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Afterdrop—Clinical and experimental perspectives
ABSTRACTS, 24th ANNUAL more generally, to a cycle of change in concentration of the impermeant solute that is present (sodium chloride in this case) such that the degree of damage suffered by the cells on exposure to a given final salt concentration and return to their original suspending solution is inversely dependent on the salt concentration in which they were initially suspended (and to which they are returned). This result does not disprove the “unfrozen fraction” hypothesis but it does provide an alternative explanation for the experimental data which is, moreover, completely consistent with the original data of Lovelock and with much subsequent work. REFERENCES 1. Mazur, P., Rail, W. F., and Rigopoulos, N. The relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes. Biophys. J. 36, 653-657 (1981). 5 1. Stabilization of Phosphofructokinase during AirDrying with Sugars and Sugar/Transition Metal Mixtures. JOHN F. CARPENTER, BETH
MARTIN, LOIS M. CROWE, AND JOHN H. CROWE(Department of Zoology, University of California, Davis, California 95616). Water is thought to be indispensible for the maintenance of the structure and function of proteins since it is intimately involved in the folding that is mandatory for a polypeptide to become a protein. Despite the sensitivity of biological macromolecules to changes in hydration, many organisms are capable of surviving removal of essentially all of their intracellular water. A common characteristic of these organisms is the presence of high concentrations (sometimes in excess of 20% of the dry weight) of sugars such as sucrose and trehalose. Furthermore, in several cases survival of desiccation has been shown to correlate directly with the level of sugar in the organism. In order to test the possibility that these sugars may serve to stabilize proteins in dehydration-tolerant organisms, we have characterized the protection afforded by organic solutes during air-drying to the extremely labile enzyme, phosphofructokinase (PFK). Since the rate of drying can be highly variable in nature, we have tested stabilization during both rapid and slow drying regimes. PFK, purified from rabbit skeletal muscle, provides an excellent model system for these evaluations because, without protection, it is completely and irreversibly inactivated during either air-drying treatment. The addition of trehalose, maltose, or sucrose to the enzyme solution prior to rapid drying results in a recovery of almost 70% of the original activity, whereas about 30% is recovered after slow drying. Similar stabilization is seen with up to 200 mM lactose, but at
561
MEETING
higher concentrations the sugar comes out of solution during drying, and there is a dramatic drop in the activity recovered. Glucose at concentrations up to 500 mM is much less effective at protecting air-dried PFK. Addition of ionic zinc to enzyme-sugar mixtures prior to drying greatly enhances the stabilization imparted by the above sugars, but zinc alone affords no protection. Several other organic solutes (proline, glycine, trimethylamine N-oxide, glycerol, and myo-inositol) afford cryoprotection to PFK, an effect enhanced by zinc, but do not stabilize the enzyme during airdrying, even if zinc is present. It is intriguing that many of these compounds are also known to stabilize proteins in solution, in a manner similar to that described for sugars, but are not able to stabilize an enzyme as water is removed. To ascertain how protection in solution is linked to that during drying, we have determined the PFK activity retained as a function of the residual water in samples. This is accomplished by preparing the enzyme samples with tritiated water. Samples are rehydrated after various degrees of drying, and aliquots are removed for determination of catalytic activity and tritium counts. We have found that PFK is inactivated by over 30% when the sample is still substantially hydrated (2900 G H,O/g PFK) and is fully inactivated when hydration is decreased to less than 70 g H,O/g PFK. In the presence of 100 mM trehalose the enzyme maintains full activity at all levels of hydration. Glycerol (400 mM), on the other hand, completely protects the enzyme down to a hydration level of 1100 g H,O/g PFK, but the enzyme is fully inactivated at I75 g H,O/g PFK. In contrast, less stabilization is seen with glucose during the initial stages of drying, but over 30% of the original activity is recovered at the end of drying. We are continuing these studies with other organic solutes. (Supported by NSF Grant DMB 85-18194.) SYMPOSIUM V-ADAPTATION LOW TEMPERATURES
TO
Resistance to Freezing. KENNETH STOREY(Department of Biology, Carleton University, Ottawa, Ontario, Canada).
