Freeze-drying has established itself as the favoured method for the stabilisation of labile biological products, especially in the pharmaceutical and biotechnology industries. Its effective and economical application depends on the complex interplay of many different factors, governed by physical, chemical and engineering principles. it is shown how the product quality can be optimised by a consideration of formulation details, dimensions of the container and the process variables employed. In particular, the physical state of the product during and after the drying process is of crucial importance.

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pBR322 plasmid DNA purified with ethidium bromide-CsCl density gradient was dried at 30℃ under fixed relative humidities of 0, 12, 33, 43, 53, 76, 84, and 92%. Single-strand breaks (ssb) were most efficiently induced under 53% relative humidity but almost no ssb were induced below 12% and above 84%. At 53% relative humidity, most ssb were induced by drying during first 24 h and further drying induced little ssb. When air was replaced with nitrogen, the ssb induction was reduced.

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Protective effect of ethylenediamine dihydrochloride (ED) in preventing the damage of bacterial cell membrane by L-drying was examined. A bacterium examined was Aquaspirillum metamorphum, which might be susceptible to damage of cell membrane occurring in the desiccation. Effective protection by addition of 30 mM ED into the basal suspending medium (BSM) was found on the survival of both the wild type strain and a surface layer protein-less mutant after drying. Vegetative cells of A. metamorphum IFO 13960 (wild type) were insensitive to 1% Triton X-100, however, the cells altered into being sensitive to the detergent after drying, and the alteration were prevented by addition of ED into the BSM. Analysis of lipopolysaccharide (LPS) and surface layer protein by deoxycholate-polyacrylamide gel electrophoresis (DOC-PAGE) revealed an apparent decrease in the amount of LPS of the cells dried without ED, whereas those profile of the cells dried with ED showed little decrease. The protective activity of ED was inhibited by addition of 100-300mM NaCl into the suspending medium containing 30mM ED and lost completely by addition of 1M NaCl. These results suggest that ED binds to outer membrane and stabilizes cell membrane, like as magnesium ions which have bridging and stabilizing effects on the cell membrane. The membrane stabilized can prevent from damage occurring in the desiccation.

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Three experiments were conducted to assess the viability of bovine blastocysts by vitrification procedures which were obtained by in vitro fertilization of oocytes matured in vitro. In experiment 1, the optimal concentrations of glycerol and 1, 2-propanediol as the basic medium (modified 199 supplemented with 20% calf serum) for producing vitrification were determined. When both glycerol and 1, 2-propanediol were present in the solution (> 45% v/v in total), vitrification of the solution was achieved. In experiment 2, blastocysts were equilibrated to the vitrification solution in step wise manners from one to 16 steps using exposure to gradually concentrated solutions. Then blastocysts were vitrified by plunging the straws containing them into liquid nitrogen, thawed and cultured. Higher survial rates were obtained for blastocysts equilibrated in 4, 8 and 16 steps (79%, 82% and 87%, respectively) than for those in 1 step (0%) and 2 steps(10%). Inexperiment 3, freeze-replica observations were carried out on blastocysts vitrified by the 16 step method. The freeze-replicas showed an absence of ice crystals throughout the cytoplasm, cavity of blastocyst and extracellular areas. In addition, little ultrastructure change in the plasma membrane of the blastocysts was observed. It was suggested that successful cryopreservation of blastocysts using the 16 step equilibration was established not only by the complete vitrification of whole specimens but also by minimization of the plasma membrane ultrastructural changes.

