Freeze-concentration- crystallization of L-phenylalanine was carried out by applying progressive freeze -concentration crystallization. Concentration effectiveness was affected by the advance rate of ice front and the stirring rate at the ice-liquid interface as expected by the concentration polarization model. When the concentration of L-phenylalanine reached the saturation concentration, it began to crystallize proving the feasibility of the simultaneous process of freeze concentration and crystallization. Crystals of L-phenylalanine thus obtained were needle-shaped and easily separable from the concentrated mother solution. The present method seems to be the best method for purification of such thermally unstable materials as medicines and food components.

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The temperature is one of the key environmental factors surrounding the living organisms in nature. In plant cells, transient increase in cytosolic Ca^<2+> concentration ([Ca^<2+>]_c) occurs as a response to an acute temperature change. Such a rise in [Ca^<2+>]_c reportedly leads to cold acclimatization. Here, we examined the effect of the temperature- shifts from ambient to the lower or higher temperatures, on induction of [Ca^<2+>]_c increases, using suspension-cultured plant cell lines expressing aequorin, a luminescent [Ca^<2+>]_c-reporter. The cultures used here include three tobacco (Nicotiana tabacum L.) cell lines, namely BY-2, Bel-W3 (O_3-sensitive) and Bel-B (O_3-tolerant), and rice (Oryza sativa L., cv. Nipponbare) AQ7 cells. Generally, rice cells and tobacco cells behaved similarly. The levels of [Ca^<2+>]_c spikes induced by cold shock were much greater than those induced by heat shock in all cell lines tested. Effects of inhibitors suggested that the cold shock-induced [Ca^<2+>]_c spike requires the uptake of extracellular Ca^<2+> via plasma membrane Ca^<2+> channels, while the heat shock response might be due to channel-independent leakages of extracellular and organelle Ca^<2+> into cytoplasm.

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The freeze-thaw behaviour of 10-60% glucose solutions was observed with the aid of Differential Scanning Calorimetry (DSC), cryomicroscopy and X-ray diffractometry. The results from DSC revealed that two exothermic peaks were observed in 51-55% glucose solution during cooling. The X-ray diffraction measurement illustrated that these two exothermic peaks in cooling are the ice crystal. The ice crystal in the first exotherm is the mixture of hexagonal and cubic ice. The recrystallization in the highly concentrated glucose solution is in the cubic form and consequently transform to hexagonal form during melting period.

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Application of near-infrared spectroscopy to the analysis of freeze-dried pharmaceutical formulation was studied. Diffuse-reflection measurement from the bottom of glass vials allowed non-destructive analysis of component crystallinity and residual water in freeze-dried solids. Transmission NIR spectra of amorphous sorbitol solid showed reduction of intermolecular hydrogen-bonding OH groups at elevated temperatures. Second-derivative NIR spectra of proteins in aqueous solutions and freeze-dried solids suggested dehydration-induced perturbation of the secondary structures.

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Vitrification provides a general solution to the problem of organ preservation, because it allows to avoid both the direct and indirect damaging effects of freezing. Generally, water in the cell crystallizes below a melting point, and ice crystals cause destruction in the cell. This is a serious problem on freeze preservation. Cryoprotectant is a material that enables cells to cryopreserve. Ethylene glycol ((HO)CH_2CH_2(OH), EG) aqueous solution is well known as cryoprotectant. Fahy et al. reported the benefit of substituting the weaker glass-former EG to the strong glass-former 1,2-propanediol on toxicity in their solution. Therefore, investigating the nature of a glass transition of EG aqueous solution is very important. Using Modulated Temperature Differential Scanning Calorimetry, MDSC, we investigated thermal relaxation around a glass transition temperature (T_g) on EG solution (20, 30, 40, 50, 60, 70, 80 mol%). The underlying heating rate was 0.5 ℃/min, modulation amplitude ± 0.5 ℃, modulated period 100, 60, 40 s.

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The terahertz spectroscopy is a powerful tool for the study of molecular motions at the terahertz region which links the dielectric relaxation and oscillatory, intermolecular motions. Terahertz spectroscopy provides an efficient method to obtain the frequency dependent optical parameters in a previously difficult region to investigate. To understand the terahertz dynamics of well known glass former, ethylene glycol, the terahertz time domain spectroscopy (THz-TDS) was applied to ethylene glycol aqueous solutions. The frequency dependent optical parameters, refractive indices, absorption coefficient and the complex permittivity in the terahertz region were measured with femtosecond terahertz pulse transmission spectroscopy. The use of various lengths of samples achieved good spectral resolution even in samples of low absorption.

