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PubMed1980

Studies on the damage to Escherichia coli cell membrane caused by different rates of freeze-thawing.

Freeze-thawing of Escherichia coli cells caused a release of cell membrane components such as protein, phospholipids and lipopolysaccharides. A greater amount of release and a lesser extent of cell survival were seen in slow freeze-thawing than in rapid freeze-thawing. Several dehydrogenases in the cells were also freed. The mode of release was also dependent on the rate of freeze-thawing. The materials released by slow freeze-thawing were found to be mostly composed of outer membrane components, whereas the materials released by rapid freeze-thawing contained cytoplasmic as well as outer membrane components. The chemical composition of these fragments differed significantly from that of the original membranes. The relative content of cytoplasmic membrane-bound enzymes in these fragments also differed from that of the cytoplasmic membrane. The fragmentation was assumed to have resulted mainly from the crystallization of external water. In slow fraeeze-thawing, it was considered that the phase separation of the membrane phospholipid bilayer increased the possibility of outer membrane fragmentation. Rapid freeze-thawing caused cytoplasmic membrane damage to the cells as well as to the outer membrane. In rapid freeze-thawing, the effect of phase separation appeared to be small because of rapid passage through the transition temperatures. The presence of 10% glycerol completely inhibited the release of cellular materials and enzymes. Cell survival was maintained at a high level in the glycerol-treated samples whether freeze-thawed slowly or rapidly.

Cell Membrane↗
PubMed1983

Delayed kinetics of recovery of haemopoiesis following autologous bone marrow transplantation. The role of excessively rapid marrow freezing rates after the release of fusion heat.

Thirty-five patients were treated by intensive chemotherapy and/or whole-body irradiation followed by reinjection of cryopreserved autologous bone marrow. In 8 patients the kinetics of recovery of haemopoiesis was delayed (recovery to 10(9) leucocytes/litre beyond day 27 and recovery to 50 X 10(9) platelets/litre beyond day 25). This delay was directly responsible for the death of 3 patients and contributed to a fatal outcome in 2 others (mortality rate 9-14%). Retrospective analysis of these 8 cases revealed that failure of autologous transplantation was associated with poor recovery of CFUc, which was in turn related to an excessively rapid freezing rate after the release of fusion heat. Recovery of CFUc to 50% or more was achieved in 100% of cases when the freezing rate was less than 5 degrees C/min, 45% for freezing rates between 5 and 10 degrees C/min and 22% when the freezing rate exceeded 10 degrees C/min (n = 71, P less than 0.001). There was an inverse linear or logarithmic relationship between CFUc recovery and freezing rate after the transition phase (r = -0.46, r = -0.43, P less than 0.001). The quantity of nitrogen introduced into the freezing chamber to annul the fusion heat must therefore be calibrated with accuracy so that the desired shortening of the transition phase will not be accompanied by an overly marked increase in the freezing rate, which would result in the destruction of stem cells. To ensure an adequate freezing rate, it is crucial to monitor the temperature continuously in each sample of bone marrow during the freezing process. This study also suggested that other factors may have interfered with the kinetics of recovery after autologous bone-marrow transplantation. These factors include myelofibrosis, the presence of an Australia antigen and administration of compounds that are toxic for the bone marrow after reinjection of cryopreserved marrow. However, the responsibility of these factors cannot be stated with certainty.

Adult↗
PubMed2004

Advanced hepatic tissue destruction in ablative cryosurgery: potentials of intermittent freezing and selective vascular inflow occlusion.

Recent studies indicate that cryosurgery represents a promising approach to treat non-resectable liver tumors. To improve parenchymal tissue destruction, a variety of modifications of the freeze-thaw procedure have been suggested, including repetitive freezing and portal-triad cross-clamping. The aim of the present study was to analyze whether intermittent freezing by application of a double freeze-thaw procedure or selective vascular inflow occlusion are more effective than a single freeze-thaw cycle to achieve complete hepatic tissue destruction. Using a porcine model, intrahepatic cryolesions were induced by freezing the hepatic tissue for a total of 15 min (n=6, SF). Additional animals (n=6) underwent a double freeze-thaw cycle of 7.5 min each (DF). A third group of animals (n=6) was treated by a single 15-min freeze-thaw cycle during selective vascular inflow occlusion (VO-SF). Seven days after freezing, DF did not change the volume of the cryolesion (25.4+/-1.7 cm(3)) compared to SF (29.9+/-3.7 cm(3)), however, resulted in enhanced destruction of hepatocyte nuclear morphology (DF-score: 2.4+/-0.2 versus SF-score: 1.1+/-0.3; p<0.05) and attenuated leukocyte infiltration within the margin of the cryolesion (DF-score: 1.5+/-0.2 versus SF-score: 2.8+/-0.1; p<0.05). VO-SF was also effective to significantly enhance destruction of hepatocyte nuclear morphology (2.8+/-0.1; p<0.05 versus SF), but, additionally, markedly increased the volume of the cryolesions (43.3+/-5.3 cm(3); p<0.05 versus SF and DF). Interestingly, VO-SF further increased the number of apoptotic cells, while leukocyte infiltration (2.3+/-0.3) was not affected compared to that after SF-treatment. Thus, our data indicate that both DF and VO-SF are effective to enhance parenchymal cell destruction within the margin of the cryolesion. VO-SF additionally increases the volume of the lesion and may therefore be most attractive for successful clinical application.

