Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves

The freezing tolerance and incidence of two forms of freezing injury (expansion-induced lysis and loss of osmotic responsiveness) were determined for protoplasts isolated from rye leaves (Secale cereale L. cv Puma) at various times during cold acclimation. During the first 4 weeks of the cold acclimation period, the LT₅₀ (i.e. the minimum temperature at which 50% of the protoplasts survived) decreased from −5°C to −25°C. In protoplasts isolated from nonacclimated leaves (NA protoplasts), expansion-induced lysis (EIL) was the predominant form of injury at the LT₅₀. However, after only 1 week of cold acclimation, the incidence of EIL was reduced to less than 10% at any subzero temperature; and loss of osmotic responsiveness was the predominant form of injury, regardless of the freezing temperature. Fusion of either NA protoplasts or protoplasts isolated from leaves of seedlings cold acclimated for 1 week (1-week ACC protoplasts) with liposomes of dilinoleoylphosphatidylcholine also decreased the incidence of EIL to less than 10%. Fusion of protoplasts with dilinoleoylphosphatidylcholine diminished the incidence of loss of osmotic responsiveness, but only in NA protoplasts or 1-week ACC protoplasts that were frozen to temperatures over the range of -5 to -10°C. These results suggest that the cold acclimation process, which results in a quantitative increase in freezing resistance, involves several different qualitative changes in the cryobehavior of the plasma membrane.     

The influence of cold acclimation on the behavior of the plasma membrane following osmotic contraction of isolated protoplasts

Summary Osmotic contraction of protoplasts isolated from cold acclimated leaves ofSecale cereale L. cv. Puma results in the formation of exocytotic extrusions of the plasma membrane. Numerous knobs or polyps were observed on the surface of the protoplasts with scanning electron microscopy. In thin sections, the extrusions were bounded by the plasma membrane with a densely osmiophilic interior. Cross-fracturing of the extrusions revealed aparticulate bodies within, a further indication that the interior of the extrusions was predominantly lipid material. Freeze-fracture of the plasma membrane suggests a possible source of this lipid material. Following osmotic contraction, the particle density on the plasma membrane protoplasmic face (PFp) increased, being reflected in both a substantial increase in paracrystalline arrays and an increase in the particle density in non-crystalline regions. This increase in particle density indicates that lipid material is preferentially lost from the plasma membrane during contraction. The density on the exoplasmic face (EFp) did not change. Together, these findings suggest that during hypertonic contraction of acclimated protoplasts, lipid material is preferentially subducted from the plasma membrane and sequestered into lipid bodies (the osmiophilic regions). The formation of lipid bodies and extrusions was readily reversible. Following osmotic expansion of acclimated protoplasts, the extrusions were retracted back into the plane of the plasma membrane.Department of Agronomy Series Paper no. 1497.     

Effect of cold acclimation on antioxidant status in cold acclimated skaters

We investigated whether cold acclimation leads to increased activity of the antioxidant defense enzymes and muscle injury. Comparisons were between short track skaters (n=6) and inline skaters (n=6) during rest and at submaximal cycling (65% VO2max) in cold (ambient temperature: 5+/-1 degrees C, relative humidity: 41+/-8%) and warm conditions (ambient temperature: 21+/-1 degrees C, relative humidity: 35+/-5%), during 60 min, respectively, and during the recovery phase. Erythrocyte superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHpx), reduced glutathione (GSH), thiobarbituric substance acid (TBARS), serum creatine kinase (CK), lactate dehydrogenase (LDH), plasma myoglobin (Mb) and cortisol were determined. Activities of CAT and GSHpx and the level of GSH and TBARS in erythrocyte and the level of LDH in serum were elevated in cold acclimated subjects. We suggested that the compensatory increase in antioxidative defense enzymes resulting from long-term cold exposure may reflect the elevated reactive oxygen species (ROS) production and muscle injury at this environment acclimation.     

