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PubMed2006

Vertebrate freezing survival: Regulation of the multicatalytic proteinase complex and controls on protein degradation.

The wood frog, Rana sylvatica, survives weeks of whole body freezing during winter hibernation, expressing numerous metabolic adaptations that deal not only with freezing but with its consequences including organ ischemia and cellular dehydration. The present study analyzes the 20s multicatalytic proteinase (MCP) complex from skeletal muscle to determine how protein degradation is managed in the ischemic frozen state. MCP was partially purified and assayed fluorometrically using three AMC-labeled substrates to compare multiple states: control (5 degrees C acclimated), 24 h frozen at -2.5 degrees C, 4 or 8 h thawed at 5 degrees C, 8 h anoxia, and 40% dehydration. MCP from frozen frogs showed significantly different K(m) and V(max) values compared with controls; e.g., K(m) Z-LLE-AMC increased by 45% during freezing and 52% under anoxia whereas V(max) decreased by 40%. After thawing, K(m) was restored and V(max) rose by 2.2-fold. Incubations promoting protein kinase or phosphatase action on MCP showed that phosphatase treatment strongly increased V(max) implicating reversible phosphorylation in MCP regulation during freeze-thaw. Western blotting showed a 36% decrease in MCP protein in muscle from frozen frogs. The 20s MCP preferentially degrades oxidatively-damaged proteins and evidence of impaired function during freezing came from a 1.4-fold increase in protein carbonyl content in muscle and liver during freezing. Ubiquitin and ubiquitin conjugate levels were unchanged in muscle but changed markedly in liver during freeze-thaw.

Animals↗
PubMed2005

Neural segregation of Fos-protein distribution in the brain following freezing and escape behaviors induced by injections of either glutamate or NMDA into the dorsal periaqueductal gray of rats.

Freezing and escape responses induced by gradual increases in the intensity of the electrical current applied to dorsal regions of the periaqueductal gray (dPAG) cause a distinct pattern of Fos distribution in the brain. From these studies, it has been suggested that a pathway involving the dPAG itself, dorsomedial hypothalamus and the cuneiform nucleus (CnF) would mediate responses to immediate danger and another one involving the amygdala and ventrolateral periaqueductal gray (vlPAG) would mediate cue-elicited responses. As electrical stimulation activates body cells and fibers of passage the need of studies with chemical stimulation of only post-synaptic fibers of the dPAG is obvious. To examine further this issue we measured Fos protein expression in brain areas activated by stimulation of the dPAG with glutamate (5 nmol/0.2 microL) and N-methyl-D-aspartate (NMDA) at doses that provoke either freezing (4 nmol/0.2 microL) or escape (7 nmol/0.2 microL) responses, respectively. The results showed that glutamate-induced freezing caused a selective increase in Fos expression in the superior and inferior colliculi as well as in the laterodorsal nucleus of the thalamus. On the other hand, NMDA-induced escape led to widespread increases in Fos labeling in almost all structures studied. Differently from glutamate, NMDA at doses provoking freezing caused significant increase of Fos labeling in the dPAG and CnF. Therefore, the present data support the notion that freezing behavior induced by activation of either non-NMDA or NMDA receptors in the dorsolateral periaqueductal gray (dlPAG) is neurally segregated: glutamate activates only structures that are mainly involved in the sensorial processing and NMDA-induced freezing structures involved in the motor output of defensive behavior. Therefore, the freezing elicited by the activation of non-NMDA receptors seem to be related to the acquisition of aversive information, whereas that resulting from the activation of NMDA receptors could serve as a preparatory response for flight.

Amygdala↗
PubMed2006

Improvement in the long-term stability of freeze-dried mouse spermatozoa by adding of a chelating agent.