Animals↗
PubMed2006

Reptile freeze tolerance: metabolism and gene expression.

Terrestrially hibernating reptiles that live in seasonally cold climates need effective strategies of cold hardiness to survive the winter. Use of thermally buffered hibernacula is very important but when exposure to temperatures below 0 degrees C cannot be avoided, either freeze avoidance (supercooling) or freeze tolerance strategies can be employed, sometimes by the same species depending on environmental conditions. Several reptile species display ecologically relevant freeze tolerance, surviving for extended times with 50% or more of their total body water frozen. The use of colligative cryoprotectants by reptiles is poorly developed but metabolic and enzymatic adaptations providing anoxia tolerance and antioxidant defense are important aids to freezing survival. New studies using DNA array screening are examining the role of freeze-responsive gene expression. Three categories of freeze responsive genes have been identified from recent screenings of liver and heart from freeze-exposed (5h post-nucleation at -2.5 degrees C) hatchling painted turtles, Chrysemys picta marginata. These genes encode (a) proteins involved in iron binding, (b) enzymes of antioxidant defense, and (c) serine protease inhibitors. The same genes were up-regulated by anoxia exposure (4 h of N2 gas exposure at 5 degrees C) of the hatchlings which suggests that these defenses for freeze tolerance are aimed at counteracting the injurious effects of the ischemia imposed by plasma freezing.

Animals↗
PubMed2006

Contribution of extracellular ice formation and the solution effects to the freezing injury of PC-3 cells suspended in NaCl solutions.

The mechanism of cell injury during slow freezing was examined using PC-3 human prostate adenocarcinoma cells suspended in NaCl solutions. The objective was to evaluate contribution of extracellular ice and the 'solution effects' to freezing injury separately. The solution effects that designate the influence of elevated concentration were evaluated from a pseudo-freezing experiment, where cells were subjected to the milieu that simulated a freeze-thaw process by changing the NaCl concentration and the temperature at the same time. The effect of extracellular ice formation on cell injury was then estimated from the difference in cell survival between the pseudo-freezing experiment and a corresponding freezing experiment. When cells were frozen to a relatively higher freezing temperature at -10 degrees C, about 30% of cells were damaged mostly due to extracellular ice formation, because the concentration increase without ice formation to 2.5-M NaCl, i.e., the equilibrium concentration at -10 degrees C, had no effect on cell survival. In contrast, in the case of the lower freezing temperature at -20 degrees C, about 90% of cells were injured by both effects, particularly 60-80% by the solution effects among them. The present results suggested that the solution effects become more crucial to cell damage during slow freezing at lower temperatures, while the effect of ice is limited to some extent.

Adenocarcinoma↗
PubMed2001

Characterization of murine monoclonal antibody to tumor necrosis factor (TNF-MAb) formulation for freeze-drying cycle development.

PURPOSE: This study was designed to characterize the formulation of protein pharmaceuticals for freeze-drying cycle development. Thermal properties of a protein formulation in a freezing temperature range are important in the development of freezing and primary drying phases. Moisture sorption properties and the relationship between moisture and stability are the bases for the design of the secondary drying phase. METHODS: We have characterized the formulation of TNF-MAb for the purpose of freeze-drying cycle development. The methods include: DTA with ER probes, freeze-drying microscopy, isothermal water adsorption, and moisture optimization. RESULTS: The DTA/ER work demonstrated the tendency to "noneutectic" freezing for the TNF-MAb formulation at cooling rates of -1 to -3 degrees C/min. The probability of glycine crystallization during freezing was quite low. A special treatment, either a high subzero temperature holding or annealing could promote the maximum crystallization of glycine, which could dramatically increase the Tg' of the remaining solution. The freeze-drying microscopy further indicated that, after the product was annealed, the cake structure was fully maintained at a Tp below -25 degrees C during primary drying. The moisture optimization study demonstrated that a drier TNF-MAb product had better stability. CONCLUSIONS: An annealing treatment should be implemented in the freezing phase in order for TNF-MAb to be dried at a higher product temperature during primary drying. A secondary drying phase at an elevated temperature was necessary in order to achieve optimum moisture content in the final product.

