Vitrification, or the conversion of fluid materials to a noncrystalline glass at low temperatures, is currently considered the most promising approach to cryopreservation because it limits the amount of injurious ice formed in the material. Glasses form when solutions are cooled to supersaturation. If these mixtures fail to separate into their pure phases, rather than crystallize, they become more viscous as the temperature falls and reach a "glass transition temperature" (T_g). Though glasses are physical solids, they are thermodynamic liquids whose viscosity has exceeded some arbitrary value. This apparent paradox that glasses are simultaneously solids and liquids is explained when one takes into consideration the time scale on which T_g was determined: to a good approximation in many well studied glasses, T_g is a function of the logarithm of the time period over which the viscosity is measured. The important implication for cryobiology is that the inherent instability of glasses makes the storage temperature a critical issue. Storing at liquid nitrogen temperature is convenient but may be impracticable as aqueous glasses are fragile and fracture if cooled far below T_g. However, if the storage temperature is increased toward T_g, glasses will decompose at exponentially increasing rates. Little systematic research has been devoted to determining the constants in the equations, though we have begun to discern what the relevant equations are. The Arrhenius equation appears not to be appropriate: all the decay processes which we have examined in glass and in cryopreserved materials are hyperexponential, of the Johnson-Mehl-Avrami type. Fortunately, T_g is easy to manipulate, as mixtures of glasses have intermediate T_g values, in proportion to their mass fraction. Changing T_g in this manner is called "plasticizing", and one of the best plasticizers is water. Removing this water elevates T_g, eventually above ambient temperatures, and is secondary drying component of freeze-drying. In seeds, intracellular glasses are an important element of storage stability and can be formed by drying at ambient temperatures without the necessity of an intermediate freezing step. Seeds are a valuable model for the relationship between storage stability and the vitreous state as the data extend back over a century.
This study was carried outto investigate the mechanism of frost crack formation in living trees of Abies sachalinensis. 1) Changes in width of the frost crack during freezing and thawing were measured using a small log (15cm in diameter and 50cm in length) of A. sachalinensis including a frost crack, which has been naturally produced. The frost crack was closed in the thawed state at room temperature of 20℃. In the freezing process, the frost crack began to re-open upon onset of freezing within the inner part of the log being at about -1℃, followed by marked increase of the width of the opening during the freezing period. Further cooling down of the log from -1℃ to -20℃, however, brought about little increase in the width of the crack. Inversely, in the thawing process the frost crack closed during the thawing in the inner part of the log at about -1℃. 2) Wood specimens including pith were taken from the inner parts of tree trunks of A. sachalinensis and Fraxinus mandshurica var. japonica, and dimensional changes in radius of the specimens during freezing and thawing were measured. The specimens collected from areas in which frost cracks had formed, swelled radially during freezing at about -1℃ and recovered to the initial dimensions during the following thawing. On the other hand, the specimens collected from areas far from frost cracks did not increase in radial dimension during the freezing at about -1℃ even in such cases where the specimens contained the same level of moisture as those of areas including frost cracks. 3) A tree trunk of A. sachalinensis including a frost crack was dissected and observed at freezing state by soft X-ray photography and cryo-scanning electron microscopy. Wetwood was observed in the inner part of the tree trunk. In the wetwood region, many internal cracks such as ring and radial-shakes, in which ice crystals segregated, had formed. In particular, it was noted that some of the ring shakes containing ice crystals developed extensively in the outer layer of the wetwood, and that the inner end of the frost cracks did not reach inwardly beyond the ring shakes. These results show that the opening of the frost crack is closely related to the internal swelling during the freezing at about -1℃, and that the swelling is related to the local ice segregation at the intercellular spaces such as ring and radial- shakes. It was suggested that tangential tension strains were induced in the peripheral parts of the tree trunk by the ice segregation occurring in the inner parts of the trunks, and thereby a radial-longitudinal splitting of frost crack was produced.