Destabilization of the plasma membrane of isolated plant protoplasts during a freeze-thaw cycle: the influence of cold acclimation

In conclusion, isolated protoplasts are an excellent arena in which destabilization of the plasma membrane can be directly observed during a freeze-thaw cycle by cryomicroscopy. Destabilization is manifested in various ways--intracellular ice formation, loss of osmotic responsiveness, or expansion-induced lysis. The incidence of any particular form of injury will depend on the freeze-thaw protocol and hardiness of the tissue from which the protoplasts were isolated. In all cases, however, cold acclimation directly increases the stability of the plasma membrane to the multiple stresses that arise during a freeze-thaw cycle. Such observations provide for functional differences in the plasma membrane that may now be used to consider the significance of any compositional changes in the membrane that might be determined.     

Mechanisms of intracellular ice formation.

The phenomenon of intracellular freezing in cells was investigated by designing experiments with cultured mouse fibroblasts on a cryomicroscope to critically assess the current hypotheses describing the genesis of intracellular ice: (a) intracellular freezing is a result of critical undercooling; (b) the cytoplasm is nucleated through aqueous pores in the plasma membrane; and (c) intracellular freezing is a result of membrane damage caused by electrical transients at the ice interface. The experimental data did not support any of these theories, but was consistent with the hypothesis that the plasma membrane is damaged at a critical gradient in osmotic pressure across the membrane, and intracellular freezing occurs as a result of this damage. An implication of this hypothesis is that mathematical models can be used to design protocols to avoid damaging gradients in osmotic pressure, allowing new approaches to the preservation of cells, tissues, and organs by rapid cooling.     

Effect of photoperiod and cold acclimation on survival of mice in cold

Male albino mice (Swiss-Webster) were raised at 5 °C under short (8L:16D) and long (16L:8D) light periods. All mice were housed in groups of three to five individuals in plastic mouse cages (16 × 12 × 28 cm) until 42 days of age with food and water ad libitum and cold exposed to ?40 °C between 10:00 am and 4:00 pm to determine survival time or time until loss of righting response occurred (CT min). Under short photo-periods, survival time was 49.3 ± 4.4 min and under long photoperiods it was 38.7 ± 1.9 min (P < 0.05). A second group of mice was maintained from birth at thermoneutral temperature (22 °C) under constant darkness, short day lengths (4L:20D), or constant light in the same fashion as mentioned above. When exposed to ?20 °C survival time was found to be 80.0 ± 5.0 min for the animals kept in constant darkness, 61.1 ± 2.3 min for animals raised in short photo-periods (4L:20D) (P < 0.01), and 52.4 ± 2.3 min for mice raised in constant light (P < 0.05). After 30 min mean rectal temperature was 32.1 ± 0.47 °C for constant-darkness animals, 30.5 ± 0.43 °C for short-day animals (P < 0.02), and 28.5 ± 0.74 °C for animals raised in constant light (P < 0.05). After 60 min mean rectal temperatures for constant-dark, 4L:20D, and constant-light animals were compared and body temperature was found to be 23.7 ± 1.6, 17.3 ± 1.5 (P < 0.01), and 12.8 ± 0.87 °C (P < 0.05), respectively. From these data, it is obvious that photoperiod influences cold resistance at both cold and thermoneutral acclimation temperatures although when considered individually, cold acclimation enhances cold survival to a greater degree than does reduced light exposure.     

Mechanism of initiation of intracellular ice formation

Intracellular ice crystallization was studied by the method of cryomicroscopy in the systems modeling a biological suspension, such as erythrocyte concentrates. Initiation of crystallization by extracellular ice through hydrophilic channels has been shown to be the most probable mechanism of intracellular ice formation.     

