• Title/Summary/Keyword: Conditioned Medium

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Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells

  • Song, Yuanda;Dilger, Emily;Bell, Jessica;Barton, William A.;Fang, Xianjun
    • BMB Reports
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    • v.43 no.8
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    • pp.541-546
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    • 2010
  • We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.

Quantitative analysis of Spirulina platensis growth with CO2 mixed aeration

  • Kim, Yong Sang;Lee, Sang-Hun
    • Environmental Engineering Research
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    • v.23 no.2
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    • pp.216-222
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    • 2018
  • The growth characteristics of Spirulina platensis were investigated using four photo-bioreactors with $CO_2$-mixed air flows. Each reactor was operated under a specific condition: 3% $CO_2$ at 50 mL/min, 3% $CO_2$ at 150 mL/min, 6% $CO_2$ at 50 mL/min, and 6% CO2 at 150 mL/min. The 3% $CO_2$ at 150 mL/min condition produced the highest algal growth rate, while the 6% $CO_2$ at 150 mL/min conditioned produced the lowest. The algal growth performance was suitably assessed by the linear growth curve rather than the exponential growth. The medium pH decreased from 9.5 to 8.7-8.8 (3% $CO_2$) and 8.4-8.5 (6% $CO_2$), of which trends were predicted only by the pH-carbonate equilibrium and the reaction kinetics between dissolved $CO_2$ and $HCO_3{^-}$. Based on the stoichiometry between the nutrient amounts and cell elements, it was predicted that depleted nitrogen (N) at the early stage of the cultivation would reduce the algal growth rates due to nutrient starvation. In this study, use of the photobioreactors capable of good light energy distribution, proper ranges of $CO_2$ in bubbles and medium pH facilitated production of high amounts of algal biomass despite N limitation.

Substrate Specificity of Mouse Glandular Kallikreins, Epidermal Growth Factor-Binding Protein Type A, B, and c against Mouse Ren 2 Prorenin (생쥐 선상칼리크레인(상피세포증식인자 결합단백질 Type A, B, 그리고 C)의 Ren 2 Prorenin에 대한 기질특이성)

  • 김화선;이희섭전병훈김원신
    • The Korean Journal of Zoology
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    • v.39 no.2
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    • pp.215-222
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    • 1996
  • In the previous studies, we have demonstrated that prorenin converting enzyme (PRECE) was identical to the epidermal grouch factor-binding protein (EGF-BP) type B, which was a member of the mouse glandular kallikrein family, To examine whether or not EGF-BP type A and C are involved in the processing of prorenin, we have cloned the CDNAS of the EGF-BP type h and C from a library of male ICR mouse submandibular gland (SMGI. And then CHO cells were transfected with the EGF-BP expression plasmids. and stable cell lines expressing a high level of the EGF-BPS precursor were obtained. The conditioned medium was then treated with trypsin, which has been knotvn to effectively convert the EGF-BP type A and C precursor to the active forms. 수ubsequentlv, the prorenin converting activity of the trypsin-treated or untreated medium was examined. PRECE converted exactly prorenin to renin, but the prorenin converting activities of EGF-BP type A and C were not detected. From these results, it seems that only type B of these EGF-BPs is involved in processing Ren 2 prorenin in mouse SMG.

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Inflammatory Responses in a Benign Prostatic Hyperplasia Epithelial Cell Line (BPH-1) Infected with Trichomonas vaginalis

