A near-infrared (NIR) spectrometer equipped with an interractance optical fiber probe, in the region 800-1100nm, was used to determine the crude fat content in frozen and defrosted southern bluefin tuna fillets. The fat content range was 0.1 to 35.1%. Multiple linear regression (MLR) analysis including 55 tuna fillets for calibration and 50 tuna fillets for prediction was used to select the optimum wavelengths for estimating the fat content. The multivariate correlation coefficients (R) between measured fat with chemical analysis and predicted fat from the NIR spectrum were obtaind for frozen one (R=0.958) and for defrosted one (R=0.944), while the standared deviations of prediction were 2.99% and 3.38%, respectively. The results show that NIR spectroscopy with a fiber-optic probe is suited to determine fat content in frozen southern bluefin tuna meat non-destructively.

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Freezing behavior of tropical and subtropical plants was examined by means of Cryo-SEM and freeze-fracture replica electron microscopy. Saintpaulia (Saintpaulia grotei mutant) leaf cells and mungbean (Vigna radiatd) seedling cells produced intracellular freezing by inoculate freezing at -2℃, whereas orchid (Paphiopedilum insigne) leaf cells responded to freezing at -2℃ by extracellular freezing. Because chilling treatment (supercooling) at -2℃ did not cause intracellular freezing, intracellular freezing in Saintpaulia and mungbean is suggested to be due to seeding of extracellular ice thorough cells. The relationships between chilling-induced plasma membrane changes and occurrence of intracellular freezing were discussed.

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The BESTCapsule2001 project was proposed for celebration of the dawn of the new Millennium, which preserves biological and environmental specimens collected from the world at the Dome F in Antarctica for more than 1,000 years. The Millennium projects around the world are outlined at first to explain the philosophical background. Significance of the BESTCapsule2001 project is then pointed out in the frame work of the world Millennium. The Dome F is the best location on the earth for natural preservation in freezing condition. The temperature, -58 degree C, is, however, too high to preserve living animal cells for ultra-long period. They can be safely preserved at the bottom of a crater in a polar region of moon.

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We have reported that the 1.8% paraformaldehyde-fixed and lyophilized human platelets retained surface Glycoprotein I b with function, and that may be one of the candidates as platelet substitutes. In this paper, we focused on HLA class I antigens on plasma membrane which are recognized by anti-HLA class I alloantibodies in alloimmunized patients. To eliminate HLA class I antigens, we treated fresh human platelets with acid buffer at pH3.0 and subsequently fixed with 1.8% paraformaldehyde. Aggregation analysis and flowcytometric analysis demonstrated that acid treated, lyophilized and rehydrated plateletes possessed ristocetin-dependent aggregation and HLA class I antigens on platelet surface were completely eliminated. These platelets may not be recognized by anti-HLA class I antibodies, and it is expected that these platelets would be applied to the patients refractory to platelet transfusions. These results suggest that acid-treated and lyophilized platelets may be useful as a platelet substitute.

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The quality control for cryopreservation of many units of placental/umbilical cord blood (PCB) is a difficult problem in cord blood banking. The BioArchive System (THERMOGENESIS Co.) is a liquid nitrogen freezer tank equipped with a controlled-rate freezer and fully automated by computer intended for the cryopreservation and storage of PCB. The BioArchive System can store 3,626 units of PCB in one tank with quality control. In this study, we evaluated the usefulness of the cryopreserved PCB by using the system and the freezing bag system (Medsep). For comparison, we froze PCB in a controlled-rate freezer (CryoMed) using the Medsep bag. The recoveries of colony-forming-units were 83.2±20.3% and 84.8±22.2% in both methods (mean±SD, n=10) and there was no statistical significance. Thus, the BioArchive System can save stock space, and all the procedures are controlled by a barcode reading. These results indicated that the BioArchive System is useful for the quality control for the cryopreservation and storage of PCB in cord blood banking.

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Progressive freeze-concentration was effectively applied to concentrate tomato juice. Effective partition constant of the solute between the solid and liquid phases was determined for the total solid and KCl-based salt content. The partition constant for the salt was higher than that of total solid. The partition constant decreased with an increase in the stirring rate at the solid-liquid interface and with a decrease in the advance rate of ice front. After the concention of tomato juice by 4.1 fold by the present method and reconstitution based on Brix, no substantial differences were observed in acid content, vitamin C concentration, and color analysis.

