• Journal of Microbiology and Biotechnology
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• v.32 no.2
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• pp.228-237
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• 2022

In this study, the effects of the immune stimulator Euglena gracilis (Euglena) in cyclophosphamide (CCP)-induced immunocompromised mice were assessed. The key component β-1,3-glucan (paramylon) constitutes 50% of E. gracilis. Mice were orally administered Euglena powder (250 and 500 mg/kg body weight (B.W.)) or β-glucan powder (250 mg/kg B.W.) for 19 days. In a preliminary immunology experiment, ICR mice were intraperitoneally injected with 80 mg of CCP/kg B.W. during the final 3 consecutive days. In the main experiment, BALB/c mice were treated with CCP for the final 5 days. To evaluate the enhancing effects of Euglena on the immune system, mouse B.W., the spleen index, natural killer (NK) cell activity and mRNA expression in splenocytes lungs and livers were determined. To detect cytokine and receptor expression, splenocytes were treated with 5 ㎍/ml concanavalin A or 1 ㎍/ml lipopolysaccharide. The B.W. and spleen index were significantly increased and NK cell activity was slightly enhanced in all the experimental groups compared to the CCP-only group. In splenocytes, the gene expression levels of tumor necrosis factor-α, interferon-γ, interleukin (IL)-10, IL-6, and IL-12 receptor were increased in the E. gracilis and β-glucan groups compared to the CCP-only group, but there was no significant difference. Treatment with 500 mg of Euglena/kg B.W. significantly upregulated dectin-1 mRNA expression in the lung and liver compared to the CCP-only group. These results suggest that Euglena may enhance the immune system by strengthening innate immunity through immunosuppression.

• Journal of Animal Science and Technology
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• v.64 no.6
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• pp.1117-1131
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• 2022

Previous studies reported that Bifidobacterium animalis ssp. lactis HY8002 (HY8002) improved intestinal integrity and had immunomodulatory effects. Lactobacillus plantarum HY7717 (HY7717) was screened in vitro from among 21 other lactic acid bacteria (LAB) and demonstrated nitric oxide (NO) production. The aims of this study were to investigate the individual and combined ex vivo and in vivo effects of LAB strains HY8002 and HY7717 at immunostimulating mice that have been challenged with an immunosuppressant drug. The combination of HY8002 and HY7717 increased the secretion of cytokines such as interferon (IFN)-γ, interleukin (IL)-12, and tumor necrosis factor (TNF)-α in splenocytes. In a cyclophosphamide (CTX)-induced immunosuppression model, administration of the foregoing LAB combination improved the splenic and hematological indices, activated natural killer (NK) cells, and up-regulated plasma immunoglobulins and cytokines. Moreover, this combination treatment increased Toll-like receptor 2 (TLR2) expression. The ability of the combination treatment to upregulate IFN-γ and TNF-α in the splenocytes was inhibited by anti-TLR2 antibody. Hence, the immune responses stimulated by the combination of HY8002 and HY7717 are associated with TLR2 activation. The preceding findings suggest that the combination of the HY8002 and HY7717 LAB strains could prove to be a beneficial and efficacious immunostimulant probiotic supplement. The combination of the two probiotic strains will be applied on the dairy foods including yogurt and cheese.

• YAKHAK HOEJI
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• v.41 no.5
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• pp.629-637
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• 1997

