Gametophytes of Equisetum arvense show interesting characteristics as experimental materials, for example, light dependent growth and apogamous production of sporophytic structure by exogenously applied cytokinin. Several attempts have been made for cryopreservation of the gametophytes, but successful results have not been obtained. In this study, we focused on the possibility of low temperature storage of Equisetum arvense gametophytes in unfrozen state. Our results showed the possibility of low temperature storage of this material as regrowth rate was retained after the gametophytes were exposed to low temperature (0℃) for 30 days. On the other hand, rice cells that were successfully cryopreserved in liquid nitrogen using prefreezing method did not show the positive results for the low temperature storage as regrowth ability was not recognized after exposure to 0℃ for 20 days.

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Experiments were conducted to confirm the importance of the hydrophobic chain length (n) of synthetic mono-tailed glycolipids for maintaining the enzymatic activity of a model protein, lactate dehydrogenase (LDH), during the freeze/thaw process. At low concentrations, mono-tailed glycolipids maintained the enzymatic activity of LDH solution after the freeze/thaw process. Above the threshold, the maintenance effect increased with an increase in n of the n-alkyl glucoside (n = 4, 6, and 8) in the presence of 0.001 to 1.0 mg ml^<-1> glycolipid concentrations, while 6-O-alkanoyl trehalose (n = 8 and 10) exhibited a similar tendency as n-alkyl glucosides. 6-O-myristoyl trehalose (n = 14) was an exception, possibly because of phase separation at subzero temperatures. When the slope of the logarithmical concentration vs. relative activity curve was calculated above the threshold concentration, a linear relationship was observed between the slope and n. This finding demonstrates the importance of designing an appropriate n for glycolipid protectants to effectively maintain enzymatic activity during the freeze/thaw process. In addition, structural dependence of the threshold on the sugar moiety was suggested.

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Nematodes have adapted to diverse ecosystems, including land, inland waters, the seabed, and even within the body of other organisms. However, as nematodes are very small (most species are less than several mm), and have limited mobility, they may be unable to escape threats to their survival, including low temperatures and desiccation. For example, the Antarctic terrestrial species of the free-living nematode Panagrolaimus davidi has developed cold tolerance strategies that enable it to withstand intracellular freezing. In the plant-parasitic potato cyst nematode Grobodera rostochiensis, the eggshell and cyst wall act as barriers to ice-nucleation so that it can withstand temperatures as low as -38℃ and survive the winter. Stem nematodes, foliar nematodes, and entomopathogenic nematodes tolerate desiccation by transitioning into a state of anhydrobiosis by producing substances such as trehalose, glycerol, and myo-inositol. Some individuals have even survived several years in a relative humidity of 0%. Studying such tolerance strategies will facilitate better understanding of the applicability of nematodes in agriculture, forestry, and fisheries.

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Choline acetate, which is one of ionic liquids, was examined for new cryoprotectant using differential scanning calorimetry. High concentration of choline acetate aqueous solutions raised the denaturation temperature of the lysozyme after the minimum at the low concentration. The fragility index and the dynamic glass transition temperature were determined and compared with those of dimethyl sulfoxide and glycerol, which are well known cryoprotectants. The fragility index of choline acetate aqueous solutions with lysozyme was nearly independent to the concentration of choline acetate. The reason was supposed to come from the ionic interaction among lysozyme, water and choline acetate molecules. From the comparison with the known cryoprotectants, choline acetate can be said to be a new good cryoprotectant and the practical experiments should be carried in the future.

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Group 3 late embryogenesis abundant (G3LEA) proteins are capable of protecting liposomes from desiccation damage. In our previous studies, a model peptide (PvLEA-22) which has two tandem repeats of the characteristic 11-mer motif found in G3LEA proteins was shown to have a similar protective function for dried liposomes. However, its underlying mechanism is still unclear. It has been hypothesized that PvLEA-22 could prevent direct membrane-membrane contacts by shielding the lipid membrane surface. In this study, to confirm the validity of this hypothesis, we performed molecular dynamics (MD) simulation combined with the so-called umbrella sampling method for a model system composed of PvLEA-22 and a membrane bilayer, and analyzed the free energy profile for the binding process of the peptide onto the membrane surface, and additionally the structure of the bound peptide. As a result, it was shown that the peptide is able to bind to the membrane with a relatively large binding free energy of 18 kcal/mol through a barrierless process, and it has a propensity to form a β-sheet-like structure on the membrane surface due to its characteristic sequence.

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Here we examined whether a short model peptide which has two tandem repeats of the 11-mer motif of a group 3 late embryogenesis abundant (LEA) protein has anti-aggregation activity against aggregation-prone proteins in cells. For this purpose, we transiently expressed GFP-fused polyQ in mammalian cells in which the model peptide had been constitutively expressed in advance. By fluorescence microscopic measurements, we counted the number of cells involving aggregated GFP-fused polyQ and evaluated the degree of anti-aggregation as a function of time. Simultaneously, we measured anti-aggregation activity of LEA proteins from the anhydrobiotic midge, Polypedilum vanderplanki. Positive control experiments were performed for cells expressing a LEA protein derived from the anhydrobiotic nematode Aphelenchus avenae. As a result, it was shown that the model peptide has the anti-aggregation activity comparable to the native LEA protein in cells.

