Investigation of the behavior of dissolved gases during freezing

This paper deals with the freezing process of aqueous solutions of gases and the nucleation of gas bubbles at the moving ice—water interface. A cryomicroscope was used to investigate the conditions of nucleation and growth of bubbles after reaching a stationary concentration profile ahead of the phase boundary. The enrichment of gases due to the distribution coefficient was detected by means of a test bubble method, i.e., the increase in the radius of a small bubble being approached by the ice front. A distribution coefficient of 0.048 (at 0 °C) was found for oxygen. Nucleation occurs when stationary growth conditions in the solution are reached. The measured oversaturation is close to 20, i.e., about the inverse of the distribution coefficient. In highly saturated gas solutions, dendritic breakdown of the planar ice-water interface due to gas enrichment could be observed. At these positions also a considerable degree of constitutional supercooling was found. Bubbles were nucleated in interdendritic spaces. Nucleation and growth of gas bubbles was seen to be a periodic process under certain circumstances which can be explained by the continuous buildup and reduction of the concentration field in the remaining solution. The growth kinetics of the bubbles and their maximum size are governed by the velocity of the ice-water interface. During growth the gas bubbles are pushed and partially encapsulated, until they reach a radius in the order of magnitude of the diffusion boundary layer of the concentration profile, and become totally engulfed by the solid phase.     

Cell partitioning during the directional solidification of trehalose solutions

Previous studies have demonstrated that ice/cell interaction influences post thaw viability and specific cryoprotective agents can affect those interactions. Trehalose, a disaccharide, has been shown to have a protective benefit during conventional slow freezing. Existing theories have been put forth to explain the protective benefit of trehalose during desiccation and vitrification, but these theories do not explain the protective benefit observed during conventional freezing protocols. The overall objective of this investigation was to characterize cell/ice interactions in the presence of trehalose using non-planar freezing conditions. To that end, lymphoblasts suspended in phosphate buffered saline solution with various levels of trehalose (0, 10, 100, and 300 mM) were frozen on a directional solidification stage. The partitioning of cells into the interdendritic space or engulfment by an advancing dendrite was determined as a function of velocity and solution composition. For a given temperature gradient, the fraction of cells entrapped into the interdendritic region increased with increasing velocity. With small additions of trehalose (10 mM), the velocity at which cells were entrapped in the interdendritic region increased. At high trehalose concentrations (100, 300 mM), interface morphology was significantly different and cells were engulfed by the advancing interface. Dehydration of cells in the region shortly before and after the interface was significant and depended upon of the type of interaction experienced by the cell (entrapped vs. engulfed). These studies suggest that one potential mechanism for the action of trehalose involves changing the ice/cell interactions during conventional slow freezing.     

Cell partitioning during the directional solidification of trehalose solutions

Previous studies have demonstrated that ice/cell interaction influences post thaw viability and specific cryoprotective agents can affect those interactions. Trehalose, a disaccharide, has been shown to have a protective benefit during conventional slow freezing. Existing theories have been put forth to explain the protective benefit of trehalose during desiccation and vitrification, but these theories do not explain the protective benefit observed during conventional freezing protocols. The overall objective of this investigation was to characterize cell/ice interactions in the presence of trehalose using non-planar freezing conditions. To that end, lymphoblasts suspended in phosphate buffered saline solution with various levels of trehalose (0, 10, 100, and 300 mM) were frozen on a directional solidification stage. The partitioning of cells into the interdendritic space or engulfment by an advancing dendrite was determined as a function of velocity and solution composition. For a given temperature gradient, the fraction of cells entrapped into the interdendritic region increased with increasing velocity. With small additions of trehalose (10 mM), the velocity at which cells were entrapped in the interdendritic region increased. At high trehalose concentrations (100, 300 mM), interface morphology was significantly different and cells were engulfed by the advancing interface. Dehydration of cells in the region shortly before and after the interface was significant and depended upon of the type of interaction experienced by the cell (entrapped vs. engulfed). These studies suggest that one potential mechanism for the action of trehalose involves changing the ice/cell interactions during conventional slow freezing.     