This study demonstrates that a small amount of chelating agent in the freeze-drying solution is necessary to prevent the deterioration of spermatozoa during freeze-drying and subsequent preservation at 4 degrees C. We freeze-dried mouse epididymal spermatozoa in the solutions containing Tris-HCl and ethylenediaminetetraacetic acid (EDTA) as a chelating agent. Spermatozoa stored for various times up to 1 year at 4 degrees C were injected intracytoplasmically into individual oocytes, and the normality of chromosomes in fertilized oocytes was analyzed. In addition, embryos derived from freeze-dried spermatozoa were transferred into recipients to determine their developmental ability. Chromosomes were maintained well when spermatozoa were freeze-dried in a solution containing 10 mM Tris-HCl and 1mM EDTA (73%), and 57% of embryos developed to term. Of embryos derived from spermatozoa stored for 1 year, 65% developed into live offspring. On the other hand, when spermatozoa were freeze-dried in a solution containing 10 mM Tris-HCl and 0 or 50 mM EDTA, spermatozoa that maintained karyotypically normal chromosomes were 64% or 22%, and only 16% or 3% of embryos were developed to term, respectively. This finding suggested that mouse spermatozoa can be freeze-dried in a simple solution containing the same composition as that used to preserve extracted DNA.

Animals↗
PubMed2005

Role of membrane transport of water and glycerol in the freeze tolerance of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae).

Overwintering larvae of the rice stem borer, Chilo suppressalis accumulate glycerol and are freezing tolerant to about -25 degrees C. However, non-diapausing larvae cannot accumulate glycerol and are killed by freezing. We compared the extent of tissue damage, the effects of glycerol concentration, and the transport of glycerol and water in fat body tissues from these larvae at selected freezing temperatures. Tissues from overwintering larvae, but not non-diapausing larvae, survive when frozen at -20 degrees C with 0.25 M glycerol, but the protection afforded by glycerol is offset by the water-channel inhibitor mercuric chloride. Glycerol in higher concentration (0.75 M) affords some protection even to the fat body of non-diapausing larvae. Radiotracer assays of overwintering larvae show that water leaves the tissues during freezing while glycerol enters, and that mercuric chloride disrupts this process. Transport is also disrupted after lethal freezing at -35 degrees C. Therefore, membrane transport of water and glycerol is involved in the avoidance of freezing injury to fat body cells of the rice stem borer, apparently by mediating the replacement of water with glycerol in freezing-tolerant tissues.

Animals↗
PubMed2005

Laboratory-based simulation of freezing profiles of beef trim for Escherichia coli O157 survival determinations.

This study was undertaken to develop a simple laboratory-based method for simulating the freezing profiles of beef trim so that their effect on E. coli O157 survival could be better assessed. A commercially available apparatus of the type used for freezing embryos, together with an associated temperature logger and software, was used for this purpose with a -80 degrees C freezer as a heat sink. Four typical beef trim freezing profiles, of different starting temperatures or lengths, were selected and modelled as straight lines for ease of manipulation. A further theoretical profile with an extended freezing plateau was also developed. The laboratory-based setup worked well and the modelled freezing profiles fitted closely to the original data. No change in numbers of any of the strains was apparent for the three simulated profiles of different lengths starting at 25 degrees C. Slight but significant (P<0.05) decreases in numbers ( approximately 0.2 log cfu g(-1)) of all strains were apparent for a profile starting at 12 degrees C. A theoretical version of this profile with a freezing plateau phase extended from 11 h to 17 h resulted in significant (P<0.05) decreases in numbers ( approximately 1.2 log cfu g(-1)) of all strains. Results indicated possible avenues for future research in controlling this pathogen. The method developed in this study proved a useful and cost-effective way for simulating freezing profiles of beef trim.

Animals↗
PubMed2004

Chromosomal integrity of freeze-dried mouse spermatozoa after 137Cs gamma-ray irradiation.

This study demonstrated that freeze-dried mouse spermatozoa possess strong resistance to 137Cs gamma-ray irradiation at doses of up to 8 Gy. Freeze-dried mouse spermatozoa were rehydrated and injected into mouse oocytes with an intracytoplasmic sperm injection (ICSI) technique. Most oocytes can be activated after ICSI by using spermatozoa irradiated with gamma-rays before and after freeze-drying. Sperm chromosome complements were analyzed at the first cleavage metaphase. Chromosome aberrations increased in a dose-dependent manner in the spermatozoa irradiated before freeze-drying. However, no increase in oocytes with chromosome aberrations was observed when fertilized by spermatozoa that had been irradiated after freeze-drying, as compared with freeze-dried spermatozoa that had not been irradiated. These results suggest that both the chromosomal integrity of freeze-dried spermatozoa, as well as their ability to activate oocytes, were protected from gamma-ray irradiation at doses at which chromosomal damage is found to be strongly induced in spermatozoa suspended in solution.