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Fluorescence correlation spectroscopy (FCS) was carried out for Rhodamine 6G (R6G) in aqueous solutions of saccharides. The autocorrelation function, G(t), exhibited binary decay. The slower and faster decays were ascribed to the diffusion of R6G molecules and to the fluorescence quenching due to intersystem crossing to the excited triplet state, respectively. The triplet state lifetime elongated in solutions with higher saccharide concentration, indicating slowdown of the oxygen diffusion.

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The dry mixtures of calf thymus DNA (CT-DNA) with trehalose, neotrehalose, maltose and sucrose have been prepared by air-drying their aqueous solution. The dry CT-DNA, which normally exists in coiled or single-stranded structure, has been found to persist in double-stranded structure when imbedded in the four disaccharides. The double-stranding of dry CT-DNA should be resulted from the 'disaccharidation' effect of disaccharides, i.e. the interaction of disaccharides with the phosphate groups and possibly other polar groups of CT-DNA.

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Trehalose acts as a good protectant against various water stresses such as desiccation and freezing. The water replacement hypothesis has been proposed as a possible mechanism by which trehalose protects biological components such as membrane and proteins in low water activity. Here, to investigate this hypothesis at atomic resolution, molecular dynamics simulations were carried out for trehalose/phospholpid bilayer systems, where the bilayer was constructed from 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC). Trehalose molecules were dissolved in the interbilayer medium at a concentration of 0.133 mol/kg or 1.220 mol/kg. After a 10 ns simulation for each system, we analyzed the hydrogen bond network formed among trehalose, water and the head group of POPC. In consequence, it was found that a part of the trehalose molecules directly hydrogen bond to the head group of POPC without being interrupted by any water molecules and that the stability of the lipid bilayer increases with an increase in trehalose concentration. These results support the validity of the water replacement hypothesis.

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Among the naturally occurring gluco-disaccharides, trehalose has the highest glass transition temperature (Tg) and the largest activation energy of enthalpy relaxation in the glassy state. These properties are very favorable as a desiccation protectant. Here, to understand at molecular level why trehalose has such unique properties, we carried out molecular dynamics simulations for the glassy states of trehalose and neotrehalose. By comparing these results, we revealed the following points: 1) there is no significant difference in the average number of intermolecular hydrogen bonds between the glassy state of trehalose and that of neotrehalose, 2) the translational motion and the void volume in the glassy matrix both are smaller in trehalose than in neotrehalose, and 3) the glycosidic bond of trehalose takes a single conformation but that of neotrehalose has more than two. These results suggested that the higher Tg and higher stability of trehalose glass is due to the increasing packing density of the glassy matrix, which originates from the conformational simplicity of this sugar, a characteristic feature of α, α-1,1-linkage.

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In the present study cellular states of dried larvae of Polypedilum vanderplanki were investigated with Fourier transform infrared spectroscopy (FT-IR) under temperature-controlled conditions. Two different types of dry samples were prepared. One was a slowly dried larva (slow sample), being able to survive desiccation, and the other one was a quickly dried larva with, as a result, little or no accumulation of trehalose and failed to acquire anhydrobiotic ability. Throughout analyzing the temperature dependence of the P=O anti-symmetric stretching band and CH_2 stretching one, respectively, it was found that the slow sample kept its cellular membrane in the liquid crystalline phase close to the ordinary viable states, which would be one of the physicochemical conditions required for successful anhydrobiosis, whereas for the quick sample the cellular membrane was in the gel phase at room temperature. A combination of these findings with our previous study which demonstrated the requirement of glassy endogenous trehalose for successful anhydobiosis leads to the conclusion that the vitrification hypothesis has priority over the water replacement hypothesis to rationalize the desiccation tolerance ability of, at least, higher animals.

