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PubMed1987

Organ-specific metabolism during freezing and thawing in a freeze-tolerant frog.

Freeze-tolerant wood frogs, Rana sylvatica, were exposed to three consecutive freeze-thaw cycles. Each 2-day freezing exposure resulted in the breakdown of liver glycogen and an accumulation of high quantities of glucose in all 10 tissues tested; during each 2-day thaw glucose was restored as liver glycogen. The data suggest that frogs do not maintain cryoprotectants throughout the winter in anticipation of freezing but only synthesize and/or maintain glucose during actual freezing episodes. The pattern of glucose accumulation during freezing suggests a peripheral vasoconstriction as freezing progresses, leaving circulation open to central organs (liver, heart, and brain) for as long as possible. Lactate and alanine contents in tissues rose during each freezing exposure and were reduced during each thaw. Based on anaerobic end-product accumulation, organs appeared to vary up to 10-fold in anaerobic energy requirements in the frozen state. Levels of adenylates and fructose-2,6-biphosphate were measured in liver and muscle and provided additional evidence of tissue-specific differences in metabolism in the frozen state.

Adenosine Triphosphate↗

PubMed1993

Physiological responses of freeze-tolerant and -intolerant frogs: clues to evolution of anuran freeze tolerance.

Freeze tolerance in the wood frog, Rana sylvatica, is promoted by multiple, integrated physiological responses to ice forming within body tissues. By analyzing the freezing responses of the sympatric, but freeze intolerant, leopard frog (R. pipiens), we sought clues to the evolution of anuran freeze tolerance. Physiological responses critical to R. sylvatica's freeze tolerance, such as the synthesis and distribution of the cryoprotectant glucose, protective dehydration of organs, and deferred cardiac failure, were present, but comparatively less pronounced, in R. pipiens. Both species were innately tolerant of hyperglycemia. Glucose supplements did not enhance the freezing viability of R. pipiens, although in vitro tests of cryoprotectant efficacy revealed that glucose and glycerol provided comparable protection to erythrocytes of both species. We conclude that the evolution of freeze tolerance in R. sylvatica is not only promoted by its desiccation tolerance and the fortuitous biophysical consequences of freezing (e.g., exothermic induction of cardioacceleration and moderation of cooling rate) but also involves a progressive enhancement of fundamental physiological stress responses.

Acclimatization↗

PubMed1997

Avoidance of intracellular freezing by the freezing-tolerant New Zealand Alpine weta Hemideina maori (Orthoptera: Stenopelmatidae).

Calorimetric analysis indicates that 82% of the body water of Hemideina maori is converted into ice at 10 degrees C. This is a high proportion and led us to investigate whether intracellular freezing occurs in H. maori tissue. Malpighian tubules and fat bodies were frozen in haemolymph on a microscope cold stage. No fat body cells, and 2% of Malpighian tubule cells froze during cooling to -8 degrees C. Unfrozen cells appeared shrunken after ice formed in the extracellular medium. There was no difference between the survival of control tissues and those frozen to -8 degrees C. At temperatures below -15 degrees C (lethal temperatures for weta), there was a decline in survival, which was strongly correlated with temperature, but no change in the appearance of tissue. It is concluded that intracellular freezing is avoided by Hemideina maori through osmotic dehydration and freeze concentration effects, but the reasons for low temperature mortality remain unclear. The freezing process in H. maori appears to rely on extracellular ice nucleation, possibly with the aid of an ice nucleating protein, to osmotically dehydrate the cells and avoid intracellular freezing. The lower lethal temperature of H. maori (-10 degrees C) is high compared to organisms that survive intracellular freezing. This suggests that the category of 'freezing tolerance' is an oversimplification, and that it may encompass at least two strategies: intracellular freezing tolerance and avoidance.

Journal Article↗

PubMed1995

Freeze-fracture, deep-etch, and freeze-substitution studies of olfactory epithelia, with special emphasis on immunocytochemical variables.