Observation and quantification of gas bubble formation on a mechanical heart valve

Clinical studies using transcranial Doppler ultrasonography in patients with mechanical heart valves (MHV) have detected gaseous emboli. The relationship of gaseous emboli release and cavitation on MHV has been a subject of debate in the literature. To study the influence of cavitation and gas content on the formation and growth of stable gas bubbles, a mock circulatory loop, which employed a Medtronic-Hall pyrolytic carbon disk valve in the mitral position, was used. A high-speed video camera allowed observation of cavitation and gas bubble release on the inflow valve surfaces as a function of cavitation intensity and carbon dioxide (CO2) concentration, while an ultrasonic monitoring system scanned the aortic outflow tract to quantify gas bubble production by calculating the gray scale levels of the images. In the absence of cavitation, no stable gas bubbles were formed. When gas bubbles were formed, they were first seen a few milliseconds after and in the vicinity of cavitation collapse. The volume of the gas bubbles detected in the aortic track increased with both increased CO2 and increased cavitation intensity. No correlation was observed between O2 concentration and bubble volume. We conclude that cavitation is an essential precursor to stable gas bubble formation, and CO2, the most soluble blood gas, is the major component of stable gas bubbles.     

A cryomicroscope for the analysis of solute polarization during freezing

A better understanding of the freezing process in the extracellular suspension medium implies the consideration of deviations from equilibrium, i.e., unsteady diffusion of heat and mass with a moving phase boundary. Such phenomena, especially solute redistribution in front of the advancing phase interface, can readily be investigated with a special cryomicroscope equipped with a spectrophotometer. A major advantage of this method is the combination of quantitative measurements in conjunction with visual observations, allowing a control of the solid-liquid interface morphology (planar-cellular-dendritic) which is crucial to the solidification process. The freezing stage designed for this purpose produces a temperature field in the sample layer resembling that within a large plate-shaped container, and hence well-defined thermal gradients (having a dominant effect on the shape of the interface). Aqueous solutions of NaMnO₄, exhibiting a maximum absorption at 525 nm and a phase diagram as well as diffusive properties very similar to NaCl in water, turned out to be a particularly suitable model for simulating of solidification of biological solutions. As long as freezing is unidimensional (planar), the concentration profiles can be scanned on-line, while multidimensional (cellular, dendritic) structures require off-line densitometric determination from photomicrographs. The experimental results agree quite well with mathematical models for both types of solidification. The observed transition points between planar freezing and higher-order structures correspond to those resulting from constitutional supercooling, a criterion roughly indicating the conditions for interface instability based on temperature and concentration gradients at the phase boundary.     

An hypothesis for survival of spermatozoa via encapsulation during plane front freezing

Plane front freezing presents the possibility of encapsulating individual cells in the ice phase. The cells may also be pushed ahead of the plane front ice interface, as is always the case for conventional dendritic freezing, where the cells are pushed ahead of the thickening dendrite arms. Cells which are encapsulated during freezing are exposed to hypotonic liquid (pure water) initially upon thawing, while cells which are pushed into the last liquid to freeze are exposed to hypertonic liquid upon thawing. Some exposure to hypertonic intercellular liquid prior to freezing may be required to build up the salt and CPA content in the intracellular liquid and thereby avoid intracellular ice formation at the given cooling rate. Encapsulation of cells by a plane front ice interface should result in three regions of cell survival in the sample: an initial region of cell death due to intracellular ice formation, a final region of cell death due to overexposure to hypertonic intercellular liquid, and an intermediate region of cell survival, where neither damage mechanism has operated to a lethal level. An advantage of plane front freezing over dendritic freezing is that the regions of cell survival and death should be geometrically separate in the sample, rather than mixed at the dendritic microstructural level, as is the case for dendritic freezing. Samples containing populations with very high or very low survival rates for spermatozoa could be obtained by simply cutting up the frozen sample.     

