Advances in Low-Temperature Biology, Volume 3

ADVANCES IN LOW-TEMPERATURE BIOLOGY Volumes • 1996 This Page Intentionally Left Blank ADVANCES IN LOW-TEMPERATURE ...

Author: P.L. Steponkus

28 downloads 928 Views 17MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

ADVANCES IN LOW-TEMPERATURE BIOLOGY

Volumes • 1996

This Page Intentionally Left Blank

ADVANCES IN LOW-TEMPERATURE BIOLOGY

Editor:

PETER L. STEPONKUS Department of Soil, Crop and Atmospheric Sciences Cornell University Ithaca, New York

VOLUME 3 • 1996

U ^ Greenwich, Connecticut

JAI PRESS rNC. London, England

Copyright © 1996 JAI PRESS INC. 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 JAI PRESS LTD. 38 Tavistock Street Covent Garden London WC2E 7PB England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher ISBN: 0-7623-0160-0 Manufactured in the United States of America

CONTENTS List of Contributors

vii

Preface Peter L Steponkus

ix

Chapter 1 Hypothermia in Relation to the Acceptable Limits of Ischemia for Bloodless Surgery Michael J. Taylor, Amr M. Eirifai, and Julian E. Bailes Chapter 2 Responses of Bark and Wood Cells to Freezing Edward N. Ashworth

1

65

Chapter 3 Extracellular Ice Formation in Freezing-Tolerant Plants Marilyn Griffith and Mervi Antikainen

107

Chapter 4 Freeze-Thaw Damage to Thylakoid Membranes: Specific Protection by Sugars and Proteins Dirk K. Hincha, Frank Sieg, Irina Bakaltcheva, Hilde Koth, and Jurgen M. Schmitt

141

Chapter 5 Crystallization and Vitrification in Aqueous Glass-Forming Solutions Patrick M.Mehl

185

Chapter 6 Cryopreservation of Drosophila melanogster Embryos Peter L Steponkus, Shannon Caldwell, Stanley P Myers, and Marco Cicero

257

INDEX

317


LIST OF CONTRIBUTORS

Mervi Antikainen

Department of Biology University of Turku Turku, Finland

Edward N. Ashworth

Department of Horticulture Purdue University West Lafayette, Indiana

Irina Bakaltcheva

Ceo-Centres Ft. Washington, Maryland

Julian E. Bailes

Cryobiology and Hypothermic Medicine Program Neurosciences Research Center Allegheny-Singer Research Institute Division of Neurosurgery Department of Surgery Allegheny University of the Health Sciences Pittsburgh, Pennsylvania

Shannon Caldwell

Department of Soil, Crop and Atmospheric Sciences Cornell University Ithaca, New York

Marco Cicero


VII

VIM

Amr M. Eirifai

LIST OF CONTRIBUTORS Cryobiology and Hypothermic Medicine Program Neurosciences Research Center Allegheny-Singer Research Institute Division of Neurosurgery Department of Surgery Allegheny University of the Health Sciences Pittsburgh, Pennsylvania

Marilyn Griffith

Department of Biology University of Waterloo Waterloo, Ontario, Canada

Dirk Hincha

Institutfur Pflanzenphysiologie und Mikrobiologie Freie Universitat Berlin, Germany

Hilde Koth


Patrick M. Mehl

Transfusion Medicine Research Program Naval Medical Research Institute Bethesda, Maryland

Stanley R Myers


Jurgen Schmitt


Frank Sieg


List of Contributors

IX

Peter L Steponkus


Michael J. Taylor

Cryobiology and Hypothermic Medicine Program Neurosciences Research Center Allegheny-Singer Research Institute Division of Neurosurgery Department of Surgery Allegheny University of the Health Sciences Pittsburgh, Pennsylvania


PREFACE The purpose of this series is to provide a selection of presentations that represent significant advances in the area of low-temperature biology. The authors have been invited to prepare a comprehensive treatise of their experimental studies, both unpublished and previously published, in order to present the reader with a comprehensive overview that is usually not possible in a journal manuscript. In this volume, Mike Taylor and his colleagues present their recent studies of clinical hypothermia and blood substitution in relation to surgical procedures. Although some degree of hypothermia (mild to moderate; 33 to 27°C) is currently used in surgical procedures in the area of cardiovascular and neurosurgery, Taylor and his colleagues are exploring the use of ultra-profound hypothermia (10 to 4°C) in order to extend the safe limits of cardiac arrest beyond the current limit of 60 minutes. Ed Ashworth presents a detailed comparison of the freezing process in woody tree species and provides new insights into the behavior of "supercooling" and "non-supercooling" species based on his extensive electron microscopy studie4s. This chapter provides the reader with an overview of the original classification of woody species based on their supercooling characteristics and geographical distribution; Ashworth then extends these studies to consider the influence of tissue organization and cell wall structure on the freezing response. Marilyn Griffith and Mervi Antikainen describe their recent studies on "Antifreeze proteins" in plants. Previously, Marilyn Griffith and Jack Duman independently discovered the existence of polypeptides that have thermal hysteresis activity in plant species. Because the activity of plant polypeptides is substantially XI

xii

PREFACE

lower than that of polypeptides isolated from fish and insects, their presence in freezing tolerant plants has prompted the question of their mechanistic significance. In their chapter, Griffith and Antikainen present evidence that some of the polypeptides have amino acid sequence homology that is similar to that of "pathogenesis-related" proteins, such as endoglucanases, endochitinases and thaumatinlike proteins. Dirk Hincha and his colleagues present an overview of cryoprotection of chloroplast thylakoids and contrast in vivo and in vitro responses in considering the cryoprotective role of sugars and soluble proteins—including their recent studies of the cryoprotective proteins that are synthesized during cold acclimation of spinach and cabbage. Patrick Mehl presents and extremely comprehensive and detailed treatise on glass transformations in aqueous solutions and their relevance to cryopreservation. In this chapter, Mehl presents many new findings and a wealth of Hterature not commonly cited in the field of cryobiology. These studies are crucial to success in the development of cryopreservation procedures for tissues and organs of mammalian species. Finally, I and my colleagues present an overview of the development and refinement of a vitrification procedure for the cryopreservation Drosophila melanogaster embryos. Not only is this procedure of practical significance for Drosophila biologists, it is the first instance in which insect embryos have been successfully cryopreserved and is serving as a model for the development of cryopreservation procedures for other insect species. Peter L. Steponkus Series Editor

chapter 1

Hypothermia in Relation to the Acceptable Limits of Ischemia for Bloodless Surgery

MICHAEL J. TAYLOR, AMR M. ELRIFAI, AND JULIAN E. BAILES

Introductory Background and Basics The Clinical Perspective and Problem—Need for Bloodless Surgery Basics of Clinical Hypothermia The Need for Profound Hypothermia and Extreme Hemodilution Historical Basis for Ultraprofound Hypothermia and Blood Substitution The Allegheny Approach to Ultraprofound Hypothermia and Blood Substitution Development of the Technique of Ultraprofound Hypothermia and Blood Substitution (UHBS). Application of UHBS with Hypothermosol to Aid Resuscitation and Surgery after Hemorrhagic Shock. Final Comments and Future Directions Future Directions and Clinical Prospects Cerebroplegia for Selective Hypothermic CNS Protection References Advances in Low-Temperature Biology Volume 3, pages 1-64. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0160-0

2 2 5 14 17 20 22 44 50 50 51 54

2

M.J. TAYLOR, A.M. ELRIFAI, and j.E. BAILES

INTRODUCTORY BACKGROUND AND BASICS The Clinical Perspective and Problem—Need for Bloodless Surgery

Today, surgeons have developed skills that allow very complex, corrective and life-saving operations to be performed—notably on the heart and brain. Many of these complicated time consuming procedures have the inherent need for temporary cessation of blood flow and have demanded protection of the different organs of the body, especially the brain, against the deleterious effects of ischemia and anoxia. Common examples of such operations include open-heart surgery, especially in infants to repair congenital defects; neurosurgical problems in adults, such as giant cerebral aneurysms and other vascular lesions; resection of tumors associated with major blood vessels and surgical repair of aneurysms of the aortic arch. Because of their high metabolic demand requiring large amounts of oxygen and glucose, the central nervous system and myocardium are especially vulnerable to the rapid onset of ischemic injury and hypothermia is frequently used as an adjunctive technique for surgical procedures that require a period of circulatory and/or cardiac arrest. It has been known for centuries that the application of cold can be protective (Swan, 1973) and potential clinical benefits of hypothermia in this century were recognized as early as 1939 when it was demonstrated that surface cooling of an ischemic limb in rats improved overall survival (Allen, 1939). However, it was not until the introduction of cardiopulmonary bypass (CPB) in the early 1950s that hypothermia became widely used clinically (Bigelow, 1950; Swan, 1955). Lowering the temperature of a euthermic subject to a temperature below that normally maintained by homeostasis reduces the metabolic rate, and hence the demand for oxygen and substrates by the tissues. On this basis, many modem day surgical procedures, particularly in the areas of cardiovascular surgery, neurosurgery, and sometimes in trauma, rely on some degree of imposed or regulated hypothermia as a relatively safe modality for effecting biologic protection during circulatory and/ or cardiac arrest (see Table 1). Nevertheless, total body hypothermic protection, or "clinical suspended animation," remains time limited by the tolerance of those tissues most sensitive to an ischemic insult, even at reduced temperatures. Currently, the accepted safe limits of cardiac arrest are less then 60 minutes at temperatures no lower than 16 to 18°C. It is well established that exceeding these limits markedly increases the risks of clinical sequelae, especially neurological complications in patients (Kramer, 1968; Baumgartner, 1983; Wells et al., 1983; Newburger, 1993). This imposes severe time constraints for a variety of surgical procedures that are otherwise technically feasible, and it remains a major goal for surgeons to extend the safe limits of hypothermic arrest beyond the present limit of 60 minutes. For convenience, clinical hypothermia is arbitrarily classified as mild (33 to 35°C), moderate (27 to 32°C), deep or profound (10 to 20°C), and ultra-profound (4 to 10°C). In this chapter we outline the principles of clinical hypothermia and

Table I .

Selective Recent Reports of Clinical Applications of Hypothermia for Bloodless Surgery

T°C

HCA or LFP

30 patients

25

LFP

71 (Heart crossclamp)

Bert & Singh 1993

10 patients 7 patients

20 15 12-15

HCA

21 -63

Crepps et al., 1987

HCA

7-56 47f 16

Szentpetery et al., 1993

18 15-20 120 18-20

HCA

Crawford et at., 1987

HCA

7-57 24-62 7-1 20 (median 31)

HCA

2-64 (mean =29)

Davis et al., 1992

18 15-18

HCA v. LFP

Newburger et al.,., 1993

HCNLFP

Greeley et at., 1993

ND*

HCA

Ekroth et al., 1989

15 20 16

HCA v. LFP

Van der Linden et al., 1993

Type o f Surgery

Duration (min)

Reference

Cardiovascular

183 patients 25 patients 5 patients 656 patients 60 patients

HCA HCA

Ergin et al., 1994 Kouchoukos et al., 1990 Svensson et al., 1993

Pediatric

171 patients 275 patients 20 patients 17 patients 8 patients 2 patients

LFP

Lichtenberg et at., 1993

HCA

McCarthy et al, 1993

8-9-13.7

HCA

Williams et al., 1991

16-20 18 16.5

HCA

Pacult et al, 1993

HCA

Ausman et at., 1993

HCA

Chaney 1993

18

LFP

Jolin et al., 1993

17f 2

HCA

Neurosurgery

10 patients 1 patient 9 patients 1 patient 1 patient 33 patients

+ Cerebral LFP

10-89

Ueda et al.. 1994 (continued)

Table 1. Type o f Surgery

14 patients 7 patients 14 patients

(Continued)

T0C

HCA or LFP

Duration (rnin)

Reference

16-20

HCA

5-51

HCA

Baumgartner et al., 1983 Spetzler 1988

HCA

Solomon 1991

Trauma

1 patient 1 patient 1 patient

LFP

ND*

Launois et al., 1989

HCA

40

Zogno et al., 1990

HCA

ND*

Hartman et al.. 1991

Oncolo~

18 patients (Renal) 7 patients 6 patients 1 patient 15 aatients Notes:

'Not Disclosed

ND* (Deep Hypothermia)

HCA

ND*

Vaislic et al., 1986

17 20 17 18(16-25)

HCA

25-45 43-75 48 8-40

Chang et al., 1988

HCA HCA HCA

HCA = Hypothermic Circulatory Arrest.

LFP = Low Flow Perfusion

Ein et al., 1981 Goh et al., 1989 Marshall et at., 1988

Hypothermic Protection During Bloodless Surgery

the modem historical background for attempts to extend the safe limits of hypothermic circulatory arrest (HCA). This will serve as a background for a description of our own recent experimental approach which, in a significant departure from techniques that rely upon moderate to deep hypothermia and hemodilution, employs ultraprofound hypothermia and blood substitution (UHBS). Basics of Clinical Hypothermia

At the cellular level, the fundamental basis of hypothermic protection is the effect of temperature on reaction rates which, according to Arrhenius' theory, are generally slowed by a reduction in temperature. Since the processes of deterioration associated with ischemia and anoxia are mediated by chemical reactions, it has proved well founded to attempt to prevent or attenuate these changes by applying hypothermia. Although our knowledge of the mechanisms of ischemic injury is far from complete, there is a considerable degree of understanding of the cascade of events that is initiated by oxygen deprivation. As shown in Figure 1 these deleterious changes begin with early onset biochemical events arising from the immediate depletion of high energy reserves (ATP and CP) and membrane depolarization, and culminating in structural changes and eventual cell death. Whilst hypothermia is known to influence reaction rates, energy metabolism, active ion transport and ion homeostasis, membrane fluidity and function, and the secretion of hormones and neurotoxins, the effects are not exclusively beneficial and harmful effects of hypothermia have to be "weighed in the balance." The detailed principles of cellular protection by applied hypothermia cannot be reviewed here but have been the subject of several useful reviews, to which the reader is referred (Pegg, 1981,1985,1986; DeLoecker, 1991; Fuller, 1991; Taylor, 1996). At the systemic level the theoretical basis for protecting the brain and vital organs during ischemia and hypoxia has also been reviewed by others (see Hickey, 1985; Hickey and Anderson, 1987; Michenfelder, 1987; Kirklin and BarrettBoyes, 1993) and in essence, relies principally upon the effect of temperature reduction upon metabolism and oxygen demand. Metabolism^ Oxygen Consumption and Hypothermia

It is well established that within the temperature range of 0 to 42°C oxygen consumption in tissues decreases by at least 50% for each 10°C decrement in temperature (Fuhram and Fuhram, 1959). Oxygen consumption (V02) is a reasonable measure of metabolic activity since for practical purposes tissue and cellular stores of oxygen do not exist and the body relies upon the circulation to bring oxygen to its tissues in quantities determined by the rate of O2 consumption. The magnitude of decrease of V02 by hypothermia is therefore regarded as an index of the degree of reduction of metabolic activity.

