Desiccation due to drought and freeze stress is a major threat to plants. Several attempts have been made to induce desiccation tolerance and cryopreservation of cultured plant cells and various plant parts. In this study, we successfully optimized the desiccation and cryopreservation conditions of cultured rice cells. Our results indicated that desiccation tolerance can be artificially induced in rice cells and that suitably controlled pretreatment is required for actively growing cultured cells containing abundant free water. We obtained highest survival when the cells were pre-cultured with 0.4 M sucrose for 5 days and desiccated at 70% relative humidity for 18 h. Our results showed that post-preservation culture medium was also important for the viability of desiccated cells. Precultured and desiccated cells could be preserved at 4℃ for 1 month but not at 26℃.

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We show that a short model peptide which has two tandem repeats of the 11-mer motif of a group3 late embryogenesis abundant protein protects proteins against aggregation by desiccation stress. Next, we measure the dissociation constant between the LEA model peptide and its partner protein (lysozyme and HybD) using quartz crystal microbalance (QCM) method. Similar experiments are performed for a native LEA protein from the anhydrobiotic nematode A. avenae. On the basis of these results, the mechanism of the anti-aggregation function of the LEA peptide is discussed.

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Here, we have performed molecular dynamics (MD) simulations of a short model peptide which has two tandem repeats of the 11-mer motif of a group3 late embryogenesis abundant protein in aqueous solution. First we showed that this peptide (LEA peptide) adopts turn-like structures in water with the hydrophilic side chains being exposed to the medium. Next, we performed MD simulations for the mixed system of the LEA peptide and lysozyme and showed that the LEA peptide binds to the surface of lysozyme through the intermolecular hydrogen bonds with three different binding patterns. Interestingly, the conformation of the LEA peptide is transformed into more extended form with binding to lysozyme. It is concluded that the LEA peptide could protect lysozyme from aggregation by shielding the protein surface in the dry state. In other words, the LEA peptide works as a chemical chaperon.

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Improving process efficiency and product quality of freeze-dried formulations is relevant for wider use of unstable protein therapeutics and drug delivery systems. Post-freeze heat treatment and controlled nucleation are getting increasing attention as methods to obtain uniform large ice crystals that allow fast ice sublimation. We examined how these freezing procedures affect morphology and physical property of freeze-dried solids. X-Ray micro-computed tomography and scanning electron microscopy (SEM) indicated variations in the ice-trace structure of microporous solids depending on the freezing methods. The freezing methods also affected profile of myo-inositol crystal forms in the lyophilized solids. The results indicated relevance of physical characterization when applying these ice-size controlling methods in freeze-drying of pharmaceuticals.

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Recently we showed that carboxylated poly-L-lysine, which is classified as a polyampholyte, has a cryoprotective effect on cells in solution without any other cryoprotectants. The recent study suggests that extra cellular environment might affect the cell viability after cryopreservation. Cryopreservation of cell-containing constructs is in high demand in tissue-engineering applications to produce the tissue-engineered products "off-the-shelf". However, cryopreservation of regenerated tissues including cell sheets and cell constructions is not easy compared to cell suspensions. In this study, we attempted to make cell scaffolds using polyampholytes for the development of the novel cryoprotective cell hydrogel scaffolds. Cells encapsulated with such in situ hydrogels can be cryopreserved well without addition of any cryoprotectants. Thus, these hydrogels can serve as scaffolds with cryoprotective properties that also provide structural integrity to tissue constructs.

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In order to discuss the applicability of ionic liquids to bioprotectants, the aqueous solutions of trehalose, ionic liquids of 1-ethyl-3-methylimidazolium chloride ([emim]Cl) and 1-butyl-3-methylimidazolium chloride ([bmim]Cl) were studied. The elastic anomaly and relaxation dynamics of supercooled liquid and glassy states were investigated by micro-Brillouin scattering spectroscopy and modulated DSC. The refractive index was also measured accurately as a function of content to determine sound velocity and attenuation from Brillouin frequency shift and peak width. The fragility index was determined by using the peak temperatures of imaginary parts of complex heat capacity. It is found that the fragility index of [bmim]Cl solution is higher than that of [emim]Cl solution.

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The cysts of Artemia franciscana (A. franciscana) exhibit strong tolerance to desiccation and freezing. However, when cysts are hydrated, advantageous resistance to environmental factors is markedly reduced. Here we report the evaluation of freezing damages after cryopreservation of decapsulated hydrated cysts, and improved hatching ratio by the treatment of cryoprotectants such as dimethl sulfoxide (DMSO). The hatching rate of decapsulated hydrated cysts declined shortly at rapid frozen (submerged in liquid nitrogen), but high hatching rates maintained at slow freezing rates (-0.5℃/min). We also observed the surface of frozen hydrated cysts using a scanning electron microscope attached with cryo-transfer system (Cryo-SEM), and found fine structural discrepancy in the section of embryos between two freezing speeds.

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We investigated the relationship between the ability of model peptides for group-3 late embryogenesis abundant (G3LEA) proteins as anti-aggregation reagents for dried liposomes and conformational features in the dried state mixed with liposomes. For this purpose, the following four peptides were tested: 1) PvLEA-22, which consists of two tandem repeats of the 11-mer motif characteristic to LEA proteins from an African sleeping chironomide, 2) its control, i.e., the peptide with the amino acid composition identical to that of PvLEA-22, although the amino acid sequence is randomly arranged, 3) Poly-L-glutamic acid, and 4) Poly-L-lysine. The peptides 1) and 2) suppressed the desiccation-induced aggregation of liposomes prepared with POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine). On the other hand, two homopolypeptides 3) and 4) exhibited no such protective effects. For all of these dried samples, Fourier transform infrared spectroscopic measurements were carried out to examine the gel-to-liquid crystalline phase transitions of the liposomes and to know the conformational structures of the mixed peptides. As a result, the G3LEA model peptide was found to be able to bind the dried liposome surface in an appropriate manner, accompanied by extension of its peptide backbone, and thereby to make itself capable of acting as a molecular shielding reagent of liposomes against desiccation-induced aggregation.

