Freezing behavior of cells in evergreen needle leaves of fir was observed by cryo-scanning electron microscopy (cryo-SEM) to determine cellular responses to subzero temperatures. Freezing resistance of needle leaves increased from summer to winter, and needle leaves in winter showed a high survival rate after freezing to -30℃ at a cooling rate of 5℃/day. Cryo-SEM observation revealed that intracellular freezing occurred in cells of the epidermis, hypodermis, mesophyll and endodermis in summer needle leaves frozen to -30℃ at a rate of 0.2℃/min. However, extracellular freezing occurred in cells of all tissues in summer needle leaves by freezing to -30℃ at a rate of 5℃/day. In winter, all cells showed extracellular freezing by freezing to -30℃ at both rapid and slow cooling rates. The results suggest that cells in needle leaves of fir adapt to subzero temperatures by extracellular freezing with development of high resistance to freeze-induced dehydration during seasonal cold acclimation.

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The stochastic nature of the value of the heterogeneous freezing point, also known as the supercooling point (SCP), is not always realized or well-defined historically. Often the SCP is measured by sealing the solution into a small capillary, and decreasing the temperature of the capillary linearly as a function of time at some preset rate until the solution freezes. This process is then usually only repeated a few times, often employing different samples from the stock solution in each successive run. This procedure misses one of the most important aspects of this phenomenon, namely the inherent width of the distribution of SCP values. In the case of whole animals, the procedure is essentially the same, but great care is taken to ensure that the animal is not seeded with ice, which would prematurely induce freezing in supercooled fluids. Freeze-tolerant animals may survive the experience, and the same sample may be used several times, but not so with freeze avoiding animals. When only a few data points are determined, the likelihood of measuring the most probable nucleation temperature is small. In fact, up to 200-300 measurements are needed on a single sample to determine accurately the nucleation temperature

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The ultrafast part of the solvation dynamics in series of imidazolium ionic liquids (IL) was investigated by femtosecond three-pulse photon echo peak shift (3PEPS) measurement utilizing an organic dye, oxazine 4 (Ox4) as a probe. The time constant, τ_1, for the ultrafast part (≤300 fs) of the 3PEPS decay exhibited a linear dependence on the square root of the anion mass with the lightest chloride anion manifesting the shortest τ_1. The ultrafast part of the solvation for Ox4 is expected to be affected by the inertial motion of IL anions because Ox4 is a cationic dye that attracts negatively charged particles. A better correlation with the effective mass and the size of both ions included was found for the second time constant, τ_2, (≤3.5 ps).

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We have investigated low temperature-induced structural reversibility of ribonuclease A (RNase A) in aqueous choline dihydrogen phoshpahte ([Chol][dhp]) solutions (X(mol%D_2O)=100〜70) by the uses of FTIR and Raman spectroscopies. From the IR spectral analyses, we found that the structural stability of RNase A remains unchanged in the concentration of [Chol][dhp] region from X=97 to 85. Moreover the secondary and tertiary structures of RNase A showed good reversibility upon cooling (77 K) in the same concentration region. On the other hand, Raman OD stretching spectra showed that the aqueous [Chol][dhp]-RNase A solutions take into the glassy state at 77 K in the region from X=90 to 70. On the basis of these results, in the concentration region from X=90 to 85, the aqueous [Chol][dhp]-RNase A solutions have a possibility of good cryoprospectant for aqueous protein solutions.

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To investigate whether or not short model peptides work as substitutes for group-3 LEA (Late Embryogenesis Abundant) proteins, two 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, and 2) its control, i.e. the peptide with the amino acid composition identical with that of PvLEA-22, although its sequence is scrambled. Liposomes were prepared with DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). Both of the two peptides were found to suppress the desiccation-induced aggregation of the liposome. When the liposome was dried in the presence of anyone of the two peptides, its gel-to-liquid crystalline temperature, measured by differential scanning calorimetry (DSC) and temperature-controlled Fourier transform infrared (FT-IR) spectroscopy, was lowered compared to that of the dried liposome without additives. The P=O stretching band (position was found to shift from that of the pure liposome in the dried state. The peptide secondary structure was composed of several different structures as well as α-helix in the dried peptide/liposome mixture, being different from the pure dried PvLEA-22, in which α-helix was predominant. These results are suggestive of the direct interactions between the added peptide and the membrane polar headgroup. Taken together, these two peptides are able to act as a kind of molecular shielding reagent by binding to the surfaces of the dried liposomes, and thereby to avoid their desiccation-induced damage.

