Preservation of the proteolytic activity of a bovine spleen lysosomal-enriched extract using various freezing conditions

The preservation of the proteolytic activity of a bovine spleen lysosomal-enriched (BSLE) extract was investigated. The BSLE extract (pH = 5.8), was subjected to storage under different conditions: refrigeration at 0 degrees C for 60 days; freezing at -20 degrees C -either directly or previously frozen in liquid nitrogen-, -80 degrees C and in liquid nitrogen; freeze-drying and stored at 0 degrees C; and freezing at -20 degrees C or in liquid nitrogen in the presence of glycerol and sorbitol as cryoprotectants. Freezing at low temperatures (-80 degrees C and in liquid nitrogen) was most effective for preserving about 100% of the initial activity of all cathepsins (B, B+L and D), as well as the activity of the extract on myofibrils, for two years. Freezing at -20 degrees C, on the contrary, led to significant (P < 0.01) losses of activity. Freeze-drying was able to preserve cathepsin activity, while it failed to maintain activity on myofibrils. Both cryoprotectants sorbitol and glycerol significantly (P < 0.01) enhanced enzyme preservation, particularly cathepsin D and the activity on myofibrils, even at a freezing temperature of -20 degrees C.     

DNA-dependent RNA-polymerase activity of isolated kinetoplasts of crithidia oncopelti]

DNA-dependent RNA-polymerase activity was found in the kinetoplasts of Crithidia oncopelti. Kinetic patterns of 14C-UTP incorporation into the acid-insoluble fraction of isolated kinetoplasts at 25 degrees, 30 degrees and 35 degrees C were estimated. The effects of different antibiotics and intercalating agents on RNA synthesis in kinetoplasts were studied. alpha-amanitin, a specific inhibitor of the nuclear enzyme, does not affect the RNA-polymerase activity of the kinetoplasts. Streptolidigin and rifampicin, inhibitors of bacterial RNA-polymerase, have little effect on RNA synthesis in the kinetoplasts even at high concentrations. Kinetoplasts preincubation in the phosphate buffer increases the permeability of their membranes for rifampicin. Intercalating agents, acriflavin and ethidium bromide, strongly inhibit the kinetoplast RNA synthesis. Similar specific effects of some antibiotics and intercalating agents on RNA synthesis in kinetoplasts and typical mitochondria may be indicative of similarity of functional properties of RNA-polymerases in those organelles.     

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.     

Leakage of a trapped fluorescent marker from liposomes: effects of eutectic crystallization of NaCl and internal freezing.

Leakage of trapped carboxyfluorescein from DL-alpha-dipalmitoylphosphatidylcholine multilamellar liposomes (diameter 1-2 microns) in NaCl solutions was measured after rapid freezing to temperatures between -15 and -55 degrees C. Leakage was low after freezing between -15 and -35 degrees C, but increased steeply between -35 and -45 degrees C. From DSC measurements it was found that the increase in leakage was associated with two crystallization processes: Eutectic crystallization of NaCl and freezing of undercooled solvent trapped in the interior of the liposomes ("internal freezing"). Damage caused by the former process could effectively be prevented by small amounts of trehalose (1% less than or equal to w less than or equal to 1.5%). Trehalose in these concentration also decreased damage due to internal freezing, but to a minor degree. In addition to these damaging transitions, a time-dependent process was found to cause leakage from the liposomes at -25 degrees C. The association between leakage and thermal activity suggests that DSC supplements cryomicroscopy and leakage measurements in the characterization of cryostability of liposomes.     

