The antifreeze glycoproteins [AFGPs] are large molecules with molecule weights of 2.6-33 kDa, which exhibit prominent antifreeze property and enable their organisms to survive in below the melting point of water. While AFGPs have gained much attention in recent years, their availability is highly limited to the isolation from natural resources. Although the total syntheses of AFGPs have been reported, the synthetic methods have been limited due to their inherent gigantic molecular structures. Thus the development of efficient synthetic alternatives of AFGPs with middle molecules is highly desirable. We herein design the novel candidates of antifreeze molecules consisting of galactose-conjugated fluorinated and non-fluorinated proline oligomers. They were designed based on the PP II helix with hydrophilic and lipophilic parts, which could induce the potential antifreeze property. The target galactose-proline oligomers were synthesized, and their physical properties were evaluated. Despite our expectation, the non-fluorinated galactose-proline oligomers showed weak antifreeze activity in contrast to the fluorinated analogues. The difference in antifreeze activity would be attributed to the fluorine gauche effect, which induces a different conformation in fluorinated prolines from that of natural proline. These results suggest that the 3D structures of the galactose-conjugated fluorinated and non-fluorinated proline oligomers are critical for their antifreeze properties.

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Water in biological or soft materials, such as cells and foods, is essential for the sustainability of materials because all the biomolecules require water to keep their molecular structures through the interaction with water molecules. “The state of water” drastically changes during freeze- or dry-preservation of these materials. Thus, the functions of such materials, including enzymatic activity, proliferation ability and texture (mechanical properties) are strongly affected by “the state of water” in the materials. In this short review, the methods for measuring the dynamic state of water molecules, the relaxation time of molecular rotation, by dielectric spectroscopy and the static state of water molecules, the number ratio of hydrogen bounds by near-infrared spectroscopy are introduced. Using ε-poly-L-lysine, Jurkat cell and gelatin gel as samples of biological or soft materials, the relationships between these measured “states of water” and the freezing characteristic of materials, including ice nucleation rate, recrystallization speed, the amount of unfreezable water and water activity are discussed.

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Hydration and dehydration of bio-macromolecules result in changes in the physical properties and thus in the structural stabilization and biological functionality. The structure and dynamics of the vicinity of water molecules with biomolecules, which is so-called hydration water, are essential to understand the hydration and dehydration processes of a biosystem, such as cell and organisms. Because neutrons have a wavelength on the order of Å and thermal energy (order of meV), we can use them to observe the structures and dynamics of biomolecules and hydration water. Neutrons are highly permeable and non-destructive probes. The large difference in neutron scattering cross sections between hydrogen and deuterium provides a powerful method for emphasizing the scattering from a biomacromolecule or hydration water via selective deuteration of hydrated biological materials. Incoherent neutron scattering (INS) and small-angle neutron scattering (SANS) can be used to examine the dynamics of hydration water in the pico-to-nanosecond time scale and the density and structure of the hydration shell around biomolecules. Neutron sources, such as the JRR-3 reactor and J-PARC accelerator, are available in Japan. The present review aims to provide the readers with brief descriptions of the neutron scattering methods and their applications to protein hydration.

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Suspension-cultured cells of Oryza sativa and Celosia cristata were cryopreserved by the pre-freezing method. Dimethyl sulfoxide (DMSO), which is often used as a cryoprotectant, is cytotoxic. Therefore, in this study, we examined the effect of carboxylate polylysine (PLL) as a cryoprotectant in replacement of DMSO. It was found that cryopreservation in Oryza sativa with 5% ethylene glycol and 3.5% PLL resulted in significantly higher survival rates than that with 5% (v/v) DMSO and 10% (w/v) glucose and in Celosia cristata with 10% (w/v) glucose and 3.5% (w/v) PLL resulted in nearly survival rates that with 5% (v/v) DMSO and 10% (w/v) glucose. Our results indicate that PLL is effective as a cryoprotectant for cryopreservation of cultured plant cells.

