A general ice block is made of infinite number of single ice crystals, whose embryos are generated at the moment of freezing. Antifreeze protein (AFP) or antifreeze glycoprotein (AFGP) accumulates on the specific surfaces of such embryonic ice crystals, which suppresses their ice growth and stabilizes a supercooled solution. This mechanism has been thought to provide cold-tolerance for an organism, and realize new freezing preservation techniques of various water-containing materials, such as processed foods, gels, noodles, doughs, seeds, fruits, vegetables, ice creams, polymers, medicines, cosmetics, cells, tissues, and organs. If the proteins can make the ice crystal size ultimately small, they will significantly improve the effectiveness of the preservations. In addition, many AFPs bind to the lipid bilayer to prolong lifetime of cells under hypothermic condition (+4°C), for which applicability to unfrozen, short-term cell preservation can be expected. The author has therefore been tried to clarify the functional mechanism of AFPs and AFGP, and further developed their mass-preparation method to find their usefulness in both industrial and medical fields. This article provides advanced knowledge obtained through the works mainly performed by the author.

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Gel-forming ability is the most important quality standard for frozen surimi. In Japan, breaking strength and breaking strain are used as indicators of gel formation. However, these values should be considered as independent indices for analyzing gel formation. Currently, the relationship between gel stiffness (breaking strength divided by the breaking strain) and breaking strength is used as a qualitative evaluation of the gelforming ability of surimi. We have attempted a novel quantitative evaluation of the gel-forming ability using the length of the line (L value) in the diagram between breaking strength and gel stiffness. This evaluation method can be used as an indicator of the overall properties of the gel, as it includes breaking strength and breaking strain components. Additionally, the gel-formation rate was determined by dividing the L value by the setting time.

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Polyvinyl pyrrolidone (PVP) has been used as a cryoprotectant agent for the cryopreservation of cells. Cell viability of C3H10T1/2 cells after freeze/thaw treatment improved with increasing PVP concentration below 50 wt%. It was revealed that the intracellular brightness became brighter as the PVP concentration increased using optical microscope observation at -70 ºC, indicating that the size of intracellular ice became smaller. The differential scanning calorimetry (DSC) experiment for cell pellets revealed that the amount of intracellular ice was reduced in PVP 50 wt% solution. The cubic-form ice (Ic) was formed in intra-cell, instead of hexagonal-form ice (Ih), as seen using the X-ray diffraction (XRD) experiment for cell pellets extracted from PVP 50 wt% suspension. And the crystallite size of intracellular ice trended to decrease as the PVP concentration increased. Furthermore, it was concluded that the crystallite size of intracellular ice was smaller than that of extracellular ice in the 50 wt% PVP solution. On the other hand, the huge ice was formed through the intra- and extra-cell in 0 wt% (without PVP) solution.

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In this short review, we introduce various aspects of insect body lipids, which have been progressively found to have many crucial physiological roles. The body-surface of insects is covered with cuticular lipids, mainly hydrocarbons. The cuticular hydrocarbons are synthesized in specialized cells called oenocytes residing beneath the epidermis. The hydrocarbons synthesized are transported to the outermost layer, wax layer, by the circulating lipoprotein, lipophorin. Although the principal role of the wax layer is the protection of internal body from desiccation, the essential features vary depending on insect species and development stage. The diapausing pupae of large and small cabbage white butterflies change the thickness and unsaturation of their cuticular hydrocarbons from their non-diapausing ones. The cuticular hydrocarbons also contribute to chemical communications. The unsaturated hydrocarbons of male crickets play an important role for sex discrimination before copulation. Modern in-situ surface analytical methods have a potential to provide more abundant and precise information about the structure and physicochemical properties of the cuticular hydrocarbons. Recent ATR FTIR spectroscopic studies indicated the necessity of correction in the phase-separation model of cuticular hydrocarbons proposed by A. G. Gibbs.

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Supercool preservation method for biological tissues has attracted attention, because the supercooled state can reduce physical damage of living cells and tissues. There are many reports that living tissues could be preserved in a solution containing cryoprotectant at a supercooled state. However, the solution with cryoprotectant has risks of cytotoxicity for living cells. Therefore, the supercool preservation method using a solution with lower cytotoxicity is required. In this study, we focused on the supercool preservation using a cell culture medium without cryoprotectant for living cell and tissue preservation. For a fundamental study, the living cells suspended in the culture medium without cryoprotectant were subjected to the supercooling condition to elucidate the effect of supercooling preservation on cell viability and proliferation. Specifically, a custom-made supercooling device to preserve cell-suspended solution was developed. Human neonatal fibroblasts were suspended in culture medium and stored inside the device at -4°C and -8°C of supercooling state for 24 hours. As a result, there was no significant difference in the survival rate after supercool preservation due to the difference in the supercooling temperature. On the other hand, both cell adhesion and cell proliferation rate were higher in a supercooled state at -4°C than stored at -8°C.