Tumor necrosis factor-${alpha}$ (TNF) induced a cytotoxic response in ME-180 cervical carcinoma cells in vitro. This cytotoxic response was accompanied by a temporal series of mitogenic stimuli : increased c-fos, c-jun and jun-B expression. Depletion of protein kinase C (PKC) by exposure of ME-180 cells to 100ng/ml phorbol myristate acetate (PMA) for 24hours almost completely abolished TNF-mediated increase in these signals, indicating that a PKC-dependent pathway is involved in TNF-mediated increases in the expression of c-fos, c-jun and jun-B. Characteristics of TNF receptors after exposure to 100ng/ml PMA or 24hours were not altered, suggesting that diminished induction of these oncogenes by TNF after PMA treatment is not due to any changes at the receptor level. To examine whether a PKC-dependent pathway is involved in TNF-mediated cytotoxicity in ME-180 cells, cytotoxicity was measured after depletion of PKC. No apparent changes in cytototoxicity after PKC depletion suggest that a PKC-dependent pathway is not involved in TNF-mediated cytotoxicity. Furthermore, results from cytotoxicity tests after exposure to staurosporine (PKC inhibitor) did not show any changes in the TNF-mediated cytotoxicity, confirming that a PKC-dependent pathway is not involved in this process. These data indicate that 1) TNF induces expression of c-fos, c-jun and jun-B oncogenes via a PKC-dependent pathway and 2) PKC-dependent expression of these three oncogenes by TNF may not be involved in TNF-mediated cytotoxicity in ME-180 cells.

• Journal of Life Science
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• v.21 no.1
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• pp.22-30
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• 2011

Apolipoprotein A-I (apoA-I) has anti-inflammatory and anti-oxidative properties. This study was designed to investigate whether apoA-I affects apoptosis and cytokine production of human blood neutrophils in an in vitro culture system. Spontaneous apoptosis of neutrophils was significantly delayed by apoA-I. In addition, high density lipoprotein containing apoA-I also delayed apoptosis of neutrophils. Apoptosis of neutrophils was inhibited by anti-scavenger receptor type B-I antibodies. The amounts of interleukin-8, interferon (IFN)-inducible protein 10 (IP-10), and tumor necrosis factor-$\alpha$ (TNF-$\alpha$) in the supernatants of cultured neutrophils treated with apoA-I were significantly increased. Combined treatment of neutrophils with IFN-$\gamma$ and apoA-I produced higher amounts of IP-10 and TNF-$\alpha$ than did treatment with IFN-$\gamma$ or apoA-I alone. The present study reveals that apoA-I activates neutrophils to produce cytokines and delays spontaneous apoptosis of neutrophils. These findings suggest that apoA-I, although a well-known negative acute-phase protein, has a pro-inflammatory effect in neutrophils.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.6115-6120
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• 2013

Introduction: Some non-small cell lung cancer (NSCLC) tumor cells are insensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -based therapy. This study was conducted to examine the effect of embelin on the sensitivity of the A549 NSCLC cell line to TRAIL receptor2 (TRAILR2) monoclonal antibodies and to investigate the potential mechanisms. Materials and Methods: A549 cells were treated with embelin, TRAILR2 mAb or a combination of both. Cell viability was measured using ATPlite assay and apoptosis rates were determined by flow cytometry with AnnexinV-FITC and propidium iodide staining, with the expression levels of proteins analyzed by Western blotting. Results: The cell survival rate of separate treatments with 100 ng/ml TRAILR2 antibody or 25 uM embelin were $81.5{\pm}1.57%$ and $61.7{\pm}2.84%$, respectively. Their combined use markedly decreased cell viability in A549 cells to $28.1{\pm}1.97%$ (P<0.05). The general caspase inhibitor Z-VAD-FMK could inhibit the embelin-enhanced sensitivity of A549 cells to TRAILR2 mAb ($75.97{\pm}3.17%$)(P<0.05). Both flow cytometry and cell morphological analysis showed that embelin was able to increase TRAIL-induced apoptosis in A549 cells. Combined treatment with embelin and TRAILR2 mAb augmented the activation of initiator caspases and effector caspase. In addition, A549 cells showed increasing levels of TRAILR2 protein and decreasing levels of Bcl-2, survivin and c-FLIP following the treatment with embelin+TRAILR2 mAb. Conclusions: Embelin could enhance TRAIL-induced apoptosis in A549 cells. The synergistic effect of the combination treatment might be due to modulation of multiple components in the TRAIL receptor-mediated apoptotic signaling pathway, including TRAILR2, XIAP, survivin, Bcl-2 and c-FLIP.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.1
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• pp.27-37
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• 2020

