Water in biological model systems was studied by the use of various physical techniques. Studies using differential scanning calorimetry found that water in polymer gels sometimes remains partially unfrozen and turns into a vitreous state during cooling, and freezes during rewarming. Ice crystallization during rewarming, observed with cross-linked polymer gels, depended on density of cross-links as well as water content and cooling rate. The density of cross-links was considered to be interrelated with the flexibility of polymer chains and water diffusivity in gels. From these studies, a concept of compartmentalized water was introduced for water in polymer gels. The concept of compartmentalized water was extended to aqueous solutions by the analysis of their freezing behavior, which provided information of hydration structures of solutes with micro-heterogeneity in aqueous systems. In search of ice nucleation-active substances, spider silk, Agelena opulenta, was found to have an ice nucleation activity. Moreover, for the substances to be strongly ice-nucleation active, it was understood that they are necessary to be hydrophilic, structurally compatible with ice crystal and large-sized as well. With the understanding of physical chemistry of water at low temperatures, cryopreservation of nematode, Caenorhabditis elegans was challenged. High rate of survival after freeze-thawing was attained with the help of dimethylsulfoxide as a cryoprotectant, ice seeding followed by slow cooling and subsequent slow heating after freezing. Finally, the present author recognized that freezing is essentially a kinetic process governed by molecular and thermal transfer.

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The sleeping chironomid, Polypedilum vanderplanki, is the only insect known to be capable of extreme desiccation tolerance, or anhydrobiosis. Here, the author briefly summarized molecular mechanisms underlying induction of the anhydrobiosis. Trehalose, a non-reducing disaccharide, which synthesized in the fat body up to 20% of the dry body mass and uniformly distributed in the entire body via trehalose transporter TRET1, is thought to replace the water in its tissues. Simultaneously, highly hydrophilic proteins called the Late Embryogenesis Abundant (LEA) proteins, are expressed in huge quantities and act as a molecular shield to protect biological molecules against aggregation and denaturation. At the late stage of induction of the anhydrobiosis, specific water channels, called aquaporins, accelerate dehydration and trehalose finally forms together with LEA proteins a glassy matrix, which protect biological molecules and the structural integrity of larvae in the anhydrobiotic state.

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Antifreeze proteins (AFPs) are a subset of ice-binding proteins that evolved to arrest ice crystal growth and also inhibit the recrystallization of ice. An accepted theory for their activity, named the adsorption-inhibition hypothesis, posits that AFP binding to ice must be irreversible because the ice crystals do not grow within a freezing hysteresis (FH) gap. This theory has been criticized due to several experimental observations such as the dependence of FH values on the AFP concentration. Here we present recent fluorescence and microfluidics experimental results of our group supporting irreversible binding of AFPs to ice surfaces. i) Photo-bleaching of GFP-tagged AFP residing on the surface of an ice crystal held in the FH gap showed that there is neither exchange nor overgrowth of the bleached AFP. ii) Ice crystals bound by AFPs showed a measurable resistance to melting (melting hysteresis) demonstrating that the AFPs remain surface-bound at temperatures above the equilibrium melting point. iii) Using a temperature controlled microfluidic apparatus we demonstrated that small ice crystals formed in AFP solution stay intact even after the surrounding solution is depleted of AFPs. Additional measurements of surface fluorescence made during and after the exchange procedure showed no decrease in the fluorescence intensity, suggesting that bound AFPs did not leave the ice surface during the process. In light of these results, this review concludes that surface-adsorbed AFPs are the core source of the freezing hysteresis activity of the tested AFPs and that their adsorption to ice is irreversible.

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We developed novel cryoprotective agents from carboxylated ε-poly-L-lysines (COOH-PLLs) for cell cryopreservation as alternatives to dimethyl sulfoxide (DMSO). The polymeric agents showed excellent cell viability profile for most cell types, including human mesenchymal stem cells. We investigated the molecular states and mobility of the solvents and solutes in ice at low temperatures by using solid-state nuclear magnetic resonance (NMR). The NMR data suggested that DMSO and COOH-PLLs differently affect the states and mobility of water and solutes in ice. The former retains the mobility at low temperature, while the latter restricts the mobility by trapping the molecules in soluble aggregates. We propose that cryoprotection of COOH-PLLs is exerted by protecting cells from stresses such as drastic changes in soluble space size and osmotic pressure during freezing.

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Leeuwenhoek first described anhydrobiosis in an aquatic microinvertebrate, the bdelloid rotifer, over 300 years ago. Initial studies suggest that the molecular mechanisms of desiccation tolerance in these fascinating animals are different to those in other organisms. Thus, bdelloids lack the non-reducing disaccharide, trehalose, and have atypical LEA proteins, although they do have powerful antioxidation systems. Bdelloids also have a remarkable capacity to acquire foreign genes by horizontal gene transfer, some of which are implicated in anhydrobiosis, suggesting that bdelloid anhydrobiosis might also involve tolerance mechanisms adopted from other organisms.

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LEA (late embryogenesis abundant) proteins are hydrophilic polypeptides, often intrinsically disordered, which accumulate concurrently with the acquisition of desiccation tolerance in higher plant seeds and other anhydrobiotes. Several hundred LEA proteins belonging to twelve classes have been identified in different organisms. Interestingly, dozens of LEA genes are found in plant genomes, which suggests the encoded proteins may have diverse functions. Analysis of the subcellular localization of the 51 LEA proteins in Arabidopsis revealed a wide distribution in most compartments. Mitochondrial LEA proteins have been indentified in seeds, and also in brine shrimp anhydrobiotic embryos, highlighting the importance of protecting the energy transducing organelle, in particular the inner membrane.