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In cryopreservation of cells, the bell-shaped cell survival curve with an optimal cooling rate is understood on the basis of two-factor hypothesis, i.e. extracellular- and intracellular-freezing injuries. The intracellular freezing injury was already modeled successfully and mathematically based on intracellular ice formation. In this study, a mathematical model with reaction kinetic formulation was newly proposed to describe the extracellular freezing injury. A parameter study of model constants was performed to evaluate the cell survival rate as a function cooling rate. The model constants were also determined by inverse problem analysis based on the experiment with human erythrocytes. The experiment and prediction by the model were compared on the cell survival curve. The result shows that the extracellular freezing-induced cell death is successfully described by the reaction kinetic model.

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Group 3 late embryogenesis abundant proteins (G3LEA) are composed of repeat sequences of characteristic 11-mer motif. This motif is likely responsible for protecting biomolecules against desiccation stress. In our previous studies, we showed that a short model peptide (LEA peptide) which has two tandem repeats of the 11-mer motif could protect proteins from desiccation-induced aggregation. However, it remains unclear whether or not it has the similar anti-aggregation effect on celluar membranes. Here, to address this issue, we prepared the solutions of giant vesicles (GV) with/without addition of the LEA peptide and measured the size distribution of GVs before and after desiccation. Moreover, to understand the protection mechanism at atomic level, we performed molecular dynamics (MD) simulation for model systems composed of membrane bilayer and a LEA peptide. These results suggest that the LEA peptide protects GVs from desiccation-induced aggregation by shielding the lipid membrane surface so as to disturb direct membrane-membrane interactions.

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Heat tolerance of suspension cultured Pogonatum inflexum and Oryza sativa cells were examined after desiccation treatment. Pogonatum cells were desiccated at 27℃ under 8% relative humidity (RH). Oryza cells were precultured in a medium containing 0.4 M sucrose for 5 days, and then desiccated at 27℃ under 70% RH. Desiccated Pogonatum cells and Oryza cells were exposed to various high temperatures for 1 h. Non-desiccated Pogonatum cells did not survive the heat treatment at 40℃ for 1 h. However, desiccated Pogonatum cells survived the heat treatment at 40℃ for 1 h. Furthermore, desiccated Pogonatum cells were showed a survival rate of approximately 50%, after heat treatment at 90℃ for 1 h. On the other hand, non-desiccated Oryza cells survived the heat treatment at 50℃ for 1 h. However, desiccated Ozyza cells did not survive the heat treatment at 50℃ for 1 h. These results suggested that desiccated Pogonatum cells acquired high temperature tolerance.

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We examined effects of different temperature treatments for rice plants before/after heading stages on water status of ripening seeds by nuclear magnetic resonance (NMR) relaxation times (T_1 and T_2). At 0 day after heading (DAH), the T_1 values of seeds grown at 30/30℃ (before/after heading stages, treated temperatures) and 30/25℃ were higher than those of 25/25℃ and 25/30℃. However, at 14 DAH, those values were less than that treated at 25/25℃ and the changes in T_1 of rice seed during an early ripening stage showed similar changes in water content suggesting T_1 values closely related to the quantity of water. The T_2 values showing less than 1 ms were observed at 30/30℃ and 30/25℃ treatments at 21 DAH, while those at 25/25℃ and 25/30℃ treatments were still over 20 ms and it sharply dropped to less than 1 ms at 28 DAH. These results indicated that the NMR relaxation times, T_1 and T_2, were useful diagnostic indicators for water status during seed ripening. Especially, free water in rice seeds at high temperature treatment after the heading stage remained for seven days longer than the treatment before the heading stage.

