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Article type: Cover
1989 Volume 35 Pages
Cover1-
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Article type: Appendix
1989 Volume 35 Pages
App1-
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Article type: Index
1989 Volume 35 Pages
i-ii
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Article type: Index
1989 Volume 35 Pages
iii-iv
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Article type: Appendix
1989 Volume 35 Pages
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Article type: Appendix
1989 Volume 35 Pages
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Michio MAEZONO
Article type: Article
1989 Volume 35 Pages
1-3
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An unopened house-style stone coffin was discovered at Fujinoki Kofun in Ikaruga-cho, Nara Prefecture. Inside the tomb a large amount of funerary equipment dating back to the latter half of the Kofun period (6th century) remained almost in perfect condition. In the coffin, in addition to inorganic materials, like swords, mirrors and beads, also woven goods remained. For understanding the society, thought and burial customs in 6th century of Japan this finding offers new materials of great importance. Moreover, regarding the historical phase preceding the Ikaruga Culture including Horyuji Temple, this excavation research has an inestimable value.
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Masaaki SAWADA
Article type: Article
1989 Volume 35 Pages
4-7
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The wooden objects had been buried underground almost completely soaked in water. Therefore, the wood was oversaturated with water, and the cellulose, the main component of wood, was nearly decomposed. If these wooden objects were let dry in a natural condition, they would quickly shrink and lose their original shapes. The freeze-drying method is principally applied to these wooden objects. First, the water in wooden objects is sufficiently replaced by tert-butyl alcohl. It is safer to treat the wooden objects with tertiary butanol of a low concentration first, and then, to impregnate them with pure tertiary butanol.
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Toshichika OHTOMO, Tetsuo YAMADA, Kosaku YOSHIDA
Article type: Article
1989 Volume 35 Pages
8-14
Published: September 30, 1989
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Seventeen strains of 7 species (Staphylococcus epidermidis, S. simulans, S. capitis, S. hominis, S. saprophyticus, S. xylosus, S. warneri) of coagulase-negative staphylococci(CNS) were stored by subculture, freezing and freeze-drying after storage for one year. They were examind by commercial identification kits and the Kloos and Schleifer method. The results showed that strains of Staphylococcus warneri, S. saprophyticus, S. hominis and S. capitis converted to different species of CNS following subculture and freeze-drying. However, no changes were shown with strains of S. xylosus, S. similans and S. epidermidis. The significant changes of biological properties were observed in aerobic acid production from lactose, turanose and N-acetylglucosamine; arginine dihydrolase activity, VP reaction; alkali phosphatase production by α-methylphosphate. Furthermore, the biological properties of the strains of 4 species (S. warneri, S. hominis, S. saprophyticus, S. epidermidis) before storage were compared with strains which had been stored for 64 hours by freezing and freeze-drying by the Kloos and Schleifer method. In these experiments, notable changes were shown in aerobic acid production from N-acetylglucosamine and lactose, and colonial morphology. Major factor of this alteration was assumed to be the effect of the process of freeze-drying.
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Takeshi SAKANE
Article type: Article
1989 Volume 35 Pages
15-20
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The damage of cell membrane as a result of L-drying and the protective effect of ethylenediamine on the damage were studied. The strain examined was Aquaspirillum metamorphum IFO 13960 which was susceptible to damage by L-drying. When this strain was dried and rehydrated with phosphate buffer, a large amount of cell components, such as RNA, proteins, and fatty acids were released into the rehydration fluid from the cells. Denatured or degradated proteins which were not found in vegetative cell envelopes appeared in the protein profiles of cell envelopes from rehydrated cells on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, the amount of a few glycoproteins which were considered to be located in cell surface markedly decreased. When magnesium sulfate (0.1%) was added to rehydration fluid, the release of cell components and damage of cell envelope proteins were prevented, whereas the degradation of glycoproteins was not. Ethylenediamine had high protective activity on the survival of this strain after L-drying. Addition of ethylenediamine (0.4%) to a medium for drying prevented the release of cell components and the damage of cell envelope protein including the glycoproteins. These results indicate that ethylenediamine prevents the damage of cell envelope protein at the drying step, and magnesium sulfate prevents the damage occurred at the rehydration step.