Freeze-fracturing and deep-etching are a well-suited set of methods to study membrane and cytoplasmic features. Various approaches are available. Possible variables include tissue preparation, fracturing only or fracturing followed by etching, modes and materials of replication, and various ways of combining freeze-fracturing and/or deep-etching with (immuno)cytochemistry. Freeze-substitution, in particular combined with embedding in methacrylate resins such as the Lowicryls, is becoming rather widely accepted for purposes of ultrastructural (immuno)cytochemistry. Most investigators active in this field agree that this combination yields superior results compared to (immuno)cytochemistry combined with more conventional means of thin section transmission electron microscopy. Yet relatively little information is available on the variations that can occur with different approaches of freeze-substitution immunocytochemistry. This review deals with some of the variations in freeze-fracturing, freeze-etching, and freeze-substitution as applied to olfactory epithelial structures and with the effectiveness of observations obtained by application of the above sets of methods in relating the special morphology of olfactory epithelial cellular structures with those obtained by other approaches. Indeed, the data obtained continue to provide an integral image in which that morphology can be related to the special biochemistry, cell and molecular biology, and electrophysiology of olfactory epithelial structures.

Animals↗

PubMed2007

Infrared microscopy for in situ measurement of protein secondary structure during freezing and freeze-drying.

A commercially available freeze-dry microscopy stage interfaced with an IR microscope is described as a method of in situ measurement of protein secondary structure in the liquid, frozen and freeze-dried states. Studies using solutions of model proteins demonstrated that spectra collected using the IR microscope have resolution and sensitivity that is comparable to techniques using a conventional infrared spectrometer. Additionally, spectra collected in triplicate on the microscope in the solution, frozen, and freeze-dried states and after reconstitution were shown to be reproducible. The limiting factor when collecting spectra on the infrared microscope appears to be the higher level of water vapor inherently present within the optical path of the microscope used in this study. Results demonstrate that the native secondary structure is perturbed in both the frozen and freeze-dried states, and bands characteristic of structural changes associated with freezing and drying stresses were observed in the Amide I region. Freeze-drying studies conducted in the presence of mannitol and sucrose demonstrated that perturbation to the native state secondary structure after freeze-drying was considerably reduced in the presence of these excipients.

Animals↗

PubMed1998

Effect of process conditions on recovery of protein activity after freezing and freeze-drying.

The objective of this research was to gain a better understanding of the degree to which recovery of activity of model proteins after freeze-drying can be maximized by manipulation of freeze-dry process conditions in the absence of protective solutes. Catalase, beta-galactosidase and lactate dehydrogenase (LDH) were used as model proteins. All of the three proteins exhibited a concentration-dependent loss of activity after freezing, with significantly higher recovery at higher concentration. The freezing method and the type of buffer were also important, with sodium phosphate buffer and freezing by immersion of vials in liquid nitrogen associated with the lowest recovery of activity. Differential scanning calorimetry was predictive of the onset of collapse during freeze-drying only for beta-galactosidase. For the other proteins, either no Tg' transition was observed, or the apparent glass transition did not correlate with the microscopically-observed collapse temperature. The time course of activity loss for beta-galactosidase and LDH was compared during freeze-drying under conditions which produced collapse of the dried matrix and conditions which produced retention of microstructure in the dried solid. Recovery of activity decreased continuously during primary drying, with no sharp drop in recovery of activity associated with the onset of collapse. The most important drying process variable affecting recovery of activity was residual moisture level, with a dramatic drop in activity recovery associated with residual moisture levels less than about 10%.

Animals↗

PubMedDate not supplied

Freeze substitution and freeze drying for stable, long-term preservation of cytologic specimens for immunostaining.