52. Natural
53. Afterdrop-Clinical and Experimental Perspecfives. G. K. BRISTOWAND G. G. GIESBRECHT
(Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada). Actively rewarming victims of modest-to-severe hypothermia (core temperatures less than 30°C) is usually associated with a further fall in the core temperature before rewarming occurs. This afterdrop of core temperature may jeopardize the victim’s life. Rewarming of these victims by shell or surface methods have been particularly implicated in the production of this afterdrop. Currently two theories of the mecha-
562
ABSTRACTS, 24th ANNUAL
nism of the afterdrop phenomenum are popular. The first mechanism explains afterdrop by increasing perfusion of core blood to the cooler shell areas which upon return to the core results in the afterdrop. The second mechanism postulated is that during the acute cooling process temperature gradients are established which upon the initiation of rewarming continues to develop with the consequent afterdrop. This latter theory therefore is totally independent of circulation. We have studied and quantified the afterdrop phenomenon in six victims of acute/subacute moderate-to-severe accidental hypothermia. Three of these victims were in cardiac arrest at the time of initiation of rewarming whereas three were not. In each of the arrested/unarrested groups two were initially rewarmed by external or shell means and one initially by a core method (peritoneal levage). Whether arrested or unarrested, shell rewarming resulted on all occasions in afterdrop of core temperature ultimately reversed by initiating core rewarming. In those episodes where core rewarming was initiated early no afterdrop was noted. To illucidate the etiology of the afterdrop phenomenon we studied the amount and rate of afterdrop in six young, fit, healthy volunteers acutely coolin in cold water (8°C) to an esophogeal core temperature of 32.8-34°C. Each subject was cooled on three occasions and then warmed by one of three techniques in random order, i.e., treadmill exercise, shivering, and application of external heat to the thorax. While cooling rates were linear and equal (approximately 2.6”C hr-I) for all techniques, the afterdrop rate in the exercise group was two to three times as fast which was significantly greater than the cooling rate (P < 0.05). Furthermore the afterdrop rates in the shivering and the thorax heating techniques were not significantly different from the cooling rates. Likewise the amount and duration of afterdrop in the exercise group was significantly greater (P < 0.05) than in the shivering or the external heat groups. We attributed these findings to the increased peripheral muscular blood flow associated with exercise. If pure conduction were the sole or most significant cause of the afterdrop of core temperature during exercise rewarming, then afterdrop rates should not have exceeded cooling rate and furthermore, there should have been little or no difference in the afterdrop characteristics among treatments. For these reasons we believe that in our study the circulation or convective etiology played a prominant role in the afterdrop of core temperature during at least the exercise group. From a practical standpoint, regardless of the contributions to the afterdrop made by either the circulation or gradient mechanisms, at core temperatures less than 30°C rewarming by a core-first method will be less hazardous to the victim. 54. Plant Freezing Tolerance: Moving Toward a Mo-
MEETING
lecular Genetic Approach. A. JOHNSON-FLANAGAN (Department of Plant Science, University of Alberta, Edmonton, Alberta, Canada). Early research documenting charges in the level of protein, RNA, DNA, and organelles involved in protein synthesis during the induction of freezing tolerance in plants is reviewed. The recent availability of new molecular biology techniques has enabled unique gene expression during cold hardening to be studied. Using in vitro translations of isolated Poly A+ mRNA or polysomes, products of new genetic messages have been identified during hardening or low temperature growth in spinach, potato, winter rape, rye, and alfalfa by several researchers. In a cell suspension culture of winter Brassica, for example, the appearance of unique mRNAs encoding a 20 kD molecular weight polypeptide and increases in a 17 kD polypeptide has been observed. The cellular function of these polypeptides, however, has yet to be elucidated. cDNA cloning is used to identify sequences specific to plants capable of hardening and to elucidate the role of unique gene expression during the development of freezing tolerance. SESSION V-ADAPTION TO LOW TEMPERATURES 55. Cryoprotectants in the Hemolymph of the Intertidal Bivalve, Mytilus edulis. STEPHEN H. LOOMIS,*+ JOHN E CARPENTER,t AND JOHN H. CROWER(*Department of Zoology, Connecticut College, New London, Connecticut 06320; and tDepartment of Zoology, University of California, Davis, California 95616). It has been known for over 30 years