• Journal of Microbiology and Biotechnology
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• v.32 no.12
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• pp.1527-1536
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• 2022

Escherichia coli can use allantoin as its sole nitrogen source under anaerobic conditions. The ureidoglycolate produced by double release of ammonia from allantoin can flow into either the glyoxylate shunt or further catabolic transcarbamoylation. Although the former pathway is well studied, the genes of the latter (catabolic) pathway are not known. In the catabolic pathway, ureidoglycolate is finally converted to carbamoyl phosphate (CP) and oxamate, and then CP is dephosphorylated to carbamate by a catabolic carbamate kinase (CK), whereby ATP is formed. We identified the ybcF gene in a gene cluster containing fdrA-ylbE-ylbF-ybcF that is located downstream of the allDCE-operon. Reverse transcription PCR of total mRNA confirmed that the genes fdrA, ylbE, ylbF, and ybcF are co-transcribed. Deletion of ybcF caused only a slight increase in metabolic flow into the glyoxylate pathway, probably because CP was used to de novo synthesize pyrimidine and arginine. The activity of the catabolic CK was analyzed using purified YbcF protein. The V_max is 1.82 U/mg YbcF for CP and 1.94 U/mg YbcF for ADP, and the K_M value is 0.47 mM for CP and 0.43 mM for ADP. With these results, it was experimentally revealed that the ybcF gene of E. coli encodes catabolic CK, which completes anaerobic allantoin degradation through substrate-level phosphorylation. Therefore, we suggest renaming the ybcF gene as allK.

• Journal of Forest and Environmental Science
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• v.26 no.2
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• pp.83-94
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• 2010

Chinese fir (Latin name: Cunninghaimia lanceolata) is one of the major commercial coniferous trees. Most of Chinese fir forests are managed in successive rotation sites, which lead productivity to decline. Autotoxicity is the important reason for soil degradation of Chinese fir plantation, especially, phenolic acids are considered as the major allelopathic toxins which induce autotoxicity in Chinese fir rotation stands. We performed here proteomic approach to investigate the response of proteins in Chinese fir leaves to salicylic acid. The tube plantlets of Chinese fir clone were treated with 120 mg/L salicylic acid for 1, 3 and 5th day. 2-DE, coupled with MALDI-TOF-TOF/MS, was used to separate and identify the responsive proteins. We found 12, 7, and 12 candidate protein spots that were up- or down-regulated by at least 2.5 fold after 1, 3, and 5th day of the stress, respectively. Of these protein spots, 16 spots were identified successfully. According to the putative physiological functions, these proteins were categorized into five classes (1) the proteins involved in protein stability and folding, including 26S proteome, Grp78, Hsp70, Hsp90 and PPIase; (2) the protein involved in photosynthesis and respiration, including OEC 33 kDa subunit, GAPDH; (3) the protein related to cell endurance to acid, F-ATPase; (4) the protein related to cytoskeleton, tubulin; (5) the protein related to protein translation: prolyl-tRNA synthetase. These results give new insights into autotoxic substance stress response in Chinese fir leaves and provide preliminary footprints for further studies on the molecular signal mechanisms induced by the stress.

• The Journal of Korean Medicine
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• v.43 no.4
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• pp.20-32
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• 2022

Objectives: Kyungok-go (KOG) is a traditional multi-herbal medicine commonly used for enforcing weakened immunity for long time. Recently, there are several reports that KOG has anti-inflammatory and immuno-stimulatory activities in many experimental models. However, the protective effects of KOG on neuronal inflammation are still undiscovered. Thus, we investigated the neuro-protective activity of KOG on lipopolysaccharide (LPS)-stimulated mouse microglia cells. To find out KOG's anti-neuroinflammatory effects on microglial cells, we examined the production of nitrite using griess assay, and mRNA expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α using real time RT-PCR. In addition, to examine the regulating mechanisms of KOG, we investigated the protein expression of mitogen-activated protein kinases (MAPKs) and Iκ-Bα by western blot. KOG inhibited the elevation of nitrite, iNOS and COX-2 on LPS-stimulated BV2 cells. Also, KOG significantly inhibited the pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α on LPS-stimulated BV2 microglial cells. Moreover, KOG inhibited the activation of c-Jun N-terminal kinase (JNK), P38 and degradation of Iκ-Bα but not the activation of extracellular signal regulated kinase (ERK) on LPS-stimulated BV2 microglial cells. These results showed KOG has the anti-inflammatory effects through the inhibition on nitrite, iNOS, COX-2, IL-1β, IL-6, and TNF-α via the deactivation of JNK, p38 and nuclear factor (NF)-κB on LPS-stimulated BV2 microglial cells. Thereby, KOG could offer the new and promising treatment for neurodegenerative disease related to neuroinflammation.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.4
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• pp.407-416
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• 2023