Effects of cooling rate and glycerol concentration on the structure of the frozen kidney: assessment by cryo-scanning electron microscopy

An experimental technique, employing a directional solidification stage for controlled freezing of tissue samples and low-temperature scanning electron microscopy for observation of the structure of the frozen-hydrated samples, was used to study freezing processes in the kidney. Parametric studies in which the cooling rate during freezing and the concentration of glycerol in the tissue were varied confirmed the results of earlier freeze-substitution studies. The results suggest a mechanism for ice propagation in the kidney similar to that already proposed for the liver, in which ice originates in, and is subsequently propagated through, the peritubular vasculature. The ice front dehydrates the cells and tubular structures encountered in its path, thus preventing intraluminal freezing. At higher rates of cooling and increased concentrations of glycerol there is less dehydration of cortical structure and intraluminal freezing occurs.     

Bubble stimulation efficiency of dinoflagellate bioluminescence

Dinoflagellate bioluminescence , a common source of bioluminescence in coastal waters , is stimulated by flow agitation . Although bubbles are anecdotally known to be stimulatory , the process has never been experimentally investigated . This study quantified the flash response of the bioluminescent dinoflagellate Lingulodinium polyedrum to stimulation by bubbles rising through still seawater . Cells were stimulated by isolated bubbles of 0 . 3–3 mm radii rising at their terminal velocity , and also by bubble clouds containing bubbles of 0 . 06–10 mm radii for different air flow rates . Stimulation efficiency , the proportion of cells producing a flash within the volume of water swept out by a rising bubble , decreased with decreasing bubble radius for radii less than approximately 1 mm . Bubbles smaller than a critical radius in the range 0 . 275–0 . 325 mm did not stimulate a flash response . The fraction of cells stimulated by bubble clouds was proportional to the volume of air in the bubble cloud , with lower stimulation levels observed for clouds with smaller bubbles . An empirical model for bubble cloud stimulation based on the isolated bubble observations successfully reproduced the observed stimulation by bubble clouds for low air flow rates . High air flow rates stimulated more light emission than expected , presumably because of additional fluid shear stress associated with collective buoyancy effects generated by the high air fraction bubble cloud . These results are relevant to bioluminescence stimulation by bubbles in two‐phase flows , such as in ship wakes , breaking waves , and sparged bioreactors . Copyright © 2015 John Wiley & Sons, Ltd.     

ADDITIONAL MECHANISMS FOR THE ORIGIN OF BUBBLES IN ANIMALS DECOMPRESSED TO SIMULATED ALTITUDES

1. A heavy ingestion of frothy emulsified fat by rats and bullfrogs does not increase susceptibility to bubble formation when the animals are decompressed 2 to 72 hours later. This indicates that gaseous films (bubble nuclei) initially present do not pass across the intestinal wall with the digested fat, and also that high fat content per se in the lymph and blood does not increase susceptibility to bubble formation. 2. Liquid caprylic acid injected into veins of bullfrogs crystallizes when the frogs are cooled. The crystallization causes bubbles to form without muscular activity on subsequent decompression. Cooling normal bullfrogs to 1–2°C. fails, however, to crystallize any substances occurring naturally in the animals that might act in a similar manner. 3. When bullfrogs are cooled (e.g. to –5° to –10°C.) until ice forms in the blood vessels, and are then warmed and decompressed, bubbles form in the absence of exercise. Crystallization of water in the body thus forms nuclei or even small bubbles that persist. If only one foot is frozen, bubbles originate in the frozen foot. In some cases visible bubbles were observed in thawed feet at sea level (i.e. without decompression). When frog''s blood is partly frozen in test tubes or in tied off sections of veins, bubbles will appear on decompression in the absence of mechanical agitation. The practical relation of this phenomenon to flight at high altitude should not be overlooked. 4. Fracturing a leg bone (tibia or femur) in a frog induces bubble formation on subsequent decompression. Bubble nuclei, which persist for ½ to 1 hour, are probably formed as a result of the intense mechanical disturbance when the bone snaps. Fracturing of bone is considerably more effective than crushing muscles for producing bubbles in frogs.     

