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Article type: Cover
1990 Volume 36 Pages
Cover1-
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Article type: Appendix
1990 Volume 36 Pages
App1-
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Article type: Appendix
1990 Volume 36 Pages
App2-
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Article type: Appendix
1990 Volume 36 Pages
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Article type: Index
1990 Volume 36 Pages
i-ii
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Article type: Index
1990 Volume 36 Pages
iii-iv
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Article type: Appendix
1990 Volume 36 Pages
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Article type: Appendix
1990 Volume 36 Pages
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Mitsuo TAKANO
Article type: Article
1990 Volume 36 Pages
1-2
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Rihachi IIZUKA, Rui AOKI
Article type: Article
1990 Volume 36 Pages
3-9
Published: October 20, 1990
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This paper consists of a general review of the cryopreservation of human embryos in IVF program and a case report of the first live birth in Japan after the intrauterine transfer of frozen-thawed embryos on the 25th of December 1989, which has been already reported by the author, Rui Aoki. The general review covers (1) principles of cryobiology, (2) comparison of cryoprotectants, (3) rapid, ultra-rapid, and slow freezing and thawing methods, (4) vitrification, and (5) synchronization of the embryos and endometrial milieu. The authors discuss a possible method for investigating the receptivity of hyperstimulated endometrium in comparison with non-stimulated one by using both fresh and frozen-thawed embryos transferred in different menstrual cycles.
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Masanori FUJITA
Article type: Article
1990 Volume 36 Pages
10-13
Published: October 20, 1990
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Dewatering of waste activated sludge by freezing was investigated in this paper. Freezing temperature of the waste activated sludge ranged ordinarily from -10℃ to -20℃, but freezing velocity depended on the freezing equipment. Frozen sludge was thawed and then dewaterd by the vacuum filter. Freezing improved the sludge dewaterability. Moreover, ice nucleating active bacteria was used to accelerate the freezing for shortening the freezing time. Addition of ice nucleating bacteria at the final concentration of 0.06 g/l decreased the freezing time remarkably. It suggested to be able to save the freezing energy in this process.
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Hisako URABE, Midori KATO, Kazuhito KAJIWARA, Yasunori TOMINAGA
Article type: Article
1990 Volume 36 Pages
14-17
Published: October 20, 1990
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Freezing processes of Tris-HCl buffer solutions and DNA gels were investigated by low frequency Raman spectroscopy and differential scanning calorimetry (DSC). The eutectic made of Tris-HCl and water are formed during the heating process, only after the solution undergoes glass transition below -50℃ during the previous cooling. DNA gels prepared by dissolving DNA in a Tris-HCl buffer solution freeze differently from salt free DNA gels. We found that when DNA gels with Tris-HCl are kept at -30℃ during heating after the previous cooling below -50℃, the eutectic of Tris-HCl and water grows slowly and separates from DNA.
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Naofumi HANAFUSA
Article type: Article
1990 Volume 36 Pages
18-21
Published: October 20, 1990
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The characteristics of unfrozen water of phospholipids, especially the comparison of lamellar phase of phosphatidylcholine (PC) and H_<II> phase of Phosphatidylethanolamine (PE) were examined using ^1HNMR. The amount of unfrozen water was reduced monotonously depending on freezing temperature. The extent of hydration was greater in lamellar phase of PC and PE than in H_<II> phase of PE. The extent of restriction of hydration water, such as molecular correlation time and self-diffusion coefficient, was greater in H_<II> phase than in lamellar phase. It seems not only the chemical structure of the head group of the lipid but also the phase and the shape of liposome affect the extent of the interaction of the hydration water and liposome.
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Yasutake SUGAWARA, Peter L. STEPONKUS
Article type: Article
1990 Volume 36 Pages
22-27
Published: October 20, 1990
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Changes in the membrane ultrastructure after freezing in protoplasts isolated from non-acclimated or cold-acclimated rye (Secale cereale L. cv. Puma) leaves or in protoplasts fused with liposomes were examined with freeze-fracture electron microscopy. In non-acclimated protoplasts, freezing to -10℃ or lower resulted in survival decrease below 10% and induced several changes in the ultrastructure of the plasma membrane and endomembranes of protoplasts. In acclimated protoplasts, these changes were not observed even at -25℃, though protoplasts were injured at this temperature. Fusion of non-acclimated protoplasts with liposomes composed of dilinoleoylphosphatidylcholine mitigated or eliminated the changes in the plasma membrane. Possible roles of membrane lipids on the changes in the membrane ultrastructure induced by freezing were discussed.
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Tsuneo A. TAKAHASHI, Mikinori KUWABARA, Wakako HIRAOKA, Kunihiko NAKAI ...