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Metabolic depression is typically correlated with extended survival of environmental challenge and energy-limitation in early life stages of various invertebrates and vertebrates. Diapause is an ontogenetically-programmed reduction of development and often metabolism seen in many invertebrates. When embryos of Artemia franciscana enter the state of diapause, the overall metabolic depression is estimated to be greater than 99%. These embryos also contain trehalose and express multiple isoforms of Late Embryogenesis Abundant (LEA) proteins, constituents often present in a number of such anhydrobiotic animals. The mRNA levels for LEA proteins are highest in diapause and post-diapause embryos that possess desiccation tolerance, but are very low in desiccation-intolerant nauplius larvae. Stable transfection of human HepG2 cells with AfrLEA2 and AfrLEA3m was performed to evaluate the possibility of improved survivorship during drying. A trehalose transporter was used for intracellular loading of this disaccharide. LEA proteins improved desiccation tolerance in mammalian cells during acute drying and immediate rehydration.

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Unique ability of larvae of the sleeping chironomid Polypedilum vanderplanki to resist complete desiccation largely relies on activity of LEA proteins. Using genome-wide analysis of the insect's mRNA diversity and expression, in addition to initially described three LEA proteins we have identified 37 new LEA-like protein-coding genes. We have demonstrated that while all genes were up-regulated in response to desiccation, other abiotic stresses, such as ionizing radiation and heat also altered Lea-like genes expression. We further found that expression of the Lea-like genes is not limited to the larvae exposed to desiccation and can be detected both in the unstressed larvae as well as in other (desiccation-sensitive) developmental stages of the chironomid.

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The primary structure of group-3 late embryogenesis abundant (G3LEA) proteins includes several times repeat of the characteristic 11-mer motif such as AKDGTKEKAGE. To get insights into the roles of the repeat sequence at molecular level, we have examined physicochemical properties of the 22- or 44-mer chemically-synthesized peptides with two or four tandem repeats of the 11-mer motif, whose sequences were derived from the G3LEA proteins originated in several anhydrobiotic organisms. As a result, it was confirmed that the model peptides belong to the family of intrinsically disordered protein as similar to the native G3LEA proteins: they are disordered in water but form α-helices upon drying. The dried model peptides are in the glassy state up to ca 100℃, and thus they play a role in reinforcing the glassy matrix of co-existing sugar, such as trehalose, in the dried state. The dried model peptides also play a role in sequestrating ionic species in the dried state, accompanied by significant backbone conformational changes. Furthermore, the model peptides have protective activity on the aggregation of proteins and membranes induced by the desiccation stress. Taken together, the model peptides are very useful for studying the functional mechanisms of native G3LEA proteins and they are promising as the protective reagents of biological molecules against desiccation stress.

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To survive at freezing temperatures, temperate plants increase their freezing tolerance by cold acclimation, during which changes of protein and lipid compositions of the plasma membrane (PM) play a key role. Brachypodium distachyon, a temperate grass species, has been recognized as a model plant and is evolutionarily closer to temperate cereals than rice. These facts suggest that B. distachyon could serve as a good system to study freezing tolerance mechanism of temperate cereals. In the present study, using non-acclimated (NA) and cold-acclimated (CA, 2℃) B. distachyon, we performed freezing tolerance test and PM proteome analysis. When freezing tolerance was evaluated by measurement of electrolyte leakage, the temperature at which 50% electrolyte leakage occurs after freezing (T_<EL50>) was lowered from -3.9℃ to -6.9℃ by cold acclimation. Furthermore based on the proteome analysis, PM proteins were found to be changed considerably by cold acclimation and the changes were similar in some parts to those found in oat and rye during cold acclimation. These results suggest that B. distachyon has an ability of cold acclimation and some common pathways responding to cold as shown in temperate cereals.

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The purpose of this study is to confirm effects on texture changes of carrot tissue accompanying formation/dissociation of gas hydrates. Fresh carrot tissues were preserved by using gas hydrate at 5℃ under 0.4MPa or 0.7MPa xenon partial pressure for a week. Texture was evaluated using two parameters, fracture stress and instantaneous elasticity related to the strength of the cell wall and cell membrane in the tissue. As a result, value of the fracture stress was slightly decreased compared to fresh tissue. Almost the same results were obtained under 0.4MPa and 0.7MPa partial pressure. The value of instantaneous elasticity was decreased inversely with increasing of partial pressure of xenon. Both parameters values were larger than that of non-xenon treatment freezing preservation. The study concludes that gas hydrate storage could make texture of carrot tissue in good condition more than non-xenon preservation. And gas hydrate technique is an effective method for fresh produces to prevent from softening tissues by freezing preservation.

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Effects of dehydration treatment on the water content of cookie dough, the melting temperature (T_m) of the crystalline amylopectin in the dough, and the non-hydrolyzed starch content of cookie baked at various temperatures were investigated. The water content of cookie dough was decreased and the T_m of the crystalline amylopectin was increased by the dehydration treatment. The non-hydrolyzed starch content obtained under a given condition increased significantly with a decrease in the initial water content of cookies. Although the pre-dehydrated cookie showed greater browning than the non-dehydrated cookie, the browning was reduced by the decreased baking temperature.

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