Extra- and intracellular ice formation in mouse oocytes

The occurrence of intracellular ice formation (IIF) during freezing, or the lack there of, is the single most important factor determining whether or not cells survive cryopreservation. One important determinant of IIF is the temperature at which a supercooled cell nucleates. To avoid intracellular ice formation, the cell must be cooled slowly enough so that osmotic dehydration eliminates nearly all cell supercooling before reaching that temperature. This report is concerned with factors that determine the nucleation temperature in mouse oocytes. Chief among these is the concentration of cryoprotective additive (here, glycerol or ethylene glycol). The temperature for IIF decreases from -14 degrees C in buffered isotonic saline (PBS) to -41 degrees C in 1M glycerol/PBS and 1.5M ethylene glycol/PBS. The latter rapidly permeates the oocyte; the former does not. The initial extracellular freezing at -3.9 to -7.8 degrees C, depending on the CPA concentration, deforms the cell. In PBS that deformation often leads to IIF; in CPA it does not. The oocytes are surrounded by a zona pellucida. That structure appears to impede the growth of external ice through it, but not to block it. In most cases, IIF is characterized by an abrupt blackening or flashing during cooling. But in some cases, especially with dezonated oocytes, a pale brown veil abruptly forms during cooling followed by slower blackening during warming. Above -30 degrees C, flashing occurs in a fraction of a second. Below -30 degrees C, it commonly occurs much more slowly. We have observed instances where flashing is accompanied by the abrupt ejection of cytoplasm. During freezing, cells lie in unfrozen channels between the growing external ice. From phase diagram data, we have computed the fraction of water and solution that remains unfrozen at the observed flash temperatures and the concentrations of salt and CPA in those channels. The results are somewhat ambiguous as to which of these characteristics best correlates with IIF.     

Cryopreservation of isolated hepatocytes: intracellular ice formation under various chemical and physical conditions.

Kinetics of intracellular ice formation (IIF) for isolated rat hepatocytes was studied using a cryomicroscopy system. The effect of the cooling rate on IIF was investigated between 20 and 400 degrees C/min in isotonic solution. At 50 degrees C/min and below, none of the hepatocytes underwent IIF; whereas at 150 degrees C/min and above, IIF was observed throughout the entire hepatocyte population. The temperature at which 50% of hepatocytes showed IIF (50TIIF) was almost constant with an average value of -7.7 degrees C. Different behavior was seen in isothermal subzero holding temperatures in the presence of extracellular ice. 50TIIF from isothermal temperature experiments was approximately -5 degrees C as opposed to -7.7 degrees C for constant cooling rate experiments. These experiments clearly demonstrated both the time and temperature dependence of IIF. On the other hand, in cooling experiments in the absence of extracellular ice, IIF was not observed until approximately -20 degrees C (at which temperature the whole suspension was frozen spontaneously) suggesting the involvement of the external ice in the initiation of IIF. The effect of dimethyl sulfoxide (Me2SO) on IIF was also quantified. 50TIIF decreased from -7.7 degrees C in the absence of Me2SO to -16.8 degrees C in 2.0 M Me2SO for a cooling rate of 400 degrees C/min. However, the cooling rate (between 75 and 400 degrees C/min) did not significantly affect 50TIIF (-8.7 degrees C) in 0.5 M Me2SO. These results suggest that multistep protocols will be required for the cryopreservation of hepatocytes.     

Characterization of intracellular ice formation in Drosophila melanogaster embryos

Cryomicroscopy and differential scanning calorimetry (DSC) were used to characterize the incidence of intracellular ice formation (IIF) in 12- to 13-hr-old embryos of Drosophila melanogaster (Oregon-R strain P2) as influenced by the state of the eggcase (untreated, dechorionated, or permeabilized), the composition of the suspending medium (with and without cryoprotectants), and the cooling rate. Untreated eggs underwent IIF over a very narrow temperature range when cooled at 4 or 16 degrees C/min with a median temperature of intracellular ice formation (TIIF50) of -28 degrees C. The freezable water volume of untreated eggs was approximately 5.4 nl as determined by DSC. IIF in dechorionated eggs occurred over a much broader temperature range (-13 to -31 degrees C), but the incidence of IIF increased sharply below -24 degrees C, and the cumulative incidence of IIF at -24 degrees C decreased with cooling rate. In permeabilized eggs without cryoprotectants (CPAs), IIF occurred at much warmer temperatures and over a much wider temperature range than in untreated eggs, and the TIIF50 was cooling rate dependent. At low cooling rates (1 to 2 degrees C/min), TIIF50 increased with cooling rate; at intermediate cooling rates (2 to 16 degrees C/min), TIIF50 decreased with cooling rate. The total incidence of IIF in permeabilized eggs was 54% at 1 degree C/min, and volumetric contraction almost always occurred during cooling. Decreasing the cooling rate to 0.5 degree C/min reduced the incidence of IIF to 43%. At a cooling rate of 4 degrees C/min, ethylene glycol reduced the TIIF50 by about 12 degrees C for each unit increase in molarity of CPA (up to 2.0 M) in the suspending medium. The TIIF50 was cooling rate dependent when embryos were preequilibrated with 1.0 M propylene glycol or ethylene glycol, but was not so in 1.0 M DMSO. For embryos equilibrated in 1.5 M ethylene glycol and then held at -5 degrees C for 1 min before further cooling at 1 degree C/min, the incidence of IIF was decreased to 31%. Increasing the duration of the isothermal hold to 10 min reduced the incidence of IIF to 22% and reduced the volume of freezable water in embryos when intracellular ice formation occurred. If the isothermal hold temperature was -7.5 or -10 degrees C, a 10- to 30-min holding time was required to achieve a comparable reduction in the incidence of IIF.     