that some intertidal invertebrates are able to survive the presence of ice in their extracellular fluids. Despite several previous attempts, cryoprotectants have not been found in these organisms. In this study we used freeze-thaw induced fusion of small unilamellar vesicles (SUVs) to test the cryoprotective capacity of components found in Mytilus edulis hemolymph. Fusion was monitored by resonance energy transfer between fluorescent probes in two populations of I-palmitoyl-2-oleoyl phosphatidylcholine: phosphatidylserine (mol ratio 85:15) SUVs. Hemolymph collected from winter-acclimatized animals reduced the amount of fusion between freeze-thawed liposomes from 30 to 10%. A l/IO dilution of the blood was needed to reduce this effect, and greater dilution led to a concentration-dependent decrease in protection. After partial purification, this material reduced vesicle fusion during freezing by the same amount as the whole blood, and also reduced freeze-induced leakage of carboxyfluorescein from 50 to 15%. Further evidence for the cryoprotective role of this material is that, in the presence of 0.6 mM zinc, it fully protects the labile enzyme phosphofructokinase from inactivation during freezethawing. Also, freeze-induced inactivation of lactate
MARTIN, LOIS M. CROWE, AND JOHN H. CROWE(Department of Zoology, University of California, Davis, California 95616). Water is thought to be indispensible for the maintenance of the structure and function of proteins since it is intimately involved in the folding that is mandatory for a polypeptide to become a protein. Despite the sensitivity of biological macromolecules to changes in hydration, many organisms are capable of surviving removal of essentially all of their intracellular water. A common characteristic of these organisms is the presence of high concentrations (sometimes in excess of 20% of the dry weight) of sugars such as sucrose and trehalose. Furthermore, in several cases survival of desiccation has been shown to correlate directly with the level of sugar in the organism. In order to test the possibility that these sugars may serve to stabilize proteins in dehydration-tolerant organisms, we have characterized the protection afforded by organic solutes during air-drying to the extremely labile enzyme, phosphofructokinase (PFK). Since the rate of drying can be highly variable in nature, we have tested stabilization during both rapid and slow drying regimes. PFK, purified from rabbit skeletal muscle, provides an excellent model system for these evaluations because, without protection, it is completely and irreversibly inactivated during either air-drying treatment. The addition of trehalose, maltose, or sucrose to the enzyme solution prior to rapid drying results in a recovery of almost 70% of the original activity, whereas about 30% is recovered after slow drying. Similar stabilization is seen with up to 200 mM lactose, but at
561
MEETING
higher concentrations the sugar comes out of solution during drying, and there is a dramatic drop in the activity recovered. Glucose at concentrations up to 500 mM is much less effective at protecting air-dried PFK. Addition of ionic zinc to enzyme-sugar mixtures prior to drying greatly enhances the stabilization imparted by the above sugars, but zinc alone affords no protection. Several other organic solutes (proline, glycine, trimethylamine N-oxide, glycerol, and myo-inositol) afford cryoprotection to PFK, an effect enhanced by zinc, but do not stabilize the enzyme during airdrying, even if zinc is present. It is intriguing that many of these compounds are also known to stabilize proteins in solution, in a manner similar to that described for sugars, but are not able to stabilize an enzyme as water is removed. To ascertain how protection in solution is linked to that during drying, we have determined the PFK activity retained as a function of the residual water in samples. This is accomplished by preparing the enzyme samples with tritiated water. Samples are rehydrated after various degrees of drying, and aliquots are removed for determination of catalytic activity and tritium counts. We have found that PFK is inactivated by over 30% when the sample is still substantially hydrated (2900 G H,O/g PFK) and is fully inactivated when hydration is decreased to less than 70 g H,O/g PFK. In the presence of 100 mM trehalose the enzyme maintains full activity at all levels of hydration. Glycerol (400 mM), on the other hand, completely protects the enzyme down to a hydration level of 1100 g H,O/g PFK, but the enzyme is fully inactivated at I75 g H,O/g PFK. In contrast, less stabilization is seen with glucose during the initial stages of drying, but over 30% of the original activity is recovered at the end of drying. We are continuing these studies with other organic solutes. (Supported by NSF Grant DMB 85-18194.) SYMPOSIUM V-ADAPTATION LOW TEMPERATURES
TO
Resistance to Freezing. KENNETH STOREY(Department of Biology, Carleton University, Ottawa, Ontario, Canada).