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We have found that desiccation tolerance of Marchantia polymorpha cells is increased by preculture in a medium with 0.5 M sucrose. During this preculture, sucrose accumulated and expression patterns of proteins changed in the cells. Desiccation tolerance had not fully developed when protein synthesis was inhibited by 10 μM cycloheximide (CHX) during preculture with sucrose. Glass transition temperature (Tg) of the precultured cells was measured using differential scanning calorimetry (DSC). Compared with that of precultured samples, Tg of precultured samples with CHX shifted to a low temperature. The results suggested that newly expressed proteins during preculture are not only involved in the development of desiccation tolerance but also influence Tg in suspension-cultured cells of M polymorpha.

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Many types of plant cells accumulate low molecular-weight soluble sugars when they acquire freezing tolerance in the process of cold acclimation. Protonema cells of the moss Physcomitrella patens develop freezing tolerance upon treatment with the phytohormone abscisic acid (ABA). We previously reported that the ABA-treated protonema cells accumulate low molecular-weight soluble sugars such as sucrose and theanderose. To clarify mechanisms underlying physiological processes leading to development of freezing tolerance, we isolated F patens mutant lines, AR1 to AR7, with reduced sensitivity to ABA. Among these lines, AR7 showed growth similar to wild type but did not develop freezing tolerance by ABA treatment. Analysis of low molecular-weight soluble sugars indicated that the AR7 cells accumulated sucrose upon ABA treatment but very little of theanderose.

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Culture system of Pogonatum inlexum was established using C-1 medium containing 3% sucrose, 5×10^<-6>M 2,4-dichlorophenoxyacetic acid and 1×10^<-5>M 6-benzyl aminopurine. The cultured cells were desiccated rapidly or slowly and then cryopreserved in liquid nitrogen. Almost 100% cells survived desiccation and cryopreservation. The desiccated cells survived room temperature for 1 month. As the preculture was not needed for the desiccation and preservation, it was considered that they have high desiccation tolerance naturally.

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Behavior of ice crystals and cells in the biological tissues during the slow-warming after rapid-cooling was visualized microscopically in time-series using a confocal laser scanning microscope with a fluorescent dye. Recrystallization of intracellular ice crystals to increase the mechanical damage of the tissues was investigated quantitatively. Size and number of the ice crystals were measured from the image-data of ice crystals and analyzed statistically to obtain in time-series the frequency, average, and standard deviation of the size of ice crystals, total amount and number density of ice crystals, etc. Influence of the aqueous solution (influence of the additive to the physiological saline) on these characteristics was made clear.

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We have screened the antifreeze material-producing fungi originated from Antarctica. Among 40 strains, 7 strains of Antarctic fungi had clearly the antifreeze activity that exhibit the change of ice crystal morphology and low level of thermal hysteresis. Among 7 strains, one strain with high level of antifreeze activity has identified as one strain of Penicillium bilaiae. The optimum culture condition for the production of antifreeze material by this strain was the culture in the NB medium at 18℃ for 2 weeks. Also, as this strain found to have low level of antifreeze activity after culturing at 30℃ for 2 weeks, we can predict that this antifreeze material is different from the structures of other antifreeze proteins. This difference was supported by the resistance of protease treatment and heat treatment, the fractionation using dialysis membrane. Furthermore, this antifreeze materials had the high stability after treating at 100℃ for 1 hr, and had instability under alkaline solution.

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For the establishment of successful cryopreservation of C. elegans, the response of the nematode to low-temperature stress was investigated in the L4 stage of the life cycle. Pumping for feeding indicated by pharynx was observed under a cryomicroscope, and its frequency was used as an indicator of mobility. Pumping frequency decreased with lowering temperature to stop below 2℃, and it showed thermal hysteresis with higher mobility in the cooling process than the heating process. When kept at 5℃ for cold acclimation, mobility at low temperatures more or less recovered with the time of preservation. However, optimum recovery was indicated at ca. 10 days, and mobility lowered with the further preservation time. On parallel with the increase in the mobility, accumulation of trehalose was indicated during cold acclimation.

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The object of this study is to clarify the responses of wintering plants that were re-grown under light / dark condition after treatment of simulated acid snow (SAS) stress with sulfuric acid solution. Fresh weight and relative water content of mature leaves were markedly decreased compared with those of younger leaves during a re-growth period of wheat pretreated by SAS (pH 2.0). Photochemical efficiency of mature leaves was decreased during a re-growth period over 24 hours, but recovered in younger leaves. The level of lipid peroxidation in mature leaves was higher than that of younger leaves during a re-growth period within 24 hours.