5


ISCHEMIC

Ischemia (02 deprivation)

CASCADE

Energy loss Transmitter release Lipo lysis . ( Free Fatty Acldsj )

Necrosis Figure 1. Schematic representation of the principal features of the cascade of events that ensues during ischemia. The pivotal event is ATP depletion that occurs within 1 to 2 minutes of oxygen deprivation. This early event leads immediately to a shift from aerobic to anaerobic metabolism, which very quickly becomes self-limiting with the production of lactate and H"^. Cell depolarization also occurs very early in the cascade leading to a breakdown of ion homeostasis and a concatenation of other intracellular and membrane-associated events that eventually culminate in necrosis and cell death. A rise in the intracellular concentration of protons and calcium is at the center of many of the mechanisms now recognized to be contributory to cell death as a result of ischemia. (For details see Siesjo, 1991; Das, 1993)

For the brain, V02 at 5°C is estimated to be 6% of the normothermic rate and Bering (1974) postulated that the brain may tolerate ischemic periods for up to three hours at temperatures below 5°C. It is also known that myocardial tissue can be preserved during three hours of global ischemia at 4°C (Swanson et al., 1980; Baumgartner, 1988; Breen et al., 1993). Since it is well estabUshed that other vital organs can tolerate anoxia for much longer periods than the heart or the brain, it has been anticipated that whole body protection may be possible during three hours of total circulatory arrest if body temperature is maintained as low as 5°C (Haneda, 1986). Nevertheless, one theoretical calculation that has subsequently been supported by the clinically determined "safe limits" of hypothermic CPB predicated a safe arrest time of only 56 minutes at 10°C (Vadot et al., 1963).

Hypothermic Protection During Bloodless Surgery Effect of Cooling on Biophysical and Biochemical Processes in Defining the Safe Limits of Circulatory Arrest

The fundamental basis of all biologic and chemical processes is molecular activity and mobility, which are governed by thermal energy, such that as temperature is lowered so molecular motion is slowed (see Taylor, 1987). The rate of biophysical processes, such as the diffusion of ions and osmosis, declines linearly with temperature by approximately 3% per 10°C (Cameron and Gardner, 1988; Hearse et al., 1981a,b). It is apparent therefore, that biophysical events are relatively little affected by the temperature changes typically imposed during the clinical use of mild to deep hypothermia. It is only at much lower temperatures that the rate of biophysical processes become significantly important, especially at subzero temperatures when phase changes lead to both ice formation and solute concentration changes (Taylor, 1984; 1987). In consideration of biochemical processes, the quantitative relationships between energy requirements of the body, reflected largely by V02, and temperature changes have been expressed mathematically in different ways: 1. The Arrhenius Relationship: Biochemical processes, in common with all chemical reactions, occur only between activated molecules the proportion of which in a given system is given by the Boltzman expression exp (-E/RT) where E is activation energy, R is the gas constant and T is the absolute temperature. According to the Arrhenius relationship, the logarithm of the reaction rate (k), is inversely proportional to the reciprocal of the absolute temperature: -logk = A(-E/2.3RT) A graphical plot of log k against 1/T yields a straight line with a slope of E/2.3R. 2. Van't Hoff Rule relates the logarithm of a chemical reaction rate directly to temperature and is commonly expressed in the form of the respiratory quotient temperature coefficient, QJQ, where Qio is the ratio of reaction rates at two temperatures separated by 10°C. Accordingly, QlO = (K2/Ki)10(T2-Ti)

For most reactions of biological interest QJQ has a value between 2 and 3, but some complex, energy-dependent reactions have a Q^Q between 4 and 6, and are more likely to stop completely at low temperatures (Hearse et al., 1981a,b). Both Qio and Arrhenius plots have been used to quantitate changes in metabolic processes occurring in biologic systems, whether they are enzyme reactions in single cells or the oxygen consumption of the entire human body. The Qio for wholebody oxygen consumption is approximately 2.0 (see Figure 2), indicating that, in general, metabolic rate is halved for each 10°C decrement in temperature. Never-

7

MJ. TAYLOR, A.M. ELRIFAI, and J.E. BAILES

_i_

HO

35

30

25

15

20

TEMPERATURE ( X )

100 90 80 70 60 50 HO

30

6"

20

MO

35

30

25

20

15

TEMPERATURE (TO b

Figure 2. Whole-body oxygen consumption as a function of temperature measured in surface cooled dogs, (a) Data from various sources compiled by Kirklin and BarrattBoyes (1993). The regression curve (with 70% confidence limits) shows the van't Hoff relation between VO2 and temperature with a slope indicating QIQ = 2.7. (b) Shows a nomogram for the same regresssion equation with VO2 expressed as a percentage of control values at 37°C. (reproduced from Kirklin and Barratt-Boyes (1993) with permission).


theless, it is doubtful that the observed decrease in V02 during cHnical hypothermia can be accounted for purely on this physicochemical basis alone. As discussed in the next section, an alternative hypothesis has been invoked to explain observed changes in cerebral metabolic rate as a function of temperature and the related calculations of the anticipated safe duration of HCA. Cerebroprotection During Clinical Hypothermia

The foregoing discussion emphasizes that it has been conmionly assumed that the basis for hypothermia-induced cerebral protection is metabolic suppression, especially as the reduction of cerebral metabolic rate (CMR) is accompanied by EEG suppression. This concept has been questioned recently as being incomplete in the light of studies that show that even mild hypothermia, with changes in brain temperature of as little as 2 to 4°C, can have a profound effect upon the extent of ischemic brain damage (Minamisawa et al., 1990; Sano et al., 1992; Todd and Warner, 1992). These studies indicate that a sigmoid curve, rather than the classically held log-linear relationship, best describes the correlation of brain temperature with histologic damage under conditions of mild hypothermia. These observations have led to the consideration of alternative hypotheses to explain the neuro-protective mechanisms of both anesthetics and hypothermia (see Todd and Warner, 1992). Alternative hypothesis of cerebroprotection. We mentioned earlier that ischemia is known to initiate a cascade of events, most of which in the early phases are biochemical in nature. Cerebral ischemia triggers a massive release of multiple neurotransmitters, one of which is glutamate, the principal excitatory amino acid in the brain (Hillered et al.,1989; Baker et al., 1991; Ginsberg, 1992). The excitatory properties of glutamate are thought to be mediated by post-synaptic depolarization and neuronal calcium influx. The entry of calcium stimulates: (a) the release of free fatty acids, which in turn results in the synthesis of various membrane-active compounds; (b) the production of oxygen and hydroxyl free radicals; and (c) the uncoupling of mitochondrial oxidative metabolism. These changes may persist into the post-ischemic phase such that facilitated calcium entry is believed to be the mediator of neuronal death (Siesjo, 1991). The high concentrations of extracellular glutamate seen during ischemia can rapidly return to normal, but either the transient increase or some other related event may lead to an enhanced sensitivity of glutamate receptors to activation. This phenomenon, which has been described by Manev (1990) as abusive stimulation of excitatory amino acid receptors, results in persistent calcium influx with the deleterious consequences outlined above. In addition, there are other changes such as altered protein synthesis and changes in gene expression (Xie et al., 1989; Nowak, 1990; Uemara et al., 1991).

9

10


With the aid of microdialysis techniques it has been shown that mild hypothermia markedly attenuates the activity of neurotoxins such as glutamate and dopamine. The growing body of evidence for the neuro-specific protective mechanisms of hypothermia, which may operate through the alteration of neurotoxin activity by the inhibition of biosynthesis, release or uptake of neurotransmitters has been reviewed recently (Ginsberg, 1992; Maher and Hachinski, 1993). Additional mechanisms involving intracellular mediators such as calcium/calmodulin-dependent protein kinase II, protein kinase C, or ubiquitin have also been implicated. Classical hypothesis in relation to cerebrotolerance of circulatory arrest. Concentrating on the classical ideas of hypothermic protection, Michenfelder and his coworkers have examined the effect of hypothermia over a much wider temperature range (14-37°C), on the cerebral metaboHc rate of oxygen consumption (CMRO2) in dogs. They have shown that the relationship is complex, resulting from the combined effect of temperature on reaction rates and the effect this has on cerebral function as reflected by electroencephalogram (EEG) changes (Steen et al., 1983, Michenfelder and Milde, 1991; 1992). As depicted in Figure 3 they determined values of '-2.2 for the QJQ of canine CMRO2 between 37 and 27°C, but a much higher value (-^4.5) at temperatures between 27 and 14°C. By correlating these measurements with the EEG activity of the brain, they showed that the changes in EEG activity are minimal between 37 and 27°C (Michenfelder and Milde, 1991), but below 27°C, EEG activity is progressively altered until ultimately an isoelectric pattern was recorded below 17°C. The temperature at which the brain is metabolically inactive varies, and its estimation depends on the location of temperature measurement. The isoelectric EEG, or electrocerebral silence, is thought to be consistent with metabolic inactivity of the brain (Coselli et al., 1988). These observations led to the hypothesis that, in the range of 37 to 27°C, the expected QJQ value of CMRO2 ('^2.2) represents the direct effect of temperature on biologic reaction rates primarily, but at lower temperatures (27 to 17°C), there is also, in addition, a significant alteration in neurofunctional status that culminates in a near isoelectric EEG. It was further hypothesized that at and below temperatures associated with an isoelectric EEG, the QJQ value should revert to near 2.0 reflecting only the direct effect of temperature on biologic reaction rates. Michenfelder and Wilde (1992) have recently demonstrated a QJQ of 2.2 at temperatures below 13°C in the presence of electrocerebral silence, thus supporting this hypothesis. Relevance to the duration of ''safe'' circulatory arrest On the commonly held basis that hypothermia-induced brain protection is mediated principally by cerebral metabolic suppression, a QJQ of 2.2 cannot explain the clinically accepted tolerance of the brain for approximately 60 minutes of circulatory arrest at temperatures in the region of 15 to 18°C (Tharion et al., 1982). A calculation based upon the known ischemic tolerance of only 6 minutes at 37°C, shows that a Q^Q of


11

Hypothermia and Cerebral Metabolism 100 ^

50 - -C\J

O cc

o

10

1 Q 10 -" 2.2

Q 10 -" 2.2

EEG:

stable

changing —1

37

- ^^***^***«^

Q 10 '" 4.5

5

27

— 17

isoelectric

Temperature (°C)

Figure 3, Relationship between cerebral metabolic rate (CMRO2) and temperature in dogs. Interpretation of calculated changes in Q^Q with observed changes in electroencephalographic (EEG) patterns are described in the text. (Redrawn from Michenfelder and Milde (1992) with permission).

2.2 can account for only 29 minutes of tolerance when temperature is reduced by 20°C. The duration of tolerance at ITC = (6 x 2.2 x 2.2) = 29 minutes. However, if QlO increased to 4.5 between 27 and 17°C, the apparent enigma is resolved since the calculation now yields a tolerable interval = (6 x 2.2 x 4.5) = 59 minutes. In summary therefore, it is apparent that the role of hypothermia in cerebroprotection is mediated principally by a temperature-induced decrease in cerebral oxygen demands sufficient to provide tolerance for extended periods of absent oxygen supply, although other mechanisms are also implicated (Busto et al., 1987; Ginsberg, 1992; Maher and Hachinski, 1993). A clear understanding of the precise mechanisms of hypothermic cerebroprotection remains equivocal, fueled by two recent bodies of evidence. On the one hand, recent studies have shown that only modest degrees of hypothermia (33 to 34°C) provide neuronal protection of a magnitude greater than can be accounted for by metabolic suppression alone (Busto et al., 1987; Ginsberg, 1992). Yet on the other, Michenfelder's group have shown that the calculated Qio for CMRO2 is influenced by both the direct effect of temperature on rates of biological reactions and the resulting influence this has on neurofunction as reflected by the EEG. They contend that these relationships can fully explain the proven tolerance of the human brain for complete global ischemia at profound levels of hypothermia on a metabolic basis alone.

M J . TAYLOR, A.M. ELRIFAI, and J.E. BAILES

12

10

20

30

MO

50

60

70

80

DURATION OF TOTAL CIRCULATORY ARREST (minutes) a l.U

H 0.9 (O UJ Q: O.b

: - i t . .

: ; ; • . .

__

-

a:

< >-

0.7

u. oc o o . b

0 h

3c^

0.5

>- _j < h2 cc

O.M

'^tsH

0.3

"ixJ 0.2 LL

Solution^ KCI

4 meq/l

NaHC03

27 meq/l

NaCi

100 meq/l

Na acetate

2 meq/l

THAM

10 meq/l

MgCl2

2 meq/l

CaCl2

3 meq/l

Dextran

30 meq/l

Glucose

10 meq/l Haff's Solution-^

Pooled homologous p lasma

4000 ml

KCI

10 meq

CaCl2

5 meq

MgS04

40 meq Kondo'i 5 Solution^

K+

107 meq/l

Na+

9 meq/l

Mg^^

28 meq/l

c\-

14 meq/l

Phosphate

54 meq/l

Bicarbonate

9 meq/l

Sulfate

28 meq/l

Glucose

25g/l

Source: ^ Neely et a!., 1963. ^Haffetal., 1975 ^Kondoetal., 1974

requirement for continued and substantial demands for oxygen during mild or moderate hypothermia. However, as outlined above, it is well established that cooling induces detrimental changes to various properties of the blood that are not effectively ameliorated by simple hemodilution; these include dramatic increases in blood viscosity, coagulopathies and the deformability and clumping of erythrocytes, which contribute significantly to the problem of multifocal blockage of the microvasculature and formation of tissue infarcts (Keen and Gerbode, 1963). The concept of totally removing the blood and replacing it with a suitable acellular substitute solution is a novel approach, the feasibility of which we have investigated in recent years at the Allegheny-Singer Research Institute and Allegh-

22

M.J. TAYLOR, A.M. ELRIFAI, and J.E. BAILES

eny University of the Health Sciences. In principle, this technique could provide a number of potential benefits over and above the obvious avoidance of the bloodrelated complications. In addition to total vascular and capillary washout and the removal of harmful catabolic products, blood substitution provides the opportunity to control the extracellular environment directly and the intracellular milieu indirectly as we describe in more detail below. Development of the Technique of Ultraprofound Hypothermia and Blood Substitution (UHBS) Phase /: Feasibility Study in a Canine Model using First-Generation^ Hypothermic Blood Substitutes

Choice of experimental model. It is readily apparent from the outline review above that the dog has been used extensively in studies of the effects of hypothermia on euthermic mammals relevant to clinical procedures. Use of the canine model therefore, provides a good deal of background data for comparison with new approaches. While the dog has been widely used as a pre-clinical model for

^^^* - • , . » - •

"^

30^ »i

1 1

1

25-

Z)

=

1°

/ 100

15 min

/

'

'—--^

/

y^

50

0 ^

^^^*^*>>^^

•

I

10

O'C

.

1

•

20

i

30

—-^

I

J

40

1/osmolal Figure 2. Packed thylakoid volume in response to different osmolalities of the suspending medium. The samples contained 2.5 mM NaCi and additional sucrose from 20 to 500 mM. Thylakoid volume, determined by hematocrit centrifugation is plotted as a function of reciprocal osmolality (Boyle-van't Hoff plot). Unfrozen controls (0°C) are compared to samples frozen to - 5 ° C for 15, 30, or 60 minutes. The straight lines at high sucrose concentrations (low reciprocal osmolalities) were fitted to the data by linear regression analysis. The data points were omitted from the figure for greater clarity.

to reach the same maximum volume as unfrozen controls (Figure 2). The changes in the membranes that lead to this effect have not been clarified yet. When thylakoids rupture, as indicated by plastocyanin release, they collapse to a small volume (Hincha, 1986; Bakaltcheva et al., 1992). Therefore, after freezing in a low osmolality solution, samples protected from damage by cryoprotective additives can be easily distinguished from unprotected samples by measuring thylakoid volume. Although freezing of thylakoids in a low osmolaUty solution leads to loss of plastocyanin, it is obvious that these conditions are not a good model of the in vivo situation since unstressed, non-acclimated spinach leaves have an osmolality of approximately 400 mOsm (Schmidt et al., 1986; Hincha, 1994). Media of varying complexity have been devised to freeze thylakoids in a solution approaching the conditions experienced by the membranes in situ and to investigate the effects of the different components of the chloroplast stroma on damage and protection (Grafflage and Krause, 1986; Santarius, 1986a,b,c; 1987b; 1990; 1991; 1992). We use a simplified stroma medium for our experiments that nevertheless yields results that are comparable to those from whole leaf experiments (Hincha and Schmitt, 1988a).

