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PubMed2002

Freezing survival, body ice content and blood composition of the freeze-tolerant European common lizard, Lacerta vivipara.

To investigate the freeze tolerance of the European common lizard, Lacerta vivipara, we froze 17 individuals to body temperatures as low as -4 degrees C under controlled laboratory conditions. The data show that this species tolerates the freezing of 50% of total body water and can survive freezing exposures of at least 24-h duration. Currently, this represents the best known development of freeze tolerance among squamate reptiles. Freezing stimulated a significant increase in blood glucose levels (16.15+/- 1.73 micromol x ml(-1) for controls versus 25.06 +/- 2.92 micromol x ml(-1) after thawing) but this increase had no significant effect on serum osmolality which was unchanged between control and freeze-exposed lizards (506.0 +/- 23.8 mosmol x l(-1) versus 501.0 +/- 25.3 mosmol x l(-1), respectively). Tests that assessed the possible presence of antifreeze proteins in lizard blood were negative. Recovery at 5 degrees C after freezing was assessed by measurements of the mean time for the return of breathing (5.9 +/- 0.5 h) and of the righting reflex (44.8 +/- 4.5 h). Because this species hibernates in wet substrates inoculative freezing may frequently occur in nature and the substantial freeze tolerance of this lizard should play a key role in its winter survival.

Adaptation, Physiological↗
PubMed2004

Strategies for exploration of freeze responsive gene expression: advances in vertebrate freeze tolerance.

Winter survival for many cold-blooded species involves freeze tolerance, the capacity to endure the freezing of a high percentage of total body water as extracellular ice. The wood frog (Rana sylvatica) is the primary model animal used for studies of vertebrate freeze tolerance and current studies in my lab are focused on the freeze-induced changes in gene expression that support freezing survival. Using cDNA library screening, we have documented the freeze-induced up-regulation of a number of genes in wood frogs including both identifiable genes (fibrinogen, ATP/ADP translocase, and mitochondrial inorganic phosphate carrier) and novel proteins (FR10, FR47, and Li16). All three novel proteins share in common the presence of hydrophobic regions that may indicate that they have an association with membranes, but apart from that each shows unique tissue distribution patterns, stimulation by different signal transduction pathways and responses to two of the component stresses of freezing, anoxia, and dehydration. The new application of cDNA array screening technology is opening up a whole new world of possibilities in the search for molecular mechanisms that underlie freezing survival. Array screening of hearts from control versus frozen frogs hints at the up-regulation of adenosine receptor signaling for the possible mediation of metabolic rate suppression, hypoxia inducible factor mediated adjustments of anaerobic metabolism, natriuretic peptide regulation of fluid dynamics, enhanced glucose transporter capacity for cryoprotectant accumulation, defenses against the accumulation of advanced glycation end products, and improved antioxidant defenses as novel parts of natural freeze tolerance that remain to be explored.

Acclimatization↗
PubMed2003

Acquisition of freezing tolerance in early autumn and seasonal changes in gall water content influence inoculative freezing of gall fly larvae, Eurosta solidaginis (Diptera, Tephritidae).

We examined seasonal changes in freeze tolerance and the susceptibility of larvae of the gall fly, Eurosta solidaginis to inoculative freezing within the goldenrod gall (Solidago sp.). In late September, when the water content of the galls was high (approximately 55%), more than half of the larvae froze within their galls when held at -2.5 degrees C for 24 h, and nearly all larvae froze at -4 or -6 degrees C. At this time, most larvae survived freezing at > or = -4 degrees C. By October plants had senesced, and their water content had decreased to 33%. Correspondingly, the number of larvae that froze by inoculation at -4 and -6 degrees C also decreased, however the proportion of larvae that survived freezing increased markedly. Gall water content reached its lowest value (10%) in November, when few larvae froze during exposure to subzero temperatures > or = -6 degrees C. In winter, rain and melting snow transiently increased gall water content to values as high as 64% causing many larvae to freeze when exposed to temperatures as high as -4 degrees C. However, in the absence of precipitation, gall tissues dried and, as before, larvae were not likely to freeze by inoculation. Consequently, in nature larvae freeze earlier in the autumn and/or at higher temperatures than would be predicted based on the temperature of crystallization (T(c)) of isolated larvae. However, even in early September when environmental temperatures are relatively high, larvae exhibited limited levels of freezing tolerance sufficient to protect them if they did freeze.

Adaptation, Physiological↗
PubMed2002

Evolution of freezing susceptibility and freezing tolerance in terrestrial arthropods.