The regeneration of myocardium following acute circulatory events remains a challenge, despite numerous efforts. Mesenchymal stem cells (MSCs) present a promising cell therapy option, but their differentiation into cardiomyocytes is a time-consuming process. Although it has been demonstrated that PSME4 degrades acetyl-YAP1, the role of PSME4 in the cardiac commitment of MSCs has not been fully elucidated. Here we reported the novel role of PSME4 in MSCs cardiac commitment. It was found that overnight treatment with apicidin in primary-cultured mouse MSCs led to rapid cardiac commitment, while MSCs from PSME4 knock-out mice did not undergo this process. Cardiac commitment was also observed using lentivirus-mediated PSME4 knockdown in immortalized human MSCs. Immunofluorescence and Western blot experiments revealed that YAP1 persisted in the nucleus of PSME4 knockdown cells even after apicidin treatment. To investigate the importance of YAP1 removal, MSCs were treated with shYAP1 and apicidin simultaneously. This combined treatment resulted in rapid YAP1 elimination and accelerated cardiac commitment. However, overexpression of acetylation-resistant YAP1 in apicidin-treated MSCs impeded cardiac commitment. In addition to apicidin, the universal effect of histone deacetylase (HDAC) inhibition on cardiac commitment was confirmed using tubastatin A and HDAC6 siRNA. Collectively, this study demonstrates that PSME4 is crucial for promoting the cardiac commitment of MSCs. HDAC inhibition acetylates YAP1 and facilitates its translocation to the nucleus, where it is removed by PSME4, promoting cardiac commitment. The failure of YAP1 to translocate or be eliminated from the nucleus results in the MSCs' inability to undergo cardiac commitment.

• Food Science of Animal Resources
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• v.44 no.1
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• pp.132-145
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• 2024

Sarcopenia, the age-related muscle atrophy, is a serious concern as it is associated with frailty, reduced physical functions, and increased mortality risk. Protein supplementation is essential for preserving muscle mass, and horse meat can be an excellent source of proteins. Since sarcopenia occurs under conditions of oxidative stress, this study aimed to investigate the potential anti-muscle atrophy effect of horse meat hydrolysate using C2C12 cells. A horse meat hydrolysate less than 3 kDa (A4<3kDa) significantly increased the viability of C2C12 myoblasts against H₂O₂-induced cytotoxicity. Exposure of C2C12 myoblasts to lipopolysaccharide led to an elevation of cellular reactive oxygen species levels and mRNA expression of proinflammatory cytokines, including tumor necrosis factor-α and interleukin 6, and these effects were attenuated by A4<3kDa treatment. Additionally, A4<3kDa activated protein synthesis-related proteins through the protein kinase B/mechanistic target of rapamycin pathway, while decreasing the expression of activity and degradation-related proteins, such as Forkhead box O3, muscle RING finger protein-1, and Atrogin-1 in dexamethasone-treated C2C12 myotubes. Therefore, the natural material A4<3kDa has the potential of protecting against muscle atrophy, while further in vivo study is needed.

• Journal of Life Science
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• v.34 no.5
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• pp.304-312
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• 2024

This study was conducted to characterize strain Y10, isolated from discarded chicken feathers. Strain Y10 was identified as Bacillus amyloliquefaciens through phenotypic and 16S rRNA gene analysis. B. amyloliquefaciens Y10 exhibited plant growth-promoting activities, including the production of fungal cell-degrading enzymes (cellulase, lipase, protease, and pectinase), siderophores, ammonia, and indoleacetic acid. Furthermore, strain Y10 was able to inhibit the mycelial growth of several phytopathogenic fungi. When 0.1% sucrose as a carbon source and 0.05% casein as a nitrogen source were added to the basal medium, adjusted to pH 10, and cultured at 35℃, the degradation rate of chicken feathers by strain Y10 was about two times higher than that of the basal medium, with the feathers almost completely degraded in four days. Strain Y10 also degraded various keratin substrates, including duck feathers, wool, and human nails. It was confirmed that the feather hydrolyzates prepared using strain Y10 exhibited antioxidant activities, such as 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (EC₅₀ = 0.38 mg/ml) and superoxide dismutase-like activity (EC₅₀ = 183.7 mg/ml). These results suggest that B. amyloliquefaciens Y10 is a potential candidate for the development of bioinoculants and feed additives applicable to the agricultural and livestock industries, as well as the microbiological treatment of keratin waste.

