• Title/Summary/Keyword: immunoblotting

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Ginsenoside Rg1 augments oxidative metabolism and anabolic response of skeletal muscle in mice

  • Jeong, Hyeon-Ju;So, Hyun-Kyung;Jo, Ayoung;Kim, Hye-Been;Lee, Sang-Jin;Bae, Gyu-Un;Kang, Jong-Sun
    • Journal of Ginseng Research
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    • v.43 no.3
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    • pp.475-481
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    • 2019
  • Background: The ginsenoside Rg1 has been shown to exert various pharmacological activities with health benefits. Previously, we have reported that Rg1 promoted myogenic differentiation and myotube growth in C2C12 myoblasts. In this study, the in vivo effect of Rg1 on fiber-type composition and oxidative metabolism in skeletal muscle was examined. Methods: To examine the effect of Rg1 on skeletal muscle, 3-month-old mice were treated with Rg1 for 5 weeks. To assess muscle strength, grip strength tests were performed, and the lower hind limb muscles were harvested, followed by various detailed analysis, such as histological staining, immunoblotting, immunostaining, and real-time quantitative reverse transcription polymerase chain reaction. In addition, to verify the in vivo data, primary myoblasts isolated from mice were treated with Rg1, and the Rg1 effect on myotube growth was examined by immunoblotting and immunostaining analysis. Results: Rg1 treatment increased the expression of myosin heavy chain isoforms characteristic for both oxidative and glycolytic muscle fibers; increased myofiber sizes were accompanied by enhanced muscle strength. Rg1 treatment also enhanced oxidative muscle metabolism with elevated oxidative phosphorylation proteins. Furthermore, Rg1-treated muscles exhibited increased levels of anabolic S6 kinase signaling. Conclusion: Rg1 improves muscle functionality via enhancing muscle gene expression and oxidative muscle metabolism in mice.

Apoptotic activity of demethoxycurcumin in MG-63 human osteosarcoma cells

  • Kang, Kyeong-Rok;Kim, Jae-Sung;Kim, Tae-Hyeon;Seo, Jeong-Yeon;Park, Jong-Hyun;Chun, Hong Sung;Yu, Sun-Kyoung;Kim, Heung-Joong;Kim, Chun Sung;Kim, Do Kyung
    • International Journal of Oral Biology
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    • v.46 no.1
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    • pp.23-29
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    • 2021
  • Demethoxycurcumin (DMC), which is a curcuminoid found in turmeric, has anti-proliferative effects on cancer cells. However, the effect of DMC on osteosarcoma has not been established. The aim of this study was to examine the effects of DMC on cell growth and apoptosis induction in MG-63 human osteosarcoma cells. This study was investigated using 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromid assay, Live/Dead cell assay, 4', 6-diamidino-2-phenylindole staining, and immunoblotting in MG-63 cells. DMC induced MG-63 cell death in a dose-dependent manner, with an estimated IC50 value of 54.4 µM. DMC treatment resulted in nuclear condensation in MG-63 cells. DMC-induced apoptosis in MG-63 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting results showed that Bcl-2 and Bcl-xL were downregulated, while Bax and Bad were upregulated by DMC in MG-63 cells. These results indicated that DMC inhibits cell proliferation and induces apoptotic cell death in MG-63 human osteosarcoma cells via the death receptor-mediated extrinsic apoptotic pathway and mitochondria-mediated intrinsic apoptotic pathway.

Effects on Goat Meat Extracts on α-Glucosidase Inhibitory Activity, Expression of Bcl-2-Associated X (BAX), p53, and p21 in Cell Line and Expression of Atrogin-1, Muscle Atrophy F-Box (MAFbx), Muscle RING-Finger Protein-1 (MuRF-1), and Myosin Heavy Chain-7 (MYH-7) in C2C12 Myoblsts

  • Joohyun Kang;Soyeon Kim;Yewon Lee;Jei Oh;Yohan Yoon
    • Food Science of Animal Resources
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    • v.43 no.2
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    • pp.359-373
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    • 2023
  • This study examined the α-glucosidase inhibitory, and apoptosis- and anti-muscular-related factors of goat meat extracts from forelegs, hind legs, loin, and ribs. The goat meat extracts were evaluated for their α-glucosidase inhibitory activity. The gene and protein expression levels of Bcl-2-associated X (bax), p53, and p21 were examined by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting in AGS and HT-29 cells. The expression levels of Atrogin-1 and MHC1b were examined by RT-PCR in C2C12 myoblasts, and the expression levels of Atrogin-1, muscle atrophy F-box (MAFbx), muscle RING-finger protein-1 (MuRF-1), and myosin heavy chain-7 were investigated by immunoblotting. α-Glucosidase inhibitory activity was higher in ethanol extract than in hydrous and hot water extracts. BAX and p53 expression levels were higher (p<0.05) in AGS cells treated with goat meat extract than those of cells treated with no goat meat extract. In HT-29 cells, the protein expression levels of BAX, p53, and p21 were higher (p<0.05) in the cells treated with goat meat extract than those of cells not treated with goat meat extract. In dexamethasone-treated C2C12 cells, goat meat extract treatment lower (p<0.05) the expression of Atrogin-1 and lower (p<0.05) the expression of MAFbx and MuRF-1. The results of the present study indicate that goat meat extracts have α-glucosidase inhibitory activity in vitro. In addition, apoptosis was induced in AGS cells and HT-29 cells treated with goat meat extract, and anti-muscular atrophy activity was also observed in C2C12 cells treated with goat meat extract.