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The effects of freeze-thaw cycles on the gel-formation ability of threadfin bream frozen surimi were studied. The rheological properties of the gel were measured by rheometer. The rates of cross-linking reaction of myosin heavy chain (MHC) of the gel were estimated by SDS-PAGE. Myosin head denaturation of surimi was assessed by measuring its myofibrillar Ca-ATPase activity. Freeze-thaw cycles caused slightly decreasing in Ca-ATPase of the threadfin bream frozen surimi. However, breaking strength, breaking strain and MHC cross linking reaction were hardly changed by freeze-thaw cycle. From these results, it was found that threadfin bream frozen surimi was less affected by the freeze-thaw cycle on gel-forming ability.

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Freezing of extracellular spaces is the first event when plants are exposed to freezing temperatures, but mechanisms of ice nucleation and propagation remain poorly understood. Japanese cold hardy dwarf bamboos grow in one of the northernmost areas of bamboo distribution and are known to employ deep supercooling in most living tissues as the freeze survival strategy. Studies have been performed on mechanisms of their deep supercooling capability. However, little is known about the ice nucleating factors which may initiate freezing at the apoplast of the vascular and epidermal tissues. This study focused on ice nucleation activity (INA) in aploplast extracts from cold hardy bamboo species, Sasa kurilensis, S. nipponica and Sasamorpha borealis. We obtained apoplast extracts from leaf blades and culms and determined their INA following size fractionation using membrane filters. The apoplast extracts from leaf blades and culms had highly extractable and particulate INA (size: 10 kDa<<0.2 μm). INA of substances larger than 300 kDa (<<0.2 μm) were similar to the freeze initiation temperatures of the vascular and epidermal tissues. Results of heat and protease treatments indicated that the major substance(s) responsible for ice nucleation in the apoplast extracts is proteinaceous, irrespective of the seasons, tissues or species.

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Carboxylated poly-L-lysine that is one of the polyampholytes, has good cryoprotective properties as a novel cryoprotectant. However, the mechanism is unknown. We investigated whether the polyampholytes could suppresses osmotic damage into cells at a high osmotic pressure condition that mimics freeze concentration. It was found that polyampholytes reduced dehydration stress when frozen at high osmotic pressure. Furthermore, by comparing the cell size at the time of freezing, it was suggested that the polyampholytes might promote cell dehydration and protect the cell membrane.

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Under external stress, many organisms accumulate sugars to protect cells and biopolymers. Sugar solutions are well known to suppress protein denaturation and aggregation. The preferential hydration of protein in sugar solution has been considered to be a major driving force toward the stabilization of protein structure. These discussions have been based on the studies using densitometry, calorimetry, spectroscopy, etc. There are few studies utilizing neutron or X-ray scattering methods, that are applicable to observe the protein hydration-shell. We have obtained the structural evidence of the effect of sugars on the protein hydration-shell by using small-angle neutron scattering and synchrotron-radiation small-angle X-ray scattering.

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Larvae of the sleeping chironomid, Polypedilum vanderplanki, have an ability of desiccation tolerance, so-called anhydrobiosis. One of the factors that contribute to desiccation tolerance is trehalose. The larvae synthesize and accumulate high levels of trehalose in the fat body during the desiccation process. TRET1 (Trehalose Transporter 1), a facilitated trehalose transporter from P. vanderplanki, transports trehalose into the hemolymph. However, the mechanism for the physiological contribution of TRET1 in the desiccation process is unknown. Here we addressed to analyze the physiological role of TRET1 in a desiccation-tolerant cell line, Pv11. We attempted to establish Pv11 cell lines knocked out Tret1 gene (Pv11-Tret1-KO cells) by a genome editing technology. As a result, Pv11-Tret1-KO cells did not proliferate in the normal growth condition. The result was attributed to the fact that Tret1 was highly expressed before the desiccation process. Thus, TRET1 is thought to play an important role not only in the desiccation process but also in the survival of Pv11 cells during normal growth. Therefore, it will be necessary to develop a time-specific conditional knockdown technology for the functional analysis of this transporter.

