• KSBB Journal
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• v.22 no.5
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• pp.313-317
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• 2007

To enhance the growth of cryopreserved cells of transgenic Nicotiana tabacum, Pluronic F-68 was supplemented in a recovery medium during post-thaw period. As cryoprotective agents, 1 M sucrose, 0.5 M glycerol and 0.5 M dimethyl sulfoxide (DMSO) were added before freezing steps. The post-thaw growth of the cells was improved with Pluronic F-68, ranged from 0.1 to 10 g/L. The interactions of Pluronic F-68 with the cells were confirmed by the changes of hydrophobicity or permeability of the cells. Pluronic F-68 did not show any effect on the activity of $\beta$-glucuronidase (GUS) in all treatments. Therefore, the addition of Pluronic F-68 in a recovery medium was found to be beneficial to enhance the post-thaw growth of cryopreserved transgenic tobacco cells without affecting the production of recombinant protein.

• Journal of Plant Biotechnology
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• v.45 no.4
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• pp.357-363
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• 2018

The objective of this study was to investigate whether exogenous proline (Pro) could confer freezing tolerance of spinach and determine fluctuations of free amino acids in spinach leaf tissues under freeze-induced stress. Treatment with Pro (10 mM) resulted in more accumulation of Pro (~2.6-fold) in Pro-treated spinaches compared to untreated ones. These Pro-pretreated spinaches were more freezing-tolerant, showing more turgid leaves and petioles compared to untreated controls. However, when spinaches pre-treated with or without Pro were subjected to freezing, there was no significant difference in overall amino acid contents, emphasizing the role of Pro as an osmoprotectant. Freezing stress prompted intensification of total amino acid contents irrespective of pretreatment with Pro. Asp, Glu, Ala, and Val were the most abundant free amino acids due to increased protein degradation and nitrogen mobilization for plant survival under freezing stress. Arg, a precursor for the synthesis of polyamines in plants, was profoundly enhanced under freezing stress. This implies that Arg plays an important role in modulating freezing tolerance. Gly, Leu, and Ile were maintained at relatively low levels in all treatments. However, Ser, Tyr, and Lys as primary constituents of dehydrins were accumulated under freezing stress, suggesting that they might play a role in increasing cryoprotective activity under freezing stress.

• The Korean Journal of Physiology
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• v.19 no.1
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• pp.15-23
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• 1985

Previous biochemical studies indicate that $(Na^++K^+)-ATPase$ is composed of two subunits, ${\alpha}$ and ${\beta}$, in a form of ${\alpha}_2{\beta}_2$ with a molecular weight of approximately 300,000 daltons. There is also suggestive evidence that the $Na^+$, $K^+$ pump in human erythrocytes occurs in a complex with some glycolytic enzymes. We assessed here in situ assembly size of the $(Na^++K^+)-ATPase$ of human erythrocytes by applying classical target theory to radiation inactivation data of the ouabain-sensitive sodium flux and ATP hydrolysis of intact cells and ghosts. Cells(in the presence of cryoprotective agent) and ghosts were irradiated at $-45^{\circ}C$ to $-50^{\circ}C$ with an increasing dose of a 1.5 MeV electron beam, and after thawing, the pump and/or enzyme activities were assayed. Each activity measured was decreased as a simple exponential function of radiation dose, from which a radiation sensitive volume (target size) was calculated. When intact cells were used, the target size of both $(Na^++K^+)-ATPase$ and $Na^+$, $K^+$ pump was found to be approximately 600,000 daltons. This target size of the ATPase was reduced to approximately 325,000 daltons if the cells were pretreated with strophanthidin. When ghosts were used, the target size of the ATPase was again approximately 325,000 daltons. Our target size measurement suggests that, in intact cells, the $(Na^++K^+)-ATPase/Na^+,K^+$ pump exists either as a dimer of $(\alpha\beta)_2$ which is a functional unit or as a monomer of $(\alpha\beta)_2$ but in tight complex with other enzyme or enzymes. The results also suggest that this dimeric or heterocomplex association is dissociated during ghost preparation and strophanthidin treatment.

• Journal of Soil and Groundwater Environment
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• v.11 no.5
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• pp.43-50
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• 2006

Bioreporter bacteria, such as recombinant bioluminescent bacteria, have been used for the detection of specific compounds in complex environmental media. In this study, optimum conditions for the preparation and application of deep-freezed and Iyophilized recombinant bioluminescent strain KG1206 were investigated for the future application on contaminated environmental sites. Genetically engineered microorganism, Pseudomonas putida mt-2 KG1206, contains TOL plasmid and the plasmid inserted $P_{m}$, promoter on the upper part of lux gone in vector pUCD615, and m-toluate and benzoate are considered direct inducers for bioluminescence. Optimum conditions determined for the preparation and application of the deep-freezed and lyophilized strain were followings: cryoprotective agent (24% sucrose), lyophilization time (12 hrs), strain concentration ($OD_{600}=0.6$), reconstitution for freezed strain (quick reconstitution at $35^{\circ}C$), reconstitution for lyophilized strain ($3{\sim}6$ hrs exposure on LB medium), carrying conditions (keep at $20^{\circ}C$ after reconstitution). These results demonstrate the feasibility of deep-freezed or lyophilized state of genetically engineered bioluminescent strain for environmental usage.