Article type: Article
1990 Volume 36 Pages
28-35
Published: October 20, 1990
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Human polymorphonuclear leukocytes (PMNs) are quite sensitive to osmotic stress. This is believed to be the main reason for the difficulty of successful cryopreservation. For the purpose of making PMNs more resistant to the osmotic stress, PMNs were treated with various chemicals. Among the chemicals, theophilline, PGE_I and hydrocortisone were the most effective at improving PMNs resistance to osmotic stress. The common character among them is the ability to enhance the amount of intracellular cAMP and cell membrane permeable dibutyryl cAMP indeed made the cells more resistant. Using these chemicals the same effect was also observed in monocytes which are quite resistant to the osmotic stress without artificial protection. Increment of intracellular cAMP is known to suppress the free radical generation in PMNs. Therefore we have examined whether free radicals were generated in PMNs by the osmotic stress and freeze-thaw cycle. Free radical generation was observed by the luminol-dependent chemiluminescence assay in the PMNs suspended in hyperosmotic solutions or frozen extracellularly. Furthermore O_2^^- production was confirmed in osmotically stressed PMNs by electron paramagnetic resonance spectroscopy using a spin trap. Free radical generation in the early stages of osmotically stressed or extracellularly frozen PMNs may explain the high sensitivity of PMNs to the osmotic stress and freezing injury.
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Yoshiyuki OMORI, Masanori TSUMURA, Taizo ICHIDA, Kazuhide YAMASATO
Article type: Article
1990 Volume 36 Pages
36-40
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Clean Cryo Blaster(CCB) is a system to disrupt cells by colliding small particles of frozen cell suspension against a metal target panel blowing with pressurised nitrogen gas flow of sonic speed. Cell suspension is rapidly frozen by spraying through nozzle into the atmosphere of ultralow temperature. CCB has hardly been applicable for most of fungi and actinomycetes which form large aggregates of filamentous cells in liquid culture, because aggregates plugged up the orifice of the spray nozzle. In order to disperse cells a modification was deviced which uses high pressure(60 kg/cm^2)for spraying. With the modified system the aggregates were dispersed into small and fairly well isolated filamentous cells, and the cells were disrupted into small fragments by the following CCB treatment. Long strands of DNA of the length over 23kbp were recovered from Penicillium chrysogenum, Streptomyces griseus and Streptomyces coelicolor. Sixteen S and 23S rRNAs were recovered from streptomysetes which appeared to be intact from electrophoregrams. The yield of RNAs from S. coelicolor was a little higher than that obtained by conventional lysis method. It was suggested that ultralow temperature throughout the disrupting process would be effective for preventing RNA from hydrolysis by indigenous or contaminating RNase.
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Masakazu KOBAYASHI, Ryoji SUNAMA, Konomi HARASHIMA
Article type: Article
1990 Volume 36 Pages
41-49
Published: October 20, 1990
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In shelf freeze-drying of pharmaceuticals, it is one of the important problems to realize "intervial uniformity" and "batch to batch uniformity or consisitency". For one of the themes on the problem, necessity of uniform shelf temperatures (e.g., within ±1℃, both across and between shelves) has been stressed by both manufacturers and users of freeze-dryers, while significant intervial variance in the sublimation rate has been pointed out by several authors, Rowe (1979), Pikal (1985), and Kobayashi (1982), (1988) for the placement of the vials put on a well controled shelf. However, all of previous works on this important problem merely described the phenomena observed in the experiments or the production processes, they did not propose clearly the way to solve the problem. This presentation discusses a method for eliminating the intervial variances in the drying conditions and to shorten drying time. In this study, authors have developed a new model of labo-scale pilot freeze-dryer having temperature controlable chamber wall, and using this system authors have made clear that the higher sublimation rate for vials placed on the shelf edge is due to an additional heat input from the edge of the shelf (Δq_h), mainly by conduction effect through the gas between the shelf surface, and also due to another additional heat input from the wall (Δq_w), mainly by radiation effect. Under the certain geometrical conditions, since these additional heat inputs, one from the shelf (Δq_h) and another from the walls (Δq_w) to each of the edge vials, have almost similar pattern (Δq_h/Δq_w ≒ const. for each vial forming the perimeter of a group of vials), it is possible to cancel the additional heat input from the shelf by maintaining the optimum wall temperature which must be a little lower than the material temperature (t_w < t_m, Δq_w < 0, & Δq_h + Δq_w = 0). In order to realize the wall temperature control successfully in a production scale freeze-dryer, it is necessary to make the wall surface reflectance for thermal radiation lower than that of bright polished stainless steel surface.
