• Title/Summary/Keyword: culture supernatant

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Study on IL -8 Expression in Peripheral Blood Monocytes (말초 혈액 단핵구에서 IL-8 발현에 관한 연구)

  • Kim, Jae-Yeol;Lee, Jae-Cheol;Kang, Min-Jong;Park, Jae-Seok;Yoo, Chul-Gyu;Kim, Young-Whan;Han, Sung-Koo;Shim, Young-Soo;Lee, Jae-Ho
    • Tuberculosis and Respiratory Diseases
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    • v.42 no.5
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    • pp.703-712
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    • 1995
  • Background: Peripheral blood monocytes are important immune effector cells that play a fundamental role in cellular immunity. In addition to their antigen-presenting and phagocytic activities, monocytes/macrophage produce a vast array of regulatory and chemotactic cytokines. Interleukin-8(IL-8), a potent neutrophil-activating and chemotactic peptide, is produced in large quantities by mononuclear phagocytes and may be an important mediator of local and systemic inflammation. Overexpression by IL-8 of such inflammation may be an important step of tissue injury frequently seen in inflammatory reaction. So it could be hypothesized that the agents which block the production of IL-8 can decrease the inflammatory reaction and tissue injury. To evaluate this, we described the effect of Dexamethasone, $PGE_2$, Indomethacin and Interferon-$\gamma$(IFN-$\gamma$) on IL-8 mRNA and protein expression from LPS-stimulated human peripheral blood monocytes(PBMC). Method: PBMC was isolated from healthy volunteers. To evaluate the effect of Dexamethasone, $PGE_2$ & Indomethacin, these drug were treated for 1 hour before and after LPS stimulation and IFN-$\gamma$ was only treated I hour before the LPS stimulation. Northern blot analysis for IL-8 mRNA and ELISA for immunoreactive IL-8 protein in culture supernatant were performed. We repeated above experiment three times for Northern blot analysis and two times for ELISA and got the same result. Results: 1) Pre- and post-treatment of Dexamethasone suppressed both the LPS stimulated IL-8 mRNA expression and IL-8 protein release in PBMC. 2) IFN-$\gamma$ pre-treatment suppressed the IL-8 mRNA expression and IL-8 protein release in unstimulated cells. 3) In LPS stimulated cells, IFN-$\gamma$ suppressed the IL-8 mRNA expression but IL-8 protein release suppression was not observed. 4) $PGE_2$ and Indomethacin exert no effect on the LPS-stimulated IL-8 mRNA and protein expression in concentration used in this experiment ($PGE_2;10^{-6}M$, Indomethacin; $10{\mu}M$). Conclusion: One of the mechanism of antiinflammatory action of Dexamethasone can be explained by the suppressing effect of IL-8 production in some extent and by this antiinflammatory effect, dexamethasone can be used to suppress local and systemic inflammation mediated by IL-8.

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Effects of Heat Shock Protein 70 (HSP70) Induction after Lipopolysaccharide Exposure on the IL-6 Production and the Cell Viability after Subsequent Lipopolysaccharide Challenge in Murine Alveolar Epithelial Cells (내독소로 전처치한 쥐 폐포상피에서 HSP70 유도가 추가 내독소 자극에 따른 IL-6 생성능 및 세포생존도에 미치는 영향)