OBJECTIVE: To develop a new method of fixing and preserving cytologic specimens for immunostaining after long-term storage at room temperature. STUDY DESIGN: The method consists of three steps: fixation, freeze substitution or freeze drying, and storage. To test the method, we used the human small cell lung carcinoma cell line Lu-135, which expresses a high level of mutant p53 protein and exhibits strong nuclear staining when reacted with an anti-p53 antibody. A smear of Lu-135 cells was fixed in a mixture of methanol and ether (50:50=vol/vol) or sprayed with a fixative containing isopropyl alcohol, methanol and polyethylene glycol. The fixed cells were freeze substituted by immersing them in a dry ice/methanol/ether bath and then were freeze dried under a vacuum. The smear was then placed in a 50-mL conical tube containing silica gel. The tube was sealed and stored at room temperature. RESULTS: Freeze substituted cells that were fixed with methanol/ether and stored for more than six months retained strong p53 positivity, as strong as that of the control cells, which had been fixed and stored in methanol. CONCLUSION: Freeze substitution and freeze drying are an alternative method of preserving cytologic specimens.

Carcinoma, Small Cell↗

PubMedDate not supplied

Water relationships in Phyllantus orbicularis and Punica granatum antiviral extracts and their influence on stability after freezing and freeze-drying.

The effect of freezing, freeze-drying and storage on inhibitory half dose (ID(50)) and on cytotoxic concentration (CC(50)) of Phyllantus orbicularis and Punica granatum extracts was studied. Selective index SI=(CC(50) /ID(50)) was considered as the antiviral criterion. Both frozen and freeze-dried extracts were analyzed by differential scanning calorimetry (DSC). The values of vitreous transition temperatures of P. granatum (Tg' and Tg) were lower than those of P. orbicularis. Sorption isotherms of freeze-dried products showed a typical sigmoidal shape, where GAB model was fitted instead of bet model. Water adsorption in freeze-dried P. prbicularis extract was higher than in the case of P. granatum. No significant changes in SI were induced by freezing or freeze-drying after a 6 months storage at -20 degrees c, but in the case of P. granatum extract, the effectiveness was highly affected after exposure to 37 degrees c or higher.

Animals↗

PubMed1975

The circumstances of freezing in the freeze-drying process of haemoderivatives.

It appears from the experiments of the authors that the macroscopic changes taken place during freezing and heating could be explained by the formation of an eutectic crystalline structure in the case of human plasma and albumin solutions. The temperature interval of eutectic spot formation showed good agreement with the temperature range of initial thawing as determined by the DTA method, i.e. with the working zone freeze-drying. It was possible to determine the maximum temperature of complete solidification from the resistance curves of the haemoderivatives. Judging from the shape of the resistance curves, the phenomenon of undercooling did not emerge with the freezing rate applied. Examining the optimal freezing rate, it appeared on the basis of some quality characteristics of the freeze-dried end-product, that the best results were obtained with --30 degrees C freezing in the case of albumin and plasma, and with --45 degrees C freezing in the case of normal and iv immunoglobulins. But the authors deem it necessary to conduct further experiments in this respect.

Blood Preservation↗

PubMed1996

Characterization of gamma-glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze-tolerant wood frog Rana sylvatica.

The freeze tolerant wood frog Rana sylvatica was studied to determine the impact of the freezing and thawing of this frog on the activity of gamma-glutamyltranspeptidase in the liver. On exposure to -2.5 degrees C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4 degrees C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at -2.5 degrees C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at -2.5 degrees C on cooling; a 10.5-fold increase after 12 h at -2.5 degrees C on the initiation of freezing; and a 22-fold increase after 24 h at -2.5 degrees C in the fully frozen state. Blood glucose levels remained elevated four-fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1.8-fold in the thawed state, while plasma ketone levels were increased 1.8-fold in the frozen state and 1.5-fold in the thawed state. Plasma total T3 levels were decreased by 22 per cent in the frozen state and normalized on thawing. In homogenates and plasma membranes isolated from the livers of Rana sylvatica, the activity of gamma-glutamyltrans-peptidase was found to be elevated at all stages of the freeze-thaw process. After 1, 12 and 24 h at -2.5 degrees C, activities were increased 2.5-, 2.3-, 2.4-fold respectively in the homogenates and 2.5-, 2.2-, 2.4-fold respectively in the plasma membranes. After thawing, activities were still increased 1.9-fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys of Rana sylvatica, the activity of gamma-glutamyltranspeptidase was increased 1.4-fold after 1 h at -2.5 degrees C after which it returned to normal. The role of thyroid hormone in producing the increase in gamma-glutamyltranspeptidase in the liver of Rana sylvatica in response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.

