X-Message-Number: 3049 Newsgroups: sci.cryonics From: (Kenneth B Storey) Subject: Re: frozen turtles live Message-ID: <> References: <343rnh$> Date: Fri, 2 Sep 1994 15:52:24 GMT In <343rnh$> writes: >I recently read a new and very interesting book called Medical >Herpetology by S. Grenard. In it he goes to great length >explaining how animals such as turtles can be completely frozen >and maintain themselves for months without oxygen and then >come to life on warm-up. There are cryoprotectants in turtle >body water (blood, tissue fluids) that can be of value in the >prez of human or mammalian cryonics experiments. This book is >available from the publisher for 19.95 +2.50pp. Their tel >#is 717-622-6050. Researchers have worked on freeze tolerance in vertebrates including frogs and turtles for some time. There is a lot of good scientific literature on this phenomenon and the cryioprotectants that these animals use for long term (months) whole body (all organs) frrezing (to ~-10 or -20oC). What follows is a list of papers from our lab which relate to this phenomenon (We list our papers for convenience since we have them on disk - not to exclude the work of others, whose papers are listed in the references within the present works). Ken Storey and Denis Joanisse FREEZE TOLERANCE IN VERTEBRATES Various species of amphibians and reptiles living in temperate and polar regions of the earth survive the freezing of extracellular body fluids during the winter. The strategy is also widespread amongst insects and other invertebrates. Adaptations supporting freeze tolerance include: 1) Protein: nucleating proteins induce & regulate extracellular freezing; thermal hysteresis proteins prevent recrystallization, 2) Cryoprotectants: high concentrations of polyols & sugars prevent excessive cell volume reduction, increase intracellular bound water content, and stabilize proteins; trehalose & proline stabilize membrane bilayer structure, and 3) Ischemia tolerance: cells and organs have adapted to tolerate the anoxia/ischemia imposed by extracellular freezing. REVIEW ARTICLES Storey KB, Storey JM (1994) Adaptations for freezing survival in ectothermic vertebrates. In: Advances in Molecular and Cell Biology, Thermobiology (Willis JS, ed) JAI Press, London Storey KB, Storey JM (1993) Cellular adaptations for freezing survival by amphibians and reptiles. In: Advances in Low Temperature Biology (Steponkus PL, ed) JAI Press, London, 2, 101-129 Storey KB & Storey JM (1992) Natural freeze tolerance in ectothermic vertebrates. Ann Rev Physiol 54:619-637 Storey KB & Storey JM (1992) Biochemical adaptations for winter survival in insects. In: Advances in Low Temperature Biology (Steponkus PL, ed) JAI Press, London, 1, 101-140 Storey JM & Storey KB (1992) Out cold. The winter life of painted turtles. Natural History 1/92:23-25 Pinder AW, Storey KB, Ultsch GR (1992) Estivation and hibernation. In: Environmental Biology of the Amphibia. (Feder ME, Burggren W, eds). University of Chicago Press, Chicago, pp. 250-274. Storey KB (1990) Life in a frozen state: adaptive strategies for natural freeze tolerance in amphibians and reptiles. Am J Physiol 258:R559-R568 Storey KB (1990) Biochemistry of natural freeze tolerance in animals: molecular adaptations and applications to cryopreservation. Biochem Cell Biol 68:687-698 Storey KB & Storey JM (1990) Frozen and alive. Scientific American 263:92-97 Storey KB & Storey JM (1989) Comment les animaux survivent au gel. La Recherche 20:332-341 Storey KB & Storey JM (1989) Freeze tolerance and freeze avoidance in ectotherms. In: Animal Adaptation to Cold. (Wang LCH, ed) Advances in Comparative and Environmental Physiology, Vol. 4, Springer-Verlag, Heidelberg, 51-82 Storey KB & Storey JM (1988) Freeze tolerance in animals. Physiol Rev 68:27-84 AMPHIBIAN AND REPTILE FREEZE TOLERANCE Storey KB, Mommsen TP 