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Mouse blastocysts were exposed to a solution containing 20% ethyleneglycol, 20% dimethyl-sulfoxide and 10% 1,3-butanediol (called as VSv) or to a 65% sorbitol solution (called as SS) with or without prior equilibration with solution in half concentration of VSv (called as ESv) for 3 min at room temperature, they were maintained for different time periods to the VSv or SS at room temperature, and they were frozen by immersion into liquid nitrogen. 1) By immersion into LN_2, no ice crystals were produced both inside and outside of mouse blastocysts which were directly (or after equilibration with ESv for 3 min) exposed to VSv or SS for more than 30 sec. 2) The survival assay of embryos after freeze-thawing showed that: a) by direct (or after equilibration with ESv for 3 min) exposure to VSv, high survival (> 90%) was obtained by exposure for 30 sec or 1 min, but survival was gradually reduced by prolonged exposure causing complete injury by exposure for more than 5 min, and: b) by direct (or after equilibra tion with ESv for 3 min) exposure to SS, no survival was provided regardless of the exposure time. 3) The light microscope observation showed that: a) by direct (or after equilibration with ESv for 3 min) exposure to VSv, no distinct shrinkage of embryos was produced, and: b) by direct (or after equilibration with ESv for 3 min) exposure to SS, distinct shrinkage of embryos was produced. 4) The freeze-etching electron microscope observation showed that: a) by direct (or after equilibration with ESv for 3 min) exposure to VSv, only slight clustering of IMPs in the plasma membrance was produced by exposure for 30 sec or 1 min, distinct IMP clustering and vesiculation in the plasma membrane and slight coagulation of cytosol were produced by exposure for 3 min, and complete denaturation of cells was produced by exposure for more than 5 min., and: b) by direct (or after equilibration with ESv for 3 min) exposure to SS, no cellular ultrastructural changes were produced by exposure for at least 10 min. From these results, it was concluded that successful cryopreservation of mouse blastocysts by one step exposure to VSv for a short period resulted from the facts that blastocysts were prevented from severe dehydration due to quick permiation of VSv inside the cells, and in a short exposure time blastocysts were also prevented from chemical toxicity which producedultras tructural changes.

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Fracture stress of soy-protein gel (tofu) was measured at temperatures between -20 and -196℃ by compression tests. Fracture stress increased as the temperature decreased until it reached a characteristic temperature; below this temperature the fracture stress remained constant. Fracture stress varied with the moisture content of the sample measured before freezing; the lower the moisture, the larger the fracture stress. When the frozen soy-protein gel was regarded as a two component system consisting of pure ice (dispersed) and concentrated amorphous solution, CAS (matrix), a calculation using a parallel model gave a reasonable value for the fracture stress of CAS.

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Although cryopreservation of platelets has been attempted for two decades, the recovery, survival in vitro function and clinical efficiency is still not satisfactory. To investigate the difficulty of cryopreserving platelets while retaining full function, we analyzed glycoproteins (GP), such as GP Ib, GP Ilb/IIIa and GP IV on frozen-thawed or osmotically stressed platelet membranes by monoclonal antibodies and flow cytometry. Cellular functions such as platelet aggregation induced by ADP, collagen and ristocetin and %HSR, etc., were also analyzed. Platelets loaded with 5% Me_2SO were frozen extracellularly to -80℃ at 2℃/min and thawed rapidly in a 37℃ water bath. The Me_2SO was removed and the expression of GP was analyzed. There was no change of expression of GP IIb/IIIa or GP IV but GP Ib was lost from about half of the population. Frozen platelets also showed about a 50% loss of ristocetin-induced platelet aggregation. Platelets frozen without Me_2SO lost GP Ib completely. Platelets osmotically stressed with plasma made hyperosmotic by addition of NaCl and resuspended in plasma were also anylyzed for GP. Again, only GP Ib was lost at osmotic stress greater than 1000mOsm/kg. Thus, GP I b is easily shed from stressed platelet membranes and may be an important reason for the difficulty in cryopreservation.

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Marchantia protoplasts (regenerated cells) which had been cultured for 18 to 22 hrs and regenerated new cell wall showed the highest tolerance to osmotic dehydration as compared with either protopalasts before culture or callus tissues from which protoplasts were isolated. The tolerance of regenerated cells decreased to the level of protoplasts when these cells were treated with cell wall digesting enzyme containing cellulase activity. However, the tolerance was maintained when the cells were treated with the enzyme in 2.0 M BSS (Balanced salt solution, NaCl:CaCl_2=9:1), although the regenerated cell wall was removed by the enzyme digestion. These results suggest that the presence of the complete form of cell wall is not necessary for the tolerance to osmotic dehydration, but some structure(s) which is not digested by the enzyme in 2.0 M BSS is involved in the tolerance mechanism.

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Ice crystal growing rate and morphology, which are the characteristics of ice crystal enlarging, depend on the degree of supercooling evaluated by the deviation from the freezing point in the equilibrium condition. However, the growth rate of ice in the solution has not been measured for various solutions. In this study, the rate of ice growing was determined under the optical microscope which has a cold stage cooled by liquid nitrogen. Ice crystal growth rate in pure water which is prepared for the high pressure liquid chromatography showed similar values and an inclination to the previous studies. Growing rates in the solutions which contained NaCl, DMSO, glucose, sucrose, maltose respectively were measured at supercooling temperatures. The empirical equations of ice crystal growing rate were expressed in the power of the degree of supercooling and concentration in lower than 0.1 mol/l. Growth rate in higher concentrations than 0.1 mol/l solution changed drastically, but the reason why such drastic changes occurred was not clarified yet in this work.