• Tuberculosis and Respiratory Diseases
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• v.49 no.3
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• pp.332-342
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• 2000

Background : The importance of pro-inflammatory cytokines, especially tumor necrosis factor $\alpha$ (INF-$\alpha$) and interleukin-1$\beta$ (IL-1$\beta$), have been extensively documented in the generation of inflammatory lung disease. Lung epithelial cells are also actively involved in initiating and maintaining inflammation by producing pro-inflammatory mediators. Understanding the mechanism of pro-inflammatory cytokine expression in lung epithelial cells is crucial to the development of new therapeutic modalities for inflammatory lung disease. Transcription of most pro-inflammatory cytokines is dependent on the activation of NF-${\kappa}B$. However, the relationship between pro-inflammatory cytokine expression and NF-${\kappa}B/I{\kappa}B$ pathway in lung epithelial cells is not clear. Methods : BEAS-2B, A549, Na-H157, NCI-H719 cells were stimulated with IL-$1{\beta}$ or TNF-$\alpha$ at various times, and then IL-8 and TNF-$\alpha$mRNA expressions were assayed by Northern blot analysis. IL-$1{\beta}$ or TNF-$\alpha$-induced NF-${\kappa}B$ activation was assessed by the nuclear translocation of p65 NF-${\kappa}B$ subunit. The degradation of $I{\kappa}B{\alpha}$ and $I{\kappa}B{\beta}$ by IL-$1{\beta}$ or TNF-$\alpha$stimulation was assayed by Western blot analysis. The phosphorylation of $I{\kappa}B{\alpha}$ was evaluated by Western blot analysis after pre-treating cells with proteasome inhibitor followed by IL-$1{\beta}$ or TNF-$\alpha$ stimulation. The basal level of IKK $\alpha$ expression was evaluated by Western blot analysis. Results: $I{\kappa}B{\alpha}$ and $I{\kappa}B{\alpha}$ was rapidly degraded after 5 minutes of incubation with IL-$1{\beta}$ or TNF-$\alpha$ in BEAS-2B, A549, and NCI-H157 cells. The activation of NF-${\kappa}B{\alpha}$ and the induction of IL-8 and TNF-$\alpha$ mRNA expression were observed by IL-$1{\beta}$ or TNF-$\alpha$ stimulation in these cells. In contrast, neither the changes in NF-${\kappa}B/I{\kappa}B$ pathway nor IL-8 and TNF-$\alpha$mRNA expression was induced by IL-$1{\beta}$ or TNF-$\alpha$ stimulation in NCI-H719 cells. IL-$1{\beta}$ and TNF-$\alpha$-induced $I{\kappa}B$ phosphorylation was observed in BEAS-2B, A549, and NCI-H157 cells, but not in NCI-H719 cells. The basal level of IKK$\alpha$ expression was not different between cell. Conclusion : NF-${\kappa}B/I{\kappa}B$ pathway plays an important role in the expression of pro-inflammatory cytokine in most lung epithelial cells. The absence of the effect on NF-${\kappa}B/I{\kappa}B$ pathway in NCI-H719 cells sæms to be due to the defect in the intracellular signal transduction pathway upstream to IKK.

• Korean Journal of Food Science and Technology
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• v.46 no.2
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• pp.198-204
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• 2014

Ultraviolet B (UV-B) irradiation is a negative factor that induces skin damage, inflammation, and aging. UVB irradiation induces the inflammatory response through interleukin (IL)-6 and IL-8 expression in keratinocytes. In addition, it induces the production of reactive oxygen species (ROS) and the activation of matrix metalloproteinase-1 (MMP-1), which plays an important role in collagen 1 degradation in the extracellular matrix. We investigated the antiaging effects of five kinds of berry in human skin keratinocyte (HaCaT) cells using juice of black raspberry (Rubus occidentalis), blueberry wild (Vacciniun angustifolium) and cultivar (Vacciniun corymbosum), black chokeberry (Aronia melanocarpa (Michx.) Elliott), and mulberry (Morus abla). HaCaT cells irradiated with UV-B exhibited increased ROS generation, as well as IL-6, IL-8, and MMP-1 gene expression, when compared to the control cells that were not irradiated with UV-B. However, pre-treatment of berry juice before UV-B irradiation significantly down-regulated the UV-B-induced ROS generation and inflammatory cytokine and MMP-1 expression. The results suggest that all berries have anti-aging effects including lowering inflammatory cytokine levels, ROS generation, and MMP-1 expression in HaCaT cells during UV-B irradiation.

