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
 

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