Animals↗
PubMed2003

Effect of freeze-thaw process on partitioning of contaminants in ferric precipitate.

The primary interest of this work was to determine the processing conditions which will provide the most water removal from the precipitate after freezing and thawing. The work examined the effect of variables such as freezing time, freezing temperature, and pre-treatment steps, e.g. agitation or concentration, on the freeze thaw process of ferric precipitates. The extent of dewatering of freeze thawed samples was not significantly affected by freezing rate or curing time, provided the floc was completely frozen. However, a pre-concentration step was found to produce a more concentrated residual. The relatively low degree of structure within the ferric flocs makes them particularly suitable for freeze-thaw conditioning. Only a slight increase in leach out of contaminant from the floc was observed. Thus, the experiments indicate that this is a very simple and effective method of dewatering precipitates, which does not require the use of additional additives.

Chemical Precipitation↗
PubMed1984

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.

Freezing↗
PubMed2001

High-speed sludge freezing.

The investigation examined the feasibility of applying a "high-speed freezing technique, using liquid nitrogen freezing, to condition activated sludges and alum sludges. Experimental results indicate that the freezing speed is much faster than that adopted in most previous works on sludge freezing. Although the filterability and settleability of the alum sludge is improved by 3-min liquid nitrogen freezing, no similar improvement is observed for activated sludge. Moreover, curing affects neither sludge after liquid nitrogen freezing, but affects the slow-freezing activated sludge.

Alum Compounds↗
PubMedDate not supplied

Freezing affects the activity and subcellular distribution profile of human placental xenobiotic- and steroid-metabolizing enzymes.

Human placentae from both smoking and non-smoking mothers were examined with respect to the activity and apparent subcellular distribution of cytochrome P-450-linked xenobiotic-and steroid-metabolizing activities. Freezing of the placental sample in pieces before preparing mitochondria and microsomes led to decreases in some catalytic activities and changed the distribution profile of activities between mitochondria and microsomes. Cholesterol side-chain cleavage enzyme and aryl hydrocarbon hydroxylase were especially sensitive to the destructive effect of freezing. Activities in the microsomes were apparently more sensitive to freezing than were those in the mitochondrial fraction. In addition to the destruction of activities by freezing, cross-contamination of microsomes and mitochondria was postulated. Freezing tended to level off the differences between induced and uninduced material. In JEG cells, the specific activities of xenobiotic-metabolizing enzymes were higher in the mitochondrial fraction. In liver biopsy samples the expected, principally microsomal, localization of xenobiotic-metabolizing enzymes was observed. It is suggested that the placenta should be processed fresh at least as far as the postmitochondrial supernatant before freezing to avoid the destructive and cross-contaminating effects of freezing.

Cell Line↗
PubMed2001

Some observations in freeze-drying of recombinant bioluminescent Escherichia coli for toxicity monitoring.

A recombinant bioluminescent bacteria, containing a fabA::luxCDABE fusion gene, has been used to characterize freeze-drying methods, which may be conveniently used as a tool for the development of a portable biosensor. Through residual water, viability, biosensing activity and scanning electron microscopy analyses, the characteristics that four cryoprotectants, trehalose, sucrose, sorbitol, and mannitol, conferred on freeze-dried samples were elucidated, including the morphology, water content and activity of the cells. It was found that trehalose showed the best freeze-drying efficiency among the tested cryoprotectants and it might have a specific capacity limitation in protection of the cells during the freeze step. Humidity might result in damage to the cells, according to the viability, when exposed to air during storage, while the water remaining post freeze-drying showed good correlation with damage to the freeze-dried cells when under air-tight storage conditions. The results with other recombinant bioluminescent bacteria indicated that these findings might be general features of the freeze-drying processes.

Biosensing Techniques↗
PubMed1997

Organic solutes in freezing tolerance.