Cryoprotection of Thylakoid Membranes

149

40

T3 0) (0 (0 0)

o CL

2 4 incubation time (h) Figure 3. Release of plastocyanin (PC) from isolated spinach thylakoids as a function of the incubation time. The membranes were suspended in an "artificial stroma medium" (5 m M MgCl2, 10 m M K2SO4, 150 m M K-glutamate, 50 m M sucrose) and were either stored on ice or in a freezer. At the indicated times, frozen samples were thawed and the membranes were removed from both frozen/thawed and unfrozen samples by centrifugation. Plastocyanin in the supernatants and in total thylakoids lysed in 2% Triton X-100 was determined by immunodiffusion (Hincha et al., 1985). Plastocyanin release is expressed as the percentage of the protein found in the supernatant compared to total thylakoid content. The values are corrected for the amount released after transfer of the membranes from the washing solution to the incubation medium (t = 0; typically about 10% of total plastocyanin content).

When thylakoids are subjected to a freeze-thaw cycle in the presence of this stroma medium, plastocyanin release shows biphasic kinetics (Figure 3) with a rapid component (< 30 min) that is directly dependent on the freezing temperature (Figure 4). After this initial loss, release continues slowly and is linearly time dependent (Figure 3). This is very similar to the in vivo situation where plastocyanin loss is also biphasic. However, the slow phase of damage develops over several days in leaves while it occurs within a few hours in vitro. Another important difference is that isolated thylakoids slowly lose plastocyanin already in an unfrozen solution at 0°C (Figure 3) while this temperature is not damaging to the membranes in situ.

150

D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and j . M . SCHMiTT

JU JO

• \

in

d CO

• •

20

O (0 CO

o

10

Q.

•

r = -0.93

-^ o^ 1

-25

1

20

1

1

15

-J

1

—1

\ 1

1

A

10

temperature (°C) Figure 4, Temperature dependence of the rapid phase of freeze-thaw damage to thylakoids suspended in the artificial stroma medium (see Figure 3 for experimental details). The straight line was fitted to the data by linear regression analysis. The regression coefficient is indicated.

Our results indicate that the slow, linearly time-dependent release of plastocyanin, both in frozen and unfrozen samples in the artificial stroma medium, is directly related to the solute loading discussed above. Similarly, the rapid, temperature-dependent phase of plastocyanin release (Figure 4) could be related to the reduced extensibility observed by hematocrit centrifugation in thylakoids after freezing. Both low osmolality solutions and the artificial stroma medium have been used to characterize possible changes in the freeze-thaw behavior of thylakoids during cold acclimation. These experiments revealed that under the same experimental conditions, thylakoids isolated from plants that were cold acclimated under natural conditions or acclimated under salt stress showed reduced loss of plastocyanin compared to thylakoids from control plants (Schmidt et al., 1986; Hincha and Schmitt, 1988b). Thylakoids from cold-acclimated plants also showed a greater preservation of light-induced proton uptake after an in vitro freeze-thaw cycle (Garber and Steponkus, 1976b). Under salt shock conditions (300 mM NaCl) the difference in plastocyanin release was already manifest after only one hour (Hincha, 1994). It could be shown that both the rapid and slow phase of plastocyanin


151

release were reduced in an artificial stroma medium when thylakoids from nonacclimated and cold-acclimated plants were compared. In agreement with that, volumetric measurements showed that the extensibility after freezing is increased and solute loading is decreased in thylakoids from cold-acclimated plants (Hincha and Schmitt, 1988b). The observed reduction in solute loading suggests a reduction in the solute permeability of the membranes during cold acclimation. This seems surprising in the light of data showing an increase in thylakoid membrane fluidity during cold acclimation of pea plants (Barber et al., 1984) and some wheat varieties (Pomeroy and Raison, 1981). It is generally assumed that an increase in fluidity leads to a higher solute permeability of a membrane (van Zoelen et al., 1978). The increased fluidity of pea thylakoids after cold acclimation was not due to changes in fatty acid unsaturation or to any pronounced changes in overall lipid composition (Chapman et al., 1983). On the other hand, the lipid composition of thylakoids from barley showed strong changes in response to an NaCl treatment with 400 mM for 3 days (Miiller and Santarius, 1978). This was attributed to a salt-dependent inactivation of the enzymes galactosyl transferase and acylase, which are located in the chloroplast envelope. This inactivation leads to a decreased content of the lipid monogalactosyldiacylglycerol in the thylakoid membranes. Whether this is also true in other plant species under salt stress, and whether this is one of the causes of increased freezing tolerance is not known. Further systematic investigations will be necessary to clarify how thylakoid membranes acquire increased freezing tolerance and what the molecular basis of this adaptation is. When the effects of cold acclimation measured with isolated, washed thylakoids were compared to the effects on thylakoids frozen in situ, it became apparent that the increased hardiness in vivo can only in part be accounted for by changes in the properties of the membranes (Hincha and Schmitt, 1988b). This suggests that soluble cryoprotectants that were removed during membrane isolation play an important role for the freezing tolerance of thylakoids in vivo. In the following sections we will discuss the effects of two classes of possible cryoprotectants, sugars and soluble proteins, on the stability of thylakoids during an in vitro freeze-thaw cycle.

CRYOPROTECTION OF THYLAKOIDS BY SUGARS In many organisms, exposure to drought, salt, or low-temperature stress leads to the accumulation of low molecular weight osmolytes. It has been noted that in all organisms, including plants, animals, and microbes, only a few classes of molecules were selected during evolution for this purpose. These include sugars and sugar derivatives, polyols (e.g., glycerol), the amino acid proline, and quaternary ammonium compounds such as glycine betaine (Somero, 1992). It is generally assumed that osmolytes act as unspecific "compatible solutes." The major requirement for compatibiUty is that a substance is non-toxic to meta-

152

D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and J.M. SCHMITT

bolic functions even at high concentrations. For the stabiUzation of soluble proteins under stress conditions, a mechanism of "preferential exclusion" of compatible solutes from the hydration shell of the proteins has been discussed (Carpenter et al., 1990). Preferential exclusion of a solute from the hydration shell of a protein leads to a preferential hydration of the protein, making unfolding and exposure of hydrophobic amino acids to the solution thermodynamically unfavorable and thereby stabilizing the native, folded structure (Timasheff, 1993). Whether this mechanism can also be operative during the stabilization of membranes is not clear at present (Crowe et al., 1990). We will show below that in the case of sugars and thylakoid membranes stabilization by direct interaction seems to play a major role. During cold acclimation of plants, leaf osmolyte concentration increases dramatically in many species (Dowgert and Steponkus, 1984; Guy et al., 1992; Koster and Lynch, 1992; Riitten and Santarius, 1992a; Ristic and Ashworth, 1993). It has been shown that transgenic tobacco plants with increased mannitol content were more resistant to salt stress than untransformed control plants (Tarczynski et al., 1992; 1993). After in vitro selection of callus cultures from winter wheat for growth in the presence of the inhibitor hydroxyproline it was found that regenerated plants with increased levels of proline showed increased salt tolerance and increased freezing tolerance after cold acclimation. Freezing tolerance in the nonacclimated state was not influenced (Dorffling et al., 1993). This would indicate that increased cellular proline concentrations are by themselves not sufficient for freezing tolerance. In combination with other changes brought about by cold acclimation, however, the higher proline content may be effective in increasing hardiness. This is emphasized by the fact that in proline-overproducing (hydroxyproline resistant) cell lines of potato (van Swaaij et al., 1986) and spring wheat (Tantau and Dorffling, 1991) no correlation between proline content and either NaCl tolerance or freezing tolerance could be found. Only a weak correlation between osmotic potential and hardiness was apparent (Tantau and Dorffling, 1991). Similarly, differences in the freezing tolerance of two barley cultivars before and after cold acclimation were not related to the levels of glycine betaine accumulated during cold hardening (Kishitani et al., 1994). Increased cellular solute concentrations could be advantageous for freezing tolerance because they reduce the freeze-induced contraction of the cells during extracellular ice formation. In some cases it could be shown that leaves were killed when the cells reached a constant residual volume during freezing while the killing temperature changed with the hardening state of the plants (Meryman et al., 1977; Schmidt et al., 1986). In other species, however, this correlation was not found (Yelenosky and Guy, 1989; Fennell et al., 1990). From the data presented above on freeze-thaw damage to thylakoids, it is clear that damage could be reduced by an increased cellular osmolality. Accumulated solutes would limit thylakoid swelling during thawing and thereby reduce rupture and loss of plastocyanin.


153

On the cellular level, some osmolytes seem to have more specific roles in salt and drought tolerance than merely to act as compatible solutes (Hanson et al., 1994). In addition, more specific factors than increased leaf osmolality must play a role in cold acclimation (O'Neill, 1983; van Swaaij et al., 1985; Riitten and Santarius, 1988; 1992b; Yelenosky and Guy, 1989; Fennell et al., 1990). One of the obstacles encountered in assessing specific functional roles of osmolytes in cellular stress resistance is the lack of knowledge about their compartmentation. Localization in the chloroplast was shown for glycine betaine in spinach under salt stress (Robinson and Jones, 1986) and for sucrose and raffinose in cabbage leaves during cold accUmation (Santarius and Milde, 1977). In addition, very little is known about possible interactions between specific sugars and membranes during freezing. Detailed studies have been conducted on the interactions of sugars, especially trehalose, with phosphatidylcholine membranes during desiccation (see Crowe et al., 1987; 1988 for reviews). The effectiveness of different sugars in protecting small unilamellar phospholipid vesicles during a freeze-thaw cycle has also been studied (see Crowe et al., 1988 for a review). Here it was generally found that the disaccharides, sucrose and trehalose, were superior to the monosaccharides and sugar alcohols studied. There were no differences in the effectiveness of sucrose and trehalose in these experiments (Anchordoguy et al., 1987). For two important reasons it is not possible to extrapolate the data cited above to our experimental system. First, in all these experiments the vesicles were frozen in liquid nitrogen. This is not relevant to the temperature range of freezing tolerance in herbaceous plants. Most importantly, one of the main factors in freeze-thaw damage both in vivo and in vitro that we described above is solute loading of membrane vesicles. This is the result of the diffusion of solutes across the membranes in the frozen state. This mechanism of freezing damage will not be operative at -196°C and any effects a solute will have on permeability would therefore not be detected under these experimental conditions. The second important difference is the lipid composition of the respective membranes. Animal membranes contain a high percentage of phospholipids with phosphatidylcholine being the predominant lipid in many membranes (Quinn, 1982). Therefore, phospholipid vesicles are a suitable experimental model to investigate the freeze-thaw stability of such membranes (Rudolph and Crowe, 1985). In contrast, thylakoids contain only about 12% phospholipid, namely phosphatidylglycerol, but no phosphatidylcholine (Dome et al., 1990). Instead, 75% of the lipids are uncharged galactolipids (50% monogalactosyldiacylglycerol (MGDG) and 25% digalactosyldiacylglycerol (DGDG)) and approximately 12% sulfoquinovosyldiacylglycerol (SQDG), a sulfoglucolipid (Quinn and WiUiams, 1983; Webb and Green, 1991). For a comprehensive review of the structure and properties of plant glycolipids see Kates (1990). We have therefore surveyed the effects of a wide range of sugars on plastocyanin release from thylakoids during freezing to -20°C. The results of these studies

154

D.K. HINCHA, F. SIEG, I BAKALTCHEVA, H. KOTH and j.M. SCHMITT

are summarized in Figure 5. It can be seen that under standard conditions in an artificial stroma medium the effects of the different sugars vary dramatically. Some structural trends can be distinguished. The three best cryoprotectants are disaccharides (1-6 digalactose > trehalose (two 1-1 linked glucose molecules) > 14 digalactose). Interestingly, the headgroup of the thylakoid membrane lipid DGDG is a disaccharide consisting of two, 1-6 linked galactose molecules. The 14 linked digalactose is much less effective. This points to very subtle structural requirements for optimal cryopreservation of thylakoids. We will present a detailed argument below that these structural requirements probably reflect the different ability of the sugars to hydrogen bond to the galactolipid headgroups. Cryoprotection was not, as suggested in other studies (Santarius, 1973), related to the polymerization grade of the sugars, as the monosaccharide galactose was superior to the disaccharides sucrose and melibiose, the trisaccharide raffinose, and the tetrasaccharide stachyose (Figure 5). The presence of a positive charge on a sugar (galactose/galactosamine; glucose/glucosamine) had no measurable influence on cryoprotection. The presence of an acetyl-group, on the other hand, virtually abolished any protective activity. The most dramatic effect resulted from the presence of a COOH-group on a monosaccharide. When the carboxyl group was linked to carbon atom C6 (glucuronic acid; galacturonic acid) the two cryoprotective sugars glucose and galactose were transformed to cryotoxic solutes. When the carboxyl was located in position CI (gluconic acid) this effect was much less pronounced. This illustrates again that the activity of the different sugars is governed by very specific structural constraints that we are just beginning to understand. Similar differences in cryoprotective efficiency between some of the sugars listed in Figure 5 have also been reported by others under different experimental conditions and by measuring light-dependent biochemical activities of the membranes (Santarius, 1973; Steponkus et al., 1977; Lineberger and Steponkus, 1980; Santarius and Bauer, 1983; Santarius and Giersch, 1983). Measurements of time-dependent plastocyanin release from thylakoids suspended in an artificial stroma medium (compare Figure 3) showed that all sugars listed in Figure 5 act exclusively on the second, slow phase of damage while the first, rapid phase is unaltered (Hincha, 1989; 1990; Hincha et al., 1993a). This points to an effect of the sugars specifically on solute loading. Volume measurements after a freeze-thaw cycle to -20°C for three hours (compare Figure 2) in the presence of different concentrations of sucrose and a constant, low concentration of another sugar verified a reduction in solute loading as the mechanism of cryoprotection or cryotoxicity. This led us to the hypothesis that the differential effects of the sugars on thylakoids are the result of their differential ability to influence the solute permeability of the membranes. We have recently started to test this hypothesis by comparing the effects of sucrose and trehalose on the glucose permeability of thylakoids (Bakaltcheva et al., 1994). We chose to compare sucrose and trehalose because it had been shown earlier that for thylakoids trehalose was a much better protectant


155

F J

glucuronic acid galacturonic acid gluconic acid N-acetylgalactosamine N-acetyiglucosamine glucose glucosamine sucrose melibiose

,

stachyose raffinose galactosamine galactose 1 -4 digalactose trehalose 1-6 digalactose 1

1

-110 protec:tion

1

1

I

•

1

C1

damage

Figure 5. Effect of different sugars on the freeze-thaw stability of isolated spinach thylakoids. Freeze-thaw damage was measured as plastocyanin release after 3 hours at -20°C. The membranes were suspended in an artificial stroma medium (see Figure 3 for details), and were frozen in the presence of up to 10 m M of the different sugars (except 1-4 and 1-6 digalactose: 3 m M ; trehalose: 2 mM). In this concentration range the effects of the sugars were a linear function of concentration. A negative slope indicates protection; a positive slope toxicity. The figure was compiled from experimental results published in Hincha, 1989; 1990; Hincha et al., 1993a, and from unpublished data.