Protective effects of Panax ginseng berry extract on blue light-induced retinal damage in ARPE-19 cells and mouse retina

  • Hye Mi Cho;Sang Jun Lee;Se-Young Choung
    • Journal of Ginseng Research
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    • v.47 no.1
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    • pp.65-73
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    • 2023
  • Background: Age-related macular degeneration (AMD) is a significant visual disease that induces impaired vision and irreversible blindness in the elderly. However, the effects of ginseng berry extract (GBE) on the retina have not been studied. Therefore, this study aimed to investigate the protective effects of GBE on blue light (BL)-induced retinal damage and elucidate its underlying mechanisms in human retinal pigment epithelial cells (ARPE-19 cells) and Balb/c retina. Methods: To investigate the effects and underlying mechanisms of GBE on retinal damage in vitro, we performed cell viability assay, pre-and post-treatment of sample, reactive oxygen species (ROS) assay, quantitative real-time PCR (qRT-PCR), and western immunoblotting using A2E-laden ARPE-19 cells with BL exposure. In addition, Balb/c mice were irradiated with BL to induce retinal degeneration and orally administrated with GBE (50, 100, 200 mg/kg). Using the harvested retina, we performed histological analysis (thickness of retinal layers), qRT-PCR, and western immunoblotting to elucidate the effects and mechanisms of GBE against retinal damage in vivo. Results: GBE significantly inhibited BL-induced cell damage in ARPE-19 cells by activating the SIRT1/PGC-1α pathway, regulating NF-kB translocation, caspase 3 activation, PARP cleavage, expressions of apoptosis-related factors (BAX/BCL-2, LC3-II, and p62), and ROS production. Furthermore, GBE prevented BL-induced retinal degeneration by restoring the thickness of retinal layers and suppressed inflammation and apoptosis via regulation of NF-kB and SIRT1/PGC-1α pathway, cleavage of caspase 3 and PARP, and expressions of apoptosis-related factors in vivo. Conclusions: GBE could be a potential agent to prevent dry AMD and progression to wet AMD.

Amygdalin Reverses Macrophage PANoptosis Induced by Drug-Resistant Escherichia coli

  • Xue Yan;Liang Jin;Huifen Zhou;Haofang Wan;Haitong Wan;Jiehong Yang
    • Journal of Microbiology and Biotechnology
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    • v.33 no.10
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    • pp.1281-1291
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    • 2023
  • Infectious diseases caused by drug-resistant Escherichia coli (E. coli) pose a critical concern for medical institutions as they can lead to high morbidity and mortality rates. In this study, amygdalin exhibited anti-inflammatory and antioxidant activities, as well as other potentials. However, whether it could influence the drug-resistant E. coli-infected cells remained unanswered. Amygdalin was therefore tested in a cellular model in which human macrophages were exposed to resistant E. coli. Apoptosis was measured by flow cytometry and the lactate dehydrogenase (LDH) assay. Western immunoblotting and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to quantify interleukin-18 (IL-18), interleukin-1β (IL-1β), and interleukin-6 (IL-6). The production of reactive oxygen species (ROS) in macrophages was detected by ROS kit. The expression of pan-apoptotic proteins in macrophages was measured by qRT-PCR and Western immunoblotting. Drug-Resistant E. coli inhibited cell viability and enhanced apoptosis in the cellular model. In cells treated with amygdalin, this compound can inhibit cell apoptosis and reduce the expression of pro - inflammatory cytokines such as IL-1β, IL-18 and IL-6. Additionally, it decreases the production of PANoptosis proteins, Furthermore, amygdalin lowered the levels of reactive oxygen species induced by drug-resistant E. coli, in cells, demonstrating its antioxidant effects. Amygdalin, a drug with a protective role, alleviated cell damage caused by drug-resistant E. coli in human macrophages by inhibiting the PANoptosis signaling pathway.