Antibodies, Monoclonal↗
PubMed1988

Hatchling turtles survive freezing during winter hibernation.

Hatchlings of the painted turtle (Chrysemys picta marginata) are unique as the only reptile and highest vertebrate life form known to tolerate the natural freezing of extracellular body fluids during winter hibernation. Turtles survived frequent exposures to temperatures as low as -6 degrees C to -8 degrees C in their shallow terrestrial nests over the 1987-1988 winter. Hatchlings collected in April 1988 had a mean supercooling point of -3.28 +/- 0.24 degrees C and survived 24 hr of freezing at -4 degrees C with 53.4% +/- 1.98% of total body water as ice. Recovery appeared complete after 20 hr of thawing at 3 degrees C. However, freezing at -10.9 degrees C, resulting in 67% ice, was lethal. A survey of possible cryoprotectants revealed a 2- to 3-fold increase in glucose content of liver and blood and a 3-fold increase in blood glycerol in response to freezing. Although quantitatively low, these responses by spring turtles strongly indicate that these may be the winter-active cryoprotectants. The total amino acid pool of blood also increased 2.25-fold in freezing-exposed turtles, and taurine accounted for 52% of the increase. Most organs accumulated high concentrations of lactate during freezing, a response to the ischemic state imposed by extracellular freezing. Changes in glycogen phosphorylase activity and levels of glucose 6-phosphate and fructose 2,6-bisphosphate were also consistent with a dependence on anaerobic glycolysis during freezing. Studies of the molecular mechanisms of natural freeze tolerance in these turtles may identify protective strategies that can be used in mammalian organ cryopreservation technology.

Animals↗
PubMed1963

Structurally caused freezing point depression of biological tissues.

When investigating the freezing behaviour (by thermal analysis) of the glycerol-extracted adductor muscle of Mytilus edulis it was observed that the temperature of ice formation in the muscular tissue was up to 1.5 degrees C lower than the freezing point of the embedding liquid, a 0.25 N KCl solution with pH = 4.9 with which the tissue had been equilibrated prior to the freezing experiment. A smaller freezing point depression was observed if the pH values of the embedding 0.25 N KCl solution were above or below pH = 4.9. Reasoning from results obtained previously in analogous experiments with artificial gels, the anomalous freezing depression is explained by the impossibility of growing at the normal freezing temperature regular macroscopic crystals inside the gel, due to the presence of the gel network. The freezing temperature is here determined by the size of the microprisms penetrating the meshes of the network at the lowered freezing temperature. This process leads finally to an ice block of more or less regular structure in which the filaments are embedded. Prerequisite for this hindrance of ideal ice growth is a sufficient tensile strength of the filamental network. The existence of structurally caused freezing point depression in biological tissue is likely to invalidate many conclusions reported in the literature, in which hypertonicity was deduced from cryoscopic data.

Cold Temperature↗
PubMed1989

In situ freezing of the urinary bladder: a trigger of rapid development of sodium o-phenylphenate-induced urinary bladder tumors in the rat.

The effect of in situ freezing of the urinary bladder on sodium o-phenylphenate (OPP-Na)-induced urinary bladder tumor development was investigated in male F344 rats. Freezing was performed at the start of the experiment by touching the serosal surface of the bladder with a frozen steel rod. As a result, three out of 27 rats (11%) developed bladder tumors within 78 weeks when 0.5% OPP-Na feeding was started 2 weeks after freezing and one out of 27 rats (4%) when the feeding was started 12 weeks after freezing. 0.5% OPP-Na alone did not induce any bladder lesions. In a second experiment, 19 out of 25 rats (76%) developed bladder tumors (carcinomas in 12 rats and papillomas in seven rats) when 2% OPP-Na was administered from 6 weeks after freezing, whereas only one rat (5%) demonstrated a bladder carcinoma in the group given 2% OPP-Na without prior freezing. In neither experiment were tumors induced by freezing alone. Enzyme histochemistry revealed no remarkable changes in enzyme activities of regenerative hyperplasia induced by freezing. The results indicate that in situ freezing of the urinary bladder acts as a trigger of rapid development of OPP-Na-induced rat urinary bladder tumors.

