• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.6
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• pp.423-427
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• 2004

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of $80.1\%$ by ammonium sulfate precipitation and a single chromatographic step involving FPLCanion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from $21.5{\~}29$ kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.

• Korean Journal of Plant Tissue Culture
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• v.27 no.3
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• pp.181-184
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• 2000

Proteins in friable and compact calli of Viola patrinii DC. were analysed. The protein contents in friable calli were lower than those in compact calli. In suspension culture, it increased to the maximum after 3 weeks culture from inoculation and decreased after 4 weeks culture. Several strong lavel signals were detected with the SDS-PAGE analysis. The polypeptides of 28, 31 and 35 KD were observed from the friable cell culture, from the compact cell culture strong band of 30 KD was determined, indicating that these polypeptides may be the major protein occurring during their cultures. Changes in amino acid contents during culture of the viola suspension cells were investigated the amino acid contents were greatest between two and three weeks culture of the viola suspension cells.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.2
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• pp.581-590
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• 2016

Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.

• KSBB Journal
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• v.30 no.3
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• pp.103-108
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• 2015

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.

• 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.

• Journal of Plant Biology
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• v.38 no.1
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• pp.55-62
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• 1995

Embryogenic cell (EC) suspension cultures derived from mature seed-embryo of rice (Oryza sativa L cv. Kye Hwa) were used for the expression patterns of isozyme and enzyme activity. EC suspension cultures were composed of cells that were densely cytoplasmic, potentially embryogenic. However, nonembryogenic cell (NEC) cultures were composed of large, elongated and vacuolated cells. These cells were analyzed for the isozyme pattern and enzyme activity of EC and NEC. Isozyme patterns of peroxidase, esterase, acid phosphatase and malate dehydrogenase exhibited striking difference in the total number of bands, specificity and intensity of band. Also, these isozymes showed very high activity in the EC. Specific band, band activity and higher enzyme activity of isozyme in EC was absent or low in NEC, which may indicate an association of these specific isozymes with morphological characterization and totipotency of embryogenic cells. These results indicate that specific pattern and activity of enzyme in EC could probably be used as a biochemical marker of EC in rice.n rice.

• Journal of Plant Biotechnology
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• v.29 no.3
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• pp.185-188
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• 2002

Off-white, friable embryogenic calluses were formed on the internal integument of mature seeds of yuzu (Citrus junos) cultured on Murashige and Skoog's basal medium at a frequency of 1.2%. Embryogenic calluses were proliferated when cultured on medium with 1 mg/L 2,4-D. Upon transfer to medium with 0.1 mg/L kinetin, embryogenic calluses produced numerous somatic embryos. Embryogenic suspension cultures were established by placing embryogenic calluses into liquid medium with 1 mg/L 2,4-D. When plated onto medium with 0.5 mg/L ABA, embryogenic cells developed into somatic embryos at a high frequency, and then regenerated into plantlets. Plantlets were successfully transplanted to potting soil and grown in a greenhouse.

• Horticultural Science & Technology
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• v.33 no.2
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• pp.251-258
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• 2015

Eurycoma longifolia is an important rare medicinal plant that contains valuable bioactive compounds. In the present study, cell suspension culture of E. longifolia was established for the production of biomass and phenolic compounds. Various medium parameters, such as concentration of auxin, salt strength of the medium, and sucrose and nitrogen concentrations, were optimized for the production of biomass at the flask-scale level. Full strength Murashige and Skoog (MS) medium supplemented with $3.0mg{\cdot}L^{-1}$ naphthaleneacetic acid (NAA), 3% (w/v) sucrose, 0:60 $NH{_4}^+:NO{_3}^-$ was found suitable for biomass accumulation. Based on the optimized flask-scale parameters, cell suspension cultures were established in balloon-type bubble bioreactors, and bioprocess parameters such as inoculum density and aeration rate were optimized. Inoculum density of $50g{\cdot}L^{-1}$ and increasing aeration rate from 0.05 to 0.3 vvm, with increases every 7 days, were suitable for the accumulation of both biomass and phenolic compounds. With the optimized conditions, $14.70g{\cdot}L^{-1}$ dry biomass, $10.33mg{\cdot}g^{-1}$ DW of phenolics and $3.89mg{\cdot}g^{-1}$ DW of flavonoids could be achieved. Phenolics isolated from the cell biomass showed optimal free radical scavenging activity.

• Journal of Plant Biotechnology
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• v.34 no.3
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• pp.207-212
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• 2007

This study describes a simple and rapid method for quantitative determination of $\gamma$-aminobutyric acid (GABA) using $^1H$ NMR spectroscopy from whole cell extracts of plant suspension cultures. When 9 cell lines derived from 8 species of higher plants maintained in liquid Marashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) were subjected to $^1H$ NMR, a cell line of Coriandrum sativum L. exhibited the highest level of GABA. The level reached up to 16.9 mg/dry wt when cells were cultured in MS medium supplemented with 0.5 mg/L 2,4-D after 3 weeks of incubation. The method for quantitative determination of GABA using $^1H$ NMR established in this study could be applied to high-throughput screening of various plant resources for GABA production and the cell suspension culture system of C. sativum could be further developed for commercial production of GABA.

• Korean Journal of Plant Tissue Culture
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• v.22 no.2
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• pp.111-114
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• 1995

To produce ${\gamma}$-linolenic acid (GLA) by cell cultures of Lithospermum erythrorhizon, we optimized medium compositions including carbon sources, nitrogen sources and growth regulators. MS basal medium supplemented with 1.0 mg/L 2, 4-D was effective for callus induction from mesophyll tissue. Addition of sucrose at 88mM concentration induced active proliferation of suspension cells and increased GLA content. Increased supplement of potassium nitrate as nitrogen source resulted in proliferous cell growth and increased total fatty acid content Abscisic acid increased cell growth and fatty acid content in callus culture, whereas as it had an inhibitory effect in suspension cell culture.