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Freezing behaviors of cells in dormant buds of katsura (Cercidiphyllum japonicum) tree were examined. Intact dormant buds showed high survival rates after freezing to -30℃ at a rate of 5℃/day (slow freezing). However, flower primordia isolated from dormant buds showed a remarkable decrease in survival rate after slow freezing to -10℃ in the presence of ice crystals adjacent to tissues, while isolated scales showed a high survival rate after slow freezing to -30℃. Katsura buds exhibited a typical pattern of extraorgan freezing, accumulation of large extracellular ice crystals within scale layers, by freezing to -30℃. Cryo-scanning electron microscopic analysis revealed that both extracellular freezing cells without freezable water in the cytoplasm and supercooling cells with freezable water in the cytoplasm of partially dehydrated cells are contained in primordial tissue and extracellular freezing cells existed in scales in dormant buds exhibiting extraorgan freezing. In isolated tissues, intracellular freezing occurred in about 50% of the cells of the flower primordia by freezing to -10℃, but extracellular freezing occurred in all scale cells by freezing to -30℃. These results suggested that katsura dormant buds adapt to subfreezing temperatures by segregation of supercooling primordial cells which are susceptible to extracellular ice crystals, from ice crystals.

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Panagrolaimus davidi is a free-living Antarctic nematode which has a high cold-tolerance. Its lipid and fatty acid compositions were compared with those of Caenorhabditis elegans in three culture temperatures (15, 20, 25℃). The ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) of P. davidi was similar to that of C. elegans and did not depend on culture temperature. On the other hand, the ratio of triacylglycerol (TAG) to phospholipid (PL) for P. davidi was significantly larger than that for C. elegans. In the PE, PC and TAG of P. davidi, the number of double bonds was small and the percentage of monounsaturated fatty acids was large as compared with C. elegans. The ratio of PE to PC was increased when P. davidi cultured at 25℃ were exposed to 5℃. In the PE and TAG of P. davidi, the number of double bonds and the percentage of polyunsaturated fatty acids were increased by the exposure to low temperature.

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Larvae of the sleeping chironomid (Polypedilum vanderplanki) exhibit a tolerance to near complete desiccation (anhydrobiosis). Previously, we have showed that desiccation of the larvae is tightly linked with over-expression of genes encoding stress-related proteins, such as HSP and LEA proteins, and a number of antioxidant proteins. In addition, there is a significant increase of ROS-scavenging capacity in the anhydrobiotic larvae, illustrating a connection between adaptation to oxidative stress and mechanisms of resistance to near complete desiccation. In order to elucidate genetic relationship between reaction of the larvae to oxidative stress and to desiccation, we have conducted wide mRNA transcription analysis of the larvae exposed to dehydration and treated with oxidative stress-generating chemical (paraquat). We found that a large number of desiccation-induced genes encoding proteins involved in cell protection, ROS stress response, and lipid synthesis were also up-regulated by treatment with paraquat. Our data suggest that oxidative stress is one of the factors defining desiccation-driven transcriptional response leading to successful anhydrobiosis.

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The cysts of Artemia franciscana (A. franciscana) are known to reserve a large quantity of trehalose and exhibit strong tolerance to desiccation and freezing. However, when cysts are hydrated, their resistance is markedly reduced. To investigate the effect of freezing rate on the hatching ratio, various levels of hydrated cysts of A. franciscana were frozen. The hatching rate of hydrated cysts declined shortly at rapid frozen (-5℃/min), but high hatching rates can be consistently observed at slow freezing rates (-0.5℃/min and -0.1℃/min). Improved DSC analysis also showed differential thermograms at two cooling rates, suggesting that ice formation inside hydrated cysts were clearly distinct between them. We hypothesized that freezing damages even from highly hydrated cysts were attenuated significantly at slow freezing rates, due at least in part to a synergistic effect of trehalose.

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The present study investigated the application of hypothermic anesthesia to the transportation of live fish without seawater. Piezoelectric elements were used to measure the frequency of gill respiratory movements in Paralichthys olivaceus maintained in seawater from +10 to -1.5℃ in order to determine which temperature was associated with the lowest resting metabolic. The frequency of gill movements decreased as the temperature decreased, until approximately 0-2℃ when gill movements increased, indicating an increase in metabolic activity. Additional experiments conducted on fish maintained outside sea water at +1, +5 and +10℃ revealed that survival was lowest at +1℃ and highest at +5℃, at which temperature some individuals survived for 27 h. The present findings suggest that the application of hypothermic anesthesia to the transportation of live fish without sea water should be performed using holding temperatures that are slightly above the lowest resting metabolic of the species being transported as this will increase the overall survival times of the fish during transit.

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