Allergenicity Change of Soybean Proteins by Thermal Treatment Methods (열처리 방법에 따른 품종별 콩 단백질의 항원성 변화)

  • Seol, Hui-Gyeong;Ko, Yu-Jin;Kim, Eun-Jung;Lee, Gyeong-Lan;Kim, Do-Gyeong;Lee, Jeong-Ok;Ahn, Kang-Mo;Ryu, Chung-Ho
    • Journal of Life Science
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    • v.22 no.4
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    • pp.524-531
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    • 2012
  • Soybean is one of the most common food materials causing food hypersensitivity reactions in Korea. In this study, we have investigated the effect of roasting and fermentation on the allergenicity of soybean. Three kinds of soybean ($Daepung$, $Daewon$, and $Taegwang$) were prepared as raw, roasted, and fermented by $Bacillus$ $subtilis$ GSK 3580, and then their proteins were extracted. The proteins were separated using SDS-PAGE, and the detection of IgE specific to soybean proteins was performed by immunoblotting using 7 sera of soybean allergy patients and non-allergic control individuals. Serum specific IgE to soybean was measured by ELISA. The SDS-PAGE of raw soybean proteins showed various-sized bands ranging from 9 to 76 kDa, which are known as major allergens. In particular, 9, 21, 34, 52, 72, and 76 kDa proteins are known as LTP, Kunits trypsin inhibitor, $Gly$ m Bd 30K, ${\beta}$-subunit, ${\alpha}$-subunit, and ${\alpha}$'-subunit of ${\beta}$-conglycinin, respectively; these are major allergens in soybean. In contrast, only peptides of less than 35 kDa were found in roasted and fermented soybeans. IgE immunoblot analysis of three roasted species of soybeans commonly detected at 38-40 kDa and 10-15 kDa. The protein bands in fermented soybean showed very weak signals or were not detected. In addition, the reactivity of most patients' sera to soybean was decreased after roasting and fermentation. With these results, it may be concluded that the allergenicity of soybeans is reduced by the roasting and fermentation processes. It is supposed that allergenic proteins in soybean were degraded by heat treatment methods and proteolytic enzymes were secreted from fermenting microorganisms.

Detection of Helicobacter pylori by Pre-embedding Immunoelectron Microscopy - Comparison with Immunoblotting Method (Pre-embedding Immunoelectron Microscopy를 이용한 Helicobacter pylori의 발견 - Immunoblotting법과 비교 -)

  • Jung, Hae-Seoung;Kim, Eun-Jung;Kim, Eun-A;Park, Ji-Hoe;Jun, Jin-Su;Seo, Ji-Hyoun;Lim, Jae-Young;Choi, Myoung-Bum;Woo, Hyang-Ok;Youn, Hee-Shang;Ko, Gyung-Hyuck;Baik, Seung-Chul;Lee, Woo-Kon;Cho, Myung-Je;Rhee, Kwang-Ho
    • Clinical and Experimental Pediatrics
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    • v.45 no.7
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    • pp.862-874
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    • 2002
  • Purpose : We tried to evaluate whether the detection rate of Helicobacter pylori in gastric biopsy specimens could be improved by using pre-embedding immunoelectron microscopy. Methods : A total of 119 children who complained of upper gastrointestinal symptoms were endoscoped at the Gyeongsang National University Hospital from July, 1996 to July, 1999. Five biopsy specimens(three for urease test, one for hematoxylin-eosin(H & E) staining, and one for preembedding immunoelectron microscopy) were obtained from each antrum and body. Immunoblotting analysis were also performed. Results : Among the 119 patients, H. pylori were found in 116 patients(97.5%) by the immunoelectron microscopy. Among three patients who were found H. pylori negative in immunoelectron microscopy, two patients showed H. pylori in H & E stained slides and one patient was urease test positive(color change within six hours). Urease tests were positive in 107 patients(89.9 %). The positive rate of immunoblotting tests was 81.5%. However, only 13 patients(10.9%) showed H. pylori on the H & E stained antrum or body tissue. Conclusion : In this study, we found H. pylori histopathologically in most of the pediatric patients who complained of upper gastrointestinal symptoms. This study showed that pre-embedding immunoelectron microscopic examinations can be used as a gold standard in the diagnosis of childhood H. pylori infection. However, this method also has limited capacity to detect widely scattered H. pylori compared to the other histopathologic diagnostic methods.