1994. Effects of temperature and freezing on hepatocytes isolated from a freeze tolerant frog. Am J Physiol 266, R1477-R1482 Rubinsky B, Wong STS, Hong JS, Gilbert J, Roos M, Storey KB (1994) 1H magnetic resonance imaging of freezing and thawing in freeze tolerant frogs. Am J Physiol 266, R1771-R1777. Rubinsky B, Hong JS, Storey KB 1994. Freeze tolerance in turtles: visual analysis by microscopy and magnetic resonance imaging. Am J Physiol Hemmings SJ, Storey KB (1994) Alterations in hepatic adrenergic receptor status underlie the freeze-induced glycemic response of the freeze tolerant frog Rana sylvatica. Can J Physiol Pharmacol King PA, Rosholt MN, Storey KB (1994) Seasonal changes in plasma membrane glucose transport in freeze tolerant wood frogs. Can J Zool Churchill TA, Storey KB (1994) Metabolic effects of dehydration on an aquatic frog Rana pipiens. J Exp Biol Churchill TA, Storey KB (1994) Metabolic responses to dehydration by liver of the wood frog Rana sylvatica. Can J Zool Churchill TA, Storey KB (1994) Effects of dehydration on organ metabolism in the frog Pseudacris crucifer: hyperglycemic responses to dehydration mimic freezing-induced cryoprotectant production. J Comp Physiol B Churchill TA, Storey KB (1994) Regulation of cryoprotectant synthesis and the role of amino acids in organ metabolism during freezing in spring peepers Pseudacris crucifer. Copeia Dalo NL, Hackman JC, Storey KB, Davidoff RA (1994) Changes in motoneuron membrane potential and reflex activity induced by sudden cooling of isolated spinal cords: differences among cold-sensitive, cold-resistant, and freeze-tolerant amphibian species. J Exp Biol Storey KB, Layne JR, Cutwa MM, Churchill TA, Storey JM 1993. Freezing survival and metabolism of box turtles, Terrapene carolina. Copeia 1993(3), 628-634 King PA, Rosholt MN & Storey KB (1993) Adaptations of plasma membrane glucose transport facilitate cryoprotectant distribution in freeze tolerant frogs. Am J Physiol 265, R1036-R1042 Vazquez-Illanes D, Storey KB (1993) 6-Phosphofructo-2-kinase and control of cryoprotectant synthesis in freeze tolerant frogs. Biochim Biophys Acta 1158, 29-32. Hermes-Lima M & Storey KB (1993) Anti-oxidant defenses in the tolerance of freezing and anoxia by garter snakes. Am J Physiol 265, R646-R652 Hermes-Lima M, Storey KB (1993) In vitro oxidative inactivation of glutathione S-transferase from a freeze tolerant reptile. Mol Cell Biochem 124, 149-158 Churchill TA & Storey KB (1993) Dehydration tolerance in wood frogs: a new perspective on the development of amphibian freeze tolerance. Am J Physiol 265, R1324-R1332 Storey KB, Bischof J & Rubinsky B (1992) Cryomicroscopic analysis of freezing in liver of the freeze tolerant wood frog. Am J Physiol 263:R185-R194 Storey KB, Baust JG & Wolanczyk JP (1992) Biochemical modification of the plasma ice nucleating activity in a freeze tolerant frog. Cryobiology 29:374-384 Storey KB, McDonald DG, Perry SF, Harris VL & Storey JM (1992) Effect of freezing on the blood chemistry of the wood frog. Cryo Lett 13: 363-370. Mommsen TP & Storey KB (1992) Hormonal effects on glycogen metabolism in isolated hepatocytes of a freeze-tolerant frog. Gen Comp Endocrinol 87:44-53 Churchill TA & Storey KB (1992) Natural freezing survival by painted turtles Chrysemys picta marginata and C. p. bellii. Am J Physiol 262:R530-R537 Churchill TA & Storey KB (1992) Freezing survival of the garter snake Thamnophis sirtalis. Can J Zool 70:99-105 Churchill TA & Storey KB (1992) Responses to freezing exposure by hatchling turtles Trachemys scripta elegans: factors influencing the development of freeze tolerance by reptiles. J Exp Biol 167:221-233 Storey KB, McDonald DG, Duman JG, & Storey JM (1991) Blood chemistry and ice nucleating activity in hatchling painted turtles. Cryo Lett 12:351-358 Churchill TA & Storey KB (1991) Metabolic responses to