• Title/Summary/Keyword: 3D cell culture system

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Implementation of an in vitro exposure system for 28 GHz

  • Lee, Young Seung;Dzagbletey, Philip Ayiku;Chung, Jae-Young;Jeon, Sang Bong;Lee, Ae-Kyoung;Kim, Nam;Song, Seong Jong;Choi, Hyung-Do
    • ETRI Journal
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    • v.42 no.6
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    • pp.837-845
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    • 2020
  • The objective of this study was to implement an in vitro exposure system for 28 GHz to investigate the biological effects of fifth-generation (5G) communication. A signal source of 28 GHz for 5G millimeter-wave (MMW) deployment was developed, followed by a variable attenuator for antenna input power control. A power amplifier was also customized to ensure a maximum output power of 10 W for high-power 28-GHz exposure. A 3-dB uniformity over the 80 mm × 80 mm area that corresponds to four Petri dishes of three-dimensional cell cultures can be obtained using a customized choke-ring-type antenna. An infrared camera is employed for temperature regulation during exposure by adjusting the airflow cooling rate via real-time feedback to the incubator. The reported measurement results confirm that the input power control, uniformity, and temperature regulation for 28-GHz exposure were successfully accomplished, indicating the possibility of a wide application of the implemented in vitro exposure system in the fields of various MMW dose-response studies.

Shikimate Metabolic Pathway Engineering in Corynebacterium glutamicum

  • Park, Eunhwi;Kim, Hye-Jin;Seo, Seung-Yeul;Lee, Han-Na;Choi, Si-Sun;Lee, Sang Joung;Kim, Eung-Soo
    • Journal of Microbiology and Biotechnology
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    • v.31 no.9
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    • pp.1305-1310
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    • 2021
  • Shikimate is a key high-demand metabolite for synthesizing valuable antiviral drugs, such as the anti-influenza drug, oseltamivir (Tamiflu). Microbial-based strategies for shikimate production have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. In this study, a microbial cell factory using Corynebacterium glutamicum was designed to overproduce shikimate in a fed-batch culture system. First, the shikimate kinase gene (aroK) responsible for converting shikimate to the next step was disrupted to facilitate the accumulation of shikimate. Several genes encoding the shikimate bypass route, such as dehydroshikimate dehydratase (QsuB), pyruvate kinase (Pyk1), and quinate/shikimate dehydrogenase (QsuD), were disrupted sequentially. An artificial operon containing several shikimate pathway genes, including aroE, aroB, aroF, and aroG were overexpressed to maximize the glucose uptake and intermediate flux. The rationally designed shikimate-overproducing C. glutamicum strain grown in an optimized medium produced approximately 37.3 g/l of shikimate in 7-L fed-batch fermentation. Overall, rational cell factory design and culture process optimization for the microbial-based production of shikimate will play a key role in complementing traditional plant-derived shikimate production processes.

Biological Synthesis of Genistein in Escherichia coli

  • Kim, Bong-Gyu
    • Journal of Microbiology and Biotechnology
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    • v.30 no.5
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    • pp.770-776
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    • 2020
  • Genistein is a type of isoflavonoid found predominantly in leguminous plants. Genistein has diverse biological activities, such as anthelmintic and antioxidant effects, as well as inhibitory effects on the growth of several cancers. In addition, genistein is well known as a phytoestrogen. In this study, we attempted to biologically synthesize genistein from either p-coumaric acid or naringenin using Escherichia coli as a biotransformation host. Four genes, Os4CL, PeCHS, RcIFS, and OsCPR, were used for genistein production. To functionally express RcIFS and OsCPR, two members of the cytochrome P450 family, in E. coli, the membrane-binding anchor domain of each gene was removed, and RcIFS and OsCPR were translationally fused to generate an RcIFS-OsCPR hybrid. Os4CL and PeCHS, or the RcIFS-OsCPR hybrid, were then transformed into E. coli BL21(DE3). Using these strains, we optimized our culture system at a laboratory scale in terms of the cell density, concentrations of substrate and isopropyl-β-D-thiogalactoside, temperature, and culture medium. Under the optimized culture conditions, genistein was produced at up to 35 mg/l and 18.6 mg/l using naringenin and p-coumaric acid, respectively.

Increased Expression of the ${\alpha}_2$ Isoform of (Na,K)ATPase in the Differentiated Murine Muscle Cell Line BC3H-1 (BC3H-1 분화세포에서의 (Na,K)ATPase ${\alpha}_2$ isoform의 표현증대)

