells. In conclusion, the novel compound MHY3655 potentially shows tyrosinase inhibitory activity, and it could be used as an ingredient in whitening cosmetics.
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