• Tuberculosis and Respiratory Diseases
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• v.52 no.6
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• pp.616-626
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• 2002

Background : The present study was performed to further improve our understanding of molecular mechanisms involved in the activation of NFkB, a major transcriptional factor involved in the inflammatory response in the lung, by particulate matter in lung epithelial cells with an aerodynamic diameter of less than $2.5{\mu}m$(PM2.5). Materials and Methods : Immediate production of reactive oxygen species (ROS) and nitrogen species (RNS), with the PM2.5 induced expression of inducible nitric oxide synthase (iNOS), $I{\kappa}B$ degradation and $NF{\kappa}B$-dependent transcriptional activity, in 549 cells, were monitored. Addition, we also examined the effect of the iNOS inhibitor, L-N6-(1-iminoethyl) lysine hydrochloride (L-NIL), on the PM2.5-induced $NF{\kappa}B$ activation in A549 cells. Results : The rapid degradation of $I{\kappa}B$ and the increase of transcriptional activity of the $NF{\kappa}B$-dependent promotor were observed in A549 cells exposed to PM2.5. The immediate production of ROS in response to PM2.5 in A549 cells was not clearly detected, although immediate responses were observed in RAW264.7 cells. A 549 cells, cultured in the presence of PM2.5, produced an increase in NO, which was noticeably significant after 15 min of exposure with the expression of iNOS mRNA. The addition of L-NIL, an iNOS inhibitor, significantly inhibited the PM2.5-induced $I{\kappa}B$ degradation and the increase of the $NF{\kappa}B$-dependent transcriptional activity. Conclusion : These results suggest that PM2.5 stimulates the immediate production of RNS, leading to the activation of $NF{\kappa}B$ in the pulmonary epithelium.

• Tuberculosis and Respiratory Diseases
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• v.55 no.5
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• pp.488-498
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• 2003

Background : Activation of the transcription factor NF-${\kappa}B$ has been shown to protect cells from tumor necrosis factor-alpha, chemotherapy, and radiation-induced apoptosis. NF-${\kappa}B$-dependent cIAP expression is a major antiapoptotic mechanism for that. NF-${\kappa}B$ activation and cIAP expression in A549 lung cancer cells which is relatively resistant to radiation-induced cell death were investigated for the mechanism of radioresistance. Materials and methods : We used A549 lung cancer cells and Clinac 1800C linear accelerator for radiation. Cell viability test was done by MTT assay. NF-${\kappa}B$ activation was tested by luciferase reporter gene assay, Western blot for $I{\kappa}B{\alpha}$ degradation, and electromobility shift assay. For blocking ${\kappa}B$, MG132 and transfection of $I{\kappa}B{\alpha}$-superrepressor plasmid construct were used. cIAP expression was analyzed by RT-PCR and cIAP2 promoter activity was performed using luciferase assay system. Results : MTT assay showed that cytotoxicity even 48 hr after radiation in A549 cells were less than 20%. Luciferas assay demonstrated weak NF-${\kappa}B$ activation of $1.6{\pm}0.2$ fold compared to PMA-induced $3.4{\pm}0.9$ fold. Radiation-induced $I{\kappa}B{\alpha}$ degradation was observed in Western blot and NF-${\kappa}B$ DNA binding was confirmed by EMSA. However, blocking NF-${\kappa}B$ using MG132 and $I{\kappa}B{\alpha}$-superrepressor transfection did not show any sensitizing effect for radiation-induced cell death. The result of RT-PCR for cIAP1 & 2 expression was negative induction while TNF-${\alpha}$ showed strong expression for cIAP1 & 2. The cIAP2 promoter activity also did not show any change compared to positive control with TNF-${\alpha}$. Conclusion : We conclude that activation of NF-${\kappa}B$ does not determine the intrinsic radiosensitivity of cancer cells, at least for the cell lines tested in this study.