  • Lee, Kyung-Lim
    • YAKHAK HOEJI
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    • v.40 no.6
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    • pp.734-738
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    • 1996
  • The development of the alpha2 isoform of (Na,K)ATPase which is high affinity ouabain receptors was studied in the differentiating nonfusing muscle cell line BC3H-1. T he differentiation process of BC3H-1 cell line was confirmed by 2-dexy-D-[$^3$H] glucose uptake experiment and the quantity of the expression of ${\alpha}_2$ isoform was measured using a whole cell [$^3$H] ouabain-binding assay. Undifferentiated growing BC3H-1 cells, myoblasts, exhibited low levels of insulin-stimulated glucose uptake and [$^3$H] ouabain-binding sites. In contrast, differentiated BC3H-1 cells, myocytes, had a 5.6-fold increase in insulin-stimulated glucose uptake and 5-fold increase in [$^3$H] ouabain-binding sites. Scatchard analysis showed that myocytes developed more [$^3$H] ouabain-binding sites than myoblasts vath a dissociation constant (kd) of 6${\times}10^{-8}$M and capacity of 6.l${\times}10^{-5}$ sites/cell. Therefore. it seems that myoblasts express low levels of ${\alpha}_2$ subunit and probably the majority of ${\alpha}_1$ subunit, whereas myocytes express high levels of ${\alpha}_2$ isoform. The results indicate that the expression of ${\alpha}_2$ isoform is developmentally regulated during differentiation and that BC3H-1 culture system provides an excellent model for the study of differentiation and mechanism of (Na,K)ATPase action in muscle which requires electrical excitability.

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Continuous Alcohol Fermentation by Cell Recycling Using Hollow Fiber Recycle Reactor (Hollow Fiber Recycle Reactor를 이용한 알콜연속 발효)

  • 이시경;박경호;백운화;장호남
    • Microbiology and Biotechnology Letters
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    • v.14 no.2
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    • pp.193-198
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    • 1986
  • Improvement of productivity in ethanol fermentation was attempted using a hollow fiber bioreactor (HFR) where Saccharomyces cerevisiac var. ellipsoideus cells were recycled to achieve a high yeast concentration. Industrial wort was used as the fermentation media without supplying any additional nutrients. The performances in hollow fiber recycle reactor (HFR) were compared with those of batch and continuous cultures. In a continuous culture with 11$^{\circ}$P and 15$^{\circ}$P wort media final ethanol concentrations were 4.71% and 5.82% (v/v) and yields 86.2% and 78.6% respectively when the dilution rate (D) was 0.1 h$^{-1}$, in contrast, the ethanol concentration and productivity in HFR were 7.64%(v/v) and 6.1g/l/h at D=0.1h$^{-1}$ with 15$^{\circ}$P media. When the dilution rate was increased to 0.2 h$^{-1}$, the concentration and the Productivity were 7.62% (v/v) and 12.2g/l/h. At D=0.3h$^{-1}$ the sugar was completely consumed and the productivity was 18.1g/l/h. This correponds to 4 times that in continuous system and 16.3 times that in the batch system performed in comparable conditions.

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Development of Stable Production Technique of Summer Spinach (Spinacia oleracea L.) in Soilless Culture in the Highlands (수경재배에 의한 고랭지 시금치의 여름철 안정생산 기술 개발)

  • Lee, Eung-Ho;Lee, Jong-Nam;Im, Ju-Sung;Ryu, Seung-Yeol;Kwon, Young-Seok;Jang, Suk-Woo
    • Journal of Bio-Environment Control
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    • v.20 no.1
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    • pp.21-26
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    • 2011
  • Differences among cultivars, conditions of nutrient solution, nursing materials, and soilless culture systems were investigated to select suitable cultivar and cultivation methods for stable production of summer spinach in the highlands. The 'Quinto' spinach showed the earliest growth and highest yield. For yield increase of summer spinach, optimal solution pH was 6.0, EC was $2.0dS{\cdot}m^{-1}$, and $NH_4-N$ ratio of nutrient solution was 30 percent Stand rate of spinach in nursing seedlings, at 200-cell-tray filled with mixed nursing soil (peat : perlite = 7 : 3), was higher than those grown in urethane sponge and rock-wool plug. Yield was also 18 to 24 percent higher than those in rock-wool plug and urethane sponge. Plant length and yield of spinach in mixed substrate (peat : perlite = 7 : 3) filled nutrient film technique (MSNFT) system were longer of 18 percent, and higher of 9 percent than those in deep flow technique (DFT) system, respectively. However, changes of root zone temperature, pH and EC showed similar pattern with DFT. Therefore, growing 'Quinto' cultivar at 6.0 of pH, $2.0dS{\cdot}m^{-1}$ of EC, 30 percent of $NH_4-N$ ratio, at 200-cell-tray filled with mixed nursing soil, and MSNFT cultivation system, was the best for production of summer spinach in the highlands.