Arthropods have evolved various adaptations to survive adverse seasons and it has long been discussed why some arthropods are freezing-susceptible and some are freezing-tolerant. However, which mode of frost resistance came first during the course of evolution? A commonly held opinion is that no choice of strategy has been offered in evolution, because each species of arthropod may have its own evolutionary and natural history, leading to cold-hardiness. Freezing tolerance is more frequent in holometabolous insect orders and partially used by certain vertebrates, like some terrestrially hibernating amphibians and reptiles. Supported by phylogenetic, ontogenetic and ecological arguments, we suggest here that freezing tolerance is more recent than freezing susceptibility in the course of arthropods evolution. In addition, we observe that three basic modes of freezing resistance in insect species exist in the field: (i) permanent or year-round freezing-susceptible species, (ii) alternative or seasonal freezing-susceptible/freezing-tolerant species, (iii) permanent or year-round freezing tolerant species.

Adaptation, Physiological↗
PubMed1997

Freezing-induced genes in wood frog (Rana sylvatica): fibrinogen upregulation by freezing and dehydration.

Differential screening of a cDNA library produced from liver of the freeze-tolerant wood frog, Rana sylvatica, was used to search for freezing-induced genes. Five freezing-responsive cDNA clones representing different genes were isolated when approximately 80,000 plaques of a cDNA library, prepared from liver of frozen frogs (24 h at -2.5 degrees C), were screened with 32P-labeled total cDNA probes from control (5 degrees C) versus freezing-exposed frogs. Two clones, pBfFR45 and pBfFR04, are reported here in detail and were found to be homologous with the genes for the alpha- and gamma-subunits of fibrinogen, respectively. The clone pBfFR45 carried a 2,305-hp cDNA sequence that was of bipartite structure, containing two open reading frames (ORFs). The first ORF potentially encoded a 332-residue polypeptide, covering a partial sequence of the NH2-terminal region of the alpha-chain. The second ORF encoded a 247-amino acid sequence, covering the whole COOH-terminal region of the alpha-chain; this was highly homologous to the FASORF (fibrinogen-alpha second ORF) of chicken alpha-fibrinogen and the extended alpha-chain of the human protein. Under control (5 degrees C) conditions, moderate levels of fibrinogen alpha- and gamma-transcripts were exclusively found in liver. When frogs were given survivable freezing exposures, levels of these transcripts in liver were highly induced. Transcription of these genes was also elevated in gut and lung during freezing, but mRNA levels in these tissues were lower than in liver. A time course assay confirmed that the transcript levels of both alpha- and gamma-subunit genes were dramatically elevated within the early hours of freezing and reached a maximum threefold increase over control levels after 8 h of freezing exposure. Two other physiological stresses, whole body dehydration and anoxia exposure, mimic individual elements of freezing stress in wood frogs. Northern blot hybridization analysis showed that the expression of both the alpha- and gamma-genes was also upregulated in response to dehydration in vivo (20% of total body water lost), but both were completely inhibited by anoxia exposure.

Amino Acid Sequence↗
PubMed1996

Freezing Characteristics of Rigid Plant Tissues (Development of Cell Tension during Extracellular Freezing).

The freezing characteristics and development of cell tension during extracellular freezing were examined in supercooling stem tissues of riverbank grapes (Vitis riparia) and cold-hardened leaves of live oak (Quercus virginiana) and mountain cranberry (Vaccinium vitis-idaea). Dormant stem xylem and pith tissues of river-bank grapes were resistant to freeze-induced dehydration above the homogeneous nucleation temperature, and they developed cell tension reaching a maximum of 27 MPa. Similarly, extracellular freezing induced cell tension in the leaves of live oak and mountain cranberry. Maximum cell tension in the leaves of live oak was 16.8 MPa and 8.3 MPa in the leaves of mountain cranberry. Following peak tensions in the leaves, a decline in the pressure was observed with progressive freezing. The results suggest that resistance to cell deformation during extracellular freezing due to cell-wall rigidity can lead to reduced cell dehydration and increased cell tension. A relationship to predict freezing behavior in plant tissues based on cell rigidity is presented. Based on cell-water relations and ice nucleation rates, cell-wall rigidity has been shown to effect the freezing characteristics of plant tissues, including freeze-induced dehydration, supercooling, and homogeneous nucleation temperatures.

Journal Article↗
PubMed1991

Freezing stress response in woody tissues observed using low-temperature scanning electron microscopy and freeze substitution techniques.