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Masakazu KOBAYASHI, Konomi HARASHIMA, Hiroichi ARIYAMA
Article type: Article
1990 Volume 36 Pages
50-58
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Up to now, in a conventional freeze-drying method for the bulk pharmaceuticals and other liquid materials, there has been employed a tray/shelf system, and the matter of complicated tray/material handling under the open space is one of the most important problems to be solved in this field (cf.Cooper,1984). Previously authors presented a paper on the closed type non-tray continuous freeze-drying system for liquid by using vertical tubes (1988). In this system, the substantial part of the liquid product poured into the drying chambers is not subjected to the freeze-drying, but is drained in the from of liquid. In the continuous multi-chamber system, the drained liquid from the chamber is charged into the next chamber together with newly supplied material so that the chambers are reiteratedly and circulatively used during continuous operation period. However, there are some materials for which a batch system is more recomendable from the quality view point. This presentation discusses a closed type batch freeze-drying system for liquid materials. By installing a simple device additionally above the bundle of tubes in the chamber, almost all of the liquid material poured into the chamber is formed into desired frozen layers, leaving behind space necessary for water vapor to flow in all the tubes, and the drain is reduced to come only "from one" of the tubes instead of "from all". The new device contacts neither the liquid nor the dried cake at all and that the chamber having no complicated structure, internal cleaning and sterillizing can be easily performed without fear of contamination.
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Article type: Appendix
1990 Volume 36 Pages
59-61
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Douglas R. MACFARLANE
Article type: Article
1990 Volume 36 Pages
63-71
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Vitrification, or glass formation, is a feature of the low temperature phase behaviour of many aqueous solutions. Here the physical conditions favouring glass formation are reviewed and discussed in terms of a number of well known aqueous solution systems. The discussion will also consider the conditions under which devitrification (crystallization during warming) and recrystallization (ie. ripening) are also important. The role of the solute in promoting glass formation has also been the subject of study in recent years, in an effort to develop improved solutes which support glass formation at lower solute concentrations. It has been observed that one of the functions of a solute which strongly promotes glass formation is to break down the highly structured regions which tend to build up in pure water at temperatures below 0℃. This occurs via strong specific interactions between the solute and water molecules, however in the extreme of very strong interactions solute hydrate compound formation can interfere with vitrification by providing an alternate crystalline phase other than ice. The nature of the solute-water interactions depend strongly on the chemical structure of the solute and detailed trends are beginning to emerge as a result of spectroscopic studies. For example, in the family of diol compounds related to ethane 1, 2 diol it has been observed, by nuclear magnetic resonance studies, that the addition of an alkyl group to the hydroxy carbon produces a more basic hydroxy group. This results in a stronger hydrogen bond between the solute and water, and hence provides the structural disruption in the solution required to promote glass formation.
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Seizo FUJIKAWA
Article type: Article
1990 Volume 36 Pages
72-75
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Plasma membrane is a primary site for freezing injury. By freezing, ultrastructure of plasma membranes shows diverse changes, such as intramembrane particle (IMP) aggregation, lamellar to hexagonal II phase transition, endoplasmic vesiculation, and formation of IMP-free patches. All these plasma membrane ultrastructural changes are closely related to the occurrence of freezing injury. It was suggested that freezing injury was caused by diverse mechanisms depending not only upon different freezing conditions but also upon difference of cell types.
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Naofumi HANAFUSA
Article type: Article
1990 Volume 36 Pages
76-80
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Dynamics of hydration water of some proteins during freeze-thawing and after freeze-drying was investigated in the presence or absence of protective substances using DSC and ^1HNMR. Protein such as myosin, which is unstable against freezing and drying, has no rigid stable hydration structure and the extent of restriction of hydration water by the protein is weaker than other stable proteins. Addition of effective protectants, the number of hydration water decreased extremely and the extent of restriction increased. It seems that destruction of hydration structure by dehydration during freezing or drying is the most important mechanism of the protein denaturation. It is considered that the effective protectants, such as sugars, amino acids, polyols or surfactants, substitute for a part of of hydration water of protein and form quasi-hydration structure and protect protein agaist denaturation. The characteristic of "quasi-liquid layer" of ice particles was also discussed in relation to the properties of hydration water of protein.