An Improved Model for Nucleation-Limited Ice Formation in Living Cells during Freezing

Ice formation in living cells is a lethal event during freezing and its characterization is important to the development of optimal protocols for not only cryopreservation but also cryotherapy applications. Although the model for probability of ice formation (PIF) in cells developed by Toner et al. has been widely used to predict nucleation-limited intracellular ice formation (IIF), our data of freezing Hela cells suggest that this model could give misleading prediction of PIF when the maximum PIF in cells during freezing is less than 1 (PIF ranges from 0 to 1). We introduce a new model to overcome this problem by incorporating a critical cell volume to modify the Toner''s original model. We further reveal that this critical cell volume is dependent on the mechanisms of ice nucleation in cells during freezing, i.e., surface-catalyzed nucleation (SCN) and volume-catalyzed nucleation (VCN). Taken together, the improved PIF model may be valuable for better understanding of the mechanisms of ice nucleation in cells during freezing and more accurate prediction of PIF for cryopreservation and cryotherapy applications.     

The importance of heat flow direction for reproducible and homogeneous freezing of bulk protein solutions

Freezing is an important operation in biotherapeutics industry. However, water crystallization in solution, containing electrolytes, sugars and proteins, is difficult to control and usually leads to substantial spatial solute heterogeneity. Herein, we address the influence of the geometry of freezing direction (axial or radial) on the heterogeneity of the frozen matrix, in terms of local concentration of solutes and thermal history. Solutions of hemoglobin were frozen radially and axially using small‐scale and pilot‐scale freezing systems. Concentration of hemoglobin, sucrose and pH values were measured by ice‐core sampling and temperature profiles were measured at several locations. The results showed that natural convection is the major source for the cryoconcentration heterogeneity of solutes over the geometry of the container. A significant improvement in this spatial heterogeneity was observed when the freezing geometry was nonconvective, i.e., the freezing front progression was unidirectional from bottom to top. Using this geometry, less than 10% variation in solutes concentration was obtained throughout the frozen solutions. This result was reproducible, even when the volume was increased by two orders of magnitude (from 30 mL to 3 L). The temperature profiles obtained for the nonconvective freezing geometry were predicted using a relatively simple computational fluid dynamics model. The reproducible solutes distribution, predictable temperature profiles, and scalability demonstrate that the bottom to top freezing geometry enables an extended control over the freezing process. This geometry has therefore shown the potential to contribute to a better understanding and control of the risks inherent to frozen storage. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1212–1221, 2013     

Protective effect of intracellular ice during freezing?

Injury results during freezing when cells are exposed to increasing concentrations of solutes or by the formation of intracellular ice. Methods to protect cells from the damaging effects of freezing have focused on the addition of cryoprotective chemicals and the determination of optimal cooling rates. Based on other studies of innocuous intracellular ice formation, this study investigates the potential for this ice to protect cells from injury during subsequent slow cooling. V-79W Chinese hamster fibroblasts and Madin-Darby Canine Kidney (MDCK) cells were cultured as single attached cells or confluent monolayers. The incidence of intracellular ice formation (IIF) in the cultures at the start of cooling was pre-determined using one of two different extracellular ice nucleation temperatures (-5 or -10 degrees C). Samples were then cooled at 1 degrees C/min to the experimental temperature (-5 to -40 degrees C) where samples were warmed rapidly and cell survival assessed using membrane integrity and metabolic activity. For single attached cells, the lower ice nucleation temperature, corresponding to increased incidence of IIF, resulted in decreased post-thaw cell recovery. In contrast, confluent monolayers in which IIF has been shown to be innocuous, show higher survival after cooling to temperatures as low as -40 degrees C, supporting the concept that intracellular ice confers cryoprotection by preventing cell dehydration during subsequent slow cooling.     

Scavenging of OH radicals produced in the sonolysis of water

The yield of hydrogen peroxide in the sonication of argon-saturated water was studied in the presence of various solutes. The efficiency of OH radical scavenging is expressed by the reciprocal value of C 1/2, the solute concentration at which the H2O2 yield is decreased by 50 per cent. C 1/2 ranges over several orders of magnitude. It is not related to the specific reactivity towards OH in homogeneous solution. However, it is correlated to the hydrophobicity of the solutes. The competition of I- and a second solute for OH was also studied. The competition between I- and HCO2- follows similar kinetics as in homogeneous solution. However, many other solutes compete in the manner which would be expected if radical scavenging occurred in different phases. The effects are explained in terms of OH radical formation in gaseous argon bubbles, combination of OH radicals to form H2O2 in an interfacial area, and enrichment of hydrophobic solutes in the bubbles.     