Geographic variation of freeze-tolerance in the earthworm Dendrobaena octaedra

Freeze-tolerance and some of the underlying biochemical defence mechanisms in the earthworm Dendrobaena octaedra was investigated. Survival after slow cooling to -2 degrees C, -4 degrees C, or -6 degrees C was analysed in D. octaedra from three geographic regions representing large differences in winter temperature (Denmark, Finland and Greenland). A large variation in freeze-tolerance between the three populations of D. octaedra was found. Earthworms from the northern populations (Finland and Greenland) tolerated lower temperatures (-6 degrees C) than earthworms from the Danish population (poor survival at -4 degrees C and -2 degrees C). In the Finnish population, freezing led to the production of high concentrations of glucose, which reached values much higher than controls (94 mg g(-1) vs. 2 mg g(-1) dry weight). Other potential cryoprotectants were not elevated after freezing. The Danish and Greenlandic populations had substantially lower mean glucose levels after freezing than the Finnish population (about 15 mg g(-1)). Danish earthworms rapidly frozen did not accumulate glucose, and did not survive freezing at -2 degrees C. Danish earthworms exposed to osmotic stress in Ringer's solutions, containing different concentrations of glycerol, showed significantly elevated glucose levels, but did not survive rapid freezing. It was determined if freezing had an influence on the reproduction of the earthworms. After warming to summer temperatures (15 degrees C), survivors of freezing produced viable cocoons. In a field experiment it was tested if natural acclimatization during autumn and winter months had an effect on freeze-tolerance in the Danish population. There was a significant increase of post-freeze survival during this period. The results of the freezing experiments are discussed in relation to the general ecology of D. octaedra.     

Freezing sensitivity in the sfr4 mutant of Arabidopsis is due to low sugar content and is manifested by loss of osmotic responsiveness

Protoplasts were tested to determine whether the freezing sensitivity of the sfr4 (sensitive to freezing) mutant of Arabidopsis was due to the mutant's deficiency in soluble sugars after cold acclimation. When grown under nonacclimated conditions, sfr4 protoplasts possessed freezing tolerance similar to that of wild type, with the temperature at which 50% of protoplasts are injured (LT(50)) of -4.5 degrees C. In both wild-type and sfr4 protoplasts, expansion-induced lysis was the predominant lesion between -2 degrees C and -4 degrees C, but its incidence was low (approximately 10%); below -5 degrees C, loss of osmotic responsiveness (LOR) was the predominant lesion. After cold acclimation, the LT(50) was decreased to only -5.6 degrees C for sfr4 protoplasts, compared with -9.1 degrees C for wild-type protoplasts. Although expansion-induced lysis was precluded in both types of protoplasts, the sfr4 protoplasts remained susceptible to LOR. After incubation of seedlings in Suc solution in the dark at 2 degrees C, freezing tolerance and the incidence of freeze-induced lesions in sfr4 protoplasts were examined. The freezing tolerance of isolated protoplasts (LT(50) of -9 degrees C) and the incidence of LOR were now similar for wild type and sfr4. These results indicate that the freezing sensitivity of cold-acclimated sfr4 is due to its continued susceptibility to LOR (associated with lyotropic formation of the hexagonal II phase) and associated with the low sugar content of its cells.     

Effects of centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and thawing rate on the post-thaw motility of equine sperm

Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm motility between semen cooled slowly in extender with or without glycerol to 5 degrees C prior to freezing to -120 degrees C and semen cooled continuously from 20 degrees C to -120 degrees C. From these experiments, a new procedure for processing, freezing and thawing semen evolved. The new procedure involved dilution of semen to 50 x 10(6) sperm/ml in centrifugation medium and centrifugation at 400 x g for 15 min, resuspension of sperm in lactose-EDTA-egg yolk extender containing 4% glycerol, packaging in 0.5-ml polyvinyl chloride straws, freezing at 10 degrees C/min from 20 degrees C to -15 degrees C and 25 degrees C/min from -15 degrees C to -120 degrees C, storage at -196 degrees C, and thawing at 75 degrees C for 7 sec. Post-thaw motility of sperm averaged 34% for the new method as compared to 22% for the old method (P<0.01).     

Cryoresistance of rat liver endoplasmic reticulum membranes]

The effects of freezing of microsomes in liquid nitrogen and those of storage of microsomal suspensions at 2-4 degrees C and -3 - -5 degrees C for 24 hrs, on the enzymatic activities and hydrophobicity of membranes were studied. The hydrophobicity was determined by fluorescence of bound 1,8-anilino-naphthalene sulfonate. Rapid freezing of the microsomal suspension in liquid nitrogen followed by rapid warming did not change the hydrophobicity of the membranes, the rate of enzymatic lipid peroxidation, the level of cytochrome P-450 and the activity of NADH- and NADPH-cytochrome c reductase. A considerable decrease in the rate of enzymatic lipid peroxidation and membrane hydrophobicity was observed in the microsomes stored for 24 hrs at 2-4 degrees C. The 24-hr storage at -3 - -5 degrees C with subsequent thawing resulted in a rapid aggregation of the microsomes.     

