Study on the cold-tolerance of Halocynthia roretzi has focused on the squirting movement of their oral and atrial siphons. These squirting movements have been classified into three patterns according to decreasing temperature. In particular, the oral and atrial siphons were closed in temperatures < -1 ℃, and only a movement that stretches and contracts the entire body was observed. With regard to the relationship between temperature and survival, no individuals died even when kept for 20 h at -1.0 to -1.5℃ if the cooling rate was -1.0 to -0.5℃/h. In the coast of Japan, marine organisms with a lower limit of habitation in the negative temperature range are only show in shellfish, such as clams and scallops, and certain fish, such as cod. Considering this, the resistance to cold temperatures exhibited by H. roretzi is probably in the high category among marine organisms.

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The ionic liquids , 1-buthyl-3-methylimidazolium chloride ([bmim]Cl) and 1-ethyl-3-methylimidazolium chloride ([emim]Cl) aqueous solutions are studied by the measurement of refractive index and micro-Brillouin scattering spectroscopy. Temperature dependence of refractive index of [bmim]Cl and [emim]Cl aqueous solutions at 532 nm is determined. The glass transition temperature is determined by the change of sound velocity. The [bmim]Cl and [emim]Cl aqueous solutions show a typical structural relaxation, and undergo a liquid-glass transition at low temperatures. Our results verify that the glass transition behavior of ionic liquids is similar to typical cryoprotectants such as glycerol.

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Inulin was employed as a typical hydrophilic polymer, and glass transition temperature (T_g) of various types of inulin was investigated by using differential scanning calorimetry. The T_g of inulin samples decreased with increasing water content and decreasing molecular weight. The effect of water content on the T_g of inulin samples could be described by the Gordon-Taylor equation. Furthermore the effect of molecular weight on the T_g of anhydrous inulin samples could be described by a stretched exponential equation. From these results, the T_g of amorphous inulin can be predicted.

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"Red-edge effect" was measured for trehalose glass, polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), and 1-propanol (PrOH) at different temperatures utilizing coumarin 153 (C153) as a fluorescent probe molecule. The featureless broad fluorescence spectrum of C153 in PrOH at 200 K exhibited no excitation dependence while that with vibrational structure at 81 K exhibited clear red-edge effect at frequencies lower than 22,500 cm^<-1>. The fluorescence of C153 in trehalose exhibited weak red-edge effect even at 298 K and it became more distinctive at lower temperatures. However, the vibrational structure was not clear and the spectral feature was closer to those in PMMA and PVA. Solvent motion in polymers seems to be more mobile compared to PrOH glass due to their larger free volume and the mobility in trehalose glass seems to lie between them.

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We have investigated temperature-induced structural changes of N-methylacetamide (NMA) in imidazolium-based ionic liquids-water mixtures by a simple differential thermal analysis (DTA) method and Raman spectroscopy. We found that hydrogen bonds among NMAs in the mixtures are preserved even in the glassy state. Our results may shed light on the possibility of ionic liquid as a cryoprotectant of proteins.

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We have developed the novel cryoprotective agents of carboxylated poly-L-lysines (COOH-PLLs) for cell cryopreservation alternative to dimethyl sulfoxide. Here in order to study the mechanisms of the cryoprotection of these polyampholytes, we have measured ice including salt and agents by NMR and evaluated the amount and dynamics of the agents, water, and salt in a freezing state. The NMR measurements revealed that COOH-PLLs have a potential to increase residual water in the ice and change the state of sodium ions into distinctive two states. The one Na component rapidly broadened and disappeared upon cooling, indicating COOH-PLLs likely reduce the contribution of Na to the osmotic pressure. We propose the mechanism of cryoprotection of COOH-PLLs is to keep cells away from the stresses like the drastic changes in the size of soluble space and osmotic pressure.