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Takeshi SAKANE
Article type: Article
1990 Volume 36 Pages
81-87
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Preservation of cells by L-drying or freeze-drying caused induction of mutation in Escherichia coli. The induction depended on the temperature during preservation. When the dried cells were preserved at temperature between 10℃ and 48℃, the number of mutants in the surviving fraction increased as the storage temperature increased. The induction of mutation could be detected neither in the cells rehydrated immediately after drying nor in the dried cells preserved at temperature below 5℃. Thiourea, adonitol and cysteine were found to be effective against the cause of mutation, which reduced the frequency of mutation during preservation. These compounds improved survival and/or prevented the induction of mutation by adding into a fluid for drying, even when the dried cells were preserved at high temperature. It seems that the roles of thiourea and adonitol in protecting the cells from mutation are to prevent DNA-strand breakage by acting as radical-scavengers during preservation of dried cells, and that of cysteine is to stimulate the activity of DNA-repair system after rehydration of dried cells and/or to act protecting on the DNA-repair system enzyme(s) during preservation of dried cells.
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Atsuo NOGUCHI, Masami FUJISAKI, Takeshi URAO, Nobuo SAKAO, Yasuo KURAO ...
Article type: Article
1990 Volume 36 Pages
88-92
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A superhigh speed cell freezing system with ultra low temperature helium gas was developed. High percentage of viability of the frozen cells was obtained by rapid thawing. Occurence of an amorphous glass formation (vitrification) in the intracellular fluid of the frozen cells by this method was discussed.
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Tsuyoshi MORINAGA, Kouichi INOUE
Article type: Article
1990 Volume 36 Pages
93-96
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The effects of oxygen and additives on the viability of 5 methanogens in freeze-drying and L-drying preservation were studied. A slight contact with O_2 as a pretreatment for freeze-drying remarkably decreased the viability of Methanosarcina barkeri DSM 800. Small amount of cysteine and adonitol added to the medium highly improved the viability of 4 species among the 5 tested.
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Shin-ichi UEDA, Tsuneo A. TAKAHASHI, Seizo FUJIKAWA
Article type: Article
1990 Volume 36 Pages
97-104
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The possibility of biological cryopreservation by ultra-rapid freezing without pretreatment by cryoprotectants was examined. Human red blood cells (RBCs) were used as a model system. Ultra-rapid freezing was achieved by metal-contact freezing using liquid helium (LHe). The freezing pattern of metal-contact frozen samples was observed by both cryo-scanning electron microscopy (Cryo-SEM) and freeze-replica techniques by using a JSM-840A new cryo-system. The possibility of cell survival by vitrification was carefully examined by comparisons between post-thawed survival of RBCs and the freezing pattern of RBCs by both electron microscopies. The results provide clear evidence that some RBCs can survive by vitrification.
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Masanori TSUMURA, Taizo ICHIDA, Kazuhide YAMASATO
Article type: Article
1990 Volume 36 Pages
105-112
Published: October 20, 1990
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The efficiency and the mode of bacterial cell disruption was investigated for a newly developed cell disrupting apparatus, Cryo Clean Blaster(CCB), which collides rapidly frozen cells onto a hard metal surface with pressurized nitrogen gas flow of sonic speed. The yield of high molecular weight DNA extracted by phenol reagent treatment followed by CCB cell disrupting procedure was used as index for cell disruption. In Escherichia coli cells from both logarithmic and stationary phases, the yield of purified DNA was twice as much as those from cells disrupted by lysis method which employed lysozyme and sodium dodecyl sulfate. Hyperchromicity of DNA was about 40% and the length of DNA strands was over 23kbp. The original shape of cells was almost maintained after CCB treatment as observed by phase contrast and electron microscopy and their appearance was like a"ghost"cell. It was supposed that the mode of cell disruption was such that the cell constituents were extruded without disrupting cells into small debris. In the cells of Bacillus subtilis and Lactobacillus plantarum from both logarithmic and stationary phases, DNA yields were as high as those by lysis methods. Lysozyme treatment of cells followed by CCB disrupting procedure improved DNA yield to about twice. Two-stepped disrupting device, which collides the once collided cells onto another metal surface gave the DNA yield thrice as much as that by lysis method in Arthrobacter globiformis cells from both logarithmic and stationary phases.
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Article type: Appendix
1990 Volume 36 Pages
113-115
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Article type: Bibliography
1990 Volume 36 Pages
116-119
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Article type: Bibliography
1990 Volume 36 Pages
120-147
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[in Japanese]
Article type: Article
1990 Volume 36 Pages
148-150
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Article type: Appendix
1990 Volume 36 Pages
151-
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Article type: Appendix
1990 Volume 36 Pages
152-153
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Article type: Appendix
1990 Volume 36 Pages
154-155
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Article type: Appendix
1990 Volume 36 Pages
156-157
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Article type: Appendix
1990 Volume 36 Pages
158-159
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Article type: Appendix
1990 Volume 36 Pages
160-161
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Article type: Appendix
1990 Volume 36 Pages
162-163
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Article type: Appendix
1990 Volume 36 Pages
164-
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Article type: Appendix
1990 Volume 36 Pages
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