The accumulation of high levels of low-molecular-weight solutes (polyhydric alcohols, saccharides) provides cryoprotection to freeze-tolerant animals by minimizing, via colligative effects, the percentage of body water converted to extracellular ice and the extent of cell volume reduction. Many freeze-tolerant insects accumulate high levels of polyols during autumn cold hardening, whereas freeze-tolerant frogs respond to ice formation in peripheral tissues by synthesizing large amounts of glucose in the liver and rapidly distributing the sugar throughout the body. Seasonal patterns of enzymatic change occur in cold-hardy insects; activities associated with cryoprotectant synthesis rise in the fall, whereas enzymes associated with polyol degradation dominate in the spring. Enzyme profiles also revealed the route of glycerol degradation via polyol dehydrogenase and the novel enzyme, glyceraldehyde kinase. Proton magnetic resonance imaging of freezing and thawing in whole frogs showed a new adaptive effect of the very high glucose levels in core organs; during thawing, organs such as liver and heart melted first, allowing recovery of their vital functions to begin while the rest of the frog thawed. New studies have examined signal transduction in the stimulation of glucose production by wood frog liver, revealing the key role of beta-adrenergic receptors and cAMP-mediated activation of glycogenolysis for cryoprotectant synthesis. The seasonal elevation of plasma membrane glucose transporters was also shown to be key to cryoprotectant distribution during freezing. Other new work has shown that frog freeze tolerance probably grew out of preexisting mechanisms of amphibian dehydration tolerance and that both freeze-tolerant and -intolerant frogs show a hyperglycemic response to desiccation at 5 degrees C.

Adaptation, Physiological↗
PubMed1999

Physical state of L-histidine after freeze-drying and long-term storage.

Liquid samples of L-histidine of varying pH values and mixed with salt, metal ions, polysorbate 80 and sucrose have been analysed by differential scanning calorimetry to evaluate the influence of these additives on the glass transition temperature and crystallisation of L-histidine during freezing and thawing. L-Histidine solutions of varying pH were freeze-dried with and without a thermal cycle and the physical state of the freeze-dried cakes, following long-term storage, were studied by powder X-ray diffraction. Amorphous L-histidine crystallised when it was exposed to moisture, and the identity of the crystalline materials is reported. The crystallisation of L-histidine during freezing and thawing is dependent on the pH of the solution and is shown to be at a minimum at pH 6, which coincides with the pK(a) of the imidazoline function. Sucrose inhibited the crystallisation of L-histidine during thawing, while sodium chloride or polysorbate 80 did not. The addition of metal ions (Ca2+ and Mg2+) up to 10% (w/w) did not depress the glass transition temperature significantly, while the addition of Zn2+ increased it. The physical state of L-histidine after freeze-drying is shown to be dependent on both the pH of the solution and the freezing cycle. The risk of crystallisation of amorphous L-histidine is low if the freeze-dried material is protected from moisture.

Calcium↗
PubMed1998

Hatchling painted turtles (Chrysemys picta) survive only brief freezing of their bodily fluids.

Neonatal painted turtles (Chrysemys picta) spend their first winter inside the shallow, subterranean nest cavity where they completed embryogenesis. Consequently, hatchlings at high latitudes may be exposed to ice and cold during the winter. This study was undertaken to determine how long hatchlings withstand freezing at temperatures slightly below 0 degree C because tolerance for freezing has been proposed to be the key to survival by overwintering animals. A thermocouple was glued to the carapace of each hatchling. The animal was dipped in water to provide a site of nucleation of ice and was then placed into a glass jar that was partially immersed in a circulating bath at -2 degrees C. Carapace temperature was monitored throughout the procedure. When a freezing exotherm was detected, timing of the freezing event began. Animals were maintained in a frozen state for 12-48 h prior to being warmed to room temperature. Of the 39 hatchlings, 22 did not survive, and mortality increased as the duration of freezing increased. Logistic regression indicates that no turtle would have survived in a frozen state for more than 54 h. These results indicate that hatchlings can survive only brief exposure to freezing of the body fluids. Thus, hatchlings cannot tolerate freezing during prolonged periods of cold.

Acclimatization↗
PubMed1997

Suppressive effects of salts on droplet coalescence in a commercially available fat emulsion during freezing for storage.

