• Title/Summary/Keyword: co-culture system

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Role of γ-glutamyltranspeptidase in osteoclastogenesis induced by Fusobacterium nucleatum

  • Kim, Aeryun;Kim, Ji-Hye
    • International Journal of Oral Biology
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    • v.46 no.3
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    • pp.127-133
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    • 2021
  • We previously showed that γ-glutamyltranspeptidase (GGT), an enzyme involved in glutathione metabolism, in Bacillus subtilis acts as a virulence factor for osteoclastogenesis via the RANKL-dependent pathway. Hence, it can be hypothesized that GGT of periodontopathic bacteria acts as a virulence factor in bone destruction. Because Fusobacterium nucleatum, which is a periodontopathic pathogen, has GGT with a primary structure similar to that of B. subtilis GGT (37.7% identify), the bone-resorbing activity of F. nucleatum GGT was examined here. Recombinant GGT (rGGT) of F. nucleatum was expressed in Escherichia coli and purified using the His tag of rGGT. F. nucleatum rGGT (Fn rGGT) was expressed as a precursor of GGT, and then processed to a heavy subunit and a light subunit, which is characteristic of general GGTs, including the human and B. subtilis enzymes. Osteoclastogenesis was achieved in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. Fn rGGT induced osteoclastogenesis to a level similar to that of B. subtilis rGGT; furthermore, osteoclastogenesis was induced in a dose-dependent manner. These results suggest that F. nucleatum GGT possesses a virulent bone-resorbing activity, which could play an important role in the pathogenesis of periodontitis.

Ibuprofen Increases the Hepatotoxicity of Ethanol through Potentiating Oxidative Stress

  • Kim, Minjeong;Lee, Eugenia Jin;Lim, Kyung-Min
    • Biomolecules & Therapeutics
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    • v.29 no.2
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    • pp.205-210
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    • 2021
  • Over 30 million prescriptions of NSAIDs (non-steroidal anti-inflammatory drugs) are issued every year. Considering that these drugs are available without a prescription as over the counter (OTC) drugs, their use will be astronomical. With the increasing use of NSAIDs, their adverse effects are drawing attention. Especially, stomach bleeding, kidney toxicity, liver toxicity, and neurological toxicity are reported as common. Ibuprofen, one of the extensively used NSAIDs along with aspirin, can also induce liver toxicity, but few studies are addressing this point. Here we examined the liver toxicity of ibuprofen and investigated whether co-exposure to ethanol can manifest synergistic effects. We employed 2D and 3D cultured human hepatoma cells, HepG2 to examine the synergistic hepatotoxicity of ibuprofen and alcohol concerning cell viability, morphology, and histology of 3D spheroids. As a result, ibuprofen and alcohol provoked synergistic hepatotoxicity against hepatocytes, and their toxicity increased prominently in 3D culture upon extended exposure. Oxidative stress appeared to be the mechanisms underlying the synergistic toxicity of ibuprofen and alcohol as evidenced by increased production of ROS and expression of the endogenous antioxidant system. Collectively, this study has demonstrated that ibuprofen and EtOH can induce synergistic hepatotoxicity, providing a line of evidence for caution against the use of ibuprofen in combination with alcohol.

Optimal evacuation route guidance system using recreational forest 3D scan data (휴양림 3D 스캔 데이터를 통한 최적 대피로 안내 시스템)

  • Jung, Sanghun;Gwon, Eunhye;Son, Hoon;Kang, Soyoung;Jung, Heokyung
    • Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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    • 2021.05a
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    • pp.258-259
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    • 2021
  • Forest lodge are divided into forest resources, auxiliary facilities, and users, and are constantly being used along with well-being culture. In addition, attention to the safety of users is also required, and this study aims to study how users evacuate within a short time (golden time) in situations of natural disasters that may occur in forests. In order to search for the current location of the user and find the best evacuation route, 3D scans of the entire forest lodge(forest resources, auxiliary facilities, etc.) are performed, and the optimal trajectory to the evacuation site is found through recognition of the current location. It is believed that it is possible to provide a quick evacuation guide through a mobile device with gps.

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Developmental Ability of Bovine Early Embryos in Chemically Defined Media (화학적 단순배양액에서의 소 초기배의 발생능력)