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Comparison of Raman spectra of aqueous sulfuric acid at room temperature and in the glassy state indicates that ionization of sulfuric acid proceeds almost completely at low temperatures. On the other hand, ionization of aqueous KOH solution decreases at low temperatures. It is inferred from these results that Lewis basicity of water increases with lowering temperature.

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Since the appearance of the paper concerning freeze-dried ground fish meat in the journal of Nissuishi (Y. Matsuda) in 1969, a number of reports have been published. These studies were performed aiming to manufacture fish meal with the properties of fresh meat by freeze-drying (FD) fish meat or frozen ground meat and to store it as a raw material for boiled fish paste for a longperiod. We searched a new application field of freeze-dried ground fish meat under joint researches since 1972. In this research process, however, we developed quite a new application as a "non-heating food binding agent" for the manufacture of copied herring roes by FD powdered ground meat. In addition, we have tried to improve the binding property. In this report, we focus on the binding ability of freeze-dried fish peptide (FDFip) as a New FD Material, and report the detailed examination of reproducibility of viscoelasticity, along with the effect of FD conditions on the reconstitution of physiological activities, effects of salt and other additives, and effects of pH and heating, when FDFip is added to foods.

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Freeze dried starch (FDSt) reported herein is one of the typical New FD Materials. Water was added to starch, boiled to be paste, and subjected to freeze-drying to obtain a new material "liquid adsorbent" with fine and porous micell structure. Properties displayed by FDSt are quite different from those of commercially available alpha starch made under atmospheric pressure through the same process of heating treatment. In the first report, it was shown that the liquid adsorption ability of FDSt is higher by several times than that of the original starch, and the evaporation and diffusion rates of adsorbed alcohol are slower than the original starch. As it has been found that the swelling rate test which is used as a simple method for the assessment of the degree of alpha rearrangement can not be used for FDSt, we report the details of the matter and the investigations we have studied concerning it.

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In a process of freeze-drying of liquid material by the use of vertical tubes, it is important that the solute concentration upon thawing of the frozen layer C_f should be equal to that of the non-frozen liquid removed away from the tubes C_I when desired amount of the frozen layer is formed. Phenomena taking place in the frozen layer formation with several aqueous solutions, mostly sucrose and dextrin, were analysed in this study. The influences of initial solute concentration C_o, freezing rate (cooling brine temperature T_h and percentage of the frozen layer to the amount of liquid poured into a tube β on C_f and C_1 have been studied experimentally. In each case for C_o=10, 20, and 30 wt % of sucrose, the difference between C_f and C_1 was observed only under slow freezing conditions, i.e. growth velocities of a couple of centimeters per hour or less (T_h≧-20℃). While for dextrin solutions, any concentration changes did not occur. Availability of "Multi-Stage Frozen Layer Formation" was confirmed in this study.

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The glacier, a moving body of snow and ice, has long been believed to be an almost non-biological world, because of its cold and severe environment. However, previously unsuspected persistent biotic communities consisting of various cold tolerant animals and microorganisms were discovered on Himalayan glaciers. All these animals are specialized to the glacier environment and complete their life cycles there. Glaciers are never abiotic, but have various type of biotic communities comparable to other fresh water environments such as lakes and rivers. Glaciers are simple and relatively closed ecosystem with specialized biotic communities.

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Aquatic mosses in Antarctic lakes were first recorded when Russian biologists carried out Antarctic studies in the International Geophysical Year (IGY), 1957-1958. Then some aquatic mosses were collected at a depth of over 30m in lakes. In Syowa Station area, two aquatic mosses Bryum pseudotriquetrum and Dicranella sp. were known, of which the latter moss with peculiar globose gemmae was found from lake beds at 3-5m depth in Skarvsnes region. Joining the 29th Japanese Antarctic Research Expedition (JARE-29, 1987-89) for a botanical study as one of the over-wintering members, the present investigator collected and determined the species of some aquatic mosses occurring on the lake beds near the Syowa Station area: i. e. Skarvsnes,Byvagasane, Breidvagnipa and Langhovde. Consequently, most specimens were a submerged form of Bryum pseudotriquetrum, but others included species of Dicranella sp. and submerged forms of Pottia heimii and Bryum amblyodon. The Dicranella sp. should be assigned to cosmopolitan species Leptobryum pyriforme on examination of cultural characters in this study. There are two theories to account for the origin of the Antarctic moss flora. One suggests that they are remnants of a preglacial climax vegetation, the other that they were carried to Antarctic by air current or sea birds from other continents. Since most terrestrial and aquatic mosses in the Syowa Station area are cosmopolitan species, the latter theory seems more reasonable.