than sucrose (Figure 5), and because trehalose was the only sugar that also reduced plastocyanin release in unfrozen samples at 0°C. This opened the possibility of investigating a potentially cryoprotective sugar-membrane interaction without the additional complexities introduced by the freezing process. Also, both sugars are thought to play an important role in the stress tolerance of plants. Sucrose is accumulated in many plants during cold acclimation (Kandler and Hopf, 1982). Trehalose is a prevalent osmolyte in many desiccation-tolerant lower animals and fungi, and in the lower desiccation-tolerant vascular plant Selaginella, and has

156


recently also been detected in the leaves of a desiccation-tolerant higher plant (Bianchi et al., 1993; Drennan et al., 1993). In order to investigate the effects of sucrose and trehalose on membrane solute permeability, we measured the flux of ^^C-glucose into isolated spinach thylakoids (Bakaltcheva et al., 1994). In the presence of 25 mM glucose, influx of the radioactive tracer was reduced by 50% when 25 mM trehalose was present as a cosolute. Under the same conditions 75 mM sucrose was required to achieve the same reduction in permeability. The effect of trehalose was not accompanied by a reduction in fluidity, estimated from fluorescence depolarization data using 1,6diphenyl-l,3,5-hexatriene (DPH) as a probe. A strong correlation between fluidity and permeability had been shown previously for other membranes (van Zoelen et al., 1978). From the fluorescence depolarization data it could also be concluded that the reduced permeability was not due to a liquid crystalline-to-gel lipid phase transition. This conclusion was corroborated by the finding that Arrhenius plots obtained from permeability measurements in the absence and presence of 25 mM trehalose between 0°C and 10°C showed no indications of a breakpoint or change in slope that might indicate the onset of a phase transition. Since we had proposed earlier that the differences in the cryoprotective effects of the sugars might be related to their ability to hydrogen bond to lipid headgroups, we used the hydrophobicity sensitive dye merocyanine 540 (MC540) to probe the solution-membrane interface. The absorbance maximum of MC540 is shifted from 540 nm to 570 nm when the dye is transferred from a hydrophilic to a hydrophobic environment (Biondi et al., 1992). We could show that the A570 to A530 absorbance ratio is linearly correlated with the dielectric constant of different solvents (Bakaltcheva et al., 1994). Changes in the spectral properties of MC540 in the presence of membranes have been related to different fractions of the dye bound to the membranes or free in solution (Lelkes and Miller, 1980). Since it had been shown that changes in lipid packing density influence the accessibility of the headgroup region of the membrane for MC540 (Stillwell et al., 1993), we used the dye to probe the accessibility of thylakoid membranes for solutes in the presence of different sugars. We found that less of the dye partitioned into the membrane surface when thylakoids had been preincubated with trehalose than after incubation with sucrose or glucose. The same was found when liposomes made of 50% DGDG were used in these experiments, but not with phosphatidylcholine vesicles or MGDG dispersions. Since MGDG does not form bilayers upon hydration (Quinn and Williams, 1983; Webb and Green, 1991), the results with this lipid have to be viewed with caution. It is not clear in how far the differences between bilayer and non-bilayer lipids influence the partitioning of the dye and therefore mask possible effects of the sugars. However, the results with DGDG confirm our earlier hypothesis that effective cryoprotection is related to binding of the sugars to galactolipid headgroups. Reduced solute permeability could be envisioned to result from a competition of


157

1,4

high

1,2 O CO

O

xy^

40 - ^ ^

Q.

20,

O

:

T^

0

control

r = 0.99

\

1

1

1

1

1 1

2 4 incubation time at 0°C (h)

Figure 7. Effect of the hemolytic, bee venom peptide melittin on plastocyanin release from spinach thylakoids. The membranes were suspended in an artificial stroma medium (see Figure 3) in the absence or presence of 100 iiig melittin/ml. The samples were incubated at 0°C and release of plastocyanin determined as described in Figure 3. Means and standard deviations of results from three experiments are shown. The lines were fitted to the data by linear regression analysis and the regression coefficients are shown.


161

80

(0 CO

c>

Q>

o

a.

0

1 2 3 4 incubation time at -20°C (h)

Figure 8, Cryotoxicity of melittin for isolated spinach thylakoids. The membranes were suspended in an artificial stroma medium (see Figure 3) in the presence of different peptide concentrations and stored frozen at -20°C. Plastocyanin release was measured after different incubation times.

mated that melittin-induced lysis of red blood cells is at least 100-fold faster than plastocyanin release from thylakoids at the same melittin concentrations. It remains to be seen in future experiments which of the differences in composition between the two types of membrane are responsible for the different rates of lysis. In freeze-thaw experiments we could show that melittin was strongly cryotoxic to thylakoids. An analysis of the time-dependent release of plastocyanin at -20°C revealed that melittin increased specifically the rapid phase of damage (Figure 8). It seems reasonable to assume that due to the freeze-induced increase in concentration the peptide partitioned more readily into the membranes and therefore led to increased lysis. Different mechanisms of melittin-induced lysis have been described for phospholipid vesicles, depending mainly on peptide and solute concentrations (Dempsey, 1990). It is not clear at present which one of these mechanisms is operative with thylakoids in the frozen or unfrozen state. Another class of amphiphilic proteins that shows effects on membrane stability are fish antifreeze or thermal hysteresis proteins (AFP) and glycoproteins (AFGP) (DeVries and Cheng, 1992). These proteins are divided into different structural classes (Hew and Yang, 1992). The class I AFPs (e.g., AFP-SF in Figure 9) have been shown by X-ray crystallography to be a-heUces (Yang et al., 1988). Class III

162

D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and j.M. SCHMITT

AFPs (e.g., AFP-AB in Figure 9) mainly consist of P strands (Sonnichsen et al., 1993), while the conformation of AFGP is most likely a y-tum structure (Drewes and Rowlen, 1993). Their biological role in arctic and antarctic fish is to non-colligatively reduce the freezing point of the body fluids by adsorption to small ice crystals, thereby inhibiting further crystal growth (Raymond and DeVries, 1977; Knight et al., 1991). They have no cryoprotective role in these fish, as the crystallization of a major part of the body water leads to the immediate death of the animals (Wang et al., 1994). It has nevertheless been proposed that a fundamental property of these proteins is the protection of animal cells or organs during frozen or cold storage (Rubinsky et al., 1991). Other investigators, however, found no evidence for protection or even reported increased damage in the presence of antifreeze proteins (Hincha et al., 1993a; Wang et al., 1994). Our own data suggest that at least some AFP and AFGP very effectively destabilize thylakoid membranes during freezing (Figure 9). Plastocyanin release was increased substantially already at very low concentrations of AFP-AB and AFGP 3/4. AFP-SF and AFGP 8 were less effective but still clearly cryotoxic. Similar to the effect of melittin (Figure 8) the fish proteins mainly increased the rapid phase of damage when time-dependent plastocyanin release was measured at -20°C (Hincha et al., 1993a). Whether the AFP and AFGP act by stably partitioning into the membrane lipid phase, as has been shown for melittin, is not known. However, the fact that some of the proteins are effective already at extremely low concentrations (Figure 9) argues against a mechanism based on changes in ice crystal morphology that have been shown to damage red blood cells at higher AFP concentrations (Carpenter and Hansen, 1992). This is corroborated by our finding that the AFGP and AFPAB also increase plastocyanin release from thylakoids at 0°C in the absence of ice crystals (Hincha et al., 1993a). This points to direct protein-membrane interactions as the cause of membrane destabilization. It has recently been shown that plants also contain thermal hysteresis proteins (Griffith et al., 1992; Duman et al., 1993). Nothing is known so far about their structure or function. It has been attempted to increase the freezing tolerance of plants by expressing genes coding for fish AFP in transgenic plants (Georges et al., 1990; Hightower et al., 1991; Kenward et al., 1993). No evidence for a successful improvement of freezing tolerance in these transformed plants has been published to date. In conclusion, the available data show that amphiphilic, a-helical proteins destabilize membranes to different extents even in the absence of an additional stress treatment. We would therefore suggest that the amphiphilic, a-helical LEA and COR proteins that have been implicated in plant stress resistance may, at best, have no effect on membrane stability. Their roles in desiccation or frost tolerance may be more indirect, as has been proposed recently by Dure (1993) for LEA proteins.


100

o

D •

AFGP8 AFGR3/4

•

AFP-AB

163

U O AFP-SF

CO

(0

o 0)

O QL

20 30 |iM protein

50

Figure 9, Effects of fish antifreeze proteins on freeze-thaw damage to spinach thylakoids. The membranes were suspended in an artificial stroma medium (see Figure 3) and were frozen at -20°C for 3 hours. Plastocyanin release was determined after thawing. The samples contained different amounts of either purified antifreeze glycoprotein 8 (AFGP 8), a mixture of AFGP 3 and 4 (all AFGPs were from the Antarctic fish Dissostichus mawsoni), the antifreeze protein from the Antarctic eel pout {Austrolycichthys brachycephalus; AFP-AB) or the Arctic starry flounder {Platichthys stellatus; AFP-SF). The molecular masses of the proteins used for the calculation of molar concentrations are: AFGP 8, 2000 Da; AFGP V4, 20 000 Da (average); AFP-AB, 6000 Da; AFP-SF, 4000 Da. The proteins were kindly provided by Prof. A. L. DeVries.

Lectins Lectins are defined as a functional group of sugar-binding proteins that are not immunoglobulins. They usually carry two or more carbohydrate recognition domains and are therefore able to agglutinate red blood cells or precipitate glycoconjugates (see Etzler, 1985; Lis and Sharon, 1986 for reviews). The three-dimensional structure of these sugar-binding domains is conserved among many carbohydrate-binding proteins, including enzymes that use sugars as their substrates (Quiocho, 1986). Lectins are found in many species of bacteria, animals, and plants, and are structurally extremely diverse (Sharon, 1993). They are usually grouped into different classes according to their specificity for different monosaccharides. Most of the proteins, however, interact much more strongly with disac-

164

D.K. HINCHA, F. SIEG, r. BAKALTCHEVA, H. KOTH and j.M. SCHMITT

charides or other more complex oligosaccharide structures (Lis and Sharon, 1986). In order to find a suitable model system to investigate the cryoprotective properties of hydrophilic proteins for biomembranes we have used commercially available, galactose-specific lectins from the seeds of several different plant species (Hincha et al., 1993b). It is well known that lectins can bind to both glycoproteins and glycolipids in membranes (Grant and Peters, 1984). Thylakoids contain no glycoproteins but a high percentage of galactolipids (Webb and Green, 1991). Therefore, interactions between this membrane and galactose-specific lectins constitute an experimental system in which the nature of the binding sites is clearly defined. We have used the release of plastocyanin from thylakoids after a three-hour-incubation at -20°C in the presence of an artificial stroma medium as an indicator for freeze-thaw damage. Of the seven lectins we investigated, three had no measurable effect on plastocyanin release up to a protein concentration of 250 [ig m P ^ Since the four protective lectins all showed a linear dependence of protection on protein concentration over this concentration range, the slopes of these lines could be used as a measure of the relative cryoprotective efficiency of the different lectins (Table 1). It can be seen that the efficiency with which the different lectins protected thylakoids varied considerably. In all cases, protection could be inhibited by the presence of up to 5 mM galactose during freezing and thawing. This indicates that binding of the protein to the galactolipid headgroups is necessary for cryoprotection. When we measured time-dependent plastocyanin release from thylakoids in the absence and presence of one of the most effective cryoprotective lectins, the Abrus precatorius agglutinin (Table 1), we found that the protein only reduced the slow phase of damage (Hincha et al., 1993b) (compare Figure 3). There was no reduction in the plastocyanin released during the first 30 minutes of freezing. The reduction in the slow, linearly time-dependent release was evident both at -20°C and at 0°C. As discussed above, this finding indicates an effect of the lectin on solute loading and therefore on the solute permeability of the thylakoid membrane. Using ^"^C-glucose as a tracer we found that the A. precatorius agglutinin reduced the permeability of the membranes by approximately 60% at a concentration of 200 |ig m P ^ As in the case of cryoprotection, the effect was a linear function of protein concentration and could be inhibited by the addition of 5 mM galactose to the incubation solution. Although membrane binding was clearly necessary, it was not sufficient for cryoprotection. The Ricinus communis agglutinin, for example, showed no cryoprotective activity but effectively agglutinated thylakoids (Table 1). Also, binding to a specific class of galactolipids was not the decisive factor, since all investigated lectins only bound to DGDG and not to MGDG when the galactolipids were separately reconstituted into phospholipid vesicles at a concentration of 20 wt% (Hincha etal., 1993b).


165

Table 1. Comparison of the Cryoprotective Efficiency of Different Galactose-Specific Lectings and Their Ability to Agglutinate Isolated Thylakoids Lectin

Relative efficiency^

Agglutination^

Abrus precatorius agglutinin

0.219

toxin A

0.108

toxin B

0.000

Bandeiraea simplicifolia

0.036

Madura

0.131

pomifera

+ + -

Ricinus communis agglutinin

0.000

toxin

0.228

Notes:

+ -

^The relative cryoprotective efficiency of the different lectins was determined in freeze-thaw experiments with isolated spinach thylakoids. The membrane vesicles were incubated for 3 hours at -20°C in the presence of an artificial stroma medium and lectins at concentrations between 0 and 250 M-g/ml. After thawing, the release of plastocyanin was determined as a measure of freeze-thaw damage. All lectins showed a linear dependence of cryoprotection on protein concentration. The slopes of these lines were therefore used to compare the cryoprotective efficacy of the different lectins (see Hincha et al., 1993b). "Thylakoids were incubated in an artificial stroma medium in the presence of 200 fig/ml of the different lectins at 0°C for 2 hours. Samples were inspected visually for agglutination relative to samples incubated under the same conditions in the absence of lectins.