freezing by garter snakes. Cryo-Lett. 12:359-366 Churchill TA & Storey KB (1991) Metabolic responses to freezing by organs of hatchling painted turtles, Chrysemys picta marginata and C. p. bellii. Can J Zool 69:2978-2984 Risman CA, David ES, Storey KB & Crerar MM (1991) Glucose and caffeine regulation of liver glycogen phosphorylase activity in the freeze-tolerant wood frog, Rana sylvatica. Biochem Cell Biol 69:251-255 Wolanczyk JP, Baust JG & Storey KB (1990) Seasonal ice nucleating activity in the freeze tolerant frog, Rana sylvatica. Cryo Lett 11:143-150 Wolanczyk JP, Storey KB & Baust JG (1990) Nucleating activity in the blood of the freeze tolerant frog, Rana sylvatica. Cryobiology 27:328-335 Storey KB, Storey JM, Brooks SPJ, Churchill TA & Brooks RJ (1988) Hatchling turtles survive freezing during winter hibernation. Proc Natl Acad USA 85:8350-8354 Crerar MM, David ES & Storey KB (1988) Electrophoretic analysis of liver glycogen phosphor-ylase activation in the freeze-tolerant wood frog. Biochim Biophys Acta 971:72-84 Storey KB (1987) Glycolysis and the regulation of cryoprotectant synthesis in liver of the freeze tolerant wood frog. J Comp Physiol 157:373-380 Storey KB (1987) Organ-specific metabolism during freezing and thawing in a freeze tolerant frog. Am J Physiol 253:R292-R297 Storey KB (1987) What contributes to freeze tolerance? News Physiol Sci 2:157-160 Storey KB & Storey JM (1987) Persistence of freeze tolerance in terrestrially hibernating frogs after spring emergence. Copeia 1987(3): 720-726 Storey KB & Storey JM (1986) Freeze tolerant frogs: cryoprotectants and tissue metabolism during freeze/thaw cycles. Can J Zool 64:49-56 Storey KB & Storey JM (1986) Freeze tolerance and intolerance as strategies of winter survival in terrestrially-hibernating amphibians. Comp Biochem Physiol 83A:613-617 Canty A, Driedzic WR & Storey KB (1986) Freeze tolerance of isolated ventricle strips of the wood frog, Rana sylvatica. Cryo Lett 7:81-86 Storey KB (1985) Freeze tolerance in terrestrial frogs. Cryo Lett 6:115-134 Storey JM & Storey KB (1985) Freeze tolerance in the grey tree frog Hyla versicolor. Can J Zool 63:49-54 Storey JM & Storey KB (1985) Triggering of cryoprotectant synthesis by the initiation of ice nucleation in the freeze tolerant frog, Rana sylvatica. J Comp Physiol 156:191-196 Storey KB (1984) Freeze tolerance in the frog, Rana sylvatica. Experientia 40:1261-1262 Storey KB & Storey JM (1984) Biochemical adaptation for freezing tolerance in the wood frog, Rana sylvatica. J Comp Physiol 155:29-36 COLD HARDINESS IN INSECTS AND OTHER INVERTEBRATES Many types of invertebrates have developed cold hardiness strategies that allow them to endure long exposures to subzero temperatures during overwintering. Two strategies are possible: freeze tolerance (an ability to endure the freezing of extracellular body fluids) and freeze avoidance (an ability to remain liquid in a supercooled state at temperatures far below the freezing point of body fluids). Many insects as well as various intertidal invertebrates are freeze tolerant (as also are various reptiles and amphibians) whereas many other types of insects and terrestrial arthropods use the freeze avoidance strategy. Adaptations supporting freeze tolerance and freeze avoidance include: 1) Protein: nucleating proteins induce & regulate extracellular freezing; antifreeze proteins inhibit ice formation, 2) Cryoprotectants: high concentrations of polyols & sugars prevent excessive cell volume reduction in freeze tolerant animals and push supercooling point to low levels in freeze avoiding animals, and 3) Metabolic arrest and ischemia tolerance: freeze tolerant animals have a well-developed tolerance for the anoxia/ischemia imposed by extracellular freezing, both types use metabolic rate suppression (e.g. diapause) for long winter survival using limited