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For different concentrate solutions of several kinds of solute, the homogeneous nucleation temperature Th and the equilibrium melting points Tm were determined by using so-called emulsion method. The obtained Th and Tm for each solute dropped with concentration increasing, however, the relationship between both temperatures showed good linearity with different gradients depending on the kind of solute. That is, the relations could be expressed by an equation Th=aTm for each solute, of which the gradients a are considered to represent the degree of supercooling ability. The correlation between the a values for each solute and the chemical structure or hydrate characters was examined. As a result, the dynamic hydration number of solute was found to have a best correlation with the a value representing supercooling ability.

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Effect of lipid alterations in the chloroplast envelope, manipulation of cytosolic sugar content, and expression of the cold-regulated (COR) genes on the cryostability of the plasma membrane was determined. All of these three factors were shown to contribute to the increase in the cryostability of the plasma membrane during freeze-induced dehydration, which consequently results in an increase in the freezing tolerance. The results suggest that the action mechanism (s) of each factor to increase the cryostability of the plasma membrane must be determined in detail.

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In this study, we examined freezing behavior of birch xylem ray parenchyma cells. In fresh samples, summer cells exhibited supercooling to around -15℃ and winter cells exhibited it to around -50℃. In samples where the plasma membranes were destructed by freeze-thawing, summer cells still exhibited supercooling to around -10℃ and winter cells exhibited it to around -30℃, showing that cell walls are responsible, at least partially, for supercooling and that cold acclimation alters the property of supercooling ability of the cell walls. As one of possible causes of the cell wall changes, we examined effects of cell wall-bounding proteins. The result showed that extraction of proteins from cell walls altered the freezing behavior of xylem ray parenchyma cells.

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A cDNA clone (SfUCPa) encoding a mitochondrial uncoupling protein (UCP) was isolated from the spadix of skunk cabbage (Symplocarpus foetidus). The cDNA contained an open reading frame of 303 amino acids (predicted molecular weight of 32.6 kDa). The predicted polypeptide had six transmembrane domains, three energy transfer protein motifs and one purine nucleotide binding domain (PNBD), which are characteristic of all known UCPs. The corresponding transcript was expressed only in the spadix and was induced by cold treatment. These results suggest that the putative SfUCPA protein may play an important role in thermogenesis in the spadix of skunk cabbage.

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Many fresh water algae form akinetes that tolerate to enviromental stresses. Yellow-green alga Tribonema bombycinum shifted from vegetative cells to akinetes with nutrient deficiency or desiccation, but cold treatment at 4℃ did not facilitate akinete formation. During akinete formation, the vegetative cells, which had a large central vacuole in the prptoplasm and thin cell walls, morphologically changed to akinetes, which had many small vacuoles and oil droplets in the protoplasm and thick cell walls. During akinete formation, the freezing tolerance (LT_<50>) also increased gradually from -3℃ in vegetative cells to far below -30℃ in akinetes. When vegetative cells were subjected to equilibrium freezing at -15℃, they shrank greatly and aparticulate domains accompanied by fracture-jump lesions occurred in the plasma membranes. Akinetes subjected to equilibrium freezing at -30℃ showed little shrinkage, and freezing-induced ultrastructural changes did not occur in the plasma membranes.

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The senescing process of the water compartments in gladiolus (Gladiolus×hybridus hort.cv. Fujinoyuki) flower petals were studied by evaluating the spin-lattice relaxation times (T_1) and the spin-spin relaxation times (T_2) using ^1H-NMR. The florets of cut gladiolus spikes showed severe wilting four days after flower opening, at 25℃ under 14L/10D conditions. Treatment with 0.1M trehalose suppressed petal senescence and prolonged its longevity by two more days. After four days, the first florets of trehalose-treated spikes maintained water more effectively than those treated with those placed in DW (distilled water) control. The changes in T_1 corresponded more closely to the water content and membrane integrity than those in T2. Furthermore, the semi-log plots of signal intensity of ^1H-NMR revealed that the water in the petal tissues consisted of at least two water components with T_1 values ranging over 1.2 s, and below about 0.7s respectively. The highly mobile water was considered to be free water derived from intact vacuoles. The free water in the petal tissues treated with DW disappeared after four days, while petals treated by trehalose maintained long T_1 values thus corresponding to significant water uptake in the petal tissues. These results suggested that trehalose helped to preserve cell viability and also enhanced the water uptake in petal tissues.