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Cells have high molecular discerning ability. Taking advantage of this ability, development of cell-adhesion systems attached to matrices has been attempted for their use in the field of biosensors. For the repeated use of biosensors, preservation of the cells bound to matrices is quite necessary. Dimethyl sulfoxide (DMSO), concentration of which is 5-10v/v%, is often used as a cryoprotective agent for animal cells. In the present study, we investigated cryopreservation of HepG2 cells in the cell-adhesion state attached to a glass matrix by applying higher concentrations of DMSO than those used in the suspension state. The rate of cell-adhesion increased, as a result, with augmentation of DMSO concentration. Cell viability also became higher with the increase in the concentration up to 20v/v% of DMSO. However, it decreased with further increase in the concentration. Then, it can be concluded that the application of 20v/v% DMSO is appropriate for cryopreservation of cells in the cell-adhesion state.

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Suspension cells of Daucus carota precultured in MS medium containing 0.4 M sucrose for 5 days showed enhanced desiccation tolerance and could be preserved in liquid nitrogen with a desiccation method. Protoplasts were isolated from precultured carrot cells by treatment with Driselase and then cultured. Although cell division rate was lower than that of the protoplasts isolated from non-precultured control cells, cell division of protoplast isolated from precultured cells was observed. Protoplasts isolated from the precultured cells were desiccated and rehydrated. Viability of rehydrated protoplasts was determined after staining with Evans blue. Some protoplasts that were not stained seemed to be alive.

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Hundreds of genes are known to respond to cold acclimation and the quantitative changes of their gene products have been investigated over many years. However, only a small number of cold-responsive genes have been subjected to the thorough analysis, and little is known about their spatial distribution and temporal regulation in planta during exposure to low temperature. Using luciferase as the reporter gene, we developed in vivo imaging and analysis method of individual plants under controlled environment to evaluate where and when the promoter is regulated. Analysis of the promoter of COR15A, a well-known cold-responsive gene, showed distinctive temporal patterns in responses to various temperatures, and the rate of its induction was highly dependent on the temperature. Moreover, the expression of COR15A promoter::ELuc corresponded to the photoperiods, indicating that the cold-inducible genes are regulated by a complex interaction of multiple environmental stimuli.

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Structural modifications of lysozyme in aqueous 1-butyl-3-methylimidazolium nitrate ([bmim][NO_3]) solutions (X=10 and 20 mol%IL) upon heating from -196℃ have been investigated by FTIR spectroscopy. From FTIR spectral analyses, the increment of disordered structure due to the unfolding and the decrease of the α-helical structure of lysozyme occurred on going from liquid to glassy states at both IL concentrations. Remarkably, lysozyme structure at X=10 showed an irreversible change, whereas that at X=20 showed a reversibility with the change of temperature. The difference in the lysozyme structural changes between X=10 and 20 may be due to the difference of the solution structure in the aqueous IL solutions. Our results suggest that high IL concentration such as X=20 has a possibility of a new simple temperature-controlled type cryoprotectant for protein.

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We investigated the effects of different temperature treatments on the ^1H-nuclear magnetic resonance (NMR) relaxation times (T_1 and T_2) before and after the heading stage of rice seeds. The T_1 values at temperatures of 25℃/30℃ and 30℃/25℃ before and after the heading stage were lower than those at 25℃/25℃ 21 days after heading (DAH). The T_2 values at 25℃/30℃ and 30℃/25℃ were abruptly shortened at 28 DAH, while that of under 25℃/25℃ treatment was hardly shortened. In addition, dehydration of rice seeds was accelerated by heat stress at 30℃ before or after the heading stage. The changes in T_1 values at 30℃ before or after the heading stage were similar to the changes in the degree of dehydration. These results indicate that T_1 and T_2 are useful diagnostic indicators for the water status during seed ripening of rice plants. A high level of expression of aquaporin gene OsPIP1;1 in seeds at 25℃/30℃ and 30℃/25℃ was detected using DNA microarray analysis at 14 DAH. Seed quality grown under 25℃/30℃ and 30℃/25℃ treatments was inferior to that of seeds under 25℃/25℃ treatment because of the occurrence of white immature kernels. These results suggested that thermal stress either before or after the heading stage led to a reduction in the quality of rice seeds as well as changes in NMR relaxation times and expression of aquaporin genes.

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Antifreeze protein (AFP) specifically binds to ice crystal surface and inhibits its growth. In order to elucidate ice-binding mechanism of fungal AFPs, we performed measurements of thermal hysteresis (TH) activity and a recently developed method called "Fluorescence-based Ice Plane Affinity (FIPA)" analysis against AFP identified from Antarctic ascomycete, Antarctomyces psychrotrophicus (AnpAFP). AnpAFP showed a maximum TH of 0.8℃ and led to an ice growth along with the c-axis, which were typical for AFP with moderate antifreeze activity. In addition, FIPA analysis showed that AnpAFP bound to only prism planes of an ice crystal. Such an ice-binding manner of AnpAFP is different from that of the other known microbial AFPs, since they can bind to both prism and basal planes.

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