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Ko IMAI
Article type: Article
1989 Volume 35 Pages
21-27
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Freezing storage at -20℃ was applied for eight bacteriophage strains for Pseudomonas aeruginosa, and their viability was examined. Of eight strains, seven were stably preserved both in the presence and absence of glycerol. An osmosensitive phage 24 (IFO 20050) was found to be sensitive to the freezing storage in the presence of 5 and 10% glycerol. Addition of sucrose or glucose made the same degree of loss in titer as that of glycerol. T-even phages of Escherichia coli, which are known to be osmosensitive, were also sensitive to the freezing storage in the presence of glycerol. When the phage lysate supplemented with 5% glycerol was cooled in dry ice-ethyl alcohol and stored at -80℃ for 1 week, the loss in titer was diminished. However, storage at -20℃ made remarkably the loss in titer. The unfrozen water content of the phage lysate with or without sugars was examined by DSC. In the presence of sugars that caused the remarkable loss in titer, the amount of unfrozen water increased at -20℃. On the other hand, the increasing of unfrozen water occurred at -7℃ in the absence of sugars. After storage at -20℃ for 1 week, the viability of lysate supplemented with both 5% glycerol and 1.7% methyl alcohol was 100 fold higher than that without methyl alcohol. Addition of 5% DMSO also markedly prevented the loss in titer by freezing storage at -20℃ in the presence of 5% glycerol. Freezing storage of phage 24 in the presence of glycerol caused leakage of DNA from phage particles. Those findings suggest that sugars can affect on hydrophobic domains of a protein of the phage particle and change the stereostructure of proteins, and this change can trigger off the leakage of DNA from particles.
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Seizo FUJIKAWA, Toshiaki SUZUKI, Shigekata SAKURAI
Article type: Article
1989 Volume 35 Pages
28-31
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It has been suggested that cellular deformation which results in direct membrane-to-membrane contact is responsible for the occurrence of slow freezing injury in tertiary hyphae of mushrooms. Present study examined the quantitative relation between deformed cell number which was observed by Cryo-scanning electron microscope and intramembrane particle aggregated cell number which was observed by freeze-replicas. The observations by both Cryo-SEM and freeze-replicas were done on same samples by using JSM-840A Cryo-system. The results showed a close quatntitative relation between the number of deformed cells and the number of IMP aggregated cells. This result provides a strong evidence that cellular deformation by slow freezing causes IMP aggregation and consequently injury takes place.
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Tsuneo A. TAKAHASHI, Sadayoshi SEKIGUCHI, Allen G. HIRSH, Robert J. WI ...
Article type: Article
1989 Volume 35 Pages
32-38
Published: September 30, 1989
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To elucidate the means by which polymer solutions protect cells from freezing injury, we cooled human monocytes to -80℃ in the presence of various polymers. Differential scanning calorimetry revealed that those polymers which protect cells best have an equilibrium glass transition temperature, Tg, of about -20℃; those with a Tg significantly higher or lower did not protect. Freeze-etch electron micrographs indicated that intracellular ice crystals had formed during freezing, but remained small in about the same proportion of cells as survived rapid thawing. We conclude that cryoprotection of slowly frozen monocytes by polymers is a consequence of a Tg of -20℃. Glass formation limits the injurious osmotic stress which cells face during freezing by preventing further osmotic stress below -20℃. At the same time, it allows cells to concentrate their cytoplasm sufficiently to restrict lethal intracellular ice formation between -20℃ and the intracellular Tg.
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Naofumi HANAFUSA
Article type: Article
1989 Volume 35 Pages
39-43
Published: September 30, 1989
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The characteristic of hydration water of ovalbumin with some surfactants was investigated. Recently, it was reported that non-ionic surfactants had excellent ability to protect the protein against the denaturation by freeze-thawing. To compare the mechanism of protection with other protectants such as sugars, polyols and amino acids, the alterations of the amount of unfrozen water, the extent of the interaction of unfrozen water and the protein were examined using ^1HNMR. Non-ionic surfactants decreased the amount, reduced self-diffusion coefficient D, and increased the rotational molecular correlation time τ_c of unfrozen water. Ionic surfactants did not show such effects. These results suggest non-ionic surfactants are substituted for some of hydration water of the protein with the polar head group and strengthen the interaction of hydration water and protein. It seems the mechanism of protection is similar with non-ionic surfactants and other protectants such as sugars.