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The sleeping chironomid Polypedilum vanderplanki is the largest animal among anhydrobiotes. The larvae can survive in an almost completely desiccated state. During desiccation, they accumulate stress-related proteins, including late embryogenesis abundant (LEA) proteins. However, there is little information about the function of LEA proteins in the larvae. Here, we focused on the function of the novel LEA protein in the P. vanderplanki, PvLEA4. The expression of PvLea4 mRNA was up-regulated by desiccation treatment and was rapidly decreased upon rehydration. As the same manner of the expression pattern of PvLea4 mRNA, PvLEA4 protein was accumulated in response to desiccation. We analyzed the protective effect of PvLEA4 protein on the enzyme activity using LDH (lactate dehydrogenase) during repetitive desiccation-rehydration treatments. LDH solution showed no activity once it was subjected to the desiccation-rehydration treatment. When PvLEA4 was added at 500 times molar ratio in LDH solution, LDH activity was maintained comparable to activity of the sample (LDH-PvLEA4 mixture) without the treatment. In addition, LDH activity of LDH-PvLEA4 mixture remained over 50% even after three drying cycles. These results indicate that PvLEA4 could play an important role in terms of the desiccation tolerance in larvae of P. vanderplanki.

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In the present study, we investigated the effects of the 22-mer model peptides of Group 3 late embryogenesis abundant (G3LEA) proteins on desiccation-induced aggregation of α-casein. Three kinds of the 22-mer model peptides were synthesized and tested, made of two repeats of the 11-mer consensus motifs characteristic of G3LEA proteins originating from plants, nematodes, and sleeping chironomide (P. vanderplanki). α-casein was aggregated when dried alone. Such aggregation was significantly suppressed in the presence of any of the 22-mer model peptides with the molar ratio of 25 relative to α-casein or higher. However, the aggregation occurred as soon as the selected 22-mer model peptide was added to the aqueous α-casein, when the molar ratio was 10 or lower. The addition of the model peptide was found to let pH values down to near the isoelectric point of α-casein (ca.4), namely its minimal solubility point. On the basis of these results, we discussed the role of the 22-mer model peptides as anti-aggregation reagents.

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Group 3 late embryogenesis abundant (G3LEA) proteins have repeating sequences composed of characteristic 11-mer motif and are likely to act as a protectant against desiccation-induced aggregation of proteins in anhydrobiotic organisms. However, its molecular mechanism remains unclear. In this study, we first show experimentally that a LEA-model peptide, composed of two repeats of the 11-mer motif, prevents the aggregation of lysozyme induced by desiccation. Next, to interpret this result at atomic level, we study the structure of the LEA-model and its interaction with lysozyme by means of both all-atom and coarse-grained molecular dynamics simulations. It is shown that the LEA model preferentially adopts a turn structure in water, but by addition of lysozyme its structure is transformed into an extended one to bind to the surface of lysozyme. In conclusion, the LEA-model can protect lysozyme from aggregation by shielding the protein surface to inhibit protein-protein direct contact.

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Xylem parenchyma cells (XPCs) of boreal trees adapt to subzero temperatures by deep supercooling. Previous studies suggested that intracellular substances were important for maintaining a supercooling state of cellular water at subzero temperatures in XPCs. In our recent studies, four kinds of flavonol glycosides and four kinds of hydrolyzable gallotannins were identified as supercooling-facilitating (SCF; anti-ice nucleation) substances in xylem extracts of katsura tree (Cercidiphyllum japonicum). In the present study, we examined SCF capabilities of the four kinds of hydrolyzable gallotannins in solutions containing different kinds of ice nucleators, including Erwinia ananas, Xanthomonas campestris, silver iodide, and 1,3,5-trihydroxybenzene (phloroglucinol). The results showed that all four gallotannins exhibited SCF capabilities in the solutions containing various kinds of ice nucleators, but capabilities were dependent on their concentrations and the type of ice nucleator used.

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Suspension cultured Pogonatum inflexum and Oryza sativa cells were desiccated and preserved at various temperatures. Pogonatum cells were desiccated rapidly and Oryza cells were desiccated slowly, then preserved at various temperatures. Desiccated Pogonatum cells were successfully preserved at 26℃ for 8 weeks. Oryza cells did not survived preservation for even one day at 26℃, although the cells could be preserved for 1 week at 4℃ and for 8 weeks at -15℃. These results suggest that Oryza cells were alive just after the desiccation but produced some deleterious alternation during the preservation at higher temperature.

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