  • Kim, Sang-Su;Kim, Jung-Hyun;Han, Ik-Hwan;Ahn, Myoung-Hee;Ryu, Jae-Sook
    • Parasites, Hosts and Diseases
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    • v.54 no.2
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    • pp.123-132
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    • 2016
  • Trichomonas vaginalis causes the most prevalent sexually transmitted infection worldwide. Trichomonads have been detected in prostatic tissues from prostatitis, benign prostatic hyperplasia (BPH), and prostate cancer. Chronic prostatic inflammation is known as a risk factor for prostate enlargement, benign prostatic hyperplasia symptoms, and acute urinary retention. Our aim was to investigate whether T. vaginalis could induce inflammatory responses in cells of a benign prostatic hyperplasia epithelial cell line (BPH-1). When BPH-1 cells were infected with T. vaginalis, the protein and mRNA of inflammatory cytokines, such as CXCL8, CCL2, IL-$1{\beta}$, and IL-6, were increased. The activities of TLR4, ROS, MAPK, JAK2/STAT3, and NF-${\kappa}B$ were also increased, whereas inhibitors of ROS, MAPK, PI3K, NF-${\kappa}B$, and anti-TLR4 antibody decreased the production of the 4 cytokines although the extent of inhibition differed. However, a JAK2 inhibitor inhibited only IL-6 production. Culture supernatants of the BPH-1 cells that had been incubated with live T. vaginalis (trichomonad-conditioned medium, TCM) contained the 4 cytokines and induced the migration of human monocytes (THP-1 cells) and mast cells (HMC-1 cells). TCM conditioned by BPH-1 cells pretreated with NF-${\kappa}B$ inhibitor showed decreased levels of cytokines and induced less migration. Therefore, it is suggested that these cytokines are involved in migration of inflammatory cells. These results suggest that T. vaginalis infection of BPH patients may cause inflammation, which may induce lower urinary tract symptoms (LUTS).

Lysophosphatidic acid enhances breast cancer cells-mediated osteoclastogenesis

  • Nam, Ju-Suk;Sharma, Ashish Ranjan;Nguyen, Lich Thi;Jagga, Supriya;Lee, Yeon-Hee;Sharma, Garima;Lee, Sang-Soo
    • The Korean Journal of Physiology and Pharmacology
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    • v.22 no.5
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    • pp.503-511
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    • 2018
  • Lysophosphatidic acid (LPA) is known to play a critical role in breast cancer metastasis to bone. In this study, we tried to investigate any role of LPA in the regulation of osteoclastogenic cytokines from breast cancer cells and the possibility of these secretory factors in affecting osteoclastogenesis. Effect of secreted cytokines on osteoclastogenesis was analyzed by treating conditioned media from LPA-stimulated breast cancer cells to differentiating osteoclasts. Result demonstrated that IL-8 and IL-11 expression were upregulated in LPA-treated MDA-MB-231 cells. IL-8 was induced in both MDA-MB-231 and MDA-MB-468, however, IL-11 was induced only in MDA-MB-231, suggesting differential LPARs participation in the expression of these cytokines. Expression of IL-8 but not IL-11 was suppressed by inhibitors of PI3K, NF-kB, ROCK and PKC pathways. In the case of PKC activation, it was observed that $PKC{\delta}$ and $PKC{\mu}$ might regulate LPA-induced expression of IL-11 and IL-8, respectively, by using specific PKC subtype inhibitors. Finally, conditioned Medium from LPA-stimulated breast cancer cells induced osteoclastogenesis. In conclusion, LPA induced the expression of osteolytic cytokines (IL-8 and IL-11) in breast cancer cells by involving different LPA receptors. Enhanced expression of IL-8 by LPA may be via ROCK, PKCu, PI3K, and NFkB signaling pathways, while enhanced expression of IL-11 might involve $PKC{\delta}$ signaling pathway. LPA has the ability to enhance breast cancer cells-mediated osteoclastogenesis by inducing the secretion of cytokines such as IL-8 and IL-11.

Pituitary Tumor-Transforming Gene (PTTG) Induces both Vascular Endothelial Growth Factor (VEGF) and Basic Fibroblast Growth Factor (bFGF)

  • Cho, Sa-Yeon
    • Bulletin of the Korean Chemical Society
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    • v.26 no.11
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    • pp.1823-1825
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    • 2005
  • Angiogenesis is tightly regulated by a variety of angiogenic activators and inhibitors. Disruption of the balanced angiogenesis leads to the progress of diseases such as cancer, rheumatoid arthritis, and diabetic blindness. Even though a number of proteins involved in angiogenesis have been identified so far, more protein factors remain to be identified due to complexity of the process. Here I report that pituitary tumor-transforming gene (PTTG) induces migration and tube formation of human umbilical vein endothelial cells (HUVECs). High levels of both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are detected in conditioned medium obtained from cells transfected with PTTG expression plasmid. Taken together, these results suggest that PTTG is an angiogenic factor that induces production of both VEGF and bFGF.