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Norio MURASE, Felix FRANKS
Article type: Article
1989 Volume 35 Pages
44-48
Published: September 30, 1989
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The freezing and thawing behaviour of the phosphate buffer mixtures was investigated by differential scanning calorimetry (DSC) in view of its practical importance in the cryopreservation and freeze-drying of biological materials. Salt precipitation from the freeze-concentrated phosphate solutions, ie., eutectic formation, depended on the kind of phosphates, cooling rate and the initial concentration of the solution. The amount of salt precipitated decreased with decreasing concentration when it comes to ca. 1M. In the case of the ternary phosphate buffers, the amount of salt precipitated depended also on the initial salt composition. It decreases especially near the composition where monobasic and dibasic salts are mixed in the molar ratio 1 : 1, or near the composition of the ternary eutectic. From these results, it was concluded that the potassium phosphate buffer is more stable at sub-zero temperatures than the sodium phosphate buffer not only under the equilibrium freezing condition but also under the non-equilibrium freezing condition.
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Article type: Appendix
1989 Volume 35 Pages
49-51
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Kazuo KOMAGATA
Article type: Article
1989 Volume 35 Pages
52-57
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Kazuo MATSUMOTO
Article type: Article
1989 Volume 35 Pages
58-61
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Etsuji HAMAYA
Article type: Article
1989 Volume 35 Pages
62-65
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Takayasu TAKIZAWA
Article type: Article
1989 Volume 35 Pages
66-68
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Yoshihiro NAKAMURA
Article type: Article
1989 Volume 35 Pages
69-76
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Makoto M. WATANABE
Article type: Article
1989 Volume 35 Pages
77-81
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The number of algal strains collected in 40 collections of algae in 16 countries is about 11,000, which are classified into 3,000 species. The most strains are maintained by subculture. Specific temperature, light intensity requirements and medium phase are variable among strains. Some algae such as blue-green algae and chlorococcalean green algae can be preserved in liquid nitrogen. Viability is measured using Morris' or Hatano's method, but viablity measurement by intracellular enzyme activity such as FDA method cannot estimate the recovery potential. Culture collection should carry out purification of unialgal strains for promoting physiological and biological and biochemical studies of algae.
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Itaru SAKAI, Shgenori YAMADA, Kana SATO, Yutaka YOSHIDA, Naosuke SETO
Article type: Article
1989 Volume 35 Pages
82-89
Published: September 30, 1989
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Frozen-stored and thawed suspensions of cells of fungus test strains kept at -80℃ were directly used as inocula for primary antimicrobial tests of pharmaceutical and cosmetic products. The test strains were Candida albicans NHL 4019, Hansenula anomala C-1 and H-1, and Aspergillus niger NHL 5088. The yeast cells and the fungal spores were suspended in phosphate buffered saline containing 0.05% Tween 80 (PBS), 10% glycerol and 5% dimethyl sulfoxide (DMSO) solutions. They were frozen at the cooling rates of -0.2, -1 and -5℃ per minute to -40℃, and then were directly preserved at -80℃ in a deep freezer. The survival values of the frozen inocula ranged from 19% to over 100% of the initial viable counts during 3 months through all the cooling conditions. The other survival values of the antimicrobial test of a model cream with p-hydroxybenzoate benzoate ester with the frozen inocula were fairly coincident with the results of the tests with unfrozen inocula at all the cooling conditions in the case of H. anomala C-1, H-1, and A. niger NHL 5088. The frozen inocula of C. albicans NHL 4019 suspended in PBS and DMSO as cryoprotectant were more sensitive to the model cream than unfrozen inocula. This discrepancy may be ascribed to some injury of the cells by the cooling conditions.
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Mihoko TAKAHASHI
Article type: Article
1989 Volume 35 Pages
90-96
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Protozoa constitute a huge group of eukaryotic microorganisms which include free-living and parasitic species. In this article, methods of preservation of a few species of ciliated protozoa which are often used for genetical researches are reviewed. The nasty problem in the preservation of genetic stocks of ciliated protozoa is aging and limited life span. To prevent aging, maintenance of cultures in low temperature and in a medium containing solid or particular nutrients are often used. Preservation in frozen conditions is only successful for Tetrahymena. Limited success was reported in Paramecium but recovery ratio is very low. Much study is needed for improving the preservation of ciliates in frozen state.