internal fuel reserves. REVIEW ARTICLES Storey KB, Storey JM (1993) Cellular adaptations for freezing survival by amphibians and reptiles. In: Advances in Low Temperature Biology (Steponkus PL, ed) JAI Press, London, 2, 101-129 Storey KB & Storey JM (1992) Natural freeze tolerance in ectothermic vertebrates. Ann Rev Physiol 54:619-637 Storey KB & Storey JM (1992) Biochemical adaptations for winter survival in insects. In: Advances in Low Temperature Biology (Steponkus PL, ed) JAI Press, London, 1, 101-140 Storey KB & Storey JM (1991) Biochemistry of cryoprotectants. In: Insects at Low Temperature (Denlinger D & Lee RE, eds), Chapman and Hall, New York, pp 64-93 Storey KB (1990) Life in a frozen state: adaptive strategies for natural freeze tolerance in amphibians and reptiles. Am J Physiol 258:R559-R568 Storey KB & Storey JM (1990) Frozen and alive. Scientific American 263:92-97 Storey KB & Storey JM (1989) Comment les animaux survivent au gel. La Recherche 20:332-341 Storey KB & Storey JM (1989) Freeze tolerance and freeze avoidance in ectotherms. In: Animal Adaptation to Cold. (Wang LCH, ed) Advances in Comparative & Environmental Physiology, Vol 4, Springer-Verlag, Heidelberg, 51-82 Storey KB & Storey JM (1988) Freeze tolerance in animals. Physiol Rev 68:27-84 INSECT FREEZE TOLERANCE Joanisse DR, Storey KB (1994) Enzyme activity profiles in an overwintering population of the freeze tolerant larvae of the gall fly Eurosta solidaginis. J Comp Physiol B Joanisse, D.R. and Storey, K.B. 1994. Mitochondrial enzymes during overwintering in two species of cold-hardy gall insects. Insect. Biochem. Molec. Biol. 24, 145-150. Joanisse, D.R. and Storey, K.B. 1994. Temperature acclimation and seasonal changes by enzymes in cold-hardy gall insects. Arch. Insect Biochem. Physiol. Storey KB, Churchill TA, Joanisse DR (1993) Freeze tolerance in hermit flour beetle (Osmoderma eremicola) larvae. J Insect Physiol 39, 737-742 Joanisse DR, Storey KB (1993) Mitochondrial enzymes during overwintering in two species of cold-hardy gall insects. Insect Biochem Mol Biol Holden CP, Storey KB (1994) 6-Phosphogluconate dehydrogenase from a freeze tolerant insect: control of the hexose monophosphate shunt and NADPH production during cryoprotectant synthesis. Insect Biochem Mol Biol 24, 167-173 Storey KB, Keefe D, Kourtz L, & Storey JM (1991) Glucose-6-phosphate dehydrog-enase in cold hardy insects: kinetic properties, freezing stabilization, and control of hexose monophosphate shunt activity. Insect Biochem 21, 157-164 Storey KB (1990) Biochemical adaptation for cold hardiness in insects. Phil Trans Royal Soc Lond B 326:635-654 Storey JM & Storey KB (1990) Carbon balance and energetics of cryoprotectant synthesis in a freeze tolerant insect: responses to perturbation by anoxia. J Comp Physiol 160:77-84 Tsumuki H, Rojas R, Storey KB & Baust JG (1987) The fate of [14C]glucose during cold-hardening in Eurosta solidaginis (Fitch). Insect Biochem 17:347-352 Storey KB, McDonald DG & Booth CE (1986) Effect of temperature acclimation on haemolymph compostion in freeze tolerant larvae of Eurosta solidaginis. J Insect Physiol 32:897-902 Storey JM & Storey KB (1986) Winter survival of the gall fly larva: profiles of fuel reserves and cryoprotectants in a natural population. J Insect Physiol 32:549-556 Storey JM & Storey KB (1985) Freezing and cellular metabolism in the gall fly larva, Eurosta solidaginis. J Comp Physiol 155:333-337 Ballantyne JS & Storey KB (1985) Characterization of mitochondria from the freezing tolerant larvae of the goldenrod gall fly, Eurosta solidaginis. Can J Zool 63:373-379 Storey KB, Micelli M, Butler KW, Smith ICP & Deslauriers R (1984) 31NMR studies of metabolism in the freeze tolerant gall fly larvae, Eurosta solidaginis. Eur J Biochem 142:591-595 Storey KB (1983) Metabolism and bound water in overwintering insects. Cryobiology 