Adaptation, Physiological↗

PubMed1989

New structural features of synapses in the anteroventral cochlear nucleus prepared by direct freezing and freeze-substitution.

The rostral anteroventral cochlear nucleus (AVCN) of the chinchilla provides a preparation in which neuronal cell bodies and synapses in the mammalian central nervous system can be examined after direct freezing and freeze-substitution of rapidly excised brain stem slices. Cell bodies and synapses in the freeze-substituted AVCN differed from those in perfusion-fixed AVCN in several interesting respects. Despite of these differences, four types of synaptic terminal were distinguished in freeze-substituted AVCN and correlated with the four well-known types of perfusion-fixed terminal. Since the transmitter at each of the four types of terminal has been tentatively identified, the structure of synaptic vesicles and junctions in the freeze-substituted terminals could be related to transmitter type. Synaptic vesicles were uniformly round, but their diameters, deployment, and related cytoskeletal elements near the synaptic junction differed in each chemical type of synapse; the synapses thought to be cholinergic, for instance, had only a few vesicles clustered at their presynaptic junctions while the rest of the vesicles were separated from the junction by a network of fine filaments. Two types of filamentous components, short vertical projections from the postsynaptic membrane and thin filaments protruding from these projections, comprised the basic structure of the postsynaptic specialization, but their sizes and distribution differed at each chemical type of terminal. For instance, the postsynaptic specialization at the glycine terminal was distinguished by numerous thin filaments which curved sideways to run parallel to the plasmalemma. Thus, freeze-substitution gives new information about structural differences between chemically different types of synapses, which may reflect differences in their transmitter storage, release, and reception. In addition, the AVCN preparation is of general interest in making cell bodies in the mammalian central nervous system available to the various structural and analytical techniques which depend on direct, rapid freezing.

Animals↗

PubMed2001

Extracellular freezing in leaves of freezing-sensitive species.

Low-temperature scanning-electron microscopy was used to study the freezing of leaves of five species that have no resistance to freezing: bean (Phaseolus vulgaris L.), tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum L.), cucumber (Cucumis sativus L.), and corn (Zea mays L.). In the leaves of the four dicotyledonous species, ice was extracellular and the cells of all tissues were collapsed. In contrast, in maize leaves ice was extracellular in the mesophyll, and these cells were collapsed, but the epidermal and bundle-sheath cells apparently retained their original shapes and volume. It is concluded that the leaves of the freezing-sensitive dicotyledonous species tested were killed by cellular dehydration induced by extracellular freezing, and not by intracellular freezing. Freezing injury in maize leaves apparently resulted from a combination of freezing-induced cellular dehydration of some cells and intracellular ice formation in epidermal and bundle-sheath cells.

Cucumis sativus↗

PubMed1986

The interaction of saccharides with lipid bilayer vesicles: stabilization during freeze-thawing and freeze-drying.

The fusion of small unilamellar vesicles of phosphatidylcholines during freeze-thawing and freeze-drying/rehydration, and the suppression of fusion under these conditions by various saccharides, was investigated by gel filtration on Sepharose 4B, quasielastic light scattering, high-resolution 1H-NMR, ESR spin labeling, and differential scanning calorimetry. Freeze-thawing and freeze-drying of aqueous small unilamellar vesicle suspensions in the presence of sufficient sucrose had no significant effect on the average size and size distribution of small unilamellar vesicles. In the presence of sucrose the structural integrity and the permeability properties of the phosphatidylcholine bilayers were retained during freeze-thawing and freeze-drying. A comparison of the stabilizing effect of sucrose with that of trehalose and glucose showed that the stabilization is not sugar-specific but is a general property of saccharides. The fraction of small unilamellar vesicles recovered after freeze-thawing depended on the saccharide/phosphatidylcholine molar ratio. The mechanism of the cryoprotective effect involves binding of the sugar to the phospholipid polar group, probably through hydrogen bonding.