The effect of water, sugars, and proteins on the pattern of ice nucleation and propagation in acclimated and nonacclimated canola leaves

Infrared video thermography was used to observe ice nucleation temperatures, patterns of ice formation, and freezing rates in nonacclimated and cold acclimated leaves of a spring (cv Quest) and a winter (cv Express) canola (Brassica napus). Distinctly different freezing patterns were observed, and the effect of water content, sugars, and soluble proteins on the freezing process was characterized. When freezing was initiated at a warm subzero temperature, ice growth rapidly spread throughout nonacclimated leaves. In contrast, acclimated leaves initiated freezing in a horseshoe pattern beginning at the uppermost edge followed by a slow progression of ice formation across the leaf. However, when acclimated leaves, either previously killed by a slow freeze (2 degrees C h(-1)) or by direct submersion in liquid nitrogen, were refrozen their freezing pattern was similar to nonacclimated leaves. A novel technique was developed using filter paper strips to determine the effects of both sugars and proteins on the rate of freezing of cell extracts. Cell sap from nonacclimated leaves froze 3-fold faster than extracts from acclimated leaves. The rate of freezing in leaves was strongly dependent upon the osmotic potential of the leaves. Simple sugars had a much greater effect on freezing rate than proteins. Nonacclimated leaves containing high water content did not supercool as much as acclimated leaves. Additionally, wetted leaves did not supercool as much as nonwetted leaves. As expected, cell solutes depressed the nucleation temperature of leaves. The use of infrared thermography has revealed that the freezing process in plants is a complex process, reminding us that many aspects of freezing tolerance occur at a whole plant level involving aspects of plant structure and metabolites rather than just the expression of specific genes alone.     

Freezing in Conifer Xylem: II. PIT ASPIRATION AND BUBBLE FORMATION

A scanning electron microscope equipped with a freezing stagewas used to examine the effects of slow freezing on pit aspirationand bubble formation in living tree stems. The size (approximately 2.0 µm diameter) and the sphericalor ellipsoidal shape of the bubbles found in the centre of frozenlumens indicated freezing rates greater than 25 µm s^–1.Both unaspirated and aspirated bordered pits were found in thefrozen xylem. The technique used did not reveal enough pitsto determine whether unaspirated pits were more prevalent thanaspirated pits. These results are compared with hypotheses and results fromprevious work on freezing in conifer xylem. Key words: Freezing, conifer xylem, bordered pits, bubbles     

Freezing of xylem sap without cavitation

Freezing of stem sections and entire twigs of hemlock (Tsuga canadensis) has been demonstrated to occur without increasing the resistance to the movement of water through the frozen part after rewarming. This was interpreted to mean that freezing did not produce cavitation in the xylem sap even though A) the sap was unquestionably frozen; B) it contained dissolved gases; and C) it was under tension before freezing and after. Freezing stem sections of some other evergreen gymnosperms during the summer again produced no evidence for cavitation of the xylem sap. On the other hand, freezing stem sections of some angiosperms invariably increased the resistance to sap flow leading to wilting and death in a few hours when the sap tension was at normal daytime values at the time of freezing. These results were interpreted to mean that the bordered pits on the tracheids of gymnosperms function to isolate the freezing sap in each tracheid so that the expansion of water upon freezing not only eliminates any existing tension but also develops positive pressure in the sap. Dissolved gases frozen out of solution may then be redissolved under this positive pressure as melting occurs. As the bubbles are reduced in size by this ice pressure developed in an isolated tracheid, further pressure is applied by the surface tension of the water against air. If the bubbles are redissolved or are reduced to sufficient small size by the time the tension returns to the sap as the last ice crystals melt, then the internal pressure from surface tension in any existing small bubbles may exceed the hydrostatic tension of the melted sap and the bubbles cannot expand and will continue to dissolve.     