The objective of the current research was to examine the response of woody plant tissues to freezing stress by using scanning electron microscopy (SEM). Nonsupercooling species red osier dogwood (Cornus stolonifera Michx.), weeping willow (Salix babylonica L.), and corkscrew willow (Salix matsudana Koidz. f. tortuosa Rehd.) survived freezing stress as low as -60 degrees C. Cell collapse of ray parenchyma cells of these species was expected but did not occur. It was concluded that ray parenchyma cells of these species do not fit into either the supercooling or extracellular freezing classifications. Tissues from flowering dogwood (Cornus florida L.), apple (Malus domestica Borkh. cv "Starking III"), red oak (Quercus rubra L.), scarlet oak (Quercus coccinea Muench.), and red ash (Fraxinus pennsylvanica Marsh) were confirmed as supercooling species, and did not survive exposures below -40 degrees C. Ray parenchyma cells of these species did not collapse in response to freezing stress, as was expected. Cell collapse along the margins of voids were observed in bark of all seven species. Voids were the result of extracellular ice crystals formed in the bark during exposure to freezing stress. Tissues prepared by freeze substitution techniques were found to be adequately preserved when compared to those prepared by conventional fixation and low temperature SEM techniques. A freezing protocol for imposing freezing stress at temperatures lower than experienced naturally in the area where the study was conducted was developed that produced responses comparable to those observed in specimens collected in the field during natural freezing events.

Journal Article↗
PubMed1985

Freeze-etching study on membrane ultrastructural changes caused by freezing: cooling rate-dependent intramembrane particle aggregation in erythrocyte stripped ghosts.

The changes of membrane ultrastructures by freezing stresses were examined on stripped ghosts which were made by removing almost all peripheral membrane proteins from human erythrocyte membranes. By freezing these stripped ghost membranes showed cooling rate-dependent intramembrane particle (IMP) aggregation. With the cooling rates at and faster than 30,000 degrees C/min, their IMPs were evenly distributed on the fracture faces. However, cooling rates at and slower than 8000 degrees C/min resulted in IMP aggregation. The degree of IMP aggregation increased in parallel with decreasing cooling rates. Without freezing, the IMP aggregation in stripped ghosts could be induced by exposing these ghosts to hypertonic salt solutions, but lowering the temperature did not affect IMP aggregation. The cooling rate-dependent IMP aggregation during freezing was suppressed by adding cryoprotective agents which were known to reduce the salt concentration of the medium during freezing. It is suggested that the IMP aggregation in stripped ghosts by freezing occurs by exposure to concentrated salt solutions during freezing. This result indicates the possibility that IMP aggregation may arise during slow freezing of some biomembranes as a result of an increase in salt concentration rather than as a result of reduction in temperature.

Erythrocyte Membrane↗
PubMed2006

Physiological responses to freezing in hatchlings of freeze-tolerant and -intolerant turtles.

Freeze tolerance is a complex cold-hardiness adaptation that has independently evolved in a diverse group of organisms, including several ectothermic vertebrates. Because little is known about the mechanistic basis for freeze tolerance in reptiles, we compared responses to experimental freezing in winter-acclimatized hatchlings representing nine taxa of temperate North American turtles, including ones that tolerated freezing and others that did not. Viability rates of hatchlings frozen to -3 degrees C for 72 h ranged from 0 to 100%. Tolerance to freezing was poor in Sternotherus odoratus, Graptemys geographica and Trachemys scripta, intermediate in Chelydra serpentina, and high in Emydoidea blandingii, Chrysemys picta bellii, C. p. marginata, Malaclemys terrapin, and Terrapene ornata, and generally reflected the winter thermal ecology of each taxon. Plasma activity of lactate dehydrogenase (LDH), a novel in vivo index of freeze/thaw damage, corroborated viability assessments and demonstrated that cryoinjury occurred even in surviving turtles. Irrespective of taxon, cryoinjury tended to be higher in smaller individuals and in those having relatively low water contents; however, bases for these associations were not apparent. Screening for certain organic osmolytes that might promote freezing survival by colligatively reducing ice content and limiting cell dehydration showed that the plasma of unfrozen (control) turtles contained small quantities of glucose (1.3-5.8 mmol l(-1)) and lactate (0.6-3.2 mmol l(-1)) and modest amounts of urea (range of mean values for all taxa 8.2-52.3 mmol l(-1)). Frozen/thawed turtles of all taxa accumulated modest amounts of glucose and lactate that jointly raised the plasma solute concentration by 30-100 mmol l(-1). We conclude that organic osmolytes accumulated both before and during freezing may promote survival in species that have evolved a tolerance to freezing, but are not necessarily accumulated for that purpose.