  • Lee, H. J.;Seo, S. W.;Jung, Y.;Byun, T. H.;Lee, S. H.;Song, H. B.
    • Journal of Embryo Transfer
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    • v.10 no.2
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    • pp.163-169
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    • 1995
  • In the experiment I for maas production of bovine early embryos, 18~20hpi fertilized eggs (756 eggs) and parthenogenic eggs (618 eggs) which were treated by 10% ethanol were cultured in both TGM and CZB. In the experiment II, suppiment effects each in CZB and CRlaa were tested by matured and fertilized oocytes which were after 18~20hpi. In the case of experiment I after 48hr, the cleavage rates of normally fertilized eggs were 66.6% in TCM treatment and 77.7% in CZB treatment, and after 240h the blastocysts were 7.5% in TCM and 14.1% in CZB. In the parthenogenic eggs, the deavage rates at 48hr were 39.6% in TCM and 57.5% in CZB, and at 240h, the blastocysts were 0.9% in TCM and 4.4% in CZB. These results showed that the effects of CZB on developmental ability to parthenogenic eggs as well as nomally fertilized eggs are relatively high. In experiment W, the effect of exposing the cleaved embryos to CZB for 30h on the blastocyst formation was examined. Similar rates of blastocyst formation were obtained both in TCM and CZB, suggesting that CZB exposure. during ealry development is critical. In experiments III ~ V, the effects of supplements were examined. The cleavage rates of CZB treatments at 48h were 83.8% in control, 78.1% in BSA+A.A+SIT, 75% in 5% FCS+A. A+SIT, 88.6% in BSA+A.A+SIT and not co-cultured BSA+A.A+SIT had 85.7% and in the case of 240h blastocysts showed 22.6, 0.0, fl.1, 6.5 and 0%, respectively. As a result, this study showed that CZB was effective culture system for in vitro development, and that CZB and CR$_1$aa had no significant differences and effects between them. It may be concluded that in the simple media containing supplements could replace the co-culture systems of bovine early embryo development.

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The Effectiveness of Emotional Safety Using PIR Sensors in Building Construction Site (센서를 활용한 건설현장 감성안전의 효용성에 관한 연구)

  • Shin, Han-Woo;Kim, Gwang-Hee;Kim, Tae-Hyung;Kim, Tae-Hui;Choi, Eung-Kyoo
    • Journal of the Korea Institute of Building Construction
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    • v.10 no.4
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    • pp.59-65
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    • 2010
  • Many Construction companies are making great efforts to prevent accidents on their work sites. Safety is one of the greatest success factors on a construction project. Nowadays, many safety tools are being applied to construction sites. In addition, an emotional safety culture is an important factor for promoting a "safety first" mindset on construction sites. Therefore, this research aims to examine the effectiveness of the emotional safety system using PIR (Pyroelectric Infrared Ray) sensors to improve the safety mindset in the building construction site. The results of this research revealed that many construction site workers are satisfied with the emotional safety system using sensors. In addition, it was found that voice safety systems give a positive impulse to the workers. By applying this system to construction sites, construction companies can improve safety and work productivity.

A Cell-Based Assay System for Monitoring NF-$\kappa$B Activity in Human Epidermal Keratinocytes: A Screening Tool of the Antioxidants and Anti-inflammatories for Dermatological Purpose

  • Moon, Ki-Young;Hahn, Bum-Soo;Lee, Jinseon;Kim, Yeong-Shik
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.27 no.1
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    • pp.17-27
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    • 2001
  • A cell-based assay system for monitoring NF-$textsc{k}$B activity was developed to determine the influence of activated NF-$textsc{k}$B in human HaCaT cells. The pNF-$textsc{k}$B-SEAP-NPT plasmid that permits expression of the secreted alkaline phosphatase (SEAP) reported gene in response to the NF-$textsc{k}$B activity and contains neomycin phosphotransferase (NPT) gene for the geneticin resistance in host cells was constructed and transfected into human keratinocyte cell line HaCaT. Human HaCaT transfectant cells secreted the SEAP enzyme into the culture medium in a time-dependent manner until 72h. NF-$textsc{k}$B activities were measured in the SEAP reporter gene assay using a fluorescent detection method. The treatment of HaCaT cell transfectants with known antioxidants [e.g., N-acetyl-L-cysteine and vitamin C] showed inhibition of NF-$textsc{k}$B activity in a time-and concentration-dependent manner. The phorbol 12-myristate 13-acetate (PMA) known as a stimulator of NF-$textsc{k}$B expression demonstrated that it increased NF-$textsc{k}$B activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-$textsc{k}$B activity in the human skin and allow the screening of anti-inflammatory agents from various synthetic chemicals and natural products for dermatological purpose. Abbrevitions used: NF-$textsc{k}$B, nuclear factor kappa B; I-$textsc{k}$B, Inhibitory kappa B; SEAP, secreted alkaline phosphatase; NPT, neomycin phosphotransferease; PCR, polymerase chain reaction: dNTP, deoxynucleoside triphosphates; DMEM, dulbecco’s modified eagle medium; FBS, fetal bovine serum; PBs, phosphate-buffered saline; MUP, 4-methylumbellifery phosphate; NAC, N-acetyl-L-cysteine; DMSO, dimethyl sulfoxide; PMA, phorbol 12-myristate 13-acetate.