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Freezing tolerance is an important character for plants living under subzero temperatures in winter season. The growth of ice crystals or the freeze-induced dehydration mechanically may disrupt the plasma membrane. Many studies have indicated that the freezing tolerance is correlated with the cryostability of plasma membrane. Although, in animal cells, the mechanically disrupted plasma membrane is rapidly repaired dependently on the extracellular calcium, no report has been published on the plasma membrane repair in plant cells. It is expected that the cryostability of plasma membrane is tightly associated with the membrane repair manner. We studied the calcium-dependent survival of plant cell for mechanical stresses using Arabidopsis protoplasts isolated from control and cold-acclimated leaves. The tolerance to electric shock by the electroporator treatment which directly disrupted plasma membrane was also dependent on the extracellular calcium. The enhanced freezing tolerance was remarkably dependent on the extracellular calcium during freezing. Interestingly, the tolerance to dehydration caused by hyperosmotic solution was hardly dependent on the extracellular calcium. Finally, we estimate that the tolerance to mechanical stress caused by the ice crystal growth is dependent on the extracellular calcium.

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In many temperate plants, cold acclimation, which is achieved by exposure to non-freezing, low temperatures, results in a significant increase in freezing tolerance as a consequence of the physicochemical stabilization of the plasma membrane, which is the primary determinant of the cell survival under freezing conditions. Although the causal relationship between an increase in freezing tolerance and changes in plasma membrane lipid composition during cold acclimation has been well established, there have been only a few studies on the role of plasma membrane proteins in acquirement of freezing tolerance To investigate it, we have determined the effect of cold-responsive plasma membrane proteins on the freezing tolerance of Arabidopsis plants. One of the cold-responsive plasma membrane proteins that has domains with high similarity to proteins in the lipocalin family (Arabidopsis lipocalin, AtLCN) was chosen. Transgenic Arabidopsis plants that overexpress AtLCN showed enhanced freezing tolerance compared to wild type plants. Using protein-lipid overlay assay, we found that recombinant AtLCN protein had a high affinity for acidic phospholipids found in the plasma membrane. Taken together, AtLCN protein may contribute to increase in freezing tolerance by accumulating adjacent to the plasma membrane and binding to the acid phospholipids.

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Effect of ascorbic acid (AsA) in germination was investigated in the seeds of two barley (Hordeum vulgare L.) cultivars, cvs. ELICE and Seijou17. Germination of ELICE seeds treated by (AsA) at 25℃ was about 80% at 7 days after treatment (DAT). On the other hand, germination of Seijou17 seeds was about 20% at 7 DAT. Germination of ELICE seeds treated at 14℃ also was about 90%, and those of Seijou17 was about 40% at 7 DAT. These results indicated that the sensitivity to AsA was significantly different in the two cultivars. The sensitivity to AsA correlated with seed dormancy in the two cultivars. AsA markedly inhibited shoot length of germinating seeds in two cultivars. NMR spin-spin relaxation time (T2) which displays the degree of endosperm degradation in germinating seeds also markedly shorted by AsA treatment. Therefore, the present results indicated that ascorbic acid not only suppressed seed germination but also inhibited the seedling growth.

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Water status in superior seeds of rice (Oryza sativa L.) panicles exposed to low temperature, 15℃ for seven days, at different maturing stages was evaluated by accumulation of dry matter, water content and NMR relaxation time (T_2). The dry weight of control seeds grown at field linearly increased after anthesis to 21 DAF and it proceeded without significant changes thereafter. On the other hand, that of seeds treated at 1st week-chilling linearly increased until 28DAF. The decay in dry matter increase was observed both at 2nd and the 3rd week-chilling treatments. The water content of control seeds linearly decreased at 7 to 14 DAF and it slightly decreased until 49 DAF while decreasing of water continent in child seeds occurred one week delay. The maintenance of higher water content in seeds was more emphasized in earlier stage of rice plants exposed to the chilling temperature. T_2 values of long and short fractions in rice seeds grown at field showed linear decrease until 21 DAF but abrupt decrease was observed in all seeds at 28 DAF. Changes in T_2 values indicated that low temperature at early stage maintained more mobile water in immature seeds seven days longer than control grown at field while the phenomena disappeared in seeds treated at middle stage. From these results, it was shown that the early stage of rice seed maturation was more sensitive to the chilling stress.

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