  • Lee, Jung Mi;Kim, Jin Sook;Kim, Young Kyoon;Kim, Seung Joon;Lee, Sook Young;Kwon, Soon Seog;Park, Sung Hak
    • Tuberculosis and Respiratory Diseases
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    • v.58 no.4
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    • pp.375-384
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    • 2005
  • Background and Aims : Pre-induction of heat shock protein 70 (HSP70) is known to effectively attenuate the lipopolysaccharide (LPS)-induced inflammatory response in lung tissue. However, it is unclear if HSP70 induction after LPS exposure attenuates the subsequent LPS-induced inflammatory response in alveolar epithelial cells. This study examined the effects of HSP70 induction after LPS exposure on the IL-6 production and the cell viability after a subsequent LPS challenge in murine alveolar epithelial cells, and investigated whether or not HSP70 itself may be involved in those effects. Methods : Murine alveolar epithelial cells were cultured and divided into two groups; the Non-Pre-LPS group without a LPS pre-treatment and the Pre-LPS group with a LPS pre-treatment. Each group was subdivided into the following four subgroups: subgroup C (control), subgroup Q (quercetin), subgroup HSP70 (HSP70 induction), and subgroup HSP70-Inh (HSP70 inhibition). HSP70 expression, which was induced by sodium arsenite and inhibited by quercetin, was analyzed by western blot analysis. The IL-6 levels in the culture supernatant were measured by ELISA, and the cell viability was measured using a simplified MTT assay. Results : The IL-6 levels were lower in subgroup HSP70 than in subgroup C (P<0.01), and were higher in subgroup HSP70-Inh than in subgroup HSP70 in both the Non-Pre-LPS and Pre-LPS groups (P<0.05, P<0.01). The cell viability tended to decrease in the Pre-LPS group compared with the Non-Pre-LPS group. While the cell viability was higher in subgroups Q, HSP70, and HSP70-Inh than in subgroup C in the Non-Pre-LPS group (P<0.05, P<0.05, P<0.01), there was no difference in cell viability among the subgroups in the Pre-LPS group. Conclusion : HSP70 induction after a LPS pre-treatment in murine alveolar epithelial cells inhibits the subsequent LPS-induced IL-6 production without affecting the cell viability, and HSP70 by itself may play an important role in this proccess.

The Extracts from Liriope platyphylla Significantly Stimulated Insulin Secretion in the HIT-T15 Pancreatic β-Cell Line (HIT-T15 췌장세포의 인슐린분비 촉진을 유도하는 맥문동(Liriope platyphylla) 추출물의 효능 및 독성분석)

  • Kim, Ji-Ha;Kim, Ji-Eun;Lee, Yoen-Kyung;Nam, So-Hee;Her, Youn-Kyung;Jee, Seoung-Wan;Kim, Sun-Guen;Park, Da-Jung;Choi, Young-Whan;Hwang, Dae-Youn
    • Journal of Life Science
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    • v.20 no.7
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    • pp.1027-1033
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    • 2010
  • Liriope platyphylla has traditionally been used in Korea and China as a therapeutic drug for the treatment of coughing, sputum, neurodegenerative disorders, obesity, and diabetes. In an effort to assess the functions of a novel extract from Liriope platyphylla in diabetes therapy, the insulin secretion abilities of 10 extracts were screened via measurements of insulin concentration in the culture supernatant using an Insulin ELISA kit. The results of this assay showed the highest levels of insulin in the LP9M80-H treated group, followed by the LP-H, LP-M, LP-E and LP9M80-C treated groups, whereas other extracts did not induce insulin secretion in the HIT-T15 cells. However, the extracts capable of stimulating insulin secretion simultaneously evidenced high apoptotic activity as compared with other extracts. Therefore, one of these extracts, LP9M80-H, was initially selected as the optimal candidate for a therapeutic drug and its optimal concentration was determined. The results of the ELISA and MTT assay demonstrated that a concentration of approximately 100-125 ug/ml of LP9M80-H was optimal with regards to cell viability and insulin secretion in the HIT-T15 cells. These results suggest that LP9M80-H could be considered as an excellent candidate for a diabetes-therapeutic drug that could induce insulin secretion in pancreatic $\beta$-cells.