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An ice-nucleating bacterium, Xanthomonas campestris pv. translucence IFO 13559 was cultured on trypticase soy broth with mitomycin C. When mitomycin C concentration was 0.10 μg/ml in cuture medium, the growth and producing xanthan gum were inhibited, but the protein concentration of cell-free extract increased in five times compared with non-addition of mitomycin C. Furthermore the productivity of an extracellular ice-nucleating material was increased with proportional to increase the protein concentration in culture medium. The freezing defference spectra in D_2O and H_2O at ice-nucleation temperature for obtained the extracellular ice-nucleating material with and without mitomycin C exhibited difference curves.

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It is known that yeast cells survive enviromental stress such as desiccation and high temperature when they accumulate trehalose. On the other hand, according to our previous studies, trehalose is an excellent promoter of water structure. Here, we investigate the relationship between trehalose content in yeast cells and their survival in response to heat shock. We prepare a mutant of yeast cell (Δnthl) which lacks trehalase called NTH1. This mutant accumulates trehalose about three times more than does the parent wild type cell (WT) in both logarithmic and stationary phases. ^1H NMR, thermogravimetry and differential scanning calorimetry are used to investigate the properties of the intracellular water in the Δnthl and WT cells. In additon, we measure the survival rate of these cells in resonse to heat shock (50℃, 10min). As a result, the trehalose content is not necessarily correlated with the viability of the cells. It is, however, found that there is a good correlation between the survival rate and the T_1 relaxation time of the intracellular water proton in both types of cells. This finding suggests that structuring of intracellular water, induced by trehalose and/or heat shock proteins, is essential for high resistance to water stress in yeast cells.

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Freezing behaviour of dextran aqueous solutions was investigated by differential scanning calorimetry (DSC). It was found, as a result, that the DSC heating traces obtained with the frozen solutions indicate a small endotherm before the appearance of a large endotherm due to ice melting at ca. 0℃. The small endotherm shifted toward a higher direction by several degrees after the equilibrium freezing, when compared with the nonequilibrium freezing after the substantial undercooling. Origin of the small endotherm was considered to be melting of small ice crystals confined within the intramolecular space of entangled dextrans which melt at lower temperatures due to Kelvin effect.

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Two crystallization exotherms during warming observed with the certain compositions of NaCl-glucose aqueous solution, were investigated by X-ray diffraction. From the results, lower and higher temperature crystallizations are identified as ice and ice/NaCl・2H_2O eutectic formation, respectively. In the case of KCl-glucose aqueous solutions, on the other hand, two kinds of crystallization during warming were observed dependent on their compositions. They are also identified by X-ray diffraction as ice/KCl eutectic formation with a lower glucose concentration solution and ice formation with a higher glucose concentration solution, respectively. A double glass transition phenomenon was observed for the certain compositions of NaCl-glucose aqueous solution vitrified during cooling. The phenomenon was taken as an evidence for the liquid-liquid immiscibility at low temperatures.

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Raman OD stretching spectra were measured for aqueous solutions of trehalose, sucrose and maltose as a function of solute concentration and temperature. Temperature covered was from -140 to 80℃. Comparison of the spectra showed that there is little spectral difference in these solutions. From the Raman results for the hydrogen bond interactions between water and sugar molecules, no specific interaction between trehalose and water molecules was found as a high bioprotector.

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We found that the colony-forming units- megakaryocyte (CFU-Meg) in human umbilical/ placental cord blood (PCB) are sensitive to freezing injuries during cryopreservation. Mononuclear cells isolated from PCB were cryopreserved by the standard method used for the PCB banking. The recovery of CFU-Meg were less than other colony-forming cells. The cells were exposed to various osmotic stresses, cooled at different cooling rates, plunged into liquid nitrogen from different temperatures. Under these stresses, CFU-Meg were more sensitive than other colony forming cells. These results indicate that CFU-Meg are sensitive to freezing injury. This may be one of the reasons that platelet engraftment is slow in PCB transplantation.

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