Neuroinflammation is an important process underlying a wide variety of neurodegenerative diseases. Carvacrol (CAR) is a phenolic monoterpene commonly used as a food additive due to its antibacterial properties, but it has also been shown to exhibit strong antioxidative, anti-inflammatory, and neuroprotective effects. Here, we sought to investigate the effects of CAR on inflammation in the hippocampus and prefrontal cortex, as well as the molecular mechanisms underlying these effects. In our study, lipopolysaccharide was injected into the lateral ventricle of rats to induce memory impairment and neuroinflammation. Daily administration of CAR (25, 50, and 100 mg/kg) for 21 days improved recognition, discrimination, and memory impairments relative to untreated controls. CAR administration significantly attenuated expression of several inflammatory factors in the brain, including interleukin-1β, tumor necrosis factor-α, and cyclooxygenase-2. In addition, CAR significantly increased expression of brain-derived neurotrophic factor (BDNF) mRNA, and decreased expression of Toll-like receptor 4 (TLR4) mRNA. Taken together, these results show that CAR can improve memory impairment caused by neuroinflammation. This cognitive enhancement is due to the anti-inflammatory effects of CAR medicated by its regulation of BDNF and TLR4. Thus, CAR has significant potential as an inhibitor of memory degeneration in neurodegenerative diseases.

• Journal of Marine Bioscience and Biotechnology
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• v.12 no.1
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• pp.29-39
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• 2020

In this study, we investigated the inhibitory potential of an ethanol extract of Carpomitra costata (EECC) (Stackhouse) Batters, a brown alga, against neuroinflammatory responses in lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results showed that EECC significantly suppressed the LPS-induced secretion of pro-inflammatory mediators, including nitric oxide (NO) and prostaglandin E₂, with no significant cytotoxic effects. EECC also inhibited the LPS-induced expression of their regulatory enzymes, such as inducible NO synthase and cyclooxygenase-2. In addition, EECC downregulated the LPS-induced expression and production of the proinflammatory cytokines, tumor necrosis factor-α and interleukin-1β. In the mechanistic assessment of the antineuroinflammatory effects, EECC was found to inhibit the nuclear translocation and DNA binding of nuclear factor-kappa B (NF-κB) by disrupting the degradation of the κB-α inhibitor in the cytoplasm. Moreover, EECC effectively suppressed the enhanced expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88, as well as the binding of LPS to TLR4 in LPS-treated BV2 cells. Furthermore, EECC markedly reduced the LPS-induced generation of reactive oxygen species (ROS), demonstrating a strong antioxidative effect. Collectively, these results suggest that EECC repressed LPS-mediated inflammatory action in the BV2 microglia through the inactivation of NF-κB signaling by antagonizing TLR4 and/or preventing ROS accumulation. While further studies are needed to fully understand the anti-inflammatory effects associated with the antioxidant activity of EECC, the current findings suggest that EECC has a potential advantage in inhibiting the onset and treatment of neuroinflammatory diseases.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.10-17
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• 2016