Calorimetry, Differential Scanning↗

PubMed2004

Investigation of freeze-drying sublimation rates using a freeze-drying microbalance technique.

This study was to investigate the effects of different freeze-drying factors on the rate of sublimation. The experiments were carried out in a custom-built freeze-drying microbalance to accurately monitor the sample temperature and control the chamber pressure. Twenty-four experiments were conducted based on a full factorial design by changing four factors: freezing rate (fast freezing or slow freezing), chamber temperature (35, 0, or -35 degrees C), chamber pressure (30 or 1000 mTorr), and the presence or absence of an annealing process. Lactate dehydrogenase (LDH), a tetrameric protein, was selected as a model protein for this study. The statistical analysis of the experimental results revealed that chamber temperature, analogous to the shelf temperature, in this experiment system, had the greatest impact on the sublimation rate. High chamber temperature resulted in high sublimation rate, regardless of the chamber pressure and thermal history of the sample. Chamber pressure was an important factor affecting the sublimation rate. In addition, both chamber temperature and chamber pressure had significant impact on sample temperature during freeze-drying. Annealing the samples was the most critical step to preserve good freeze-dried cake structure.

Air Pressure↗

PubMed2003

Freeze-induced expression of a novel gene, fr47, in the liver of the freeze-tolerant wood frog, Rana sylvatica.

The ability to endure the freezing of body fluids is well developed as an adaptation for winter survival in several species of woodland frogs. Recently, the mechanisms supporting natural freeze tolerance have been shown to include the expression of novel genes. One such novel gene, fr47, codes for a 390-amino acid protein present in the livers of freeze-tolerant anurans (Rana sylvatica, Pseudacris crucifer, Hyla versicolor) but not in freeze-intolerant species (Rana pipiens, Scaphiopus couchii). Regulatory influences on gene and protein expression were investigated using R. sylvatica. Northern blot analysis showed that transcript levels were increased following 24 h of freezing (5.1-fold), 24 h of anoxia exposure (6.4-fold), or the loss of 20% of total body water (2.7-fold). Immunoblotting with anti-FR47 antibody indicated that protein levels increased during freezing and thawing, but decreased somewhat during anoxia or dehydration exposure, although rebounding during recovery. These results suggest that (i) FR47 function is important for freeze survival, and (ii) that control at the protein level may be exerted posttranscriptionally. Finally, assessment of putative signal transduction pathways regulating fr47 gene expression, via in vitro incubations of liver slices, indicated the involvement of a protein kinase C-mediated pathway.

Amino Acid Sequence↗

PubMed2003

Enhanced aqueous dissolution of a poorly water soluble drug by novel particle engineering technology: spray-freezing into liquid with atmospheric freeze-drying.