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E. coli cold resistant growth (Crg) mutants, MET 1 and MET 2, having the ability to grow at 7.5℃, were isolated from E. coli strain K-12 which shows rapid loss of viability under the same condition (cold sensitive growth, Csg). Strain MET 3, the trp B derivative of stain K-12 constructed by genetic manipulation using strain PA3092 as a donor of genetic markers, showed remarkable and rapid loss of viability at temperatures between 8 and 10℃ (cold super sensitive, Css). Cell elongation was observed at these temperatures of cell death, while strain MET6 constructed from MET3 by transduction using MET1 (crg-1) as a donor was able to grow normally at and above 7℃. The crg gene (s) was located very close to the tdk gene at 27.5 min on E. coli chromosome by the conjugation and transduction experiments. The gene responsible for the Css phenotype was deduced to be identical or very close to the crg locus. The DNA fragment which complemented the Css phenotype of MET3 was cloned from E. coli wild-type strain. The complementing DNA, adjacent to thymidine kinase-like gene, was found to encode histone-like protein H-NS. The role of H-NS gene in pleiotropic regulation was discussed.

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As the insects are poikilothermic, body temperature fluctuates with ambient temperature and there is a possibility of freezing during overwintering. Against freezing, insects are devided into two groups; Freeze tolerant species which tolerate extracellular freezing and freeze-intolerant species which indicates freeze-avoidance. With the exception of the freeze-tolerant species with very high degree of supercooling the main difference between the two strategies (freeze-tolerance and freeze-intolerance) is the winter loss or masking of nucleators on freeze-intolerant species, and synthesis or unmasking of nucleating agents in freeze-tolerant species. Ice nucleation active bacteria (INAB), Erwinia herbicola were found in diamond-back moth (Plutella xylostella) pupae. The bacteria may be the origin of ice nucleation activity in the pupae. Because INAB exist on the leaves of many plant species and are distributed all over the world, it is highly possible that phytophagous insects take INAB on feeding and this may cause a death on hibernating.

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Effects of temperature on the kinetic and equilibrium parameters of the binding reaction of a gonadotropic hormone (follicle stimulating hormone, FSH) to its receptor in both homeothermic and poikilothermic vertebrates were studied, since we found that FSH could bind to its receptors at low temperatures in poikilotherms but not in homeotherms. The association rate constant in poikilotherms was significantly less affected by temperature than in homeotherms. The equilibrium constant of association was high at around 37℃ and decreased by lowering the temperature. That of amphibians was maximum between 15 and 25℃ and decreased as a whole at higher temperature. The affinity in a turtle did not vary significantly in the temperature range between 0 and 40℃. These optimal temperatures coincided well with the temperatures at which gonadotropin acts on the gonad in each species under the natural condition.

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Our hibernation studies on mammals started from the physiological finding that Ca ion regulatory system of cardiac muscles from chipmunks, a mammalian hibernator, was markedly changed for preparation for hibernation. From this evidence, the existence of an endogenous factor related to hibernation was assumed and four types of protein specific for hibernation were identified in the blood of chipmunks. These proteins (HP-20, 25, 27 and 55) were markedly reduced from the blood before, and during hibernation. HP-20, 25 and 27 are homologous and a novel type of protein, while HP-55 is similar to α_1-antitrypsin. These novel proteins were detected only in hibernating species in the squirrel family. In the present paper, our recent studies on the heart function and the blood proteins specific for hibernation were reviewed.

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Many plant species indigenous to tropical and subtropical low lands are sensitive to chilling environment. Protracted exposure to low temperatures ranged above zero to 12℃ leads eventually cell death, although the cell injury is reversible at the initial stage. In mung bean seedlings, vacuolar membranes are found to be the most sensitive cellular site for chilling temperature. Dysfunction of the vacuolr proton pumps under low temperature due to the strong temperaturedependency of the enzymes and the time-dependent inactivation of the H^+-ATPase appears to cause the leakage of protons from vacuoles and leads an acidosis of the cytoplasms in the initial stage of chilling treatment, which is considered to be a determinant of cell injury.

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