Enzyme immunoassay to analyze specific binding activity of antibody to antigen uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). Chemical methods are usually used for coupling of these enzymes to antibody, which is complicated and random cross-linking process. As results, it causes decreases or loss of functional activity of either antibody or enzyme. In addition, most enzyme assays use secondary antibody to detect antigen binding activity of primary antibody. Enzymes coupled to secondary antibody provide a binding signal by substrate-based color development, suggesting secondary antibody is required in enzyme immunoassay. Additional incubation time for binding of secondary antibody should also be necessary. More importantly, non-specific binding activity caused by secondary antibody should also be eliminated. In this study, we cloned AP isolated from Escherichia coli (E. coli) chromosome by PCR and fused to) hAY4 single-chain variable domain fragment (ScFv) specific to death receptor (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand (TRAIL). hAY4 ScFv-AP expressed in E. coli showed 73.8 kDa as a monomer in SDS-PAGE. However, this fusion protein shown in size-exclusion chromatography (SEC) exhibited 147.6 kDa as a dimer confirming that natural dimerization of AP by non-covalent association induced ScFv-AP dimerization. In several immunoassay such as ELISA, Western blot and immunocytochemistry, it showed antigen binding activity by color development of substrates catalyzed by AP directly fused to primary hAY4 ScFv without secondary antibody. In summary, hAY4 ScFv-AP fusion protein was successfully purified as a soluble dimeric form in E. coli and showed antigen binding activity in several immunoassays without addition of secondary antibody which sometimes causes time-consuming, expensive and non-specific false binding.

• BMB Reports
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• v.40 no.1
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• pp.88-94
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• 2007

Matrix metalloproteinase-9 (MMP-9) plays a pivotal role in the turnover of extracellular matrix (ECM) and in the migration of normal and tumor cells in response to normal physiologic and numerous pathologic conditions. Here, we show that the transcription of the MMP-9 gene is induced by lipopolysaccharide (LPS) stimulation in cells of a macrophage lineage (RAW 264.7 cells). We provide evidence that the NF-$\kappa$B binding site of the MMP-9 gene contributes to its expression in the LPS-signaling pathway, since mutation of NF-$\kappa$B binding site of MMP-9 promoter leads to a dramatic reduction in MMP-9 promoter activation. In addition, the degradation of l$\kappa$B$\alpha$;, and the presences of myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated kinase 6 (TRAF6) were found to be required for LPS-activated MMP-9 expression. Chromatin immunoprecipitation (ChIP) assays showed that functional interaction between NF-$\kappa$B and the MMP-9 promoter element is necessary for LPS-activated MMP-9 induction in RAW 264.7 cells. In conclusion, our observations demonstrate that NF-$\kappa$B contributes to LPS-induced MMP-9 gene expression in a mouse macrophage cell line.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.19
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• pp.8069-8073
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• 2014

Background: Hepatocellular carcinoma (HCC) is one of the most frequent cancers worldwide, with a high mortality. Most patients present with late stage disease, when the treatment options are limited to systemic chemotherapy. The purpose of our study was to evaluate the significance of p53 and EGFR expression in HCC, and to determine whether these two markers correlate with conventional parameters of prognosis. Materials and Methods: Our study included a total of 45 patients, diagnosed histopathologically with HCC. Clinicopathological data including sex, age, tumor necrosis, tumor size, histologic grading, tumor stage, the presence of cirrhosis and chronic hepatitis, were recorded from the Institute database. Three independent microscopic fields were selected for each sample and all the tumor cells within each microscopic field were counted, and then the positive percent of p53 cells were calculated. Three staining patterns were recognized: diffuse, heterogenous and focal. The intensity of EGFR staining was scored on a scale of 0-3+: 0 no staining; 1+ when a weak membrane staining was observed; 2+ when membrane staining is more intense than in 1+, but less than 3+, and 3+ when intense dark brown staining delineated the membrane. To determine the relationship between EGFR expression and p53, we performed double staining in the same HCC specimens. Results: By immunohistochemical staining, p53 protein was detected in tumor cell nuclei in 20 HCCs (44%). We found a significant correlation between the intensity of p53 expression and the histological grade (p=0.008). EGFR expression was detected in 17 (38%) cases, linked to histological grade (p=0.039). Moreover, the intensity of p53 expression was significantly correlated with EGFR intensity (p=0.014). Conclusions: Our results suggest that overexpression of p53 and EGFR plays an important role in hepatocarcinogenesis and contributes to more advanced disease. These markers are not only valuable predictors of prognosis in HCC, but they are also rational targets for new anti-tumor strategies.