Animals↗
PubMed2006

Additional freeze hardiness in wheat acquired by exposure to -3 degreesC is associated with extensive physiological, morphological, and molecular changes.

Cold-acclimated plants acquire an additional 3-5 degrees C increase in freezing tolerance when exposed to -3 degrees C for 12-18 h before a freezing test (LT50) is applied. The -3 degrees C treatment replicates soil freezing that can occur in the days or weeks leading to overwintering by freezing-tolerant plants. This additional freezing tolerance is called subzero acclimation (SZA) to differentiate it from cold acclimation (CA) that is acquired at above-freezing temperatures. Using wheat as a model, results have been obtained indicating that SZA is accompanied by changes in physiology, cellular structure, the transcriptome, and the proteome. Using a variety of assays, including DNA arrays, reverse transcription-polymerase chain reaction (RT-PCR), 2D gels with mass spectroscopic identification of proteins, and electron microscopy, changes were observed to occur as a consequence of SZA and the acquisition of added freezing tolerance. In contrast to CA, SZA induced the movement of intracellular water to the extracellular space. Many unknown and stress-related genes were upregulated by SZA including some with obvious roles in SZA. Many genes related to photosynthesis and plastids were downregulated. Changes resulting from SZA often appeared to be a loss of rather than an appearance of new proteins. From a cytological perspective, SZA resulted in alterations of organelle structure including the Golgi. The results indicate that the enhanced freezing tolerance of SZA is correlated with a wide diversity of changes, indicating that the additional freezing tolerance is the result of complex biological processes.

Acclimatization↗
PubMed1998

Vacuolar membrane lesions induced by a freeze-thaw cycle in protoplasts isolated from deacclimated tubers of Jerusalem artichoke (Helianthus tuberosus L.).

The processes of freezing injury in Jerusalem artichoke (Helianthus tuberosus L.) tubers were studied using protoplasts isolated from cold-acclimated and deacclimated tubers. Prior to freezing, protoplasts were preloaded with 10 microM fluorescein diacetate (FDA) in an isotonic sorbitol solution. After freeze-thawing at various temperatures, cell viability was evaluated under a fluorescence microscope. In cold-acclimated tubers, more than 80% of protoplasts survived freezing to -20 degrees C. By contrast, in deacclimated tubers, the cell survival abruptly declined after freezing to temperatures below -5 degrees C. Thus, freezing tolerance differed significantly between protoplasts isolated from cold-acclimated and deacclimated tubers. Two distinct types of cell injury, which were caused by either damage to plasma membrane (cell-lysis type) or by damage to the vacuolar membrane (abnormal-staining type), were observed, depending on the cold hardiness and freezing temperature. In the cells of the abnormal-staining type, shrinkage of the central vacuolar space and simultaneous acidification of the cytoplasmic space were characteristically observed immediately before complete cell-rehydration during thawing. The decrease in freezing tolerance of protoplasts after deacclimation was suggested to be due mainly to destabilization of the vacuolar membrane by freeze-induced dehydration stress.

Freezing↗
PubMed1983

Segregation of proteins and sodium in human plasma upon freezing.

The proteins and sodium in human source blood plasma are segregated and concentrated on freezing, particularly in plasma that is frozen slowly in a large container. The temperature and the concentration gradients at the freezing front induce a convection in the liquid phase and thus play the primary role in the segregation of proteins and sodium in the frozen mass. Unidirectional freezing, where the freezing front moves against gravity, minimizes segregation because the density gradient-induced convection is minimized at the solid-liquid interface. Microscopic observation of the freezing front of the plasma indicates that ice crystals are the primary solid formed. Their shape is generally irregular, and they become highly dendritic as the freezing rate increases. Apparently, the liquid phase at the freezing front is trapped between the ice crystals; it then undergoes a complex secondary freezing process at a lower temperature behind the primary freezing front of the ice crystals.

Blood Proteins↗
PubMed2006

Stabilization of frozen Lactobacillus delbrueckii subsp. bulgaricus in glycerol suspensions: Freezing kinetics and storage temperature effects.

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at -196 degrees C and -20 degrees C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (-196 degrees C and -80 degrees C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at -20 degrees C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.