Effect of cold acclimation on norepinephrine stimulated oxygen consumption in muscle

Summary The purpose of this study was to determine the effect of norepinephrine (NE) on oxygen consumption ( ) of perfused (constant flow) muscle in cold acclimated (CA) and control rats. Infusion of NE for a five minute period caused an increase in of similar magnitude in both groups. Infusion of NE for 30 min resulted in an elevated steady state in the cold acclimated group, while the control group showed only an initial increase in followed by a continual decline during the remainder of the 30 min infusion period. These results suggest that when rats are challenged by cold exposure, the magnitude of the initial muscle response to NE by control and acclimated rats is the same, but a useful sustained higher muscle oxygen consumption is found only in the cold acclimated animals.Abbreviations CA cold acclimated - NE norepinephrine     

The formation of wall-like envelopes by isolated tomato-fruit protoplasts

Summary Formation of a new cell wall around tomato protoplasts was confirmed by optical microscopy, electron microscopy and X-ray diffraction. This wall is composed of three layers; (a) an outer ring, which seems to be composed of diffuse, amorphous material, (b) an intermediate space, crossed by radial fibers, (c) a thicker, inner band composed of dense, highly consolidated material which may have sub-layers within it. Occasionally, cells are observed with only the dense consolidated layer about them. The origin of this wall and its component layers is not yet understood.National Research Council Post-doctoral Fellow, 1967–1969.     

低温驯化对阿里山潜蝇茧蜂的影响

研究了低温驯化对阿里山潜蝇茧蜂Fopius arisanus (Sonan)的影响,结果显示,阿里山潜蝇茧蜂雌雄成蜂SCP、FP均随驯化时间的延长呈先升高后下降的趋势,当处理时间为24 h时,雌雄成蜂SCP、FP均达到最高值,（SCP分别为雌-7.66±0.27℃、雄-7.22±0.31℃,FP分别为雌-2.95±0.16℃、雄-2.58±0.23℃）与对照差异显著;处理时间为72 h时,雌雄成蜂SCP、FP均达到最低值（SCP分别为雌-9.76±0.30℃、雄-8.98±0.28℃,FP分别为雌-5.08±0.23℃、雄-4.28±0.28℃）,但与对照无显著性差异;相同处理时间,雄虫SCP值高于雌虫。从头年的12月份到次年的3月份,随着时间的推移,阿里山潜蝇茧蜂越冬雌雄蜂的SCP、FP均低于室内饲养的对照组,到次年3月份雌雄虫SCP、FP均降至最低值（SCP分别为雌-12.24±0.23℃、雄-11.37±0.26℃,FP分别为雌-6.65±0.24℃、雄-5.81±0.24℃）,且同月份雌蜂的SCP、FP均低于雄蜂。阿里山潜蝇茧蜂越冬代成蜂寄生率及其子代的羽化率均随时间的推移先下降后上升,但均低于对照组;寄生率及其子代的羽化率均在次年2月份时降至最低（寄生率为37.12±1.04,羽化率为9.68±0.47）,且与对照组相比差异显著。