Effect of freezing temperature,at which straws were plunged into liquid nitrogen,on the post-thaw motility and acrosomal status of ram spermatozoa

The present study was conducted to observe the effect of initial freezing temperature on subsequent survival and acrosomal integrity of Malpura and Bharat Merino ram spermatozoa during post-thawing incubation. Semen samples were diluted in TEST-yolk-glycerol extender, loaded in 0.25 ml straws and cooled down to -25, -75 or -125 degrees C freezing temperature using a programmable cell freezer. Computer assisted sperm analysis and acrosomal integrity of thawed samples were assessed after thawing and at hourly intervals during incubation at 37 degrees C for 4 h. The percentage of motile cells in samples frozen at -125 degrees C were 80.3 and 63.7 after post-thawing and -thawing incubation, compared to 75.9 and 39.7 at -25 degrees C or 73.9 and 51.8 at -75 degrees C temperatures, respectively. The spermatozoa with normal acrosome were also significantly, respectively, higher in samples frozen at -125 degrees C, compared to -25 and -75 degrees C temperatures. There were no significant breed variations on percentage of motile, percentage of rapidly motile cells, percentage of normal acrosomes, curvilinear velocity and lateral head displacement except straight line velocity and average path velocity of spermatozoa. The results indicated that -125 degrees C initial freezing temperature conferred the best cryopreserving ability to ram spermatozoa for post-thawing thermoresistance test compared to -25 or -75 degrees C freezing temperature.     

Changes in phospholipid composition of mitochondria after freezing-warming in the presence of Na2S204

The phospholipid composition of mitochondria membranes subjected to rapid freezing (300-400 degrees C per 1 min) to -196 degrees C and subsequent slow warning (at 20 degrees C) was determined by the method of chromatography in the thin layer of silicagel. Under such conditions of freezing and warming a significant decrease in lecithin and ethanolamine phosphatide content is observed in the mitochondria membranes. When freezing the suspension of mitochondria in the medium containing Na2S2O4 in a concentration of 0.05 M only the lipid component changes slightly.     

Relation of several antioxidant enzymes to rapid freezing resistance in suspension cultured cells from alpine Chorispora bungeana

Chorispora bungeana Fisch. & C.A. Mey (Crucifer) is a rare alpine subnival plant surviving sudden snowstorms. In this paper, we have attempted to explore possible roles of autoxidation rate (AR) and the antioxidant enzymes associated with cryoprotective mechanisms in the plant cells. The results showed that when the suspension cultures growing at 25 degrees C were suddenly exposed to -8 degrees C for 15 days, 2,3,5-triphenyltetrazolium chloride reduction was not affected within 9 days and AR remained at a low level in comparison with controls. This indicated that the cells maintained considerable amounts of soluble protein and the integrity of the cell membranes was intact during the whole freezing test. Furthermore, on average, the activity of antioxidant enzymes such as superoxide dismutase, dehydroascorbate reductase, ascorbate peroxidase and glutathione reductase were prominently enhanced in the freezing-stressed cells. Peroxidase activity significantly increased soon after freezing, possibly to make up for the early decrease of catalase activity in the cells. Statistical analysis showed negative correlations between resistance to rapid freezing and antioxidant enzyme activity in the cultured cells after exposure from 25 to -8 degrees C, indicating that the reduction of cell viability with freezing activates a combination of antioxidant enzymes that results in intact cells. All of these findings suggest a synergy between these antioxidant enzymes, leading to a low autoxidation rate that contributes to the protection of the cell membranes and plays an important role in the resistance of suspension cultured cells of C. bungeana to sudden freezing.     