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Toshiyuki KOJIMA, Kenji ENDO, Yukio UZISATO, Yukihiro FUJINO, Tsuneo T ...
Article type: Article
1989 Volume 35 Pages
97-108
Published: September 30, 1989
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The aim of this article is to introduce a part of the current study on frozen storage of pig embryos which have not yet been successfully preserved at -196℃. The hypothesis of the present study was that low temperature injury would be the primary cause of freezing injury for pig embryos. With the intention of avoiding low temperature injury, egg yolk at the proportion of 5%(v/v)and 10%(v/v) was added to solutions for 4℃ exposure experiment and for freezing experiment (-196℃). Embryos of the range from early blastocyst to hatched blastocyst were collected surgically from several breeds on Day 5 to 6 (Day 0=estrus). In the experiment of 4℃ exposure, 10 out of 21 embryos developed in vitro when 5% egg yolk was added to PBS supplemented with 10% fetal calf serum. In the experiment of freezing, 7 out of 14 embryos, which were frozen and thawed by the standard procedures for cow embryos in the presence of 10% glycerol and 10% egg yolk, obviously continued to maintain the viability after 24 h in vitro. These results suggest that low temperature injury may be one of the cause of freezing injury for pig embryos, and that the injury may result from cell membrane, especially from phospholipid bilayer. In this study, no pregnancy was obtained after transferring the frozen-thawed embryos to recipient pigs.
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Tadao OHNO
Article type: Article
1989 Volume 35 Pages
109-112
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We developed a newserum-free medium suitable for crypreservation of serum-free cultured animal cells by utilizing methylcellulose. We also developed in situ freezing method for anchorage-dependent cultured cells.
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Keiji TERAO, Shigeo HONJO
Article type: Article
1989 Volume 35 Pages
113-117
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Masaya ISHIKAWA
Article type: Article
1989 Volume 35 Pages
118-124
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Culture conditions involved in the induction of cold hardiness by abscisic acid (ABA) and associated cellular changes were investigated using bromegrass cultured cells. The highest freezing tolerance (below -30℃) after 7 days of incubation was induced at the ABA concentrations between 75 and 500 μM and at temperatures between 25 and 30℃. This process was independent of the presence of light. Under these conditions, the growth was not depressed by ABA. High levels of cold hardiness were achieved at temperatures where growth was most remarkable. The decrease in the cellular water content was parallel to the level of cold hardiness induced. ABA induced several sets of cellular proteins and only two of them (25 and 200 kD) were also induced by the growth of cells at low temperature (3℃). As a method of preconditioning for cryopreservation, ABA treatment was more effective than cold or osmotic treatment in the case of bromegrass culture. Freezing of ABA-treated cells in 10% sucrose resulted in high rates of survival at -40℃. However, it is not always applicable to other cultured cells as it is affected by the genetic potential of cold hardiness of the plant and also some unknown factors like the origin of cultures or cell morphology.
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Motoyoshi SATAKE
Article type: Article
1989 Volume 35 Pages
125-128
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Syo KUROKAWA
Article type: Article
1989 Volume 35 Pages
129-132
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Article type: Appendix
1989 Volume 35 Pages
133-136
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Article type: Bibliography
1989 Volume 35 Pages
137-140
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Article type: Bibliography
1989 Volume 35 Pages
141-168
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[in Japanese]
Article type: Article
1989 Volume 35 Pages
169-170
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Article type: Appendix
1989 Volume 35 Pages
171-
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Article type: Appendix
1989 Volume 35 Pages
172-173
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Article type: Appendix
1989 Volume 35 Pages
174-175
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Article type: Appendix
1989 Volume 35 Pages
176-177
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Article type: Appendix
1989 Volume 35 Pages
178-179
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Article type: Appendix
1989 Volume 35 Pages
180-181
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Article type: Appendix
1989 Volume 35 Pages
182-183
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Article type: Appendix
1989 Volume 35 Pages
184-
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Article type: Appendix
1989 Volume 35 Pages
App2-
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