Effects of solution composition on the theoretical prediction of ice nucleation kinetics and thermodynamics

Predictions by various mathematical models of intracellular ice formation (proposed by Mazur, Pitt, Toner, and Karlsson, respectively) were compared to the known thermodynamic and kinetic behavior of ice formation in supercooled aqueous systems. The older models (Mazur, Pitt, and Toner) significantly underestimated the magnitude of colligative nonequilibrium freezing point depression in response to increased concentration of solutes, such as salts or cryoprotectants. Furthermore, kinetics predicted using phenomenological models (by Mazur and Pitt) exhibited implausible temperature-dependence, with the probability of intracellular ice formation being allowed to increase even at temperatures below the glass transition point. The Toner model, on the other hand, produced invalid results at temperatures below −48 °C. The Karlsson model was the only model that consistently yielded realistic predictions over a wide range of temperatures and solute concentrations, especially in the presence of cryoprotectant additives. To facilitate adoption of the Karlsson model of intracellular ice nucleation, the complete set of model equations has been collected and described in detail.     

High ice nucleation temperature of zebrafish embryos: slow-freezing is not an option

Although fish embryos have been used in a number of slow-freezing cryopreservation experiments, they have never been successfully cryopreserved. In part this is because little is known about whether ice forms within the embryo during the slow-freezing dehydration process. Therefore, we examined the temperature of intraembryonic ice formation (T(IIF)) and the temperature of extraembryonic ice formation (T(EIF)), using a cryomicroscope. We used both unmodified zebrafish embryos and those with water channels (aquaporin-3 or AQP3) inserted into their membranes to increase permeability to water and cryoprotectants, examined at 100% epiboly to the 6-somite stage. In these experiments we examined: (1) the spontaneous freezing of (external) solutions; (2) the spontaneous freezing of solutions containing embryos; (3) the effect of preloading the embryos with cryoprotectants on T(IIF); (4) whether preloading the embryos with cryoprotectant helps in survival after nucleating events in the solution; and (5) the damaging effects of extracellular nucleation events versus solution toxicity on the embryos. The solutes alone (embryo medium--EM, sucrose culture medium, 1 M propylene glycol in EM, and 1 M propylene glycol in a sucrose culture medium) froze at -14.9 +/- 1.1, -17.0 +/- 0.3, -17.8 +/- 1.0, and -17.7 +/- 1.4, respectively. There was no difference amongst these means (P > 0.05), thus adding cryoprotectant did not significantly lower the nucleation point. Adding embryos (preloaded with cryoprotectant or not) did not change the basic freezing characteristics of these solutes. In all these experiments, (T(EIF)) equaled (T(IIF)), and there was no difference in the freezing point of the solutions with or without the embryos (P > 0.05). Additionally, there was no difference in the freezing characteristics of embryos with and without aquaporins (P > 0.05). The formation of intraembryonic ice was lethal to the zebrafish embryos in all cases. But this lethal outcome was not related to solution injury effects, because 88-98% of embryos survived when exposed to a higher solute concentration with no ice present. Taken together, these data suggest that slow-freezing is not a suitable option for zebrafish embryos. The mechanism of this high temperature nucleation event in zebrafish embryos is still unknown.     