PURPOSE: The purpose of this work was to investigate spray-freezing into liquid (SFL) and atmospheric freeze-drying (ATMFD) as industrial processes for producing micronized SFL powders with enhanced aqueous dissolution. Micronized SFL powders dried by ATMFD were compared with vacuum freeze-dried SFL powders. METHOD: Danazol was formulated with polyvinyl alcohol (MW 22,000), polyvinylpyrrolidone K-15, and poloxamer 407 to produce micronized SFL powders that were freeze-dried under vacuum or dried by ATMFD. The powders were characterized using Karl-Fischer titration, gas chromatography, differential scanning calorimetry, X-ray diffraction, scanning electron microscopy, surface area, and dissolution testing (SLS 0.75%/Tris 1.21% buffer media). RESULTS: Micronized SFL powders containing amorphous drug were successfully dried using the ATMFD process. Micronized SFL powders contained less than 5% w/w and 50 ppm of residual water and organic solvent, respectively, which were similar to those contents detected in a co-ground physical mixture of similar composition. Micronized SFL powders dried by ATMFD had lower surface areas than powders produced by vacuum freeze-drying (5.7 vs. 8.9 m2/g) but significantly greater surface areas than the micronized bulk drug (0.5 m2/g) and co-ground physical mixture (1.9 m2/g). Rapid wetting and dissolution occurred when the SFL powders were introduced into the dissolution media. By 5 min, 100% dissolution of danazol from the ATMFD-micronized SFL powder had occurred, which was similar to the dissolution profile of the vacuum freeze-dried SFL powder. CONCLUSIONS: Vacuum freeze-drying is not a preferred technique in the pharmaceutical industry because of scalability and high-cost concerns. The ATMFD process enables commercialization of the SFL particle-engineering technology as a micronization method to enhance dissolution of hydrophobic drugs.

Calorimetry, Differential Scanning↗

PubMed2004

Upregulation of the mitochondrial phosphate carrier during freezing in the wood frog Rana sylvatica: potential roles of transporters in freeze tolerance.

Screening of a cDNA library prepared from liver of the freeze-tolerant wood frog (Rana sylvatica) identified a freeze-responsive clone containing a 1370-nt sequence with an open reading frame of 360 amino acids. Sequence analysis revealed 84-86% identity with the mammalian inorganic phosphate carrier (PiC) that spans the inner mitochondrial membrane. Northern blot analysis showed that pic transcript levels increased over a time course of freezing, reaching 60-fold upregulation after 24-h frozen. Transcript levels were also assessed under freezing-related stresses with results showing a strong increase in pic transcript levels in response to dehydration (elevated 9.0-fold in 40% dehydrated frogs) but not under anoxia. Western blotting revealed elevated PiC protein over a time course of freeze-thaw whereas other mitochondrial carriers (dicarboxylate carrier, oxoglutarate transporter) of the same family were not affected by freezing. This modulation of PiC protein levels may play a role in mitochondrial ionic and/or osmotic balance during freeze-induced cell volume reduction.

Acclimatization↗

PubMed2002

Cytochemical investigation of gastric gland component cells with high-pressure freezing followed by freeze-substitution and hydrophilic resin embedding.

Gastric gland component cells were electron-microscopically and immunoelectronmicroscopically examined with high-pressure freezing followed by freeze substitution and a low-temperature embedding resin method and compared to that of the conventional chemical-fixation method. The rat gastric gland was high-pressure frozen, freeze-substituted with acetone-containing osmium or acrolein, and embedded in Epon 812 or Lowicryl K4M, respectively. Using the high-pressure freezing method, the vitreous freezing range reached the depth of 150 microns from the surface. The ultrathin sections from both procedures embedding in Epon 812 and Lowicryl K4M were doubly stained with uranyl acetate and lead acetate, and histochemically or immunohistochemically stained, respectively. In comparison to the conventional chemical fixation method, excellent results were obtained with respect to ultrastructural preservation. The stainings performed in this experiment included periodic acid-thiocarbohydrazide-silver proteinate staining, cationic colloidal cold at pH 2.5 staining, Helix pomatia lectin-staining, anti-alpha or -beta subunit antibodies of H+K(+)-ATPase immunostaining and pepsinogen immunostaining. The staining intensity of those was stronger than that of the conventional immersion-chemical fixation method. In addition to these results, the labels also showed good specific localization. In this paper, we provide a description of the high-pressure freezing followed by freeze substitution and low-temperature embedding resin method compared to the conventional chemical-fixation method. Our results suggest that this method is a suitable tool for ultrastructural and histochemical/immunohistochemical studies at high resolution.

Animals↗