Changes in Antigenicity of Gliadin from Medium Flour by Autoclave and Microwave Treatments (가압가열 및 Microwave 처리에 의한 중력분 Gliadin의 항원성 변화)

  • Kwak, Ji-Hee;Kim, Koth-Bong-Woo-Ri;Lee, Chung-Jo;Kim, Min-Ji;Kim, Dong-Hyun;SunWoo, Chan;Jung, Seul-A;Kang, Ju-Youn;Kim, Hyun-Jee;Choi, Jung-Su;Kim, Seong-Won;Ahn, Dong-Hyun
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.40 no.10
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    • pp.1423-1429
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    • 2011
  • This study was conducted to evaluate the effect of physical treatments on the antigenicity of gliadin in medium wheat flour. The wheat flour was treated with an autoclave (5, 10, 30, 50 min), a microwave (1, 5, 10 min), and both (10, 30, 50 min/ 5, 10 min), and investigated by SDS-PAGE, immunoblotting and Ci-ELISA using anti-gliadin IgG. The results showed that the binding ability of anti-gliadin IgG to gliadin in wheat flour was slightly decreased when autoclaved or when autoclaved and microwaved. Especially, it was reduced to about 69% after autoclaving for 50 min and 73% after autoclaving for 50 min and microwaving for 5 min. In addition, gliadin bands in the 50 min autoclaved group disappeared in both SDS-PAGE and immunoblotting. On the other hand, the antigenicity of gliadin was unaffected by microwaving alone. Consequently, there were no considerable changes in using an autoclave alone or in combination with a microwave. These results suggest that autoclaving may affect the reduction of the antigenicity of gliadin in medium wheat flour.

Effect of Digestive Enzymes on the Allergenicity of Autoclaved Market Pork Sausages (가압가열 처리한 시판 돈육 소시지의 항원성에 미치는 소화효소의 영향)

  • Kim, Seo-Jin;Kim, Koth-Bong-Woo-Ri;Song, Eu-Jin;Lee, So-Young;Yoon, So-Young;Lee, So-Jeong;Lee, Chung-Jo;Ahn, Dong-Hyun
    • Food Science of Animal Resources
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    • v.29 no.2
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    • pp.238-244
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    • 2009
  • Food allergy is a serious nutritional problem in both children and adults. Therefore, food allergenicity reduction methods are greatly needed. The allergenicity is altered by various manufacturing processes, and the digestibility of food proteins can be affected by food processing. This study was conducted to investigate the effect of in-vitro digestibility on the allergenicity of autoclaved market pork sausages using pepsin (30min) and trypsin (5, 30, 60, 90, and 120min). The binding ability of the porcine serum albumin (PSA) from sausages A and B significantly decreased by about 30 and 23%, respectively, after autoclave treatment (121; 5, 10, and 30 min). After the pepsin and trypsin treatments, the binding ability of products A and B at 30 min decreased. These competitive indirect enzyme-linked immunosorbent assay (ci-ELISA) results corresponded well with the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting results. The results demonstrated that the allergenicity of pork sausages considerably decreased after autoclave treatment, and were also maintained or decreased after enzyme treatment. Accordingly, autoclave treatment represents a promising processing technology for the reduction of the allergenicity of diverse food products.

Effects of Autoclave and Microwave Treatments on the Antigenicity of Gliadin in Medium Wheat Dough (가압가열 및 microwave에 의한 중력분 반죽 gliadin의 항원성 변화)

  • Kwak, Ji-Hee;Kim, Koth-Bong-Woo-Ri;Lee, Chung-Jo;Kim, Min-Ji;Kim, Dong-Hyun;SunWoo, Chan;Jung, Seul-A;Kim, Hyun-Jee;Choi, Jung-Su;Kim, Seong-Won;Ahn, Dong-Hyun
    • Korean Journal of Food Science and Technology
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    • v.44 no.1
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    • pp.55-60
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    • 2012
  • The aim of this study was to determine the optimal physical treatment to reduce the antigenicity of gliadin in wheat dough. Medium wheat dough was treated with an autoclave (5, 10, 30, and 50 min at $121^{\circ}C$, 1 atm), a microwave (1, 5, and 10 min) or both (10, 30, and 50 min/5, 10 min). The proteins in the dough extracts were analyzed by SDSPAGE and the binding ability of anti-gliadin IgG to gliadin was examined by ci-ELISA and immunoblotting. Results showed that the ability of anti-gliadin IgG to bind to gliadin in wheat dough treated with an autoclave alone or in combination with a microwave was decreased. Especially, it declined to ~77% after autoclaving for 30 min and 35% after both autoclaving for 50 min and microwaving for 5 min. In addition, the intensity of gliadin bands in SDS-PAGE were weakened and anti-gliadin IgG did not recognize gliadin in immunoblotting. However, microwaving alone did not affect the antigenicity of gliadin in wheat dough. These results indicate that autoclaving may affect the reduction of the antigenicity of gliadin in medium wheat dough. Moreover, autoclaving in combination with microwaving is more effective for reducing the antigenicity of wheat dough.