Insect antifreezes and ice-nucleating agents

Cold-tolerant, freeze-susceptible insects (those which die if frozen) survive subzero temperatures by proliferating antifreeze solutes which lower the freezing and supercooling points of their body fluids. These antifreezes are of two basic types. Lowmolecular-weight polyhydroxy alcohols and sugars depress the freezing point of water on a colligative basis, although at higher concentrations these solutes may deviate from linearity. Recent studies have shown that these solutes lower the supercooling point of aqueous solutions approximately two times more than they depress the freezing point. Consequently, if a freeze-susceptible insect accumulates sufficient glycerol to lower the freezing point by 5 °C, then the glycerol should depress the insect's supercooling point by 10 °C.Some cold-tolerant, freeze-susceptible insects produce proteins which produce a thermal hysteresis (a difference between the freezing and melting point) of several degrees in the body fluids. These thermal hysteresis proteins (THPs) are similar to the antifreeze proteins and glycoproteins of polar marine teleost fishes. The THPs lower the freezing, and presumably the supercooling, point by a noncolligative mechanism. Consequently, the insect can build up these antifreezes, and thereby gain protection from freezing, without the disruptive increases in osmotic pressure which accompany the accumulation of polyols or sugars. Therefore the THPs can be more easily accumulated and maintained during warm periods in anticipation of subzero temperatures. It is not surprising then that photoperiod, as well as temperature, is a critical environmental cue in the control of THP levels in insects.Some species of freeze-tolerant insects also produce THPs. This appears somewhat odd, since most freeze-tolerant insects produce ice nucleators which function to inhibit supercooling and it is therefore not clear why such an insect would produce antifreeze proteins. It is possible that the THPs have an alternate function in these species. However, it also appears that the THPs function as antifreezes during those periods of the year when these insects are not freeze tolerant (i.e., early autumn and spring) but when subzero temperatures could occur. In addition, at least one freeze-tolerant insect which produces THPs, Dendroides canadensis, typically loses freeze tolerance during midwinter thaws and then regains tolerance. The THPs could be important during those periods when Dendroides loses freeze tolerance by making the insect less susceptible to sudden temperature decreases.Comparatively little is known of the biochemistry of insect THPs. However, comparisons of those few insect THPs which have been purified with the THPs of fishes show some interesting differences. The insect THPs lack the large alanine component commonly found in the fish THPs. In addition, the insect THPs generally contain greater percentages of hydrophilic amino acids than do those of the fish. Perhaps the most interesting insect THPs are those from Tenebrio molitor which have an extremely large cysteine component (28% in one THP). Studies on the primary and higher-order structure of the insect THPs need to be carried out so that more critical comparisons with the fish THPs can be made. This may provide important insights into the mechanisms of freezing point and supercooling point depression exhibited by these molecules. In addition, comparative studies of the freezing and supercooling point depressing activities of the various THPs, in relation to their structures, should prove most interesting.It has become increasingly apparent over the last few years that most freeze-tolerant insects, unlike freeze-susceptible species, inhibit supercooling by accumulating ice-nucleating agents in their hemolymph. These nucleators function to ensure that ice formation occurs in the extracellular fluid at fairly high temperatures, thereby minimizing the possibility of formation of lethal intracellular ice. Little is known of the nature of the insect ice-nucleating agents. Those few which have been studied are heat sensitive and nondialyzable and are inactivated by proteolytic enzymes, thus indicating that they are proteinaceous. Studies on the structure-function relationships of these unique molecules should be done.     

Factors contributing to inactivation of isolated thylakoid membranes during freezing in the presence of variable amounts of glucose and NaCl.

During freezing of isolated spinach thylakoids in sugar/salt solutions, the two solutes affected membrane survival in opposite ways: membrane damage due to increased electrolyte concentration can be prevented by sugar. Calculation of the final concentrations of NaCl or glucose reached in the residual unfrozen portion of the system revealed that the effects of the solutes on membrane activity can be explained in part by colligative action. In addition, the fraction of the residual liquid in the frozen system contributes to membrane injury. During severe freezing in the presence of very low initial solute concentrations, membrane damage drastically increased with a decrease in the volume of the unfrozen solution. Freezing injury under these conditions is likely to be due to mechanical damage by the ice crystals that occupy a very high fraction of the frozen system. At higher starting concentrations of sugar plus salt, membrane damage increased with an increase in the amount of the residual unfrozen liquid. Thylakoid inactivation at these higher initial solute concentrations can be largely attributed to dilution of the membrane fraction, as freezing damage at a given sugar/salt ratio decreased with increasing the thylakoid concentration in the sample. Moreover, membrane survival in the absence of freezing decreased with lowering the temperature, indicating that the temperature affected membrane damage not only via alterations related to the ice formation. From the data it was evident that damage of thylakoid membranes was determined by various individual factors, such as the amount of ice formed, the final concentrations of solutes and membranes in the residual unfrozen solution, the final volume of this fraction, the temperature and the freezing time. The relative contribution of these factors depended on the experimental conditions, mainly the sugar/salt ratio, the initial solute concentrations, and the freezing temperature.