20:365-9 Storey JM & Storey KB (1983) Regulation of cryoprotectant metabolism in the overwintering gall fly larva, Eurosta solidaginis: temperature control of glycerol and sorbitol levels. J Comp Physiol 149:495-502 Storey KB (1982) Phosphofructokinase from the overwintering gall fly larva, Eurosta solidaginis: control of cryoprotectant polyol synthesis. Insect Biochem 12:501-505 Storey JM & Storey KB (1982) Kinetic properties and regulation of glycerol-3-P dehydrogenase from the freezing-tolerant gall fly larva, Eurosta solidaginis. Cryobiology 19:185-194 Male KB & Storey KB (1982) Purification and properties of glutamate dehydrogenase from the cold hardy gall fly larva, Eurosta solidaginis. Insect Biochem 12:507-514 Storey KB & Storey JM (1981) Biochemical strategies of overwintering in the gall fly larva, Eurosta solidaginis: effect of low temperature acclimation on the activities of enzymes of intermediary metabolism. J Comp Physiol 144:191-199 Storey KB, Baust JG & Storey JM (1981) Intermediary metabolism during low temperature acclimation in the overwintering gall fly larva, Eurosta solidaginis. J Comp Physiol 144:183-190 Storey KB, Baust JG & Buescher P (1981) Determination of water bound by soluble subcellular components during low temperature acclimation in the gall fly larva, Eurosta solidaginis. Cryobiology 18:315-321 INSECT FREEZE AVOIDANCE Holden CP, Storey KB (1994) Purification and characterization of aldolase from a cold hardy insect: enzyme role in glycerol biosynthesis. Insect Biochem Mol Biol 24, 265-270 Holden CP, Storey KB (1994) Fructose1,6bisphosphatase from a cold-hardy insect: control of cryoprotectant glycerol catabolism. Arch Insect Biochem Physiol Joanisse, D.R. and Storey, K.B. 1994. Enzyme activity profiles in an overwintering population of freeze avoiding gall moth larvae Epiblema scudderiana. Can. J. Zool. . Holden CP, Storey KB (1993) Purification and characterization of glycogen phosphorylase a and b from the freeze avoiding gall moth larvae Epiblema scudderiana. J Comp Physiol B 163, 499-507. Holden CP, Storey KB (1993) Regulation of phosphofructokinase and the control of cryoprotectant synthesis in a freeze-avoiding insect. Can J Zool 71, 1895-1899 Churchill TA & Storey KB (1989) Seasonal variation in the temperature-stimulated intercon-version of glycogen and glycerol pools in a freeze avoiding larva. Cryo Lett 10:127-136 Churchill TA & Storey KB (1989) Metabolic correlates to glycerol biosynthesis in a freeze avoiding insect, Epiblema scudderiana. J Comp Physiol 159:461-472 Churchill TA & Storey KB (1989) Metabolic responses during long term exposure to subzero temperatures by a freeze avoiding insect. Cryo Lett 10:197-204 Churchill TA & Storey KB (1989) Metabolic consequences of rapid cycles of temperature change for freeze avoiding versus freeze tolerant insects. J Insect Physiol 35:579-586 Rickards J, Kelleher MJ & Storey KB (1987) Strategies of freeze avoidance in larvae of the goldenrod gall moth Epiblema scudderiana: Winter profiles of a natural population. J Insect Physiol 33:443-450 Kelleher MJ, Rickards J & Storey KB (1987) Strategies of freeze avoidance in larvae of the goldenrod gall moth Epiblema scudderiana: Laboratory investigations of temperature cues in the regulation of cold hardiness. J Insect Physiol 33:581-586. MARINE INVERTEBRATE FREEZE TOLERANCE Churchill TA, Storey KB (1994) Metabolic responses to freezing and anoxia by the periwinkle, Littorina littorea. Can J Zool Churchill TA, Storey KB (1994) Metabolic responses to freezing and anoxia by the freeze tolerant marine mussel, Geukensia demissus. J Exp Mar Biol Ecol Russell EL, Storey KB (1994) Anoxia and freezing exposures stimulate covalent modification of enzymes of carbohydrate metabolism in Littorina littorea. J Comp Physiol B Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=3049