Adaptation, Physiological↗
PubMed1989

Changes in chemical structure and function in Escherichia coli cell membranes caused by freeze-thawing. I. Change of lipid state in bilayer vesicles and in the original membrane fragments depending on rate of freezing.

The effect of different rates of freezing on the character of lipids in unilamellar lipid bilayer vesicles and in the original membrane fragments of Escherichia coli B cells was investigated by measuring the temperature-dependent fluorescence polarization ratio changes of cis- and trans-parinaric acids. In lipid bilayer vesicles, both slow and rapid freezing brought about significant alterations in fluorescence polarization ratios in the specimens derived from both logarithmic and stationary-phase cells. In the original membrane fragments derived from logarithmic-phase cells, slow freezing gave rise to a similar alteration in fluorescence polarization ratio change, but no such alteration was found in the case of rapid freezing. Logarithmic-phase cells suffered from a membrane permeability change during slow freezing, which subsequently resulted in low cell viability. The cells suffered only slight impairment in membrane function during rapid freezing, and maintained higher viability. These results suggest that the primary site of damage due to freezing of the cells is the cellular membranes, and this destruction is due to a lipid state change in the membranes brought about by freezing.

Cell Membrane↗
PubMed2005

Up-regulation of acidic ribosomal phosphoprotein P0 in response to freezing or anoxia in the freeze tolerant wood frog, Rana sylvatica.

Natural freezing survival by the wood frog, Rana sylvatica, involves multiple organ-specific, freeze-responsive changes in gene expression. The present study provides the first report of freeze-responsive genes in brain. Differential screening of a cDNA library made from brain of frozen wood frogs revealed a freeze-responsive clone encoding a protein of 315 amino acids that was identified as the acidic ribosomal phosphoprotein, P0 (GenBank Accession No. AF176302). The amino acid sequence showed 91-92% identity with the protein from other vertebrates. Thirteen unique amino acid substitutions occurred as compared with mammalian or avian P0 sequences; these may represent structural differences that support protein function at low body temperature. Transcripts of P0 rose by 8-fold in brain of frogs frozen for 24 h at -2.5 degrees C, compared with controls at 5 degrees C, and reached 12-fold higher in 24 h thawed frogs. Immunoblotting showed that P0 protein increased by approximately 3-fold in brain during freezing and remained high after thawing. Freeze up-regulation of P0 was largely brain-specific; transcript levels were unaffected in skeletal muscle and skin and, although transcripts rose approximately 2-fold in liver of frozen frogs, liver P0 protein was unchanged (although P0 protein was much higher overall in liver than in brain). P0 transcripts in wood frog brain were also elevated during anoxia exposure (by approximately 4-fold), but did not change under dehydration stress. The gene was similarly up-regulated under anoxia in brain of the freeze intolerant leopard frog, Rana pipiens. This suggests that P0 expression responds to anoxia stress during freezing. Changes in P0 content in ribosomes may contribute to altered patterns of protein synthesis under anoxia or ischemia.

Amino Acid Sequence↗
PubMed2004

Influence of chemical and freezing fixation methods in the freeze-fracture of stratum corneum.

A comparison between two fixation techniques for freeze-fracture was established. Stratum corneum (SC) samples from pig epidermis were fixed using high-pressure freezing (HPF) and using plunging in propane freezing; the latter after chemical fixation. Then, frozen samples were freeze-fractured, coated with platinum-carbon, and visualized using a high-resolution low-temperature scanning electron microscope and a transmission electron microscope. Our results indicate that the plane of freeze-fracture was different depending on the fixation and freezing methodology used. In the samples frozen by HPF without chemical fixation, the fracture plane laid mainly between the lipid lamellae. However, when chemical fixation and plunging in propane freezing was used, the fracture plane did not show preference to a specific way. Plunging in propane freezing of chemically fixed samples, on the other hand, provides a more homogeneous fracture behaviour. Thus, depending on the methodology used, we can favour a visualization of either lipid or protein domains of the SC. These results could be very useful in future ultrastructural studies in order to facilitate the microscopic visualization and interpretation of the complex images such as those of SC and even of other samples in which different domains coexist.

Animals↗
PubMed2004

Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods.

