Pulse combustion drying was used for dry preservation of lactic acid bacteria. An antioxidant i.e., 0.01% ascorbic acid or 1.0% glutathione was added to skimmed milk (5%) and trehalose (5%) to protect against desiccation of bacterial cells. The survival rate of dried lactic acid bacteria was > 90% on addition of the antioxidants, while addition of only skimmed milk and trehalose resulted in a 27% survival rate. Neither oxidized ascorbic acid nor glycine demonstrated this effect. Therefore, addition of an antioxidant to prevent peroxidation of the lipid membrane of microorganisms is effective in maintaining the viability of lactic acid bacteria subjected to pulse combustion drying. This the first study to achieve a sufficiently high survival rate of lactic acid bacteria in order to develop a microbiological agent using pulse combustion dryer.

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Ice nucleation is the primary event that a plant undergoes at subfreezing temperatures. How plants controls the freezing of their tissues is an important mechanism in cold hardiness. Ice nucleation activity (INA), the ability to cause heterogeneous ice nucleation of water, in plant tissues seems to be a key element contributing to this mechanism. Here I summarize the results of surveying INA in various tissues from more than 600 plant species using a newly revised test tube INA assay. Cold tender plants (tropical and subtropical) had low levels of INA whilst cold hardy plants had high levels of INA in specific tissues. The localization of INA in cold hardy plants corresponded closely to the intrinsic freezing behaviors of the tissues: bark tissues which undergo extracellular freezing and bud scales which act as an ice sink in extraorgan freezing had high levels of INA whilst florets which remain deeply supercooled had low levels of INA. INA was classified into heat-stable and -labile types with regard to the resistance to autoclaving (121℃, 15 min), which were specific to tissues and species. This allowed us to determine the distributions of these types of INA within a plant family and the inheritance of this trait using interspecific hybrids. The supernatant of autoclaved plant tissues tended to show anti-nucleation activity (the ability to suppress INA) at low osmolality (< 5 mOsm) irrespective of plant origins (cold hardy or sensitive). The high INA may act as a subfreezing temperature sensor to avoid excess supercooling of the tissues and to readily initiate freezing in the specific portion of the tissues, which likely allows the tissues to properly undergo the intrinsic freezing behaviors (e.g., extracellular freezing, extraorgan freezing). The applicability and precautions required in utilizing the revised test tube INA assay are discussed.

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We here report the results of MD simulations for several systems including a model peptide which has two tandem repeats of the characteristic 11-mer motif found in group 3 late embryogenesis abundant (G3LEA) proteins and its partner protein (lysozyme and HybD) in aqueous solution. And for comparison, we performed MD simulations for a control peptide which has the same amino acid composition as that of the G3LEA model but a scrambled sequence. As a result, the G3LEA model was found to spontaneously approach its partner and form the association complex. According to detailed analysis, the acidic residues (Glu and Asp) and basic residues (Lys) in the G3LEA model contribute to binding to lysozyme and HybD, respectively. Similar results were obtained for the control peptide.

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We investigated whether a model peptide for group 3 late embryogenesis abundant (G3LEA) proteins that we had developed previously is capable of protecting liposomes against their fusion under desiccation stress. Four different peptides were tested for comparison: 1) PvLEA-22, which consists of two tandem repeats of the 11-mer motif characteristic of LEA proteins from an African sleeping chironomid; 2) a peptide with amino acid composition identical to that of PvLEA-22, but with its sequence scrambled; 3) poly-L-glutamic acid; and 4) poly-L-lysine. Based upon the results of the particle size distribution measurement of liposomes and the leakage test of a fluorescent probe inside liposomes, we argue that the synthetic 22-mer peptide that derived from its parent G3LEA protein sequence is superior to simple homopeptides as the anti-fusion reagent of liposomes and can achieve the ability of protecting liposome that is comparable to or higher than several native LEA proteins reported previously.

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We developed various novel synthetic polyampholytes via reversible addition fragmentation chain transfer (RAFT) polymerization for cryopreservation of cells. The polymers, in spite of being structurally analogous with each other, showed very different cryoprotective properties. Copolymer of methacrylic acid (MAA) and N, N-dimethylaminoethyl methacrylate (DMAEMA) showed excellent cryoprotective property and the hydrophobic modification of the copolymer enhanced the cell viability significantly. On the other hand, another polymer with similar structure, poly-carboxymethyl betaine (poly-CMB), a zwitterion-type polyampholyte, exhibited no cryoprotective property and the addition of hydrophobicity did not have much effect on the cell viability, whereas a similar zwitterion type polyampholyte, poly-sulfobetaine (poly-SPB), exhibited intermediate cryoprotective property. These findings suggest that cryoprotective property depends to a great extent on the polymer structure and the structural differences affect the interaction among polymer chains in solution.

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Cells have a high molecular discerning ability. Recently, taking advantage of this ability as a kind of biosensors, cells attached on matrix have been developed in the field of biosensors. In the previous study, we reported cells attached to the glass matrix can be cryopreservated using 20v/v% concentration of DMSO as a cryoprotective agent, which usually allows 10v/v% DMSO concentration in cryovials. However, Poly-Dimethyl Siloxane (PDMS) is preferably used as a matrix for biosensors instead of the glass matrix. In the present study, we investigated cryopreservation of HepG2 cells attached on PDMS matrix by applying DMSO as a cryoprotectctive agent. As a result, the rate of cell adhesion increased with the DMSO concentration. Cell viability was highest at 20v/v% DMSO concentration. However, 30v/v% DMSO concentration had a cytotoxic effect to cell viability. Thus, similar to the glass matrix, 20v/v% DMSO concentration is suitable for the cryopreservation matrix.