Biosynthesis of trifolin, a bioactive flavonoid by biotransformation (생물전환으로 생리활성물질인 trifolin의 생합성)

  • Noh, Hye-Ryeong;Kang, Ju-Yeong;Kim, Bong-Gyu
    • Journal of Applied Biological Chemistry
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    • v.64 no.3
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    • pp.309-316
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    • 2021
  • Kaempferol 3-O-galactoside (Trifolin), a member of the flavonol group, has been reported to have anticancer effects against promyelocytic leukemia, histocytic lymphoma, skin melanoma and lung cancer. Trifolin has been extracted and used from several plants, but the extraction process is complicated and the final yield is low. Biotransformation is an alternative tool to produce high value-added chemicals from inexpensive compounds. To synthesis trifolin from naringenin, three genes (PeFLS and OsUGE-PhUGT) were introduced into Escherichia coli, respectively. In order to synthesis trifolin from naringenin, a co-culture fermentation system was established by optimizing the cell concentration, biotransformation temperature and medium, isopropyl-β-D-thiogalactoside (IPTG) concentration, substrate supply concentration, and recombinant protein induction time. The established optimal conditions for trifolin production were a 3:1 ratio of BL-UGTE to BL-FLS, induction of recombinant protein at 25 ℃ for 4 h after addition of 2.0 mM IPTG, biotransformation at 30 ℃, and supply of 300 μM naringenin. Through the optimized co-culture fermentation system, trifolin was biosynthesized up to 67.3 mg/L.

Antioxidant Activity with Flavonoidal Constituents from Aerva persica

  • Ahmed Ejaz;Imran Muhammad;Malik Abdul;Ashraf Muhammad
    • Archives of Pharmacal Research
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    • v.29 no.5
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    • pp.343-347
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    • 2006
  • A new flavanone Persinol (1) and the new flavanone glucosides persinosides A (2) and B (3), along with known flavonoids (4 and 5) have been isolated from the ethyl acetate soluble fraction of the whole plants of Aerva persica. Their structures were elucidated on the basis of extensive analysis of nuclear magnetic resonance (1D & 2D-NMR) spectral data. All of them showed profound antioxidative activities by DPPH and cytochrome-c-reduction assays using the HL-60 cell culture system.

Enhanced Production of hCTLA4Ig by Suppressing Cell Death in Transgenic Rice Cell Suspension Cultures (형질전환 벼 현탁세포 배양에서 세포 사멸 억제를 통한 hCTLA4Ig 생산성 증대)

  • Kim, Myong-Sik;Nam, Hyung-Jin;Kim, Min-Sub;Kwon, Jun-Young;Kim, Dong-Il
    • KSBB Journal
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    • v.28 no.4
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    • pp.260-268
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    • 2013
  • Transgenic plant cell cultures are an attractive expression system for the production of industrial and pharmaceutical proteins because of their advantages in safety and low production cost. Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced and secreted when sugar was depleted in culture medium by transgenic rice cell lines (Oryza sativa L.) using RAmy3D promoter. Due to the production of the target protein by sugar depletion, concomitant occurrence of cell death is inevitable. For that reason, inhibition of cell death for enhancing productivity was necessary for the production period without energy sources. Supplementation of 0.1 mM sodium nitroprusside improved cell viability by 1.4-fold and maximum hCTLA4Ig production by 1.3-fold compared to those of control. Addition of 1 and 10 mM glutathione, N-acetylcysteine (NAC), and nicotinamide inhibited apoptotic-like programmed cell death by decreasing the activity of reactive oxygen species. Production hCTLA4Ig was enhanced 1.4-, 1.25-, and 1.15-fold with 10 mM NAC, 1 mM NAC, and 1 mM glutathione, respectively. In addition, it was found that the supplementation of NAC enhanced the cell viability.

Responses of Tobacco Photomixotrophic Cultured Cells to Various Herbicides (다양한 제초제에 대한 담배 Photomixotrophic 배양세포의 반응)

  • 권혜경;권석윤;이행순;윤의수;김진석;조광연;곽상수
    • Korean Journal of Plant Tissue Culture
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    • v.26 no.3
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    • pp.183-187
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    • 1999
  • To establish an efficient screening system for new herbicides using plant cultured cells, responses of tobacco photomixotrophic cultured (PH) cells to various herbicides with different modes of action were surveyed by measuring the cell growth and ion conductivity in medium. The cells were cultured in Murashige and Skoog (MS) medium containing 0.7mg/L 2,4-D, 0.3mg/L kinetin and 30 g/L sucrose at $25^{\circ}C$ in the light (100 rpm). Chemicals were treated to suspension cultures of tobacco PH cells at the time of subculture. The cell growth and ion conductivity in the medium were investigated on 12 days after chemical treatment. The ion conductivity assay gave well correlated results to the cell growth inhibition data. The responses of tobacco PM cells were dependent on the modes of action of chemicals tested. Atrazine, an inhibitor of photosynthetic electron transport (PET), strongly inhibited both the cell membrane and cell growth ($IC_{50}$/, about 1 $\mu$M). Butachlor (an inhibitor of cell division), glufosinate (an inhibitor of amino acid biosynthesis), and fluridone (an inhibitor of carotenoid biosynthesis) showed a dose-dependent inhibition. However, Quinclorac, a herbicide with an auxin activity, did not affect the cell growth and ion leakage. These results suggested that tobacco PM cells is suitable materials for the simple screening of new herbicides such as PET, amino acid biosynthesis, ceil division inhibitors by measuring the cell growth and ion conductivity.

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