Freeze-induced membrane damage in ram spermatozoa is manifested after thawing: observations with experimental cryomicroscopy.

Cryoinjury in ram sperm was investigated by direct observation, using cryomicroscopy, to validate model hypotheses of freezing injury in such a specialized cell. Fluorescein diacetate was used to determine when during the freeze-thaw cycle the sperm membrane became permeable. In noncryoprotected sperm plasma membrane, integrity was maintained throughout the cooling and freezing process, but fluorescein leakage occurred during rewarming. The temperature of post-thaw permeabilization varied in relation to the minimum temperature reached during freezing; cells cooled to -10 degrees C retained fluorescence into the post-thaw temperature range of 9-24 degrees C (mean +/- SEM; 13.25 +/- 0.91 degrees C), whereas cells cooled to -20 degrees C lost fluorescence shortly after thawing (mean +/- SEM; 2.62 +/- 0.91 degrees C). Sperm cooled to 5 degrees C, but not frozen, retained fluorescence during rewarming up to 20-30 degrees C. The inclusion of glycerol and egg yolk in the freezing medium significantly and independently increased the post-thaw permeabilization temperature. Maintenance of fluorescence was also correlated with ability to resume motility after thawing. Sperm reactivation experiments were undertaken to examine deleterious effects of freezing upon the flagellar microtubular assembly. No direct evidence for such effects was obtained. Instead, a highly significant correlation between minimum freezing temperature and post-thaw temperature of initial reactivation was detected.     

Expression of a beetle, Dendroides canadensis, antifreeze protein in Drosophila melanogaster

Antifreeze protein 1 (DAFP-1), from the beetle Dendroides canadensis, was expressed in Drosophila melanogaster. Mean thermal hysteresis values (the difference between freezing and melting points), indicative of antifreeze protein activity, in the hemolymph of transgenic flies were found to be as high as 6.23+/-0.10 degrees C (using the nanoliter osmometer). Direct comparisons of the capillary and nanoliter osmometer techniques for measuring THA were made, illustrating the much higher values obtained by the latter. Transgenic Drosophila had supercooling points, both in contact with ice and not, that were slightly, but significantly, lower than wild-type controls (1.5-2.0 degrees C and 2.0-4.0 degrees C, respectively). The results indicate functionality of DAFP-1 in Drosophila melanogaster (the ability of DAFP-1 to inhibit both inoculative freezing across the cuticle and freezing initiated by endogenous ice nucleators). The much larger effects of DAFPs in inhibiting inoculative freezing and ice nucleation in Dendroides canadensis relative to the transgenic Drosophila may partially result from the lower DAFP concentrations and activities in Drosophila, however the absence of multiple types of DAFPs and absence of tissue specific expression may also contribute. Transgenic Drosophila were also able to live significantly longer than controls at 0 degrees C and 4 degrees C, indicating that DAFP-1 is able to increase cold tolerance at above freezing temperatures.     

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.     

The role of freezing in trypsin activity oscillations.

The importance of the frozen phase in the formation of cryooscillations of trypsin activity has been shown in experiments conducted at -10 degrees C under frozen and supercooled conditions, respectively. A solution containing trypsin obtained by trypsinogen activation and 0.1 M MnCl2 was distributed in test tubes with or without previous freezing and kept at -10 degrees C and pH 8.4. At given time intervals the frozen and supercooled samples were tested simultaneously for tryptic activity. Although a temporal motion of trypsin activity was produced by the frozen samples, the activity of the supercooled samples began to oscillate only after spontaneous freezing of the solutions. This phenomenon suggests the importance of compartmentalization of the frozen heterogeneous system, which results in an increase in concentration vs a decrease in diffusion rate of the components.     

