• Journal of Plant Biology
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• 제34권1호
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• pp.31-35
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• 1991

Induction of symbiosis between N. muscorum and cultured tobacco cells associately cultured on nitrogen-free medium and effects of polyamines on culture condition were carried out. Analysis of amino acid composition in associate cultures showed increase in total amino acid amounts than single culture of cultured tobacco cells and methionine was markly increased in associative culture on N-free medium treated with 1 mM spermine. These results indicated that the composition of amino acids increased effectively in associative cultures by nitrogen fixation of N. muscorum.scorum.

• KSBB Journal
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• 제18권6호
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• pp.435-439
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• 2003

Plant cell culture can be divide into two classes non-organic culture and organic culture. Non-organic culture such as suspension culture has many researches, however organic culture about recombinant protein production has little researches. Recombinant protein produced through organ culture is quite stable and it can make proteins by itself without any grow regulators. Therefore organ culture is much easier than other methods. In this research, we used transformed tobacco seed. At first we germinated the seed then separated stems and leaves from the grown plant. And raised in liquid medium by in vitro vegetative reproduction. Continuing most suitable conditions, we compared the Quantities of recombinant protein from intra cellular with from extra cellular. And adding some permeabilizing agents (Pluronic F-68, Triton X-100, DMSO, PEG8000), we increased the productivity of the recombinant protein.

• Korean Journal of Plant Tissue Culture
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• 제27권2호
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• pp.133-136
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• 2000

To evaluate the biological effects of dykellic acid, a novel apoptosis inhibitor, isolated from microorganism on the plant cells, the cell growth, protein contents, and superoxide dismutase (SOD) activity were investigated in suspension cultures of tobacco photomixotrophic cultured (PM) cells on 12 days after different concentration of chemical treatment. The cells were cultured in MS medium containing 0.7 mg/L 2,4-D, 0.3 mg/L kinetin, 30 g/L sucrose and 200 mM NaCl at $25^{\circ}C$ in the light (100 rpm). Dykellic acid strongly inhibited the cell growth by evaluating the cell fresh wt and the ion conductivity in the medium ($IC_{50}$/, about 20 $\mu$M). The results as inhibition of cell growth and cell wall damage were same. The compound significantly increased the protein contents and the SOD specific activity in proportion with the dosage. The results suggested that dykellic acid may have biological activity in plant cells and tobacco PM cells may be suitable biomaterials for in vitro evaluation of the biological activity of natural products.

• KSBB Journal
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• 제5권2호
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• pp.151-158
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• 1990

Investigations on the liability of nitrogen usuage by Nostoc muscorum that has nitrogen fixing ability, and cultured tobacco cells as they were associately cultured on nitrogen-free media and effects of polyamine on the associated culture condition were carried out. In addition, measurement on the activity of nitrate reductase, glutamine synthetase, glutamate dehydrogenase and glutamate synthase that take part in the metabolic pathway of nitrogen fixation product were performed. Among enzymes participating in the metabolic pathway of nitrogen fixation products, the activity of nitrogen reductase stimulated five times in associated culture, and that of glutamine synthetase of N. muscorum increased two times after heterocyst differentiated. Activity of glutamate dehydrogenase increased markedly when cultured tobacco cells were solely incubated on nitrogen-free media, but inhibited when cultured associately. And, glutamate synthase was showed the highest activity in 0.1 mM of spermine treated group.

• Applied Biological Chemistry
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• 제43권4호
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• pp.231-235
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• 2000

The changes of calmodulin levels, calmodulin-binding proteins, and $Ca^{2+}$/calmodulin-dependent glutamate decarboxylase during the growth of tobacco suspension cells were investigated. Tobacco cells exhibited a typical growth curve, including an exponential growth phase between 3 and 5 days after inoculation, and an apparent stationary phase occurring after 5 day. Although slight changes were observed from sample to sample, calmodulin protein levels remained similar during the phases of culture growth. Several $Ca^{2+}-dependent$ calmodulin-binding proteins including 56, 46, 36, and 32-kDa proteins were detected in tobacco cell extracts. The 56-kDa protein was identified as glutamate decarboxylase by Western-blot analysis using an anti-GAD monoclonal antibody. The levels of GAD protein and the specific activity of GAD enzyme were highest during the middle exponential phase of the culture growth cycle. These data suggest that $Ca^{2+}$/calmodulin-dependent glutamate decarboxylase is modulated during the growth of tobacco suspension cells.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.391-392
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• 2000

Tobacco cells transformed with hGM-CSF gene produced $162\;{\mu}g/L$ of hGM-CSF, a valuable therapeutic protein in seven days after inoculation. The protein concentration decreased after the maximum point. It is evidenced that the environment of cell culture was inappropriate for the stability of the protein(e.g., low osmolarity which can cause cell lysis).

• Journal of Plant Biotechnology
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• 제31권2호
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• pp.175-178
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• 2004

Transgenic tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cell lines expressing a human histone H1.5 (referred to as hH1.5), which suppress collagen-induced rheumatoid arthritis, were developed under the oxidative stress-inducible peroxidase (SWPA2) promoter. Tobacco BY-2 cells were transformed by Agrobacterium-mediated method. The kanamycin-resistant calli were selected on the modified MS medium containing 150mg/L kanamycin and 300mg/L claforan. Transgenic cell lines were confirmed by PCR and northern blot analysis. Recombinant hH1.5 (rhH1.5) protein (42 kDa) was also detected by Western blot analysis, showing a different molecular weight of human hH1.5 (32 kDa). These results suggested that a hH1.5 gene was properly introduced in tobacco cultured cells under the control of SWPA2 promoter. The further characterization of rhH1.5 protein remains to be studied.

• Korean Journal of Plant Tissue Culture
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• 제26권3호
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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.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.284-287
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• 2003

Effects of ultrasound on cell growth and the production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) were investigated using transgenic Nicotiana tabacum cell suspension cultures. The culture suffered a slight growth depression immediately after the sonication, but gradually recovered to normal growth within 2 days. When the cells were exposed to ultrasound, the level of secreted hGM-CSF was 2.14 times higher than that in normal condition. From the beginning to 6 days of culture, production of secreted hGM-CSF was higher than that of control and then decreased. At the end of culture, however, hGM-CSF was considerably increased up to 36.7%. In the case of intracellular hGM-CSF, the level was slightly higher than that obtained in normal condition. Total hGM-CSF production was 31.5% higher than that of control culture after 6 days. The highest amount of hGM-CSF was $34.9\;{\mu}g/L$.

• Korean Journal of Plant Tissue Culture
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• 제25권3호
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• pp.141-146
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• 1998

Feeding experiment of [$^3$H] 5-epi-aristolochene (5-EAS) demonstrated in suspension cultures of tobacco (Nicotiana tabacum L.) that 5-EAS hydroxylase activity was absent from control cells, but induced to a maximum level within 18 h after the addition of cellulase, and was very similar to induction pattern of sesquiterpene cyclase. This result suggest that the conversion of 5-EAS to capsidiol is catalyzed by at least one elicitor-inducible hydroxylase. Cytochrome P450 inhibitors, ancymidol and ketoconazole, suppressed the elicitor-induced capsidiol accumulation by inhibiting hydroxylase activity, suggesting that the hydroxylase may be a Cyt P450 type enzyme.