Suspension-cultured cells of wheat (Triticum monococcum L.) were cultured in media containing abscisic acid (ABA) and high concentrations (0.44 or 0.88M) of mannitol or sucrose and changes in their freezing tolerance were investigated. In a medium with ABA, freezing tolerance of the cells increased rapidly and achieved a maximal level after 7-10 days of culture. Optimal concentration of ABA for increasing the freezing tolerance was 10 to 20 mg/l. Survival rate of the cells which were cultured in a medium containing 10mg/l ABA for 7 days was very high even at the freezing temperature of -50℃, whereas that of control cells was very low (<10%) at -15℃. Increase in the freezing tolerance was also observed in a mannitol or sucrose medium but the effect of these substances was less than that of ABA. The osmolarity of cell extracts from the cells increased with these treatments. However, there was no correlation between the degree of freezing tolerance and the osmolarity of cell extract. Protein analysis using SDS-PAGE showed that distinct changes in protein profile were induced by ABA in the medium but not induced by high concentrations of sucose or mannitol. These results suggest that different mechanisms are involved in the development of freezing tolerance of these cells for the two treatments.
The occurrence of freezing injury in cortical parenchyma cells of mulberry trees was closely related with formation of aparticulate domains in the plasma membranes, which was produced by close membrane to membrane approach by freezing-induced cellular dehydration and deformation. The avoidance of the formation of the aparticulate domains in the plasma membranes in the cortical parenchyma cells in midwinter was related to the changes in the cytoplasmic structures which were produced rapidly by freezing to near subzero temperatures. The ER in vesicular form was rapidly altered to multiplex layers surrounding beneath the plasma membranes by freezing to near subzero temperatures. It was suggested that conversion of ER to the multiplex layers might play important role to prevent formation of aparticulate domains in the plasma membrane and providing a mechanism for extreme freezing tolerance.
The liquid-solid thermal equilibrium and the glass transition behaviour for glycine solution and sucrose-glycine mixture were investigated by using DSC. For glycine solutions the glass transition was not observed at all, but instead, it was found that a eutectic mixture of glycine and ice is formed during freezing. On reheating the eutectic mixtures, the crystal form of glycine changed from β type to α type. For sucros-eglycine blends, a typical glass transition was recognized on DSC heating curves, and the transition temperature decreased as the weight ratio of glycine increased. This shows that glycine effects on glassy sucrose as a plasticizer. These results, concerning the relation between the glass transition temperature and the weight fraction of glycine, could be represented by a semi-empirical equation. It was possible to figure a ternary state diagram by the combination of three binary state diagrams, sucrose-glycine, sucrose-water and glycine-water. Although the ternary state diagram is not complete, it could predict the behaviour during freezing of the ternary system.
A phenomenon of ice crystallization during rewarming observed with a gel made of Sephadex G-25, a kind of cross-linked dextran, and water was investigated by DSC (differential scanning calorimetry). The exotherm due to ice crystallization during rewarming in the DSC scan is always preceded by a drift toward an endothermic direction. It was indicated that the drift cannot be ascribed to a glass transition of the polymer-water system but melting of ice crystals as the extent of the drift is too large for the change in heat capacity due to a glass transition. Further DSC measurements were conducted, where a rewarming scan was interrupted when the exotherm appeared and completed (ca. -8℃), followed by the second cooling to -50℃ and the rewarming. During the second rewarming, the exotherm disappeared and the temperature where the DSC curve begins to drift toward an endothermic direction became higher by several degrees. Different thawing behaviour observed during the first and second rewarmings can be explained by the different size distribution of ice crystals formed in the gel during precedent cooling.
The volume change of aqueous solutions on freezing was measured for various alkali metal chlorides. For potassium chloride, the volume change of the aqueous solution on freezing was larger than that of water, and it became larger with the increase in the concentration of KC1. On the contrary, the change was smaller for sodium chloride than that of water, and it became smaller with the increase in the concentration of NaCl.
DTA traces of emulsified aqueous mixed alkali halide solution were obtained after the emulsions were cooled down to liquid nitrogen temperature. There is a clear trend that melting behavior for the solutions containing cations of similar ionic radii is more complex than that for the solutions containing cations of different ionic radii.