Freeze-fixation and freeze-gelation methods are presented in this paper which can be used to prepare highly porous scaffolds without using the time and energy consuming freeze-drying process. The porous structure was generated during the freeze of a polymer solution, following which either the solvent was extracted by a non-solvent or the polymer was gelled under the freezing condition; thus, the porous structure would not be destructed during the subsequent drying stage. Compared with the freeze-drying method, the presented methods are time and energy-saving, with less residual solvent, and easier to be scaled up. Besides, the problem of formation of surface skin can be resolved and the limitation of using solvent with low boiling point can be lifted by the presented methods. With the freeze-extraction and freeze-gelation methods, porous PLLA, PLGA, chitosan and alginate scaffolds were successfully fabricated. In addition to the presentation of the morphologies of the fabricated scaffolds, preliminary data of cell culture on them are as well included in the present work.

Alginates↗
PubMed2002

Effect of freezing parameters (freeze cycle and thaw process) on tissue destruction following renal cryoablation.

BACKGROUND AND PURPOSE: Renal cryoablation is a successful nephron-sparing treatment alternative for selected patients with small renal tumors. The purpose of this study was to compare the effects of the number of freeze cycles (one v two) and the thaw process (active v passive) on renal tissue following cryodestruction. MATERIALS AND METHODS: Sixteen female mongrel dogs (19.9 +/- 2.1 kg) were randomly divided into four groups and underwent transabdominal laparoscopic access by standard techniques. Tissue freezing was performed using argon gas following interstitial cryoprobe (3 mm) placement into the upper and lower poles of the left kidney. Single active (SA), single passive (SP) double active (DA) or double passive (DP) 15-minute treatment cycle(s) were carried out via the CRYOcare Cryosurgical Unit (Endocare, Irving, CA) on eight kidneys each. An active thaw process with helium gas or a passive thaw process was initiated after each freeze period. The cryoprobe was removed when the temperature reached 0 degrees C. Four weeks following cryosurgery, animals were sacrificed, and the renal tissue was evaluated grossly and histologically. RESULTS: Interstitial cryoprobe temperatures decreased from 31.3 degrees C +/- 1.4 degrees C to -142 degrees C +/- 1.0 degrees C following the 15-minute freeze cycle. The temperature reached 0 degrees C significantly faster following active thaw than with the passive process (2.13 +/- 0.24 min/freeze cycle and 15.18 +/- 2.97 min/freeze cycle, respectively; P < 0.0001). Grossly, each lesion consisted of a central area of necrosis surrounded by a rim of white tissue. On microscopic examination, each lesion consisted of a central area of liquefaction necrosis (LN) surrounded by various degrees of fibrosis and granulation tissue admixed with residual parenchyma. The size of the LN was significantly different in tissues subjected to double and single freeze cycles when compared across both thaw processes (active and passive). There was no significant difference in the overall lesion volume following DA, DP, SA, or SP. CONCLUSIONS: Renal cryodestruction via laparoscopic access achieves complete tissue ablation without complications. The double freeze cycle produced significantly larger areas of LN than the single freeze regardless of the thaw process. The type of thaw process did not affect the amount of tissue damage. Utilizing a double 15-minute freeze cycle with the faster active thaw process will effectively cryoablate renal tissue as well as significantly reduce overall operative time.

Animals↗
PubMed2005

Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying.

AIMS: The effects of protective agents, rehydration media and freezing temperature on the viabilities of Lactobacillus brevis and Oenococcus oeni H-2 when subjected to freeze-drying were investigated. METHODS AND RESULTS: Several protectants and rehydration media were tested to improve the survival after freeze-drying. The cells were also frozen at -65 and -20 degrees C to check the effect of freezing temperature on the viability. CONCLUSIONS: The best protectant and rehydration medium to obtain the highest viability after freeze-drying varied with the species of bacteria. Yeast extract (4.0%) and sodium glutamate (2.5% ) gave maximum viability of L. brevis and O. oeni (67.8% and 53.6% respectively). The highest survival of L. brevis and O. oeni were obtained when rehydrated with 10% sucrose and MGY medium respectively. When the bacterial cells were frozen quickly (-65 degrees C) than slowly (-20 degrees C), L. brevis and O. oeni both showed increased viability after freeze-drying. SIGNIFICANCE AND IMPACT OF THE STUDY: The viabilities of L. brevis and O. oeni after freeze-drying were shown to be strain specific and dependent on protective agents, rehydration media and freezing temperature.

Amino Acids↗
PubMed2004

Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions.

Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.

Aquaporins↗