It had been shown in previous studies with a variety of plant lectins that they all possess, to a different extent, hydrophobic domains that are accessible to watersoluble fluorescent dyes (Roberts and Goldstein, 1982; 1983; Loganathan et al., 1992). Toluidinylnaphtalenesulfonic acid (TNS) is one of the dyes that can be used to quantitate the hydrophobicity of proteins, as its fluorescence emission increases upon binding to a hydrophobic domain on a protein surface. TNS titration experiments with the lectins listed in Table 1 showed a linear correlation between lectin hydrophobicity and cryoprotective efficiency (Hincha et al., 1993b). From the results described above we propose that the cryoprotective effect of the lectins is mediated by a hydrophobic interaction between the protein and the membrane. Binding of the lectin to a DGDG headgroup is a necessary prerequisite as it probably brings a hydrophobic domain on the protein surface close enough to the hydrophobic core region of the lipid bilayer to make an effective interaction possible. This is depicted schematically in Figure 10. This hydrophobic interaction could influence the physical state of the lipids in a way that results in reduced solute permeability. The available literature on lectinmembrane interactions provides only a few clues on possible effects of lectin binding on the physical properties of the membrane lipids. Most of these studies have been conducted with RCA^Q, the Ricinus toxin. RCA^Q is a cytotoxin made up of an A and a B chain covalently linked by a cystine bridge. The B chain contains the

166


Aqueous Phase

Figure 10. Schematic representation of the putative action of a cryoprotective lectin. The protein Is bound to the headgroup of a digalactolipid. Hydrophobic sites on the protein surface interact with the hydrophobic core region of the membrane. This is thought to lead to the observed reduction in membrane solute permeability (see text for details). Note that the different components are not drawn to scale.

sugar binding sites while the A chain has an enzymatic activity that very efficiently and irreversibly inactivates eucaryotic ribosomes (Etzler, 1985; Montfort et al., 1987). It has been shown that the uptake of A chain into mammalian cells depends on the binding of B chain to the cell membrane (Houston, 1982). Nevertheless, the isolated A chain partitions into pure phospholipid membranes independently of a sugar binding activity (Utsumi et al., 1984; 1989). The same has also been shown


167

for the lectin concanavalin A (van der Bosch and McConnell, 1975). In both cases, vesicle fusion was observed as a result of the lectin-membrane interaction. For liposomes made from a mixture of phosphatidylcholine and galactocerebrosides it has been shown that RCA^Q binds to such membranes (Utsumi et al., 1987) and that this leads to an increased acyl chain ordering of the lipids (Picquart et al., 1989). It seems possible that the reduced solute permeability we observed in thylakoids in the presence of the Abrus agglutinin (Hincha et al., 1993b) could be the result of similar changes in the state of the membrane lipids. These proposed changes in the lipid phase of thylakoid membranes are the subject of current experiments in our laboratory. Another open question is whether cryoprotection of cellular membranes is one of the functions that lectins have in plants. It is obvious that the seed lectins we used in the experiments described above (Table 1) cannot be involved in the freezing tolerance of chloroplasts in leaves. Information on plant leaf lectins, however, is scarce in the literature, and no such lectins are commercially available. Recent investigations with lectins isolated from the leaves of mistletoe {Yiscum album; kindly provided by Dr. K. Pfiiller) indicate that these proteins also have cryoprotective properties for thylakoids (unpublished results). Mistletoe lectins show sequence similarity to RCA50 (Dietrich et al., 1992) and are galactose specific (Lee et al., 1992). Unfortunately, it is not known whether these lectins are present in the chloroplasts of mistletoe leaves. There is no reason to assume that the cryoprotection of membranes by lectins must be confined to thylakoids. Most membranes in plant cells, such as the chloroplast envelope (Block et al., 1983), tonoplast (Haschke et al., 1990), and plasma membrane (Lynch and Steponkus, 1987) contain glycolipids. The same is true for most membranes in the cells of animals and microorganisms (Quinn, 1982; Kates, 1990). Interactions of the approriate lectins with these membranes are possible, and there is no reason at present to assume that they could not lead to the same changes in the physical properties of the membranes that we have observed in thylakoids. This would open a new field of study on a novel class of highly specific cryoprotectants for a variety of natural and possibly also artificial membranes. Cryoprotectins

The existence of proteins that can protect a biological membrane against freezethaw damage was first reported by Heber and Kempfle (1970). They isolated a protein fraction from cold-acclimated spinach and cabbage leaves that prevented the inactivation of cycUc photophosphorylation in spinach thylakoid membranes during a freeze-thaw cycle to -25°C. These results were later corroborated and extended by the same group (Volger and Heber, 1975). A similar activity has also been reported from the leaves of Nothofagus dombeyi (Rosas et al., 1986). As discussed above, photophosphorylation is a biochemical activity that requires an unimpaired functioning of several membrane components. It is there-

168

D.K. HINCHA, F. SIEG, r. BAKALTCHEVA, H. KOTH and j . M . SCHMrTT

O^C I -20^C - + - Cryoprotectin

m

1 cm

Figure 11. Volumetric assay for cryoprotective proteins. Thylakoids were incubated in the presence of 2.5 mM NaCi, 5 m M sucrose, and where indicated cryoprotective protein from cold-acclimated cabbage leaves, for three hours at either -20°C or 0°C. After thawing, hematocrit capillaries were filled with the respective thylakoid suspensions and sealed with plastic caps at the lower end. The capillaries were then centrifuged and the differences in pellet height (packed thylakoid volume; see Figure 2) can be used to quantitate the cryoprotective effect of a protein fraction. The 0°C control corresponds to 100% protection, the -20°C control without added protein to 0% protection. The cryoprotection afforded by the protein assayed was close to 100%.

fore not possible to determine from such data w^hether inactivation and protection during freezing in vitro take place at membrane sites that are relevant for freezethaw damage in vivo. Since we had shown that the release of plastocyanin from


169

thylakoids occurs during freezing and thawing both in vitro and in leaves, we have used this marker to evaluate the activity of possible cryoprotective proteins. These experiments showed that a protein fraction partially purified by the method of Heber and Kempfle (1970) from the leaves of cold-acclimated cabbage plants was highly effective in reducing plastocyanin release from thylakoids isolated from non-acchmated spinach (Hincha et al., 1989b). Unfortunately, these immunological assays for plastocyanin release are very time consuming. In order to facilitate the purification of cryoprotective proteins by chromatographic methods, where large numbers of samples have to be tested after fractionation, we have developed a volumetric assay (Hincha and Schmitt, 1992b). It makes use of the fact that the rupture of thylakoids that leads to the release of plastocyanin also results in a collapse of the membrane vesicles. This can be detected as a reduced packed volume after hematocrit centrifugation (Figure 11), when unfrozen control samples are compared to frozen-thawed samples. The presence of cryoprotective proteins leads to a preservation of packed thylakoid volume after freezing (Figure 11). When the two assay systems, plastocyanin release and hematocrit centrifugation, were compared directly using dilution series of a cryoprotective protein fraction, it was found that in both cases the measured cryoprotection was a linear function of protein concentration. Also, the protection values were linearly correlated with each other, indicating that plastocyanin release and hematocrit assays can be used interchangeably (Hincha and Schmitt, 1992b). We therefore now mostly use the volumetric method, since it is much faster and cheaper than the immunological assay. Since thylakoids can be protected from freeze-thaw damage by many other substances besides proteins, one of our first objectives was to make sure that the activity we measured was indeed based on a protein. We therefore subjected a crude cryoprotectin fraction from cabbage leaves to tryptic digestion and found that cryoprotection was abolished (Hincha et al., 1990). Protection was, however, not an unspecific effect of the presence of protein in the samples during freezing. Bovine serum albumin (BSA) only provided a very low degree of protection (typically around 10%) even at much higher concentrations than those used for the cryoprotectins (Hincha et al., 1990). In addition, protein fractions isolated by the same procedure (see below) from non-acclimated spinach and cabbage showed no protection beyond that afforded by BSA at the same concentrations. This also indicates that cryoprotectins are cold-inducible, at least at the level of cryoprotective activity. Whether they are also inducible at the protein and mRNA concentration levels remains to be shown. Cryoprotectins act on thylakoid membranes in a highly specific way. Our first estimates from crude preparations indicated that they were at least 20,000-fold more effective than sucrose when compared on a molar basis (Hincha et al., 1989b). Calculations from our most highly purified samples (see below) showed that cryoprotectins are about 10^-fold more effective than sucrose, and 1000-fold

170

D.K. HINCHA, F. SIEG, I. BAKALTCHEVA, H. KOTH and j.M. SCHMITT

more effective than the Abrus precatorius agglutinin, one of the most efficient cryoprotective lectins (Table 1). This rules out a non-specific, colligative mode of action. We found that our earlier, crude preparations of cabbage cryoprotectins reduced solute loading of thylakoids during freezing and also increased the extensibility of the membranes after thawing to an extent that made the volumetric behavior of thylakoids in a Boyle-van't Hoff plot (compare Figure 2) indistinguishable between unfrozen controls and samples frozen for three hours at -20°C (Hincha et al., 1990). These results were corroborated by experiments with thylakoids suspended in an artificial stroma medium. Plastocyanin release was reduced both during the first, rapid phase of damage and during the slow, time-dependent phase (compare Figure 3). The slow release of plastocyanin in unfrozen samples at 0°C, however, was not influenced by the presence of cryoprotectins at the concentrations employed in these experiments. This suggests that the higher concentrations achieved by freeze-induced dehydration are necessary to observe effects on plastocyanin release in this experimental system. Since apparently one of the functions of cryoprotectins was to reduce solute loading of thylakoids during freezing, presumably by reducing the solute permeability of the membranes, and the same had also been found for cry oprotective lectins (Hincha et al., 1993b), we were interested to see whether cryoprotectins also act by a sugar-binding mechanism. Our experiments have shown that cryoprotectins are strongly inhibited in the presence of free galactose, but not in the presence of glucose at the same concentrations (unpublished results). This makes a protective mechanism mediated by a binding of the proteins to galactolipid headgroups very likely. Further experiments with lectins and cryoprotectins will have to show how similar their mode of action on the membranes is. Cryoprotectins also have some features in common with the COR and LEA proteins previously discussed. All of these proteins are very hydrophilic, water soluble molecules and most strikingly are not precipitated by boiling (Heber and Kempfle, 1970; Jacobsen and Shaw, 1989; Lin et al., 1990; Ried and Walker-Simmons, 1990; Hincha and Schmitt, 1992b; Neven et al., 1993; Rao et al., 1993). Because no sequence information is available for the cryoprotective proteins, it is unclear whether this common property is due to common structural features. Since no specific activity could be assigned to any of the COR or LEA proteins, it is also not clear whether or not these proteins are inactivated by boiling. For the cryoprotectins, on the other hand, it has been shown that their cryoprotective activity was completely unimpaired by a 10-minute incubation in a boiling water bath (Hincha and Schmitt, 1992b). Solubility during heat treatment was of course an important feature in our attempts to purify cryoprotective proteins, because the major part of the soluble proteins in a leaf extract coagulates and precipitates during boiling. The soluble fraction after boiling still contains a large number of polypeptides, which makes further purification steps necessary. The fact that cryoprotectins remain soluble at pH 4 was used to remove acid-labile contaminants. A further purification and con-


171

120 ^^ 100 o k. c

o

o

80

"O

— O

o

(J) ^

\

100

i

Y^

LU J

10 -i

o o o

4.9

Vw=2.5°C/mln

-4-

Vw=40*C/mln

X

/

-/

\/vf=^0''Clm\n

5.1

5.2 1000/Tg

f

5.3

5.4

5.5

Figure 18, Variations of the glass transition temperature Tg for glycerol as a function of the warming rate V ^ for various cooling rates.

O'Reilly and Hodge, 1991). With the use of Tf, it has been shown that the best conditions for the analysis of Tg are when cooling and heating rates are both equal. For these experimental conditions, the determination of Tg is similar to the determination of the fictive temperature Tf due to a minimal effect of the overshoot at the glass transition on the determination of the intersection of the extrapolated enthalpy of the supercooled liquid and that of the glassy state. Glycerol is a good example for that purpose as shown in Figure 18. The dependence of Tg on cooling and warming rates determines an apparent activation energy used to describe the kinetics of the glass transition within the scheme of the Narayanaswamy-Tool model (Narayanaswamy, 1971; 1988). In Figure 18, minimum values of Tg are observed for the condition: cooling rate = heating rate. The glass configuration achieved during cooling will relax weakly during warming at the same rate. If the cooling rate is slower, the configuration will be more stable and the overheating of the glass will result in larger overshoots and higher values of Tg. Slower warming rates allow the glass to relax before the glass transition and also results in higher Tg values. The Narayanaswamy-Tool method is, however, too complex and needs various fitting processes to the thermal curves to allow calculation of the various parameters (Moynihan et al., 1974; 1976; 1993; Rekhson, 1987; 1989; Scherer, 1988; Hodge, 1991; 1994; O'Reilly and Hodge, 1991). Another fundamental approach has been developed by using the percolation theory to follow the packing of amorphous clusters above a threshold defining the

Crystallization and Vitrification in Aqueous Glass-Forming Solutions

215

glass transition (Cyrot, 1980; Chen, 1981) in order to study the spectrum of the relaxation times that describe the glass transition in order to explain the VogelFulcher-Tamman behavior of the highly supercooled liquid and glassy state. This approach is also too complex for a rapid estimate of the behavior of aqueous glassforming solutions. With the approximation that the heat capacity of the glass below Tg is constant or varies smoothly, a simpler model has been developed to fit the glass transition thermal curve (Hicter and Desre, 1983). The variation of enthalpy during warming is decomposed as a continuous transition from liquid to glass with a complementary energetic term as a concept of glass cluster formation: — - r^ + Y(r^ -r^)+o^^^^ o

1

dT-

^P'-^^^P

^P^^^dT/dt

[91

^^^

where Cp and Cp are respectively the specific heat of the glass and the Uquid, X is the fraction of liquid within the glassy state and Q is an energy describing the sample thermal history. Usually a first-order reaction can be chosen for the kinetic constant with: dX = K(l-X) dt

[10]

with K = K^ exp o

[11]

The VFT dependence of K can be approximated to an Arrhenius dependence to limit the number of parameters. Resolution of the differential equation then includes an exponential integral, which can be solved numerically. This model has been successfully applied to the glass transition of 60% (w/w) ethylene glycol in water without annealing below the glass transition (Figure 19). The model did not, however, yield a perfect fit with glass transition after annealing, and still needs to be extended to these more complex conditions. An indirect method has been developed to enable quantification of ice nucleation during storage of aqueous, glass-forming solutions close to or below Tg (Mehl, 1993b,c,g; 1995c,d,e; Mehl and Shi, 1994; 1995). The glass transition is described more rigorously by the variation of the relaxation times. These relaxation times from the a-relaxation are theoretically related to the self- and mutualdiffusion constants of water molecules in the glassy matrix. Annealing experiments have been monitored in calorimetry and cryomicroscopy studies to estimate variations of excess enthalpy with temperature and to correlate it with eventual ice nucleus formation (Mehl, 1993b,g). Annealing at various temperatures T^ during periods t^ are performed after cooling. The effects of t^ and T^ are analyzed by cal-

PATRICK M. MEHL

216

O

o i

'150

-140

-130

-120

-no

TEMPERATURE ( X ) Figure 19. Reconstruction of the glass transition thermal curve for 60% (w/w) ethylene glycol in water using the Hicter and Desre (1983) model for the glass transition. The variation of the measured specific heat ( ^ ) is best fitted with the calculated ( r ) C° which is the given by C° = Cp+Q[(dX/dt)/(dT/dt)] where the real specific heat of the sample is corrected with a term including an energy Q reflecting the thermal history of the sample and X being the supercooled liquid fraction defined by Cp=Cjf+X(Cpl-Cp); the notations are reported in the text.

culating the excess enthalpy recovery Hexcess(Ta'ta) during anneahng (Mehl, 1993b; 1995d). This recovery is then analyzed using the non-exponential Kohlraush-Williams-Watts function or stretched relaxation function (Hunt, 1993; 1994): HT^;t^)

= exp[-[r/T]P]

[12]

with T =

T^ e x p [ - A / r j

[13]

assuming that the relaxation time x is Arrhenius type as a first approximation. This relaxation time T is in fact an apparent relaxation time function of the distribution of glassy state relaxation times at the corresponding temperature (Ngai and Wright, 1988; Mehl, 1995d). The exponent p represents the nonlinearity of the