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Cryopreservation to preserve biomaterial in a living state over the long term is an essential technique in various fields including medical and food engineering. However, intracellular freezing after extracellular freezing during cryopreservation of fresh vegetables with often used cooling rate causes lethal cell damage due to intracellular ice formation. Therefore, it is important to understand the intracellular freezing mechanism to improve the cryopreservation technique. This study was performed to clarify the characteristic intracellular ice crystal formation behavior in plant tissues using a high speed camera. The results showed that behavior clearly varied with cooling rate. The high speed camera images revealed the quantitative differences in the growth rate and grain diameter of intracellular ice crystals with respect to the cooling rate.

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A water solution placed in a freezer changes into a polycrystalline state of ice in keeping with the exclusion of the solute. This exclusion is attributed to growth of the ice crystals, as the growth only consumes water molecules, leading to concentration of the excluded solute into a lastly frozen area. This phenomenon was named "progressive freeze-concentration (PFC)", which is sometimes the problem for quality preservation of water-containing materials, such as foods and tissues. Here we examined whether antifreeze protein (AFP) works to prevent PFC, since AFP can bind specifically to ice crystals to inhibit their growth. For this purpose, we froze the AFP solution containing red-colored ink in a house-made PFC device consisting of a glass vessel sandwiched by heat insulating materials. The ink was concentrated near the bottom of a placoid ice prepared in the PFC device without AFP, while it was dispersed with AFP. A level of dispersion was dependent on the AFP concentration and ice crystal shape. These results suggest that AFP possesses inhibitory effect on PFC, which was presumably correlated with the known abilities of AFP, such as ice-shaping, thermal hysteresis, and ice recrystallization inhibition.

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The cryopreservation of nerve cells benefits the study with cells for medicine and poison screening. Recently, a carboxylated ε-poly-L-lysine (COOH-PLL) was found to have a remarkable effect as a cryoprotectant without any additional agents. This study investigated the effect of COOH-PLL on the post-thaw morphological changes of differentiated PC 12 cells adherent to substrate in physiological saline (PS) with different concentration of COOH-PLL. The results were compared with those of the authors' previous study for PS alone and PS with dimethyl sulfoxide, focusing on the post-thaw morphological changes of cells. The results showed that COOH-PLL is considerably effective to reduce the post-thaw morphological changes of neurites, depending on the concentration of COOH-PLL.

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Some polyphenolic compounds isolated in xylem tissues of woody plant, Katsura tree, showed anti-ice nucleation (supercooling-facilitating) activity in a solution containing ice nucleator such as silver iodide or ice-nucleation bacterial cells, resulting in maintenance of a supercooling state of the solution for a long time and decline of freezing temperature of the solution. Levels of these supercooling-facilitating activities were dependent on types of both the polyphenolic compounds and ice-nucleators used in the assay solution. To study the mechanism of supercooling-facilitating activity by these polyphenolic compounds, characterization of ice-nucleators should be necessary in addition to these polyphenolic compounds resulting in decline of freezing temperature. In the present study, a gene of ice-nucleation inaA protein, of Erwinia ananas was cloned and recombinant inaA protein with histidine-tag was expressed in Escherichia coli cells for further characterization. Freezing temperature of a cell suspension culture of E. coli cells transformed with recombinant inaA gene was higher than that of E. coli cells transformed with recombinant lacZ gene with histidine-tag, a negative control sample, suggesting acquirement of ice-nucleation activity by expression of recombinant inaA gene in transformed E. coli cells.

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Dextran turns into a gel form by an addition of potassium chloride. However, it does not gel by an addition of sodium chloride. The dextran gelation mechanism was investigated from observation of gelation by an addition of various chlorides such as CaCl_2, NaCl, SrCl_2, BaCl_2, KCl, RbCl, CsCl_2 and viscosity measurements, sol-gel transitions. From the results, gelation was observed by an addition of potassium chloride or rubidium chloride. Viscosity of KCl+dextran solutions was increased suddenly when potassium chloride concentrations were within the gel concentration. KCl+dextran gel was found to observe the sol-gel transitions.

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For the investigation of cryoprotective effect of glycine betaine (GB) on the nematode Caenorhabditis elegans and the Antarctic nematode Panagrolaimus davidi, we observed the freezing behavior and examined survival rate after freeze-thawing of the nematodes. When the nematode P. davidi was cooled slowly to -75℃ in the presence of GB (0.2 M - 1.0 M), most nematodes remained unfrozen and survived after thawing. However, when the nematodes were quenched with liquid nitrogen from -75℃, damaged nematodes were observed after thawing and survival rates were decreased. In the case of nematode C. elegans that was cooled slowly, percentage of the unfrozen nematodes and the survival rate after thawing increased as the GB concentration increased to 1.0 M. While the Antarctic nematode P. davidi was quenched by dipping into LN_2 from ambient temperature, the cryoprotective effect of GB was observed. When they dipped in 1.0 M GB were quenched with a cryo-plate, 73% of nematodes could survive after thawing. Thus, it was clear that GB has a cryoprotective effect on the nematodes.

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Effect of abscisic acid (ABA) on chloroplast relocation in gemmae cells of the liverwort Marchantia polymorpha was examined. Cold incubation of gemmae of wild-type plants in a liquid medium under dim light caused relocation of chloroplasts from the periclinal to anticlinal position, but presence of 1 μM or greater concentrations of ABA inhibited this movement as observed by light and fluorescent microscopy. Gemma cells of the transgenic MT-2 line overexpressing MpABI1A encoding a group A protein phosphatase 2C known as a negative regulator of ABA signaling were also used for chloroplast observation. It was found that the cold-induced chloroplast relocation was observed at any ABA concentration tested in MT-2, indicating that the cells exhibited ABA insensitivity. These results indicated that ABA specifically acts on inhibition of the chloroplast movement.

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