Beneficial effect of intracellular free high-mannose oligosaccharides on cryopreservation of mammalian cells and proteins

The cryoprotective effect of intracellular free high-mannose oligosaccharides (HMOS) on mammalian cells and proteins was examined by monitoring PC-12 cell viability and assaying protein kinase C (PKC)-epsilon activity. 1-Deoxymannojirimycin, an inhibitor of alpha-mannosidase, to cause an increase in intracellular free HMOS, significantly rescued PC-12 cells with 2-h freezing insult at -15 degrees C in a concentration (1-50mM)- and pretreatment time (48-72h)-dependent manner, as compared with unpretreated cells; full rescue from freezing injury was obtained with 1-deoxymannojirimycin at more than 25mM for 48-h pretreatment and more than 3mM for 72- and 96-h pretreatment. For PC-12 cells pretreated with 1-deoxymannojirimycin at 1mM for 72h, thawed cell viability after more than 8-w cryopreservation at -80 degrees C in 10% (v/v) dimethyl sulfoxide was much higher than that for cells without pretreatment. PKC-epsilon activity was well preserved after 16-h cryopreservation at -20 degrees C in the presence of mannose 9-N-acetylglucosamine 2 (Man9-GlcNAc2) (1 mM), an HMOS, while the activity was reduced to 15% without Man9-GlcNAc2. Collectively, the results of the present study suggest that intracellular free HMOS is a key molecule to protect mammalian cells and proteins from freezing injury; in other words, HMOS could be a new target for cryopreservation of mammalian cells and proteins.     

Cryopreservation of semen in the dog: use of ultra-freezers of -152 degrees C as a viable alternative to liquid nitrogen

This experimental work was carried out to validate the use of a -152 degrees C ultra-low temperature freezer to freeze and store canine semen. The semen of three dogs was pooled and processed to obtain a final dilution with a concentration of 100 x 10(6) spermatozoa/mL, glycerol at 5% and Equex at 0.5%. Then, four freezing protocols were tested to evaluate the cryosurvival of sperm at 1, 7, 30, 60 and 120 days after freezing: (I) semen was frozen and stored in liquid nitrogen; (II) semen was frozen in liquid nitrogen and stored in the ultra-low freezer at -152 degrees C; (III) semen was frozen in the vapour of liquid nitrogen and stored in the ultra-low freezer at -152 degrees C; (IV) semen was frozen and stored in the ultra-low freezer at -152 degrees C. Data were statistically analyzed by repeated measures analysis of variance to determine the effect of the freezing protocol and time on the sperm characteristics assessed. The percentages of sperm motility and of dead/live spermatozoa were similar throughout the experimental period, with no significant differences (P < 0.05) to be observed between four different freezing techniques tested. At 120 days after freezing, the percentage of abnormal cells and the percentage of sperm cells with abnormal acrosome were not significantly different between the freezing techniques. Although the number of dogs used was slightly low, in vitro results of this preliminary study showed that the use of ultra-freezers at -152 degrees C to freeze and store canine semen could be a viable alternative to liquid nitrogen.     

Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C

We developed a procedure to culture microorganisms below freezing point on solid media (cellulose powder or plastic film) with ethanol as the sole carbon source without using artificial antifreezes. Enrichment from soil and permafrost obtained on such frozen solid media contained mainly fungi, and further purification resulted in isolation of basidiomycetous yeasts of the genera Mrakia and Leucosporidium as well as ascomycetous fungi of the genus Geomyces. Contrary to solid frozen media, the enrichment of liquid nutrient solutions at 0 degrees C or supercooled solutions stabilized by glycerol at -1 to -5 degrees C led to the isolation of bacteria representing the genera Polaromonas, Pseudomonas and Arthrobacter. The growth of fungi on ethanol-microcrystalline cellulose media at -8 degrees C was exponential with generation times of 4.6-34 days, while bacteria displayed a linear or progressively declining curvilinear dynamic. At -17 to -0 degrees C the growth of isolates and entire soil community on 14C-ethanol was continuous and characterized by yields of 0.27-0.52 g cell C (g of C-substrate)(-1), similar to growth above the freezing point. The 'state of maintenance,' implying measurable catabolic activity of non-growing cells, was not confirmed. Below -18 to -35 degrees C, the isolated organisms were able to grow only transiently for 3 weeks after cooling with measurable respiratory and biosynthetic (14CO2 uptake) activity. Then metabolic activity declined to zero, and microorganisms entered a state of reversible dormancy.