217

response of the glassy state and is a measure of the width of the relaxation time distribution of the glassy state at T^. The Kohlraush-Williams-Watts relaxation function or stretched exponential relaxation function is the result of the frequency response of the glassy state at the annealing temperature. Mathematically, this function is the Laplace transform of the frequency response function of the glassy state. The function describing the relaxation of an intensive thermodynamic function such as the enthalpy is the ratio: ,.j. .f \ _ excess^ a'a^~ excess^ a' ^^ [14] ^^ a^ J - CH (T ',oo)-H (T ;0)) ^ excess^ a' ^ excess^ a' ^^ The maximum Hexcess(Ta»'^) ^^s been first assumed as the difference between the continuation of the variation of the enthalpy of the supercooled liquid below the glass transition and the enthalpy of the glassy state (Mehl, 1993b; 1995d). For more precise calculations, the maximum excess enthalpies have been experimentally determined for accessible experimental times with annealing close to the glass transition temperature where the relaxation times of the glassy state are less than 100 minutes in comparison to annealing periods of 1000 minutes (Mehl, 1993b). It has been shown that the linear extrapolation of the supercooled liquid enthalpy below the glass transition is at the limit of the experimental calculations. Therefore, experimental determination of the maximum excess enthalpy is needed for a more precise determination of the kinetics of the relaxation below the glass transition temperature (Mehl, 1995d). The recent theory linking the glass transition T^(W^) to the cooling and warming rate V^, enables determination of the lowest glass transition temperature T^ with the relation (Shi, 1994; Mehl and Shi, 1994; 1995): Ln[VJ = - — — ^ — — + Ln[C] [15] ^ T (V ) — T g^ c^ s where T^ is determined with a best fit. The effect of thermal conduction has been shown to be negligible (Mehl, 1995d). Analysis and determination of the different parameters for the function (^ are performed using a simple transformation of Eq. [12] and using linear regression and least square regression techniques to fit the data (Mehl, 1995d). T^ defines the two thermal domains where the maximum excess enthalpy can vary and reaches its utmost value (Mehl and Shi, 1994). The maximum excess enthalpy as a function of the temperature allows for calculation of the remaining parameters within the Kohlraush-Williams-Watts model. Usually the variation of the specific heat at the glass transition is considered to be a hyperbolic function of temperature to recover the VTF law using the Gibbs expression for the relaxation times (Angell, 1988a,b; 1991a,b; Hodge, 1991; 1994). However, it has been recently discussed that this variation is described closely between an

218

PATRICK M. MEHL

hyperbolic function and a constant function of the temperature (Hodge, 1994). In the present model, the specific heat of the glass is assumed constant (Mehl, 1993b,c,g; 1995c,d,e; Mehl and Shi, 1994; 1995). The difference between the two variations results in a variation of a few percent in the determination by the best fit of the maximum excess enthalpy at T^. Determination of all the parameters allows for the analysis of the kinetics of the enthalpy relaxation in the glassy state. Knowledge of the relaxation time spectrum with temperature will characterize completely the relaxation of the glassy state and its configurational state with time and temperature with the condition of knowing the thermal history of the sample. Reconstruction of the direct thermal curve will then be related to the inverse problem using the experimental relaxation times to pass to the determination of the enthalpy of the glassy state as a function of the temperature with x(T) -> H(T). Knowledge of the characteristics of the glassy state will permit one to differentiate between the possibility of ice cluster formation and phase separation below the glass transition in vitrified aqueous solutions. Fractures in the Glassy State Determination of the fracture thermal domain is difficult due to its dependence on the sample geometry and on the intrinsic nature of the glassy state. Fracturing of vitrified samples has been observed in many various systems such as vitrified embryos (Rail and Meyer, 1989). These fractures have been shown to be related to the decrease in survival in vitrified cells suspensions. The fractures lead to mechanical damage that cannot be healed by the biological system after rewarming. Glass fractures have initially been studied by observing the healing process of cracks at the glass transition (Kroener and Luyet, 1966). This healing has been observed as related to the increase of diffusion at the glass transition (Mehl, 1989; 1990). The healing is not complete for aqueous solutions because ice nucleation is induced by the heat released at the tip of the fracture during cracking. Therefore, ice nucleation and ice crystal growth with the nucleation of gas bubbles can be observed during warming if the cracks are sufficiently large (Mehl, 1990; Williams and Camahan, 1990; Williams et al., 1990). Microscopic gas bubbles have also been observed to coalesce during warming depending on the viscosity of the liquid (Mehl, personal observations). Even if sufficiently rapid warming rates limit crystal growth, crack healing will hardly preserve the cellular organization of vitrified organs especially if interstitial ice growth occurs. At temperatures below Tg, redissolution of ice nuclei is highly improbable as tested experimentally with fracture healing (Mehl, unpublished results). Therefore, fractures formation must be avoided. The formation of a fracture is regarded as intrinsic to the nature of the glassy state. The definition of T^ for the glass as a parameter that is independent of the cooling/warming rate leads to the definition of a maximum of excess enthalpy that can be stored by the glass during cooling (Mehl and Shi, 1994). These values are


219

obviously dependent on the initial cooling rate and are physically important for characterization of the glass relaxation. Relaxation below the temperature T^ will theoretically be directed towards the same configurational state presently defined as the ideal glass. Between Tg and T^, the experimental dependence of the maximum excess enthalpy recovery during annealing as a function of the annealing temperature supports the hypothesis that the glassy state will relax towards the supercooled liquid state (Mehl, 1993b,c,g; 1995c,d,e; Mehl and Shi, 1994; 1995) and not towards the crystalline state as described in classical models. This conclusion supports the model of defect diffusion (Perez, 1994). This relaxation process is related to the abihty of the glass to overcome thermal stresses within it. The creation of a fracture is similar to a thermally activated process such as bond breaking, and it has been suggested that the activation energy for fracture formation is decreased by a factor proportional to the thermal stress applied to the glass (Curran et al., 1987). Therefore, the dissipation of thermal stresses will be associated with the relaxation of the local energy through the elastic energies stored in the intermolecular bonds. Therefore, the enthalpy relaxation time of the glass as a function of the temperature is of great importance for characterizing the glassy state. The dynamics of the nucleation and growth of fractures in materials has been reviewed within different theories (Curran et al., 1987). All define a threshold stress that is proportional to the ratio of the temperature to the bond breaking strain (Tatsumisago et al., 1990). The glass strength has been assumed to be related to a fragility factor provided by the ratio of the activation energy from the shear viscosity to the glass transition temperature. The activation energy represents the energy of breaking bonds at the glass transition. This ratio is the inverse of that which defines the threshold stress for the nucleation of fractures. A higher ratio leads to an Arrhenius-like representation and a lower value to a Vogel-FulcherTamman-like representation (Angell, 1988a,b; 1991a,b) with a higher fragility for the VFT- like liquids. Angell has shown a correlation between the variation of the specific heat at the glass transition and the strength of the liquids (Angell, 1988a,b). However, other authors disagree with this definition (Murthy, 1989a,b; Sokolov et al., 1993). The notion of strong/fragile glasses, as for the notion of strong/fragile liquids introduced by Angell, is characterized by the strength of the bridging network between molecules. Both arguments from the liquid side of the glass transitions provide ambiguous views of the strength of glasses compared to the definition of the thermal activation theory of the nucleation of fractures (Murthy, 1989; Angell, 1991a,b; Sokolov et al., 1993). For the binary system of propylene glycol/D20, the definition of the strength of the glassy state as presented by Angell is at least related to the ability of the glassy state to store a maximum energy. The ratio of the apparent activation energy to the temperature can then be calculated. The dependence of T^ and its activation energy D on the solute concentration is determined. A similar fragility coefficient F to that from the shear viscosity is provided by:

220

PATRICK M. MEHL

[16]

F = -D/T,

The curve of the optimal T^ is shown in Figure 20 as a function of the propylene glycol concentration using polynomial regression methods for the bestfit.The maximum of the maximum excess enthalpies are deduced from the values of T^ and the variation of the maximum excess enthalpy with the annealing temperature T^ (Mehl, 1995d). This last variation has been approximated to a linear function such as: H::L.iTJ=CT^H^ excess^

a^

a

o

[17]

This equation is similar to an approximate representation of the difference of enthalpy between the glassy state after cooling at V^ and the ideal glassy state as: H^'^'^'iT^) - H ' ^ - ' ^ ' « - ( r ^ ) = ^S(c;«'^'')[r^ - T^]

[18]

where g is a factor taking into account the overshoot at the glass transition and 5CjJ^^'^^ being the variation of the specific heat at the glass transition Tg and assuming that this variation is constant over the thermal range between Tg and T^ (Mehl and Shi, 1994). The values of the fragility F are shown in Figure 21 as a function of propylene glycol concentration. The maximum of these values is observed for a molar ratio of propylene glycol: D2O that is close to 1:1. Assuming that the activation energy is proportional to the mean bond-breaking energy at the glass transition, the threshold stress that can be applied to the glass before fractures are nucleated is proportional to the inverse of F and to the molar number of bonds (Tatsumisago et al., 1990). The maximum of F then corresponds to the minimum in the stress threshold. The decrease of the glass transition temperature Tg from the pure solute to the 1:1 molar corresponds also to a plasticization of the glassy state of the pure solute by the water molecules. Therefore, the glassy state is destabilized as water is added to the solute. Therefore, the 1:1 molar ratio apparently corresponds to a maximum weakening of the glassy state by the water molecules. A similar maximum weakening has been reported for dilute aqueous solutions that are hyperquenched (Hallbrucker and Mayer, 1989). Therefore, the definition of F is apparently in agreement with the theory of the fracture nucleation theory and can be used for the definition of the strong/fragile character of the glasses. The thermal stresses are related to the energy stored and the thermal gradient seen by the glassy state. The formation of a fracture is a kinetic process resulting from the inability of the glass structure to release the stress energy through relaxation with a volume and temperature recovery. For the maximum value of F, the relaxation times are observed to be maximum as for concentrations close to 1:1 for a chosen temperature (Mehl, 1995d). This supports the definition of the fragility coefficient as a good parameter to characterize the fragility of glasses. The variations of the


221 68

200

67 66

5"

•^•^

65 O) H 64 LU 63 62

a:

2

LU 61 Q. LU 60 h59 58

35

45

55

65

75

85

95

PROPYLENE GLYCOL CONCENTRATION (%W/W)

Figure 20. Variation of the optimal glass transition temperature Tg as a function of the solute concentration for the binary system 1,2-propanediol/D20. The calculated value determined from experimental points are connected with a polynomial regression curve corresponding to a second-order regression.

50

60

70

80

90

100

PROPYLENE GLYCOL CONCENTRATION %mol/mol

Figure 21. Fragility values F = S/T^ calculated from optimal values \ and from activation energies S determined simultaneously with Tg for the system 1,2propanedlol/D20.

222

PATRICK M. MEHL

enthalpy relaxation times establish the fragile or strong nature of the glassy state (Mehl, 1995a). Thermal stresses in the glassy state are dependent on the geometrical design of the sample. They are functions of the thermal conductivity of the glass and the associated mechanical response. Thermal stress calculations need the support of algorithms using finite element theory for complex geometry. This geometric approach to the problem has been initiated, but is without definitive answers (Fahy et al., 1990). Knowledge of thermal conductivities and their temperature dependence close to the glass transition are needed, but are generally missing. More basic studies are still needed for understanding fracture kinetics in vitrified samples. The temperature at which visible fractures are created is weakly dependent on the cooling rate between 2 and 40°C/min for thin films of glycerol (Mehl, personal observations). Experimental Studies of the Glass Transition

The method of enthalpy relaxation uses calorimetric annealing experiments where formation of fractures is avoided. Using the Kohlraush-Williams-Watts model, the parameters can be determined successively by various plots (Mehl, 1993b). The effect of annealing is shown in Figure 22 for 89% (w/w) 1,2-propanediol in D2O. The glass transition overshoot provides a measure of the excess enthalpy of the glass that is released during warming. The results of various measures of excess enthalpies are shown in Figure 23 for various values of T^ and t^. The dependence of Tg on the cooling and warming rates is shown in Figure 24 with the determination of T^, the apparent activation energy and its constant. T^ defines the isoconfigurational thermal domain T < T^ where the maximum excess enthalpy remains constant. Calculations of the maximum of excess enthalpy allow for determination of the enthalpy relaxation function (j) shown in Figure 25. Linear regressions enable the calculation of enthalpy relaxation times x and the non-linearity exponent p. A comparison of these enthalpy relaxation times T with published results is presented in Figure 26 and with the exponent P in Figure 27 (Birge and Nagel, 1985; Birge, 1986). A good accord is observed with the general behavior of the relaxation times and also with the behavior of the non-linear exponent p (Ngai and Wright, 1988). Similar results are observed for propylene glycol and glycerol when a temperature-jump is used for determination of the enthalpy relaxation in calorimetry (Fujimori et al., 1992; 1995; Fujimori and Oguni, 1994). The results support the existence of an intermediate state between the glassy state below Tg and the liquid similar to the rubbery state in polymer solutions. The definition of relaxation times allows the inverse reconstruction of the glass transition thermal curve with the relation: dHif)=^"excess(T)^^^/^^^^''dt

,

[19]


223

where [20]

excess^ ^

Application of these equations is expected to present a good fit of experimental data. Effect of Other Physical Parameters Similar to the definition of Tf (Narayanaswami, 1977; 1988), Gupta presented the notion of afictivepressure for the glassy state from previous studies (Davis and Jones, 1953; Gupta, 1988). This notion emphasizes the degrees of freedom of the glass. Indeed, the configurational state of the glassy state requires more than the fictive temperature to be thermodynamically defined. In the thermodynamic description of the glass, the nature of the glass transition is the kinetic result of the freezing-in of the configurational states of the supercooled liquid during cooling (Jackie, 1986; Aagren, 1988). The excess enthalpy and entropy between the supercooled liquid and the equilibrium crystal are then progressively frozen in place

R9%>W/W L2.PROPANEDIOL

IN

D20

THERMAL CURVES DURING WARMING AT lO'C/min AFTER ANNEAUNG AT DIFFERENT TEMPERATURES DURING 40 MIN COOUNG RATE '^ 40*C/miM

no

.100

-90

SCANNING TEMPERATURE C O

Figure 22, Thermal curves showing the glass transition during warming (1 C'C/min) of a sample of 89% (w/w) 1,2-propanediol in D2O after annealing at various temperatures for 1 hour.

PATRICK M . MEHL

224

-109.3X O). i

o

1

(0

1

^ r

^;^

-m.rc

^^'^^NaCi^^^^HCi^^^^NaOH- This means that ice crystal growth is more rapid for NaCl than for HCl and more rapid than for NaOH even if ice nucleation is higher for HCl solvent than for NaCl. Therefore, this result suggests a real effect of the presence of OH" and H30'^ ions on ice crystal growth. A partial substitution of a neutral salt by its base will strongly increase the glassforming tendency by limiting crystal growth and nucleation. Even a partial substitution of the neutral salt by an acid form will slightly increase the glass-forming tendency by limiting crystal growth only. This is an important result for the design of the carrier solutions for the vitrification technique. This is especially important for lowering the critical warming rates, which are strongly dependent on the ice crystal growth rates for the purpose of cryopreservation of organs (Mehl, 1993e). Practical Applications of Vitrification Solutions Definitions of Critical Cooling and Warming Rates

The vitrification technique must be supported by the definition of the concentration range of the doubly unstable domain (Angell et al., 1981; Fahy et al., 1984). The kinetics of ice crystallization then must be defined especially for characterization of crystal growth, which is the critical step in the calculation of the critical warming rates (Mehl, 1993e). Calculation of the isothermal parameters allows for determination of the critical cooling and warming rates (Mehl, 1993e) with a supplementary condition of non-coalescence of the crystals to be included. This condition of precoalescent stages for crystal growth has been investigated by Shi and Seinfeld (1994). It has been previously discussed that the number of ice crystals within the sample is not as critical as the size of the ice crystals (Mazur, 1984; Takahashi et al., 1986; Mehl, 1993e). Therefore, the method used for the determination of the critical rates was first to measure the nucleus density or nucleation rate and then to deduce from calorimetry the growth rates within the sample. Deduction of the size of the crystal during cooling and during warming was then possible with a correlation with direct observation of the growth rate by cryomicroscopy.