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Cells and organisms respond to various stresses when exposed to the heat and pressure, and physical stimulation. Response to the stress can be regarded as a role of a defense strategy for maintaining homeostasis. In plants, tolerance to low temperature was carried out by the removal of ROS (Reactive Oxygen Species), accumulation of sugar, and controlling hormones. On the other hand, there is little report about the cold stress response in mammals, because they are homeothermic animals. In this study, we elucidated a possible mechanism for response to cold stress in HaCaT cell as a human epidermal cell model. First, we evaluated the viability of HaCaT cells by staining calcein-AM and propidium iodide after 4℃ cultivation. Increase of dead cells was observed from 12 h after cultivation at 4℃. In addition, increase of intracellular Ca^<2+> concentration and mitochondrial ROS was observed from 12 h after cultivation at 4℃. Proportion of cell death was reduced when mitochondrial ROS generation was inhibited using antioxidants NAC (N-acetyl-L-cysteine) but intracellular Ca^<2+> concentration was not changed at all. Overall, induction by cold stress-mediated, cell death may be related to by mitochondrial ROS in HaCaT cell.

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In this study, we found that a vacuum drying under the room temperature for few minutes vitrifies the thin liquid film of trehalose aqueous solution that formed on plasma treated plate. The results of infrared absorption spectroscopy by the transmission method and the ATR method clarified that the films with various thicknesses and of various trehalose concentrations were vitrified by the rapid desiccation. The moisture content for all the rapidly desiccated films in this study were less than 10 wt% which corresponds to that of trehalose dehydrate. In addition, it was found that the amount of water can be quantified from the areas of ν2 bend + libration combination IR absorption peaks of water molecule.

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The thermal denaturation of hen egg white lysozyme in the solution with/without Choline Acetate ionic liquid has been investigated by Small-Angle Neutron Scattering (SANS) and Differential Scanning Calorimetry (DSC). The gyration radius does not show noticeable change by the addition of the ionic liquid in a native state, while on further heating the remarkable increase of the radius of gyration after the denaturation is suppressed by the ionic liquid, probably owing to the aggregation of lysozyme molecules. The fractal dimension slightly decreases by the ionic liquid, and the remarkable drop is observed in the vicinity of denaturation temperature for both cases.

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Antarctic nematode Panagrolaimus davidi has a high cold tolerance. In order to clarify the relationship of this tolerance and the lipid of the nematode, we investigated the phospholipid composition around body surface and the fatty acid composition of surface phospholipids. We also observed the distribution of fat droplets in the nematodes and studied thermal behavior of the total lipids extracted from the nematodes by the use of a differential scanning calorimeter. Around the surface of P. davidi, the ratio of phosphatidylcholine to phosphatidylethanolamine, and the proportion of unsaturated fatty acids were larger than that of C. elegans. Fat droplets exist throughout the body of P. davidi. Thermal studies revealed that triacylglycerol in fat droplets of P. davidi is mostly in a liquid state at the low temperatures around 0℃.

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We have investigated the states of water molecule in the 1-butyl-3-methylimidazolium nitrate ([bmim][NO_3])-D_2O mixtures in the wide concentrations (X(mol%IL)) by Raman spectroscopy. From Raman OD stretching spectral analyses of D_2O at 298K and 77K, we found that water molecules in the [bmim][NO_3]-D_2O mixtures have three different states against the [bmim][NO_3] concentration; (i) the bulk-like water (X〜10), (ii) strong hydrogen bonded water in [bmim][NO_3] (X=10〜30), and (iii) the hydrated water with [bmim][NO_3] (X>30), respectively.

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Cryopreservation technique is very essential to the various fields, such as medical and food engineering. Considering the worldwide food shortage due to the explosive increase of world population and frequently occurring extraordinary climate in near future, the development of cryopreservation technique is desired. Under the above background, we have continued to investigate the load effect of electric current on plant cell in the cooling and freezing processes in order to discuss the possibility of new cryopreservation technique. We had found that the load of electric current influenced on the ice crystal behavior and temperature at the occurrence of intracellular freezing and reported about the results previously. In this research, we investigated how the load of electric current influenced on intracellular pH in the cooling process, focusing on intracellular pH which is one of indicators of cell activity. Helianthus tuberosus L. and Allium cepa L. were used as plants for testing. Intracellular pH was measured using fluorescence intensity ratiometry. As a result, it is clear that the load of electric current causes the reduction in the lowering of intracellular pH in plant with the progress of cooling.

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Calcium ion (Ca^<2+>) acts as a second messenger in plants. Cold stress causes cytosolic Ca^<2+> elevation, which is expected to act as a signal to finally regulate gene expression. Because transient elevation of cytosolic Ca^<2+> concentration was observed in cold stress but the detail is still unknown, we developed an experimental system to detect intracellular Ca^<2+> dynamics with the combination of the confocal cryomicroscopy and Ca^<2+>-sensitive fluorescent probe, Yellow Cameleon 3.6, which is a fluorescence resonance energy transfer (FRET)-based Ca^<2+> sensor. This system can detect changes in cytosolic Ca^<2+> concentration in living Arabidopsis roots in a non-destructive way. Using this system, we observed Ca^<2+> signals at three cooling rates: 2℃/min, 4℃/min and 8℃/min. A faster cooling rate resulted in a higher and more rapid peak elevation of cytosolic Ca^<2+>, suggesting that cooling rate is a key factor in the temperature sensing mechanism. By the same token, it is inferred that the sensing of cooling rate in plant cells may lead to changes in gene expression appropriate to temperature changes.

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