Cryoprotective Agents↗
PubMed1990

Evaluation of freeze-substitution and conventional embedding protocols for routine electron microscopic processing of eubacteria.

Freeze-substitution and more conventional embedding protocols were evaluated for their accurate preservation of eubacterial ultrastructure. Radioisotopes were specifically incorporated into the RNA, DNA, peptidoglycan, and lipopolysaccharide of two isogenic derivatives of Escherichia coli K-12 as representative gram-negative eubacteria and into the RNA and peptidoglycan of Bacillus subtilis strains 168 and W23 as representative gram-positive eubacteria. Radiolabeled bacteria were processed for electron microscopy by conventional methods with glutaraldehyde fixation, osmium tetroxide postfixation, dehydration in either a graded acetone or ethanol series, and infiltration in either Spurr or Epon 812 resin. A second set of cells were simultaneously freeze-substituted by plunge-freezing in liquid propane, substituting in anhydrous acetone containing 2% (wt/vol) osmium tetroxide, and 2% (wt/vol) uranyl acetate, and infiltrating in Epon 812. Extraction of radiolabeled cell components was monitored by liquid scintillation counting at all stages of processing to indicate retention of cell labels. Electron microscopy was also used to visually confirm ultrastructural integrity. Radiolabeled nucleic acid and wall components were extracted by both methods. In conventionally embedded specimens, dehydration was particularly damaging, with ethanol-dehydrated cells losing significantly more radiolabeled material during dehydration and subsequent infiltration than acetone-treated cells. For freeze-substituted specimens, postsubstitution washes in acetone were the most deleterious step for gram-negative cells, while infiltration was more damaging for gram-positive cells. Autoradiographs of specimens collected during freeze-substitution were scanned with an optical densitometer to provide an indication of freezing damage; the majority of label lost from freeze-substituted cells was a result of poor freezing to approximately one-half of the cell population, thus accounting for the relatively high levels of radiolabel detected in the processing fluids. These experiments revealed that gram-positive and gram-negative cells respond differently to freezing; these differences are discussed with reference to wall structure. It was apparent that the cells frozen first (ie., the first to contact the cryogen) retained the highest percentage of all radioisotopes, and the highest level of cellular infrastructure, indicative of better preservation. The preservation of these select cells was far superior to that obtained by more conventional techniques.

Bacillus subtilis↗
PubMedDate not supplied

The effects of freeze-drying and ethylene oxide sterilization on the mechanical properties of human patellar tendon.

Human patellar tendon allografts are used to replace injured anterior cruciate ligaments. They are often stored by freeze-drying or freezing before use and have been sterilized by gamma irradiation or ethylene oxide gas. Studies comparing the mechanical properties of tendons preserved by freeze-drying and sterilized by ethylene oxide have yielded conflicting results. This study examined the effects of freeze-drying and ethylene oxide sterilization on the mechanical properties of human patellar tendons obtained from 11 human cadavers; all were free of musculoskeletal disease. The 22 patellar tendons were halved longitudinally; 7 were frozen at -70 degrees C for 8 weeks and served as controls; 8 were freeze-dried; and 7 were freeze-dried and sterilized with ethylene oxide gas. Ultimate tensile stress and longitudinal strain were greater for frozen than for freeze-dried, but not greater than freeze-dried, ethylene oxide-sterilized tendons (P < 0.05). The modulus of elasticity was similar in all tendon groups. The study suggests that some in vitro mechanical properties of frozen tendons may be marginally superior to the properties of freeze-dried tendons, even after correction for size difference. Ethylene oxide gas sterilization had no adverse effect on tendon mechanical properties.

Aged↗
PubMed2006

Increased susceptibility to repeated freeze-thaw cycles in Escherichia coli following long-term evolution in a benign environment.