243

For practical vitrification solutions, ice nucleation may occur during cooling through the heterogeneous process as crystal growth is avoided for having a thermal range lower than that of the homogeneous nucleation. However, for calorimetric samples, the smaller volume will induce a lower probability for an heterogeneous event than for large volume (Fahy et al., 1990). Calorimetric measurements are essential for determination of crystal growth when homogeneous nucleation is predominant over heterogeneous nucleation. It is, however, limited under conditions of heterogeneous ice nucleation in highly concentrated solutions such as vitrification solutions. Therefore, measuring the amount of ice is not sufficient for understanding the kinetics of ice crystallization. TTT-curves can be determined as a first step for the determination of the critical cooling rates (MacFarlane, 1982;MacFarlaneetal., 1983a,b; Sutton, 1989; 1991a,b; 1992). However, the comparison between the TTT-curves determined during isothermal annealing and similar TTT-curves constructed from constant cooling or heating rate conditions present different patterns. This is due to the non-linearity of ice nucleation and the existence of an induction time for nucleation, which is dependent on temperature and the nature of the heterogeneous loci for nucleation (Khamskii, 1969). The nose method of TTT-curves will therefore provide larger critical cooling rates than required. However, the more difficult step for preservation of biological material by vitrification is the rewarming. During the initial cooling, stable and unstable ice nuclei form. During storage, stabilization of the unstable ice nuclei is possible. The critical warming rates are therefore determined by the ice crystal growth rates (Mehl, 1993e). These rates can be calculated from calorimetry with the knowledge of the ice nuclei density within the samples. All these calculations are, however, limited by the assumption of very limited heterogeneous ice nucleation that must occur at a lower thermal range than that of the ice crystal growth. This is usually the case for small dilute samples but not for large volume samples (Mehl, 1989; 1990; 1991; Fahy et al., 1990). Critical cooling rates can be calculated directly from the crystal growth rate U(T) determined by cryomicroscopy using the direct integral providing the radius of the ice crystals with the cooling rate Vc (Vc90% of the eggs in 5 min with 5 minutes) after the loading step.

Cryopreservation of Drosophila Melanogaster

311

5. Dehydration in Vitrification Solution Aspirate off most of loading solution; blot off any excess with a Kimwipe. Add 300 jiil of cold (0°C) vitrification solution (42 wt% ethylene glycol + 6.125 wt% BD20 solutes) and place the tube in ice for 10 minutes. Well-permeabilized eggs will initially float then sink as they dehydrate. The proportion of eggs that sink and the rate at which they sink indicate the efficacy of permeabilization. 6. Quenching in Nitrogen Slush (Vitrification) Nitrogen slush is prepared by placing a small container (500 ml) of liquid nitrogen under a vacuum; under a vacuum, the liquid nitrogen will freeze and form a slush. Start the vacuum pump at a time so that formation of slush coincides with the end of the dehydration time; allow at least 30 seconds for the slush to form. Before preparation of the nitrogen slush, place two 60 mm petri dishes on ice; one is initially empty, the other contains 100 mesh copper EM grids (available from Electron Microscopy Sciences, catalog number G-IOO-CU). When nitrogen slush starts to form, wipe any condensation off the empty petri dish with a Kimwipe and place drops of eggs in the vitrification solution ('-5 |il) on the dish using a 0.25-cc polypropylene straw and syringe. After the slush is formed, pick up a copper grid with chilled forceps and dip it into the dehydrating solution, then scoop the grid through and under the drop of the vitrification solution containing the eggs. Rapidly plunge the grid and forceps into the nitrogen slush, drawing the grid through the slush; release the grid. If done properly, the eggs will remain on the grid, and the grid will sink in the nitrogen slush. Continue placing the remaining grids in the slush; this takes about 1.5 minutes for 9 grids. Leave grids in the nitrogen slush for 5 minutes during which time the slush will melt to liquid nitrogen after which the grids can be transferred to cryogenic vials for long-term storage in a liquid nitrogen dewar. 7. Warming and Removal of Ethylene Glycol Remove the grids containing the vitrified embryos from liquid nitrogen with forceps and drop them into 2 ml of a dilution solution containing 1.0 M sucrose in BD20 at '-21.5°C. Vortex the tube after the addition of each grid. After 2 minutes, pour the eggs (and grids) into a permeabilization basket; discard the solution. Place the basket in a 60 mm petri dish filled with BD20. After 8 minutes, transfer basket to a second petri dish containing BD20. After 10 minutes, empty the eggs from the basket into the same petri dish, and transfer the eggs to a test tube containing 2 ml BD20. 8. Culture of Eggs After dilution of the vitrification solution and unloading of the ethylene glycol, the cryopreserved eggs are placed in watch glasses (50 mm dia) and covered with a light, mineral oil (Fisher 0121-1). Operationally, transfer -'100 to 200 eggs to the watch glasses with a straw. For this step in the procedure, it is critical that excess BD20 is removed from the sample before addition of the mineral oil and that the eggs be spread apart rather than being left in clumps—otherwise development and hatching is greatly impaired. For this, a folded Kimwipe

312

P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CrCERO

is used to absorb the excess BD20 before covering the eggs with 1 to 2 ml of mineral oil, after which the eggs are gently spread eggs apart with a camel-hair brush. The watch glass is then placed in a petri dish with wet filter paper and covered; the petri dish is then placed in a covered, plastic box and incubated at 25 °C. Hatching begins '-9 to 10 hours after removal of the eggs from liquid nitrogen and dilution of the ethylene glycol; the final hatching percentage is determined after 2 days. Consistently higher hatching percentages are attained by culturing the eggs in oil, but it is necessary to remove the larvae from the oil as soon as they hatch. 9. Culture of Larvae A few hours before the larvae are to be recovered, prepare modified yeast-glucose food (7 g baker's yeast, 3.5 g glucose, 2.9 g agar in 100 ml H2O + 15 ml of acid mix). Place -'10 ml of the food in shell vials (25 m m x 95 mm) while it is hot. Cool the vials, chop-up the food, and plug the vials with cotton or rayon plugs. Gently recover larvae from the oil with a brush, and place -^20 larvae in each food vial. Incubate at 25°C in chamber with high humidity. Adult eclosion occurs in -'2 weeks. It is critical to transfer the larvae from the oil to the food as soon as they hatch. Prolonged exposure to oil after hatching reduces the number of larvae that will pupariate.

ACKNOWLEDGMENTS Numerous individuals have contributed to the development of the cryopreservation procedure for D. melanogaster embryos and the contributions of Ross J. Maclntyre, Stan Leibo, Ron Pitt, Ta-Te Lin, Dan Lynch, Doug Knipple, Bill Rail, and Viktor Bronshteyn are gratefully acknowledged. Special thanks is given to Cheryl Wisniewski and Anne Stone, who worked on the project for three years during their undergraduate studies. Finally, the interest, support, and encouragement of Dr. Irene Eckstrand (NIH), who together with Dr. DeLill Nasser (NSF), were strong advocates of the need for the development of a cryopreservation procedure for D. melanogaster germ plasm is sincerely acknowledged. This project was supported by grants from the U.S. Department of Health and Human Services, National Institute of General Medical Sciences (Grant No. ROl GM37575) and the National Science Foundation (Grant No. DMB-9009425).

REFERENCES Arking, R., & Parente, A. (1980). Effects of RNA inhibitors on the development of Drosophila embryos permeabilized by a new technique. J. Exp. Zool. 212, 183-194. Auerbach, C. (1959). Spontaneous mutations in dry spores of Neurospora crassa. Z. Vererbungslehre 90, 335-346. Birkina, B.N. (1938). The effect of low temperature on the mutation process in Drosophila melanogaster. Biol. Zh. 7, 653-660. Bronshteyn, V.L., & Steponkus, P.L. (1992). Differential scanning calorimetry studies of ice formation in Drosophila melanogaster embryos. Cryobiology 29, 764-765. Bronshteyn, V.L., & Steponkus, P.L. (1993). Characterization of dehydration of Drosophila melanogaster embryos in vitrification solutions. Cryobiology 30, 617.

Ctyopreservation of Drosophila Melanogaster

313

Bronshteyn, V.L., & Steponkus, RL. (1994). Amino acids and carbohydrates limit permeation of ethylene glycol in Drosophila melanogaster embryos. Cryobiology 31, 569. Charlesworth, B., & Charlesworth, D. (1985). Genetic variation in recombination in Drosophila. I. Response to selection and preliminary genetic analysis. Heredity 54, 71-83. Cicero, M., Caldwell, S., & Steponkus, RL. (1992). Permeabilization of Drosophila melanogaster embryos. Cryobiology 29, 762-763. Cicero, M., & Steponkus, RL. (1993). Permeabilization of Drosophila melanogaster embryos: To dry or not to dry, that is the question. Cryobiology 30, 616. Cowley, C.W., Timson, W.J., & Sawdye, J.A. (1961). Ultra rapid cooling techniques in the freezing of biological materials. Biodynamica 8(170), 317-329. Dowgert, M.F., & Steponkus, RL. (1983). Effect of cold acclimation on intracellular ice formation in isolated protoplasts. Riant Rhysiol. 72, 978-988. Fahy, G.M., MacFarlane, D.R., Angell, C.A., & Meryman, H.T. (1984). Vitrification as an approach to cryopreservation. Cryobiology 21, 407-426. Fahy, G.M., Levy, D.L, & Ali, S.E. (1987). Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions. Cryobiology 24, 196-213. Franks, F , & Bray, M. (1980). Mechanism of ice nucleation in undercooled plant cells. Cryo-Letters 1,221-226. Franks, F, Mathias, S.F., Galfre, P., Webster, S.D., & Brown, D. (1983). Ice nucleation and freezing in undercooled cells. Cryobiology 20, 298-309. Gustafsson, A. (1960). Mutations in agricultural plants. Hereditas 33, 1-100. Houle, D., Kondrashov, A.S., Yampolsky, L.Y., Morikawa, B., Caldwell, S., & Steponkus, RL. (1995). Effect of cryopreservation on the lethal mutation rate in Drosophila melanogaster embryos. Cryobiology 32, 567-568. Hunter, F.R., & DeLuque, O. (1959). Osmotic studies of amphibian eggs, IL Ovarian eggs. Biol. Bull. 468-481. Kerkis, J. (1941). The effect of low temperature on the mutation frequency in D. melanogaster with consideration about the causes of mutations in nature. Drosophila Inform. Serv. 15, 25. Leibo, S.R (1976). Nucleation temperatures of intracellular ice formation in mouse ova. Cryobiology 13, 646. Leibo, S.R (1980). Water permeability and its activation energy of fertilized and unfertilized mouse ova. J. Membr. Biol. 53, 179-188. Leibo, S.R, Mazur, P., & Jackowski, S.L. (1974). Factors affecting survival of mouse embryos during freezing and thawing. Exp. Cell Res. 89, 79-88. Leibo, S.R, Myers, S.R, & Steponkus, RL. (1988). Survival of Drosophila melanogaster embryos cooled to subzero temperatures. Cryobiology 25, 545-546. Leopold, R.A., Nelson, D.R., & Atkinson, RW. (1995). Permeabilization of Muscid and Calliphorid embryos. Cryobiology 32, 579. Limbourg, B., & Zalokar, M. 1978. Permeabilization of Drosophila eggs. Dev. Biol. 35, 382-387. Lin, T.-T, Lynch, D.V., Myers, S.R, Pitt, R.E., & Steponkus, RL. (1987). Volumetric behavior and hydraulic conductivity of Drosophila embryos. Cryobiology 24, 542-543. Lin, T.-T, Pitt, R.E., & Steponkus, RL. (1988). Permeability of Drosophila melanogaster embryos to ethylene glycol and glycerol. Cryobiology 25, 527. Lin, T.-T, Pitt, R.E., & Steponkus, RL. (1989). Osmometric behavior of Drosophila melanogaster embryos. Cryobiology 26, 453-471. Luyet, B.J. (1961). A method for increasing the cooHng rate in refrigeration by immersion in liquid nitrogen or in other boiling baths. Biodynamica 8( 171), 331 -352. Lynch, D.V., Myers, S.R, Leibo, S.R, Macintyre, R.J., & Steponkus, RL. (1988). Permeabilization of Drosophila eggs using isopropanol and hexane. DIS 67, 89-90. Lynch, D.V, Lin, T.-T, Myers, S.R, Leibo, S.R, Macintyre, R.J., Pitt, R.E., & Steponkus, RL. (1989). A two-step method for permeabilization of Drosophila eggs. Cryobiology 26, 445-452.

314

P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO

MacFarlane, D.R., Forsyth, M., & Barton, C.A. (1992). Vitrification and devitrification in cryopreservation. In: Advances in Low-Temperature Biology, Volume 1 (Steponkus, P.L., ed.), pp. 221-278. JAI Press, Ltd., London. Margaritas, L.H., Kafatos, F.C., & Petri, W.H. (1980). The eggshell of Drosophila melanogaster. J. Cell Sci. 43, 1-35. Massip, A., Van Der Zwalmen, P., Scheffen, B., & Ectors, F. (1986). Pregnancies following transfer of cattle embryos preserved by vitrification. Cryo-Letters 7, 270-273. Mathias, S.F., Franks, R, & Trafford, K. (1984). Nucleation and growth of ice in deeply undercooled erythrocytes. Cryobiology 21, 123-130. Mazur, P. (1977). The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology 14,251-272. Mazur, P., Rail, W.F., & Leibo, S.P. (1984). Kinetics of water loss and the likelihood of intracellular freezing in mouse ova. Cell Biophys. 6, 197-213. Mazur, P, Cole, K.W., Schreuders, RD., & Mahowald, A.R (1991). Confirmation of the ability of permeabihzed 12-hr Drosophila embryos to survive cooling to -200°C. Cryobiology 28, 524525. Mazur, P., Schneider, U., & Mahowald, A.R (1992a). Characteristics and kinetics of subzero chilling injury in Drosophila embryos. Cryobiology 29, 39-68. Mazur, P., Cole, K.W., & Mahowald, A.R (1992b). Critical factors affecting the permeabilization of Drosophila embryos by alkanes. Cryobiology 29, 210-239. Mazur, R, Cole, K.W., Hall, J.W., Schreuders, RD., & Mahowald, A.R (1992c). Cryobiological preservation oi Drosophila embryos. Science 258, 1932-1935. Mazur, P., Cole, K.W., Schreuders, RD., & Mahowald, A.R (1993). Contributions of cooling and warming rate and developmental stage to the survival of Drosophila embryos cooled to -205 °C. Cryobiology 30, 45-73. Miles, J.E., & Bale, J.S. (1995). Steps towards the cryopreservation of the biological control agent Aphidoletes aphidimyza Rond. (Diptera, Cecidomyiidae). Cryo-Letters 16, 329338. Mitchison, T.J., & Sedat, J. (1983). Localization of antigenic determinants in whole Drosophila embryos. Dev. Biol. 99, 261-264. Myers, S.P, Lin, T.-T., Pitt, R.E., & Steponkus, P.L. (1987a). Cryobehavior of immature bovine oocytes. Cryo-Letters 8, 260-275. Myers, S.R, Lynch, D.V., Pitt, R.E., & Steponkus, P.L. (1987b). Cryobehavior of Drosophila embryos. Cryobiology 24, 549. Myers, S.P, Lin, T.-T., Pitt, R.E., & Steponkus, P.L. (1988a). Tolerance of Drosophila melanogaster embryos to ethylene glycol. Cryobiology 25, 545. Myers, S.R, Lynch, D.V., Knipple, D.C., Leibo, S.R, & Steponkus, P.L. (1988b). Low temperature sensitivity of Drosophila melanogaster embryos. Cryobiology 25, 544-545. Myers, S.R, Gardner, L.C., & Steponkus, P.L. (1989a). Tolerance of Drosophila melanogaster embryos to permeating cryoprotectants. Cryobiology 26, 550. Myers, S.R, Pitt, R.E., Lynch, D.V, & Steponkus, P.L. (1989b). Characterization of intracellular ice formation in Drosophila melanogaster embryos. Cryobiology 26, 472-484. Myers, S.R, & Steponkus, PL. (1990). Subzero chilling sensitivity of Drosophila melanogaster embryos. Cryobiology 27, 651-652. Ostergren, G. (1944). An efficient chemical for the induction of sticky chromosomes. Hereditas 30, 213-216. Parsegian, A., Rau, D., & Zimmerberg, J. (1986). Structural transitions induced by osmotic stress. In: Membranes, Metabolism, and Dry Organisms (Leopold, A.C. ed.), pp. 306-317. Comstock Publ. Assoc, Ithaca, NY. Pitt, R.E., & Steponkus, P.L. (1989). Quantitative analysis of the probability of intracellular ice formation during freezing of isolated protoplasts. Cryobiology 26, 44-63.

