Cryopreservation of five marine microalgae (NRIA strains; Chaetoceros ceratosporum, Chaetoceros gracilis, Chaetoceros simplex, Phaeodactylum tricornutum, Isochrysis galbana) which are widely used in the aquaculture industry as food organisms by the two-step cooling method was attempted. The solution A (50%seawater + 5 %DMSO +0.5M sorbitol) for P. tricornutum and solution B (100%seawater +10%glycerol +0.5M sorbitol) for C. ceratosporum, C. gracilis, C. simplex and I. galbana were employed as cryoprotective solutions in the present study. The cells from a anaexponential phase of growth in P. tricornutum, the cells from a pro-stationary phase in C. gracilis and I. galbana and the cells from a meta-stationary phase in C. ceratosporum and C. simplex were found to be resistant to stresses of freezing and thawing, respectively. The appropriate Cryopreservation procedures were as follows: The cryoprotectants were added gradually over a period of 15 min followed by an equilibration period of 45 min. The cells were then prefrozen to -40℃ at 0.1-1.0/min prior to immersion in LN_2 After storage in LN_2, the cell suspension was thawed quickly by agitation of the vial in a water bath at 40℃, and the cryoprotectants were washed off by gradual dilution with seawater. The best survivals evaluated by the MPN-method were 18.0% in C. ceratosporum, 25.5% in C. gracilis, 21.2% in C. simplex, 40.5% in P. tricornutum and 13.8% in I. galbana, respectively. These survivals were independent of the period of storages at least 150 days.

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High pressure treatment has been expected for the sterilized effect upon foods and for less effect upon the properties of foods than thermal treatment. In the present paper big-eyed tuna meat was treated by high pressure among 0.1-500MPa for 10 minutes and subsequent stored for 7 days at 5℃, and the changes in the properties of it were investigated. Immeadiately after treatment high pressure had not any effect upon the freshness of meat, while had a distinct effect upon the color of it according to the intensity of pressure. During the post-pressured storage for 7 days at 5℃, however, high pressure has an important effect upon the changes in the freshness of meat measured by K-value. K-value in high pressure treated (300-500MPa) meat was better than that in non- or low pressure treated (0.1-200MPa) ones, which suggests an inactivation of enzymes related to ATP-breakdown at the pressure of around SOOMPa. The color change measured by metmyoglobin was somewhat occured in every meat after storing for 4 days, but after storing for 7 days it was not occured in high pressure treated (300-500MPa) ones, while a distinct change in low pressure treated (0.1-100MPa) ones was occured, which suggests an acceleration of autoxidation from oxy- and deoxymyoglobin to metmyoglobin in the meat treated by high pressure and subsequent inactivation of enzyme related to the autoxidation. From the lowest value in 200MPa treated meat after storing for 7 days it seems that 200MPa pre-treatment has the most effect on the red color retention of tuna meat.

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Enzymic digests of bovine hemoglobin were fractionated by reverse-phase liquid chromatography. The antifreeze activities of the fractions were analyzed by determing the amount of unfreezable water in their solutions. Fractions with high antifreeze activity were further purified by reverse- and normal-phase liquid chromatography. As the result, four peptides with high antifreeze activity were isolated. The amino acid sequences of them were identified as Gly-Gln-Ala-Gly-Ala, Val-Gly-Gly-Glu, Gly-Ser-Asp-Gln-Val-Lys and sodium salt of Ser-Ala-Glu-Glu. From the antifreeze activities of them and of commercial peptides, it is assumed that peptides mainly consisted of hydrophilic amino acids or low molecular weight amino acids and sodium salt of peptides mainly consisted of acidic amino acids have high antifreeze activities.

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This paper presents a brief introduction to coupled membrane transport theory and parameter estimation methods which have been applied to quantify membrane transport properties using data generated from osmotic perturbation experiments. Technologies developed to quantify such membrane transport are identified and described briefly. Emphasis in the present paper is placed on one of these technologies, the micro diffusion chamber method, as a means of providing representative examples of applications.