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Autophagy plays an important role in maintenance of cellular homeostasis and nutrient translocation under normal condition and is up-regulated under abiotic and biotic stress for degradation of damaged or dysfunctional cell constituents in plants. Several lines of evidence indicated that treatments of salt and heat stress on plants induce ROS production leading to activation of autophagy for removal of potentially highly toxic denatured proteins in mesophyll cells. In this study, we examined whether autophagy is involved in protection of photosynthesis activity of soybean leaf under chilling by monitoring a photosynthesis parameter and ROS production. Photosynthesis System II activity (Fv/Fm) and SPAD values significantly decreased under chilling and light irradiation whereas those values were affected marginally under chilling and dark. Significant reactive oxygen species (ROS) production were detected by diaminobenzidine (DAB) and nitroblue tetrazolium chloride (NBT) staining in soybean leaf under chilling and light irradiation but not under chilling and dark, indicating that excess ROS production including H2O2 and O2 - damaged photosynthesis activities in soybean leaf. Reduction of Fv/Fm levels in chilling and light irradiation-treated soybean leaf were enhanced in the presence of an autophagy inhibitor 3-metyladenine, but alleviated in the presence of an autophagy activator, acetyl carnitine, compared with that in the absence of those reagents. These results indicated that autophagy is involved in maintenance of photosynthesis activities in soybean leaf damaged by ROS produced under chilling and light irradiation.

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Cryopreservation (also called as cryobanking) of gel-embedded shoot tips on the cryo-plate dehydrated by vitrification solution (V cryo-plate method) was examined for saving plant resources of hybrid aspen. Cold-hardened shoot tips of in vitro grown hybrid aspen were precultured for 1 day at 25℃ on solidified 1/2 Murashige and Skoog (MS) medium containing 0.3 M sucrose. Pretreated shoot tips were placed in elliptical wells on an aluminium cryo-plate and embedded in calcium alginate gel. Osmoprotection treatment was performed in a loading solution containing 2.0 M glycerol and 1.0 M sucrose for 30 min at 25℃. After the shoot tip samples on the plates were treated with the plant vitrification solution 2 for 20 min at 25℃, the samples on the plates were directly plunged into liquid nitrogen. For rewarming, shoot tip samples on the cryo-plates were immersed in 1.0 M sucrose solution at 25℃. Then, shoot tips in the gels were regrown at 25℃ on solidified 1/2 MS medium. The regrowth rate of cryopreserved shoot tips was almost 100%, and normal shoot growth was observed without callus formation. This protocol appears to be a promising technique for cryobanking of hybrid aspen genetic resources.

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Cold acclimation (CA) results in alterations of plasma membrane (PM) and cell wall proteins in plants, which is considered to be crucial for increasing their freezing tolerance. Fasciclin-like arabinogalactan proteins (FLAs) are abundant glycoproteins in the PM and cell wall and have potential roles in abiotic stress responses via transducing signaling and modulating cell wall functions. In present study, we investigated temporal expression of 21 FLA genes during CA in Arabidopsis thaliana. We found that there are a variety of changing patterns among FLAs in response to CA. Principal component analysis clearly showed that expression of FLAs and its responses to CA varied considerably between shoot and root. FLAs2, 8 and 13, the most abundant FLA genes, exhibited increases of their expression in the early stage of CA, but its extent was greatly different between shoot and root. Taken together, we will discuss about the potential role of FLAs in response to low temperature in plants.

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Three pure trehalose fatty acid monoesters (TMs) with different fatty acid substitutions and their mixtures were assessed for their ability to preserve a lactate dehydrogenase (LDH) activity during freeze/thaw processes. 6-O-dodecanoyl trehalose (TM12) exhibited a prominent effect for preserving LDH activity. 6-O-hexadecanoyl trehalose (TM16) and 6-O-oleoyl trehalose (TM18:1), were less effective, possibly because of the tendency for TM16 to precipitation during freeze/thaw cycles and a lower surface tension reduction ability for TM18:1. TM18:1 was about as effective as TM16. Notably, the gradual replacement of TM12 with TM18:1 dose-dependently reduced the ability of TMs to preserve LDH activity, and no significant mixing effect was observed, whereas replacement with TM16 afforded the positive deviation from the expected curve. Inhibition of TM16 precipitation during freeze/thaw processes by TM12 might facilitate TM16 performance. The effectiveness of a mixing strategy of the stabilizer was confirmed.

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Cowpea [Vigna unguiculata (L.) Walp.] is highly adapted to drought conditions and avoids water loss from leaf by closing stomata via abscisic acid (ABA) signaling. Recently, molecular genetic studies in Arabidopsis have revealed that the ABA responses are mediated by various long-distance signals and ABA transport mechanisms. However, it remains unknown whether the transport mechanisms of ABA are involved in significant drought tolerance of cowpea. Here we identified a set of ATP-binding cassette (ABC) transporter gene candidates, VuABCG22, VuABCG25 and VuABCG40 by searching for ABA transporter homologs on cowpea genome database. One VuABCG25, two VuABCG22 and four VuABCG40 were identified in cowpea genome, while those corresponding homologs are single genes in Arabidopsis genome. Semi-quantitative RTPCR analysis indicated that water stress including drought and salinity significantly induced up-regulation of VuABCG25 expression but not VuABCG22 and VuABCG40. Three genes out of four VuABCG40 genes are found in a gene cluster at the chromosome 1 in the cowpea genome. These results strongly suggest that ABArelated ABC transporter genes are highly conserved between cowpea and Arabidopsis and that VuABCG25 is involved in the ABA signaling pathway in cowpea under water stress.

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