Growth Stimulation and Inhibition of Differentiation of the Human Colon Carcinoma Cell Line Caco-2 with an Anti-Sense Insulin-Like Growth Factor Binding Protein-3 Construct

  • YoonPark, Jung-Han
    • BMB Reports
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    • v.32 no.3
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    • pp.266-272
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    • 1999
  • The insulin-like growth factor (IGF) system consisting of IGF-I, IGF-II, IGF-receptors, and IGF-binding proteins (IGFBP) regulates the proliferation of a variety of cancer cell types. To examine whether a decrease in endogenous IGFBP-3 stimulates proliferation or inhibits differentiation, Caco-2 cells, a human colon adenocarcinoma cell line, were stably transfected with an anti-sense IGFBP-3 expression construct or pcDNA3 vector as control. Accumulation of IGFBP-3 mRNA and secretion of IGFBP-3 into serum-free conditioned medium, 9 days after plating, were significantly lower in Caco-2 cell clones transfected with anti-sense IGFBP-3 cDNA compared to the controls. The anti-sense clones grew at a similar rate to the controls for 8 days after plating, but achieved a higher final density between days 10 and 12. The levels of sucrase-isomaltase mRNA, a marker of enterocyte differentiation of Caco-2 cells, were lower in the anti-sense clones examined on day 9. In conclusion, proliferation of Caco-2 cells can be stimulated by lowering endogenously-produced IGFBP-3.

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A Study on Warping of Panel Products (목재판상류 변형에 관한 연구)

  • 김수원;강호양
    • Journal of the Korea Furniture Society
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    • v.13 no.1
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    • pp.19-25
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    • 2002
  • Modern furniture is mostly made of panel products such as a glue-up panel, a particle board and a medium density fiberboard(MDF). Warping is a major defect of these panel products resulting in degrading final products. In this study the factors related to the warping of a glue-up panel and MDF were investigated by comparing the physical properties of warped specimens with those of the unwarped. The differences between the moisture contents measured on the both surfaces of specimens were found to mainly influence the warping of glue-up panel specimens whether conditioned or not. The average oven-dry density of warped glue-up panel specimens was definitely higher than that of the unwarped at l% significance level. For MDF the influencing factor on warping has not been revealed yet, however it was found that the conditioning reduced the number of warped specimens.

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Posterior density estimation for structural parameters using improved differential evolution adaptive Metropolis algorithm

  • Zhou, Jin;Mita, Akira;Mei, Liu
    • Smart Structures and Systems
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    • v.15 no.3
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    • pp.735-749
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    • 2015
  • The major difficulty of using Bayesian probabilistic inference for system identification is to obtain the posterior probability density of parameters conditioned by the measured response. The posterior density of structural parameters indicates how plausible each model is when considering the uncertainty of prediction errors. The Markov chain Monte Carlo (MCMC) method is a widespread medium for posterior inference but its convergence is often slow. The differential evolution adaptive Metropolis-Hasting (DREAM) algorithm boasts a population-based mechanism, which nms multiple different Markov chains simultaneously, and a global optimum exploration ability. This paper proposes an improved differential evolution adaptive Metropolis-Hasting algorithm (IDREAM) strategy to estimate the posterior density of structural parameters. The main benefit of IDREAM is its efficient MCMC simulation through its use of the adaptive Metropolis (AM) method with a mutation strategy for ensuring quick convergence and robust solutions. Its effectiveness was demonstrated in simulations on identifying the structural parameters with limited output data and noise polluted measurements.