To assess possible improvements in the practical use of long-term freezing storage for commercially available fat emulsions, the effects of salts on coalescence (fusion) of emulsions during freeze-thawing cycles were studied. For this purpose, apparent size changes were measured by dynamic light scattering, and the dispersed droplets were directly observed by freeze-fracture electron microscopy before and after the freeze-thawing processes. Intravenous high-calorie nutrient fluid, based on soybean oil in water (2.2 wt% glycerin) and emulsified by egg phosphatides, was selected as a representative commercially available fat emulsion. Possible mechanisms of the suppressive effects of various salts (Li, Na, K, Rb, and Cs chlorides) on coalescence during repeated freezing with liquid nitrogen and subsequent thawing were examined in terms of the zeta-potential of the emulsion droplets and differential scanning calorimetry heating curves of the water-salt-glycerin ternary solution. The suppressive effects of salts on the coalescence of the emulsion droplets during freeze-thaw cycles could be ascribed to the suppression of ice crystal formation and the condensation of the droplets in unfrozen aqueous channels between the ice crystals during the freezing cycle.

Calorimetry, Differential Scanning↗
PubMedDate not supplied

Fish antifreeze protein and the freezing and recrystallization of ice.

Antifreeze glycopeptide and peptides from the blood of polar fishes prevent the growth of ice crystals in water at temperatures down to approximately 1 degree C below freezing point, but do not appreciably influence the equilibrium freezing point. This freezing point hysteresis must be a disequilibrium effect, or it would violate Gibbs' phase rule, but the separate freezing and melting points are experimentally very definite: ice neither melts nor freezes perceptibly within the 'hysteresis gap', for periods of hours or days. We report here unusual crystal faces on ice crystals grown from solutions of very low concentrations of the anti-freeze glycopeptides and peptides. This is a clue to the mechanism of freezing inhibition, and it may be the basis of a simple, very sensitive test for antifreeze material. Very low concentrations of the antifreeze protein are also remarkably effective in preventing the recrystallization of ice.

Antifreeze Proteins↗
PubMed2004

The plasma membrane-bound phospholipase Ddelta enhances freezing tolerance in Arabidopsis thaliana.

Freezing injury is a major environmental limitation on the productivity and geographical distribution of plants. Here we show that freezing tolerance can be manipulated in Arabidopsis thaliana by genetic alteration of the gene encoding phospholipase Ddelta (PLDdelta), which is involved in membrane lipid hydrolysis and cell signaling. Genetic knockout of the plasma membrane-associated PLDdelta rendered A. thaliana plants more sensitive to freezing, whereas overexpression of PLDdelta increased freezing tolerance. Lipid profiling revealed that PLDdelta contributed approximately 20% of the phosphatidic acid produced in wild-type plants during freezing, and overexpression of PLDdelta increased the production of phosphatidic acid species. The PLDdelta alterations did not affect the expression of the cold-regulated genes COR47 or COR78 or alter cold-induced increases in proline or soluble sugars, suggesting that the PLD pathway is a unique determinant of the response to freezing and may present opportunities for improving plant freezing tolerance.

Arabidopsis↗
PubMed2003

Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples.

Specimen preparation methods based on high-pressure freezing and freeze-substitution have enabled significant advances in the quality of morphological preservation of biological samples for electron microscopy. However, visualization of a subset of cellular membranes, particularly the endoplasmic reticulum and cis Golgi, is often impaired by a lack of contrast. By contrast, some efforts to increase membrane staining may lead to excessively granular staining. No one freeze-substitution method has emerged that both overcomes these limitations and is suitable for all types of analysis. However, one or more of the following protocols, perhaps with minor modifications, should yield satisfactory results in most cases. Freeze-substitution in glutaraldehyde and uranyl acetate in acetone, followed by embedding in Lowicryl HM20, generates samples suitable for both immunolocalization and high-resolution structural studies. Membranes are typically lightly stained but very well defined. Initial freeze-substitution in tannic acid and glutaraldehyde in acetone prior to exposure to osmium tetroxide significantly enhanced contrast on mammalian cellular membranes. Finally, initial trials indicate that freeze-substitution in potassium permanganate in acetone can provide strong contrast on endoplasmic reticulum and Golgi as well as other membranes. The tendency of permanganate to degrade cytoskeletal elements and other proteins when employed in aqueous solutions at room temperature is apparently curtailed when it is used as a freeze-substitution reagent.

Cell Membrane↗