Cell suspension of Nicotiana tabacum L. cv. BY2 was cultured with a modified LS medium. The cells were examined to vitrify through two methods. The 7-days culture was replaced in the medium with 1M sucrose and cultured for 1 day. In the method 1, the cells were vitrified with PVS2 (by SAKAI) in liquid nitrogen. In the method 2, the cells were transferred to the medium containing 2M glycerol plus 0.4M sorbitol, and then to that containing 4.5M glycerol plus 1.46M sorbitol successively. Cells in the concentrated medium were vitrified similarly to the method 1. Plasmolysis of the cells was observed in both methods, but more mildly in the method 2. The ratio of heat of devitrification to that of thawing was 0.82 or 0.67 in the two methods, respectively. The thawed cells in both methods were hard to grow on the solid medium. Only the growth was observed on a solid medium through nursing culture plate of the cells from methed 2.
The polyamine composition in the cells of Aquaspirillum metamorphum IFO 13960 which is sensitive to desiccation was exmined by high-performance liquid chromatography(HPLC). A total of 2254 nmol polyamines which consisted of hydroxyputrescine, putrescine, cadaverine and spermidine were contained in 0.1 g of the wet cells. When the cells were incubated in a basal suspending medium (BSM) with 30 mM ethylenediamine (ED) for 3 hrs at room temperature, 1952 nmol of bound ED were found as another polyamine component in 0.1 g of the cells. The polyamine content in the cells was decresed to 282nmol/0.1 g after drying with BSM. The decrease of cellular polyamines was not prevented by the addition of 30 mM ED to BSM. However, ED was found to bind to the cells with an amount of 3470 nmol/0.1 g of cells. Thus the total amount of polyamines in the cells did not change during desiccation. By the addition of NaCl, both protective effect of ED and binding of ED to the cells were inhibited. When the cells were dried with 30 mM ED and 1 M NaCl, survival rate decreased to 1% from 53%, and the bound ED in the whole cells decreased to 49% of that dried without NaCl. The binding of ED to envelopes during desiccation was inhibited completely by the addition of 1 M NaCl. These results indicate that the decrease in cellular polyamine content during desiccation may cause the death of the cells. ED may compensate the loss of polyamines by its binding property to the cell membranes and prevent the desiccation damage.
Exposure of E. coli cells to high temperature at 50 to 55℃ results in sublethal or lethal stress on cells. Bacterial membranes have a role in the cellular resistance to heat, as suggested by the effect of membrane fluidity on the heat resistance. The membranes are damaged by heat, as exemplified by the blebbing and vesiculation of the outer membrane and by the leakage of intracellular ions and other small molecules and the loss of capability of substrate transport resulting from the injury of the cytoplasmic membrane. However, the cells repair the damaged functions and structures of the membranes as well as other sites, if the damages are not serious, and they recover from the injured situations under proper conditions during the post-heating incubation period. In addition, so-called heat shock proteins and "the repair proteins" may have a role in the recovery process.
Various responses are observed in bacteria to hyperosmotic pressure. Here, possibilities of some hypotheses for signals and sensors of osmotic pressure, and osmoprotectants were reviewed. Two-component regulatory system of sensor-effector seems plausible to detect any change in extracellular osmotic pressure and transduce it into various adaptational systems in the cells. Although there are many reports of osmoprotectants, it is remained as resolved question how these compounds can support cell growth. And, it was pointed out that gross analysis for them is not helpful for consideration of real situation in the cytoplasm. 'Ectoine', one of excellent osmoprotectants, was found in a moderate halophile, Halomonas sp. Unlike glycine betaine it is not known about the regulatory systems of ectoine accumulation with response to hypersalinity. But, all of the three enzymes, 2,4-diaminobutyric acid (2,4-DABA) transaminase, 2,4-DABA acetyltransferase, and ectoine synthase, were confirmed. The first and the third enzymes were purified and characterized. The results indicated that as a whole the pathway seems capable of synthesis of ectoine at approx. 0.5M NaCl.
World ocean covers about 70% of the earth's surface, with about 3,800m of the average depth and more than 11,000m of the deepest trench areas. Deep-sea area is characterized by low temperature, high hydrostatic pressure, low concentration of organic matter (available nutrients) and complete darkness, which restrict existence of most terrestrial and marine surface microorganisms. However, there have been finding and isolation of living microorganisms, which are called as barophile(pressure-loving microorganisms) and have probably adapted to such extreme environmental conditions with long duration of time. In this paper, characteristic nature of barophilic microorganisms so far studied has been reviewed.