Characterization of a Novel Fibrinolytic Enzyme Produced from Bacillus subtilis BK-17 (Bacillus subtilis BK-17 유래 혈전용해 효소의 특성)

  • Hyun Bek;Lim Hak-Seob;Chung Kyung Kae;Choi Yung Hyun;Choi Byung Tae;Seo Min-Jeong;Kim Ji-Eun;Ryu Eun-Ju;Huh Man Kyu;Joo Woo Hong;Jeong Young Kee
    • Journal of Life Science
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    • v.15 no.6 s.73
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    • pp.987-993
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    • 2005
  • A bacterium, producing a fibrinolytic enzyme, was screened from a decaying rice plant. The bacterium was identified as Bacillus subtilis by morphological, biochemical, and physiological properties and named Bacillus subtilis BK-17. The fibrinolytic enzyme (BK) was purified from supernatant of Bacillus subtilis BK-17 culture broth. The molecular weight was 31 kDa as determined by SDS-PAGE. The effect of temperature, pH, and plasminogen on the activity of the bacillokinase (BK) was analysed and the activity was compared with urokinase. The optimal temperature and pH were $50^{circ}C$ and pH 7, pH 8, respectively. The BK activity was inhibited to $45\%$, $35\%$, and $23\%$ with 1mM EDTA, $Zn^{2+}$, and $Ca^{2+}$, respectively. However, $Mg^{2+}$, $Mn^{2+}$, and $Co^{2+}$ ions did not have any significant effect on the enzyme activity The BK showed the artivity in the both plates, plasminogen-free fibrin plate and plasminogen-rich fibrin plate. The result indicates that the BK can directly act the fibrin. In comparison of fibrinolytic activity with urokinase on the fibrin plate, the BK shows about 20 folds higher activity than that of the urokinase.

Removal of Red Tide Organisms -2. Flocculation of Red Tide Organisms by Using Loess- (적조생물의 구제 -2. 황토에 의한 적조생물의 응집제거-)

  • KIM Sung-Jae
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.33 no.5
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    • pp.455-462
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    • 2000
  • The objective of this study was to examine the physicochemical characteristics of coagulation reaction between loess and red tide organisms (RTO) and its feasibility, in developing a technology for the removal of RTO bloom in coastal sea. The physicochemical characteristics of loess were examined for a particle size distribution, surface characteristics by scanning electron microscope, zeta potential, and alkalinity and pH variations in sea water. Two kinds of RTO that were used in this study, Cylindrothen closterium and Skeietonema costatum, were sampled in Masan bay and were cultured in laboratory. Coagulation experiments were conducted using various concentrations of loess, RTO, and a jar tester. The supernatant and RTO culture solution were analyzed for pH, alkalinity, RTO cell number. A negative zeta potential of loess increased with increasing pH at $10^(-3)M$ NaCl solution and had -71.3 mV at pH 9.36. Loess had a positive zeta potential of +1,8 mV at pH 1.98, which resulted in a characteristic of material having an amphoteric surface charge. In NaCl and $CaCl_2$, solutions, loess had a decreasing negative zeta potential with increasing $Na^+\;and\;Ca^(+2)$ ion concentration and then didn't result in a charge reversal due to not occurring specific adsorption for $Na^+$ ion while resulted in a charge reversal due to occurring specific adsorption for $Ca^(+2)$ ion. In sea water, loess and RTO showed the similar zeta potential values of -112,1 and -9.2 mV, respectively and sea sand powder showed the highest zeta potential value of -25.7 mV in the clays. EDLs (electrical double-layers) of loess and RTO were extremely compressed due to high concentration of salts included in sea water, As a result, there didn't almost exist EDL repulsive force between loess and RTO approaching each other and then LVDW (London-yan der Waals) attractive force was always larger than EDL repulsive force to easily form a floe. Removal rates of RTO exponentially increased with increasing a loess concentration. The removal rates steeply increased until $800 mg/l$ of loess, and reached $100{\%}$ at 6,400 mg/l of loess. Removal rates of RTO exponentially increased with increasing a G-value. This indicated that mixing (i.e., collision among particles) was very important for a coagulation reaction. Loess showed the highest RTO removal rates in the clays.

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