BACKGROUND: In order to study the dynamics of evolutionary change, 12 populations of E. coli B were serially propagated for 20,000 generations in minimal glucose medium at constant 37 degrees C. Correlated changes in various other traits have been previously associated with the improvement in competitive fitness in the selective environment. This study examines whether these evolved lines changed in their ability to tolerate the stresses of prolonged freezing and repeated freeze-thaw cycles during adaptation to a benign environment. RESULTS: All 12 lines that evolved in the benign environment for 20,000 generations are more sensitive to freeze-thaw cycles than their ancestor. The evolved lines have an average mortality rate of 54% per daily cycle, compared to the ancestral rate of 34%. By contrast, there was no significant difference between the evolved lines and their ancestor in mortality during prolonged freezing. There was also some variability among the evolved lines in susceptibility to repeated freeze-thaw cycles. Those lines that had evolved higher competitive fitness in the minimal glucose medium at 37 degrees C also had higher mortality during freeze-thaw cycles. This variability was not associated, however, with differences among lines in DNA repair functionality and mutability. CONCLUSION: The consistency of the evolutionary declines in freeze-thaw tolerance, the correlation between fitness in glucose medium at 37 degrees C and mortality during freeze-thaw cycles, and the absence of greater declines in freeze-thaw survival among the hypermutable lines all indicate a trade-off between performance in minimal glucose medium at 37 degrees C and the capacity to tolerate this stress. Analyses of the mutations that enhance fitness at 37 degrees C may shed light on the physiological basis of this trade-off.

Adaptation, Physiological↗
PubMed2003

Geographic variation in energy storage and physiological responses to freezing in the gray treefrogs Hyla versicolor and H. chrysoscelis.

The physiological responses supporting freeze tolerance in anurans are well known, but the evolution of this trait remains little studied. This is the first common-garden study of geographic variation in cryoprotective responses to freezing and the degree of freeze tolerance. We studied the gray treefrogs (Hyla versicolor and H. chrysoscelis) from sympatric sites in Minnesota, Indiana and Missouri. Patterns in the literature suggest that northern frogs produce more cryoprotectants upon freezing, but we found no geographic variation in cryoprotective responses or degree of freeze tolerance. The concentration of glucose produced upon freezing was higher than previously reported for this species (liver: 475 micro mol g(-1) dry mass). Unfrozen frogs had high levels of glycerol (liver: approx. 150 micro mol g(-1) dry mass), and did not produce more upon freezing. Liver glycogen content (concentration multiplied by liver mass) was highest in frogs from Minnesota and Missouri, and was stored in preference to lipids in Minnesota frogs, possibly to provide energy for the longer northern winters. Minnesota frogs accumulated more ice (53.4+/-1.8%) after freezing to-2.5 degrees C than Indiana frogs (45.5+/-3.3%). The two species differed in body size but not in any of the physiological parameters measured. We conclude that these populations show no adaptive variation in freeze tolerance and that comparing published studies may be misleading because of different acclimation and feeding regimes.

Acclimatization↗
PubMed1998

Freezing survival by isolated Malpighian tubules of the New Zealand alpine weta Hemideina maori.

The ability of isolated Malpighian tubules from a freeze-tolerant insect, the New Zealand alpine weta (Hemideina maori), to withstand freezing was assessed by measuring post-freeze membrane potentials and rates of fluid secretion. The hemolymph of cold-acclimated Hemideina maori was found to contain relatively high concentrations of the cryoprotectants trehalose (>300 mmol l-1) and proline (41 mmol l-1). Survival of isolated Malpighian tubules was correspondingly high when a high concentration of trehalose was present in the bathing saline. Tubules allowed to recover for 20 min from a 1 h freeze to -5 degrees C in saline containing 400 mmol l-1 trehalose had a basolateral membrane potential of -53 mV compared with a potential of -63 mV in tubules not exposed to a freeze/thaw cycle. Fluid secretion in tubules that had experienced a freeze/thaw cycle in saline containing 400 mmol l-1 trehalose was 9.9+/-2.6 nl h-1 compared with 18.7+/-5.0 nl h-1 (means +/- s.e.m., N=18) in tubules that had not been frozen. Tubules frozen in saline containing a lower concentration of trehalose (200 mmol l-1) or in glucose (400 mmol l-1) showed a similar ability to survive freezing to -5 degrees C. In contrast, freezing for 1 h at -5 degrees C in saline containing 400 mmol l-1 sucrose produced a 57 % decrease in membrane potential and an 88 % decrease in secretion rate. Tubules held in saline lacking high concentrations of sugars showed no survival after freezing to -5 degrees C for 1 h. When frozen to -15 degrees C, tubules appeared to survive best in saline with the highest trehalose concentration (400 mmol l-1). Freezing damage was not simply the result of exposure to cold, since tubules chilled (unfrozen) to -5 degrees C for 1 h were not compromised even when the bathing saline lacked a high sugar concentration. Exposure of tubules to a combination of low temperature and high osmolality mimicked damage caused by actual freezing: the membrane potential showed a 60 % recovery when the test was performed in saline containing trehalose, but showed no recovery in saline containing sucrose.

Amino Acids↗