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In Japan the late Dr. Hiroshi Iizuka is a pioneer in the field of the investigation of Antarctic microorganisms. We have reported on the diversity and originality of Antarctic microbial resources, for examples, yeasts and bacteria. At present time, I report that a microorganism from Antarctica has an abnormal diversity of morphological expression, and propose a hypothesis on the origin and evolution of life, and moreover, point out the importance of the symbiosis, diversity and environment of organisms. Microorganisms were isolated from the lake water samples of the Lake Vanda, Antarctica. These water samples were presented by Dr. Genki Matsumoto. The single colony isolations were done four times. The nuclei of cells were stained with DAPI (4', 6-diamidino-2-phenylindole), a fluorochrome specific for DNA, and were observed by a fluorescence microscope (Olympus BHS-RFK). The strain were cultured on Yeast-Malt slant or plate agar medium. The microorganisms (the J strain) grew even at 4℃ under aerobically or anaerobically. When the J strain grew at 20℃ on Yeast-Malt plate or slant agar for 7 days, the abnormal morphological diversity of cells was observed. The J strain showed various sizes and shapes of cells, for examples, spherical, triangle, irregular and filamentous and so on. The J strain had the diversity of the growth pattern, and also had the multi-cells and big structured cells. The cells of the J strain were different clearly from the the cells existing in the present time. The cells of the J strain had prokaryotic nuclei and eukaryotic nuclei according to observation of the nuclei of cells with fluorescence microscopy (DAPI stain). Moreover, the J strain had the diversity of the number and shape of nuclei in cells. From these results, it was concluded that the J strain had no ability of self-reproduction. I propose the following hypothesis. There may be remained the primitive environment on the origin of life in Antarctica. The pattern of start growth is not the pattern of self-reproduction, but caostic non self-reproduction. I speculate that an organism may have not been able to adapt to the environment but to only specialize to the one, and that the specialization to the environment is the same with the restriction to the one. Accordingly, it is concluded that in the existence of organisms the harmonical diversity of organisms is very important, and also the organism should have the diversity and originality.

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Permittivity was measured for rice (Oryza saliva L. japonica) cell suspension in the frequency range from 0.2 to 20 MHz. A good linear correlation (R=0.999) was observed between the permittivity at 0.41 MHz and the cell density. A decrease of the permittivity of cell suspension through a freezing-thawing process was observed and this seemed to reflect a mechanical damage on the plasma membrane of the cell. The intact cell ratio without the damage on the plasma membrane was estimated from the permittivity and was compared with a viability calculated from TTC (2, 3, 5-triphenyltetrazolium chloride) reduction assay at various freezing conditions. Effects of dimethylsulfoxide (DMSO) concentration, as a cryoprotectant, and cooling rate agreed between the intact cell ratio and TTC viability in their trends, although a quantitative discrepancy was observed between the two indices. Water permeability was also measured by permittivity to be 0.008 and 0.02 [pm/s/Pa] at 25℃ for rice and grape (Vitis sp) cells, respectively.

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Effects of glycerol, which is known as a poorly permeating cryoprotectant, on the cryopreservation of cultured plant cells of wheat (Triticum monococcum) were investigated. Survival of cells rapidly decreased when cryoprotective solutions containing glycerol or glycerol plus sucrose were added to the cells. However, an apparent cryoprotective effect of the combination of glycerol and sucrose was observed in the cells frozen to -30℃ or -196℃ although it was less effective than that of DMSO and glucose.

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Glass-forming composition regions were determined by a simple DTA method for aqueous phenyltrialkylammonium and benzyltrialkylammonium chloride solutions. Raman spectra of these solutions were also measured. Aryl groups are shown to act as "structure breaker" in these solutions.

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Thermal analysis of freezing behaviour of polymer gels was carried out by differential scanning calorimetry (DSC) together with the observation of their frozen state by scanning electron microscopy (SEM). As a result, it was indicated by DSC that small ice crystals, whose melting temperature is lowered because of the size effect, are formed trapped by the polymer network, and that vitrification is an extreme case of the formation of small ice crystals. However, no such small ice crystals were observed by the SEM observation.

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The procedures to determine parameters of pre-freezing process of biological cells are discussed. In pre-freezing process, the concentration of cryoprotectant is raised stepwise to avoid osmotic stress. Raising rate of concentration of cryoprotectants can be chosen by the relaxation time of biological cell. Optimum parameters can be obtained by estimating volume percent of the cell.

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To examine the cryoprotective role of glycerol in preserving frozen red blood cells, we used a differential scanning calorimeter (DSC) to study the thermal behavior of the clinically used glycerol-based cryoprotective solutions (complex system) as well as the glycerol-water (binary) system. We found two peculiar zones in which freezing occurs without the formation of ice crystals: an amorphous state (glycerol concentrations between 45% and 60% for the complex system and between 40% and 55% for the binary system) and a gelation state (for glycerol concentration greater than 60% for the complex system and 55% for the binary system). However, clinically used cryoprotectants are not used in either of these states, but are used in the zone with cryoprotectant concentration lower than that for which the amorphous state occurs. This attributes to an increase in the effective glycerol concentration inside and outside the cells because ice forms during freezing.

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