52. Natural
53. Afterdrop-Clinical and Experimental Perspecfives. G. K. BRISTOWAND G. G. GIESBRECHT
(Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada). Actively rewarming victims of modest-to-severe hypothermia (core temperatures less than 30°C) is usually associated with a further fall in the core temperature before rewarming occurs. This afterdrop of core temperature may jeopardize the victim’s life. Rewarming of these victims by shell or surface methods have been particularly implicated in the production of this afterdrop. Currently two theories of the mecha-
562
ABSTRACTS, 24th ANNUAL
nism of the afterdrop phenomenum are popular. The first mechanism explains afterdrop by increasing perfusion of core blood to the cooler shell areas which upon return to the core results in the afterdrop. The second mechanism postulated is that during the acute cooling process temperature gradients are established which upon the initiation of rewarming continues to develop with the consequent afterdrop. This latter theory therefore is totally independent of circulation. We have studied and quantified the afterdrop phenomenon in six victims of acute/subacute moderate-to-severe accidental hypothermia. Three of these victims were in cardiac arrest at the time of initiation of rewarming whereas three were not. In each of the arrested/unarrested groups two were initially rewarmed by external or shell means and one initially by a core method (peritoneal levage). Whether arrested or unarrested, shell rewarming resulted on all occasions in afterdrop of core temperature ultimately reversed by initiating core rewarming. In those episodes where core rewarming was initiated early no afterdrop was noted. To illucidate the etiology of the afterdrop phenomenon we studied the amount and rate of afterdrop in six young, fit, healthy volunteers acutely coolin in cold water (8°C) to an esophogeal core temperature of 32.8-34°C. Each subject was cooled on three occasions and then warmed by one of three techniques in random order, i.e., treadmill exercise, shivering, and application of external heat to the thorax. While cooling rates were linear and equal (approximately 2.6”C hr-I) for all techniques, the afterdrop rate in the exercise group was two to three times as fast which was significantly greater than the cooling rate (P < 0.05). Furthermore the afterdrop rates in the shivering and the thorax heating techniques were not significantly different from the cooling rates. Likewise the amount and duration of afterdrop in the exercise group was significantly greater (P < 0.05) than in the shivering or the external heat groups. We attributed these findings to the increased peripheral muscular blood flow associated with exercise. If pure conduction were the sole or most significant cause of the afterdrop of core temperature during exercise rewarming, then afterdrop rates should not have exceeded cooling rate and furthermore, there should have been little or no difference in the afterdrop characteristics among treatments. For these reasons we believe that in our study the circulation or convective etiology played a prominant role in the afterdrop of core temperature during at least the exercise group. From a practical standpoint, regardless of the contributions to the afterdrop made by either the circulation or gradient mechanisms, at core temperatures less than 30°C rewarming by a core-first method will be less hazardous to the victim. 54. Plant Freezing Tolerance: Moving Toward a Mo-
MEETING
lecular Genetic Approach. A. JOHNSON-FLANAGAN (Department of Plant Science, University of Alberta, Edmonton, Alberta, Canada). Early research documenting charges in the level of protein, RNA, DNA, and organelles involved in protein synthesis during the induction of freezing tolerance in plants is reviewed. The recent availability of new molecular biology techniques has enabled unique gene expression during cold hardening to be studied. Using in vitro translations of isolated Poly A+ mRNA or polysomes, products of new genetic messages have been identified during hardening or low temperature growth in spinach, potato, winter rape, rye, and alfalfa by several researchers. In a cell suspension culture of winter Brassica, for example, the appearance of unique mRNAs encoding a 20 kD molecular weight polypeptide and increases in a 17 kD polypeptide has been observed. The cellular function of these polypeptides, however, has yet to be elucidated. cDNA cloning is used to identify sequences specific to plants capable of hardening and to elucidate the role of unique gene expression during the development of freezing tolerance. SESSION V-ADAPTION TO LOW TEMPERATURES 55. Cryoprotectants in the Hemolymph of the Intertidal Bivalve, Mytilus edulis. STEPHEN H. LOOMIS,*+ JOHN E CARPENTER,t AND JOHN H. CROWER(*Department of Zoology, Connecticut College, New London, Connecticut 06320; and tDepartment of Zoology, University of California, Davis, California 95616). It has been known for over 30 years that some intertidal invertebrates are able to survive the presence of ice in their extracellular fluids. Despite several previous attempts, cryoprotectants have not been found in these organisms. In this study we used freeze-thaw induced fusion of small unilamellar vesicles (SUVs) to test the cryoprotective capacity of components found in Mytilus edulis hemolymph. Fusion was monitored by resonance energy transfer between fluorescent probes in two populations of I-palmitoyl-2-oleoyl phosphatidylcholine: phosphatidylserine (mol ratio 85:15) SUVs. Hemolymph collected from winter-acclimatized animals reduced the amount of fusion between freeze-thawed liposomes from 30 to 10%. A l/IO dilution of the blood was needed to reduce this effect, and greater dilution led to a concentration-dependent decrease in protection. After partial purification, this material reduced vesicle fusion during freezing by the same amount as the whole blood, and also reduced freeze-induced leakage of carboxyfluorescein from 50 to 15%. Further evidence for the cryoprotective role of this material is that, in the presence of 0.6 mM zinc, it fully protects the labile enzyme phosphofructokinase from inactivation during freezethawing. Also, freeze-induced inactivation of lactate