It is a well known fact that the living realm respond to heat by synthesizing a set of specific proteins, so called heat shock proteins (hsps). These hsps in addition to have protective role against the initial temperature insult, also induce resistance to subsequent much higher temperature stress, referred as thermotolerance. We are using yeast cells as a model system to study various kinds of heat shock induced tolerance and have so far demonstrated that the prior heat shock confer not only thermotolarance but also freezing as well as high hydrostatic pressure tolerance in yeast. When cells were heat shocked before freeze-thawing, the viability was increased significantly in comparison to directly frozen cells. The increase in viability was coincide with the induction of hsp synthesis. Heat shock was also found to be significantly effective against baroinjury. It was observed that the content of unfreezing state of cell water was increased whereas spin-spin relaxation time decreased during heat shock treatment and these changes were correlated with the increase in viability as reported in this journal last year. These observations led us to study the viscosity of cell water for which we treated yeast cells under high gravity acceleration. The heat shocked cells were subjected to ultracentrifugation at 25x10^4G for 15 to 120 min. Non heat shocked and 0.5-2.0M glycerol suspended cells were used as control and reference, respectively. We observed 10 to 100 times higher viability in the cells treated with high gravity after heat shock or in the presence of glycerol suspension compared to the control. The morphology of the directly centrifuged cells were found to be atypical, but prior heat shocked or glycerol suspended cells showed normal shape. We propose that heat shock protein induction affect cell water to result in viscous as reflected by protection against freeze-thawing, high hydrostatic pressure and ultra-centrifugal force injury.
Escherichia coli cells respond to increased flux of active oxygens by activating a set of coregulated genes. In response to increased flux of hydrogen peroxide and other organic peroxides, the expression of at least 30 proteins becomes elevated over the basal levels. The peroxide stimulon includes eight proteins in E. coli that are prositively regulated by the locus, oxyR. Recently, the E. coli oxyR gene has been cloned and its nucleotide sequence has been determined. The amino acid sequence of OxyR protein deduced from the nucleotide sequence reveals a high degree of homology to those of several bacterial activator proteins called LysR family. The OxyR protein specifically binds to the upstream regulatory regions of the oxyR and katG genes as demonstrated by the DNase I footprinting experiment. It is suggested that the reduced form of OxyR protein is converted to its oxidized from by peroxide stress, which can initiate the transcription of the regulon. On the other hand, upon exposure to elevated levels of superoxide, E. coli cells respond to the superoxide stress by invoking different stimulons from the peroxide stress. At least nine proteins are produced by a regulon controlled by two regulatory genes, soxR and soxS, which position head to head with an intervening sequence of 85 bp. The soxRS regulon includes the sodA, nfo, zwf and micF genes. In addition, several new superoxideinducible genes (soi genes) have recently been isolated and characterized in our laboratory.
Roles of the unsaturation of membrane lipids in low-temperature adaptation were studied using strains of cyanobacteria, Synechocystis PCC6803 and Anacystis nidulans R2-SPc that were gene-technologically manipulated with a desaturase gene. In the case of Synechocystis PCC6803, the unsaturation of membrane lipids was eliminated stepwise and discretely. The wild type contained mono-, di- and triunsaturated lipid molecules; a mutant, designated Fad6, contained mono- and diunsaturated lipid molecules; and a transformant of Fad6 with a disrupted gene for desaturation, designated Fad6/desA::Km^r contained only monounsaturated lipid molecules. Fad6/desA::Km^r was the most susceptible to low temperature among these strains with respect to growth and photoinhibition, whereas Fad6 and wild type were apparently indistinguishable in terms of sensitivity to growth and photoinhibition at low temperature. The introduction of a desaturase gene from Synechocystis PCC6803 into A. nidulans R2-SPc increased the unsaturation of membrane lipids and the tolerance of the recipient to low temperature. These results suggest that the unsaturation of membrane lipids is important for the tolerance toward low temperature.
Over 60 species of insects are tolerant to freezing. About half of them have supercooling points above -10℃. The high supercooling points are frequently associated with the presence of endogenous ice-nucleating agents. Start of freezing at high subzero temperatures is believed to protect freeze-tolerant insects against lethal intracellular freezing. In some other insects supercool below -10℃ , their potential freeze tolerance is occasionally realized by the inoculation of ice at temperatures nearly below 0℃. The present paper deals with the initiation mechanisms of freezing in three types of freeze-tolerant insects.
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