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Larvae of the anhydrobiotic midge Polypedilum vanderplanki show the extreme desiccation tolerance. Recently, Pv11 cell line was established from Polypedilum vanderplanki embryos. Pv11 cells exhibit the desiccation tolerance when treated with trehalose before desiccation. During trehalose treatment, the expression level of various genes drastically changes. It is suggested that trehalose stimulation is transmitted to cells by signaling activation. Nevertheless, it is unclear which second messengers work during trehalose treatment. We can evaluate the second messenger behavior by live-cell imaging using sensor proteins. However, the live-cell imaging for Pv11 cells is difficult because Pv11 cells are floating cells. Here, we introduce the live-cell calcium imaging method in Pv11 cells by immobilization with Biocompatible Anchor for cell Membrane (BAM). We established the Ca²⁺ indicator stable expressing Pv11 cell line (Pv11-GCaMP6f). Then, we immobilized Pv11-GCaMP6f with BAM and added the calcium ionophore by using perfusion system. Finally, we succeeded to develop the method for real-time Ca²⁺ imaging at a single cell level.

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Although cowpea (Vigna unguiculata (L.) Walp.) is known as one of the drought-tolerate crops with various strategies coping with water deficit, it is very sensitive to the drought stress in the flower bud developing stage. In this study, we planned to explore how these flower bud abscission in cowpea under drought stress is regulated through the ABA and ethylene. Drought stress enhanced the rate of flower bud abscission and ethylene functions to promote it vice versa ABA. Gene expression of ABA responsible element binding factor 1, AREB1 was induced at 6 days after drought treatment and ethylene responsive factor 1 (ERF1) was induced at 9 days after drought treatment. Drought stress enhanced both endogenous ABA and ethylene productions, and especially endogenous ABA content was enhanced by ethylene inhibitor treatment. In addition, ABA treatment suppressed endogenous ethylene production in both the well-watered flower bud and the drought stressed one. These results indicate that ABA and ethylene interaction regulates responses in intact flower buds to drought stress.

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Human pluripotent stem cells are expected to be a valuable resource in the field of cell therapy and drug discovery. Cryopreservation is a key technique to realize these applications which will require large cell numbers. However, the common protocols for the cryopreservation including dissociation into single cells have many steps with technical difficulties. Here, we report on an efficient method for the cryopreservation with the cells maintained adherent on culture dishes to save work. In order to establish the method, we employed the processes for the avoidance of extreme undercooling and determined the optimum cooling rate.

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Amber is a unique biopreservation organic material in which ancient insects, animals, and plants were fossilized for a few tens of millions of years. The pressure-dependence of the elastic properties of the Baltic amber was studied using a micro-Brillouin scattering system and a diamond anvil cell up to 12 GPa. As the pressure increases, the sound velocity of longitudinal acoustic (LA) wave rapidly increased in the low-pressure regime and became gradual increase at high-pressures. The pressure dependence of the LA velocity is discussed based on the excess vibrational density of states of a glass.

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The aim of this study was to investigate the vapor pressure measurement method of foods at sub-zero temperature to understand the process of drying of foods in frozen storage. Bovine serum albumin (BSA) was used as a sample in this study. Freeze-dried (FD) BSA and heat-denatured (HD-FD) BSA were also prepared for the measurement. Further, a humidity probe that can function at sub-zero temperature was added to the prepared measurement system. The vapor pressure of the BSA samples was measured at 25°C and –18°C. The moisture adsorption isotherm of BSA obtained at –18°C was similar to the one at 25°C. Moreover, the water vapor pressure of the samples at sub-zero temperature showed the same behavior as at 25°C. The moisture adsorption isotherms of FD and HD-FD samples also exhibited the same characteristics as those of the BSA. Therefore, a basic method can be devised to explain the drying mechanism of frozen foods by combining the results of this study.

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The cysts of Artemia franciscana (A. franciscana) exhibit intense tolerance to desiccation and freezing. However, when cysts are hydrated, these resistances to the extreme environments are drastically impaired. In this report, we investigated into improved membrane permeability of the decapsulated cysts by the treatments of synthetic cationic polymer and several amphipathic anionic surfactants. We found here that poly-allylamine, synthetic cationic polymer, was to be relatively effective on the embryonic membrane permeability of hydrated cysts. This reagent could prove useful when introducing alternative endogenous factors that assist their stress-resistant functions in future studies.

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