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Production and Purification of Single Chain Human Insulin Precursors with Various Fusion Peptides

  • Cho, Chung-Woo;Park, Sun-Ho;Nam, Doo-Hyun
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.6 no.2
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    • pp.144-149
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    • 2001
  • For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

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Study on Anti-oxidant and Anti-inflammatory Activity of the Tissue-cultured Shoot Clumps from Raoulia australis (라울리아 신초 추출물의 항산화 및 항염 효능에 관한 연구)

  • Park, Chang-Min;Han, Na-Kyeong;Joung, Min-Seok;Paek, Kee-Yoeup;Choi, Jong-Wan
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.40 no.4
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    • pp.349-357
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    • 2014
  • In this study, the shoot clumps extract of tissue-cultured Raoulia australis using the bioreactor culture system was tested for use a natural cosmetic ingredient. Tissue-cultured R. australis shoot clumps extract was tested anti-oxidant and anti-inflammatory activity for a cosmetic application. R. australis is a wild herbaceous plant of the asteraceae growing in New Zealand and Australia. Previous studies have reported anti-viral activity of the inhibitory effects for the growth of viruses induced meningitis, bronchitis and respiratory diseases but other biological effects are unknown. The shoot clumps extract of tissue-cultured R. australis showed higher anti-oxidant effect and anti-inflammatory effect than the natural R. australis extract. In DPPH, NBT and ABTS assay, the shoot clumps extract of tissue-cultured R. australis enhanced radical scavenging activity (up to 10~25% at $50{\mu}L/mL$) more than the natural R. australis extract. Also, the shoot clumps extract of tissue-cultured R. australis inhibited expression of iNOS and COX-2 protein in LPS-stimulated Raw 264.7 macrophages more than the natural R. australis extract. From this study, the shoot clumps extract of tissue-cultured R. australis displayed strong possibility as a new natural cosmetic ingredient for skin-care products.

Impaired Functions of Lymphocytes on Nitric Oxide Production in Endotoxin- Tolerant Mice (내독소내성 마우스에서 Nitric Oxide 생성에 미치는 림프구 부전)

  • Gil, Young-Gi;Kang, Mi-Kyung
    • Journal of Life Science
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    • v.18 no.11
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    • pp.1471-1478
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    • 2008
  • In this study, nitric oxide (NO) production in a macrophage-lymphocyte co-culture system was used to assess the cytokine producing capability of cells during endotoxin tolerance in mice. Incubation of peritoneal macrophages with interferon-$\tau$ (IFN-$\tau$) in the presence of lipopolysaccharide (LPS) augmented NO synthesis. Exogenous tumor necrosis factor-$\alpha$(TNF-$\alpha$) could also replace LPS for the stimulation of NO production. Macrophages co-cultured with splenic lymphocytes showed augmented NO synthesis by LPS alone. However, pretreatment of mice with 2.5 mg/kg LPS completely prevented the lethality and the increase of blood TNF-$\alpha$ and IFN-$\tau$ after the second challenge with a lethal dose of LPS. In addition, when macrophages prepared from LPS-tolerant mice were co-cultured with normal splenocytes, LPS also could not induce the production of NO, even in the presence of exogenous TNF-$\alpha$. Moreover, when normal macrophages were co-cultured with splenocytes obtained from LPS-tolerant mice, stimulation with LPS could not evoke the NO production enhancement. However, this down-regulation was able to reverse by exogenous IFN-$\tau$ or concanavalin A (ConA), a stimulator of IFN-$\tau$ production. Our results indicate that not only macrophages but also lymphocytes contribute to LPS tolerance. As INF-$\tau$ can enhance the expression of TNF-$\alpha$, the decrease of INF-$\tau$synthesis from lymphocytes may orchestrate with the decrease of TNF-$\alpha$ synthesis from LPS-tolerant macrophages for the production of tolerant state and the prevention of excessive inflammation. Therefore, LPS tolerance may be exploited for prophylaxis of severe sepsis in patients at risk.

Study on Biological Activities of Adventitious Roots Cultured from Echinacea angustifolia (에키네시아 조직 배양체 추출물의 생리활성에 관한 연구)

  • Park, Chang-Min;Joung, Min-Seok;Ko, Du-Jin;Paek, Kee-Yoeup;Choi, Jong-Wan
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.37 no.3
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    • pp.219-226
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    • 2011
  • The Echinacea, which has been commonly known as a species of composite herb of dicotyledonous plant, has been used in native American traditional medicine for the treatment of diseases like colds or other infections in North America. We artificially cultured the adventitious roots of Echinacea angustifolia using the bioreactor culture system from Echinacea angustifolia and evaluated the efficacy as a cosmetic ingredient for skin care. Several studies previously have reported neogenesis, wound healing and inflammatory inhibition effect of Echinacea angustifolia but other efficacies were not well known. In the present study, we investigated the cosmetic efficacy to know applicable value of adventitious roots cultured from Echinacea angustifolia as a cosmetic ingredient. The adventitious roots extract of Echinacea angustifolia has superior anti-oxidant effect and matrix metalloproteinase-2 inhibitory effect, compared to natural Echinacea angustifolia. These results indicate that the adventitious roots extract cultured from Echinacea angustifolia presents a new possibility of being applicable to skin care and anti-wrinkle products as a cosmetic ingredient.