Cryopreservation of Drosophila Melanogaster

315

Pitt, R.E., Lin, T.-T., Myers, S.P., & Steponkus, P.L. (1989). Intracellular ice formation in Drosophila melanogaster embryos: Implications for conventional cryopreservation. Cryobiology 26, 550-551. Pitt, R.E., Myers, S.P, Lin, T.-T., & Steponkus, P.L. (1991). Subfreezing volumetric behavior and stochastic modeling of intracellular ice formation in Drosophila melanogaster embryos. Cryobiology 28, 72-86. Rail, W.F. (1987). Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 24, 387-402. Rail, W.F., & Fahy, G.M. (1985). Ice-free cryopreservation of mouse embryos at -196°C by vitrification. Nature 313, 573-575. Rasmussen, D.H., MacCaulay, M.N., & MacKenzie, A.P. (1975). Supercooling and nucleation of ice in single cells. Cryobiology 12, 328-339. Rendel, J.M., & Sheldon, B.L. (1956). The effect of cold treatment on mutation in Drosophila melanogaster. Aust. J. Agr. Res. 7, 556-573. Scheffen, B., Van Der Zwalmen, P., & Massip, A. (1986). A simple and efficient procedure for preservation of mouse embryos by vitrification. Cryo-Letters 7, 260-269. Steponkus, PL., & Caldwell, S. (1992). Cryopreservation of Drosophila melanogaster Qmbryos by vitrification. Cryobiology 29, 763-764. Steponkus, PL., & Caldwell, S. (1993). An optimized procedure for the cryopreservation of Drosophila melanogaster Qmbryos. Cryo-Letters 14, 377-382. Steponkus, PL., & Dowgert, M.F. (1983a). Ice nucleation temperature of isolated protoplasts. Plant Physiol. 72, S-44. Steponkus, PL., & Dowgert, M.F. (1983b). Influence of cold acclimation and suspending medium composition on the ice nucleation temperature of isolated protoplasts. Cryobiology 20, 735. Steponkus, PL., Myers, S.P, Lynch, D.V., Pitt, R.E., Lin, T.-T., Maclntyre, R.J., Leibo, S.P, & Rail, W.F. (1989). Cryopreservation of Drosophila melanogaster embryos. Cryobiology 26, 575-576. Steponkus, PL., Myers, S.P, & Gardner, L.C. (1990a). Cryopreservation of Drosophila melanogaster embryos. Cryobiology 27, 651. Steponkus, PL., Myers, S.P, Lynch, D.V., Gardner, L., Bronshteyn, V., Leibo, S.P, Rail, W.F., Pitt, R.E., Lin, T.-T., & Maclntyre, R.J. (1990b). Cryopreservation of Drosophila melanogaster embryos. Nature 345, 170-172. Steponkus, PL., Myers, S.P, Lynch, D.V., Pitt, R.E., Lin, T.-T., Maclntyre, R.J., Leibo, S.P, & Rail, W.F. (1991). Cryobiology of Drosophila melanogaster embryos. In: Insects at Low Temperature (Lee, R.E. Jr., & Denlinger, D.L. eds.), pp. 408-423. Chapman and Hall, New York. Steponkus, PL., Langis, R., & Fujikawa, S. (1992). Cryopreservation of plant tissues by vitrification. In: Advances in Low-Temperature Biology, Volume 1 (Steponkus, P.L. ed.), pp. 1-61. JAI Press, Ltd., London. Steponkus, PL., Caldwell, S., & Wisniewski, C. (1993). Cryopreservation of Drosophila melanogaster embryos: optimization of the procedure for maximum survival. Cryobiology 30, 617. Steponkus, PL., Uemura, M., & Webb, M.S. (1993). A contrast of the cryostability of the plasma membrane of winter rye and spring oat—two species that widely differ in their freezing tolerance and plasma membrane lipid composition. In: Advances in Low-Temperature Biology, Volume 2 (Steponkus, PL. ed.), pp. 211-312. JAI Press, Ltd, London. Steponkus, PL., Bronshteyn, V.L., & Caldwell, S.C. (1994). Cryopreservation of Drosophila melanogaster embryos: Formulation of improved vitrification solutions. Cryobiology 31, 568. Takahashi, T., Hirsh, A.G., Erbe, E.F., Bross, J.B., Steere, R.L., & Williams, R.J. (1986). Vitrification of human monocytes. Cryobiology 23, 103-115. Valencia, M.P., Miller, L.H., & Mazur, P. (1996a). Permeability of intact and dechorionated eggs of the Anopheles mosquito to water vapor and liquid water: A comparison with Drosophila. Cryobiology 33, 142-148.

316

P.L STEPONKUS, S. CALDWELL, S.P. MYERS, and M. CICERO

Valencia, M.R, Miller, L.H., & Mazur, P. (1996b). Permeabilization of eggs of the malaria mosquito Anopheles gambiae. Cryobiology 33, 149-162. Widmer, B., & Gehring, W.J. (1974). A method for permeabilization of Drosophila eggs. DIS 51, 149. Zamenhof, S.H., Eichhom, H.H., & Rosenbaum-Oliver, D. (1968). Mutability of stored spores of Bacillus subtilis. Nature 220, 818-819.

INDEX

1,2-propanediol, 197, 199-202, 206208, 221-224, 226, 228-230, 235,237-239,242,246,251, 252 1,3-butanediol, 190, 192, 198, 204, 205, 227, 228, 232, 240, 251, 252 2,3-butanediol, 227, 228, 240, 246, 247, 251, 252 2,4-pentanediol, 195, 198, 203, 252 Abies balsamea, 119 Acetamide, 238 Acidosis, 14, 24, 32, 45, 54 Adenosine, 32, 41, 56, 63, 176 Amorphous layer, 133 Annealing, 191-193, 198, 203, 205, 208,211-213,215-217,219, 220, 222-224, 226, 227, 229, 232, 235-239, 243, 252, 253 Anopheles gambiae, 308, 315 Anoxia, 2, 5, 6, 27, 42, 108 Antifreeze activity, 107, 118, 119, 121, 122, 126, 128 Antifreeze proteins, 107, 116-120, 122, 124, 125-127, 129, 130, 134, 136, 137, 161-163, 177, 197 Aphidoletes aphidimyza, 308, 314 Apoplastic extracts, 120, 122, 125, 126, 129

Apple, 65, 68, 72-78, 80, 94, 103, 105, 109, 135 Aqueous blood substitutes, 27, 28 Arabidopsis thaliana, 118, 137, 159, 176, 178, 179, 183 Arabinoxylans, 107,131,132,137,139 Arrhenius'theory, 7,156,192,195,200, 204, 210, 215, 216, 219, 227 Asanguineous extracorporeal perfusion, 24 ATP synthase complex, 144 Azalea, 133, 135 Bittersweet nightshade, 119, 120, 122, 124, 126, 129, 136 Black currant, 110 Bloodless surgery, 1, 2, 14, 17, 20, 34, 53, 56 Boyle-van't Hoff plot, 7, 8, 147, 148, 170 Butanediol, 190, 192, 198, 204, 205, 227, 228, 232, 240, 246, 247, 251,252 Cabbage, 153, 167-171, 181 Canine model, 22, 23, 28, 33, 34,43, 45-47, 54, 61, 63 Cavitation, 95, 105, 106 Cell walls, 77, 81, 82, 84-86, 88, 89, 93, 96, 98, 99, 101-104, 109, 113, 131-134, 136 317

318

Cerebral blood flow velocity, 47 Cerebral metabolic rate ,9-11 Cerebroplegia, 1, 51, 52, 60 Cerebroprotection, 9, 11 Chilling injury, 260, 280-287, 289291,293,314 Chitinases, 126, 131, 137 Circulatory arrest, 5-7, 10, 12, 13, 15, 18,20,28,41,42,46-48,5052, 54-64 Clinical hypothermia, 1, 2, 5, 9, 16, 28, 55, 62 Clinical suspended animation, 2, 45 Clubmoss, 119 Coagulopathies, 15, 16, 21, 27, 60 Cold acclimation, 66-68, 81, 84, 90, 92,94,98,99, 109, 115, 117121, 123, 125-133, 136-139, 141-144, 150-153,155, 158, 159, 167, 173, 176-182,313, 315 Cold shock, 289 Cold-inducible genes, 143 Cooling protocols, 257, 280, 281, 289 Cooling rate, 70, 93, 187, 197, 199, 214, 219, 222, 234, 240, 243, 244, 252, 255, 259, 269, 276,277, 279, 281, 283, 286288,290-293,313 COR proteins, 159, 162, 170 Corkscrew willow, 78, 80, 81, 109 Cornusflorida, 78, 105, 109 Cornus sericea, 78, 104, 105 Cornus stolonifera, 109 Creatine kinase, 38, 39, 47, 48, 51, 56 Cryofixation, 65, 70, 71, 93, 103-105, 109 Cryomicroscopy, 194-197, 215, 235, 242-244,251,277

INDEX

Cryopreservation, 137, 154, 174, 180, 181, 186, 229, 235, 238, 240, 242, 243, 248, 250-255, 257261,263,264,269,270,276, 278,281-283,285-287,289, 291,293-295,306-308,312315 Cryoprotectants, 108,137, 144, 148, 151, 154-158, 162, 164-173, 174, 176, 177, 179, 180, 182, 186, 187, 203, 240, 246, 247,251,254,257,259,261, 262, 270, 271, 278, 280, 285, 297,314 Cryoprotectins, 141, 158, 167, 169173 CryoSEM, 82 Cryotoxic, 144, 154, 157, 161, 162, 173, 177 Crystal growth, 109, 116, 120, 122, 127, 162, 174, 185-200, 202205, 218, 234, 237, 241245,247, 248, 250, 252-254, 291-293 Crystallization, 107, 112, 131, 142, 162, 185-192, 196-200, 202205,207,229,231,233237,239-243, 245-255 Crystallization kinetics, 190, 200, 233, 234, 240, 245, 247-250, 255 Cubic ice, 196, 236, 246, 254 Deep supercooling, 67-69, 78, 80, 81, 84, 88, 89, 94, 101-106, 133, 139 Dehydration, 65, 78, 80-82, 84, 86, 88,95,98, 105, 108, 132, 142, 146, 159, 170, 174, 175, 181,186, 228, 245, 253, 258, 260, 261, 263, 270, 273, 280, 283-287, 291-293, 296-299, 301-303,305,307,310-312

Index

Dendritic crystal growth, 193 Devitrification, 192, 197, 199, 201, 206, 207, 233, 237, 238, 247, 250,251,254,289,292, 293,314 Dextran-40, 24, 30-32 Diamines, 239 Differential thermal analysis, 66-69, 72, 78, 91, 102, 109, 110, 129, 130, 137, 250, 254, 276 Directional crystallization, 207 DMSO, 186, 190, 194, 261, 262, 271, 278, 280 Double glass transitions, 226-229, 232 Drosophila melanogaster, 257-261, 269-273, 275-283, 285-289, 291, 293, 294, 297, 301, 305308,312-314 Embryo, 112, 159, 180,218,253, 257-308, 310-315 Erwinia herbicola, 114 Espeletia, 131 Ethylene glycol, 172, 173, 211, 213, 215,216,227,231,234-237, 246,247,249,251,252, 258,262, 271-273, 279-287, 289, 290, 296-303, 306, 307, 310-314 Eutectic, 74, 231-233 Extracellular ice, 67, 69, 71, 73-76, 78-80, 84, 88, 89, 95, 98, 99, 103,105, 107-110, 112, 115, 117, 119, 120, 129-135, 138, 142, 147, 152, 186,277,281 Fictive pressure, 223, 249 Fictive temperature, 210, 211, 213, 214, 223, 252 Flowering dogwood, 68, 78-84, 87, 88,90,94,96, 101, 102,109, 133

319 Fluidity, 5, 151, 156, 174, 179, 182, 230 Fluorescein diacetate, 262-265, 268, 310 Forsythia, 110-112, 132,135 Forsythia viridissima, 110 Fractures, 82, 185, 188, 198, 207, 218-220, 222 Fragility of glasses, 220 Fraxinus pennsylvanica, 78, 109 Freeze-induced dehydration, 98, 260, 280, 283, 284, 286 Freeze-substitution, 65, 66, 71-73, 75-81,89,91,93,98, 100, 104, 105 Freezing damage, 142, 144, 153, 176, 177, 180 Freezing-sensitive plants, 108, 134 Freezing-tolerant plants, 108, 112115, 119, 120, 127,129, 131, 132, 134 Gas bubbles, 95, 205, 207, 218 Gene expression, 9, 143, 176, 181 Genetic stability, 258, 307 Glass fractures, 218 Glass stability, 233, 255 Glass strength, 219 Glass transition temperature, 187, 199,207,208,210,211,214, 217,219-221,225,230, 231,234,236,240,252,290 Glass transitions, 185-188, 190, 192, 193, 199, 200, 207-237, 240, 241, 244-252, 254, 290 Glass-forming tendency, 189, 190, 229, 233-235, 239, 242, 246, 247, 251, 252, 291 Glucanases, 126, 131 Glutamic acid, 298-302, 306, 307, 310 Glutathione, 32, 41, 55, 58, 63

320

INDEX

Ice crystallization, 107, 112, 131, 142, 185, 186, 188, 190-192, 196, 197, 199, 202, 205, 229, 233, 234, 236, 239-243, 245, 246, 249, 252-254 Ice formation, 7, 66, 67, 69, 73, 76, 78,80,84,91,93-95,98,99, Hemodilution, 1, 5, 14-18, 20, 21, 56, 103, 105, 107-116, 120, 124, 60,63 127-129, 131, 132, 134, 135, Hemorrhagic shock, 1, 44-49, 61, 63 Heterogenous nucleation, 185,189,277 138, 147, 152, 179, 190, 253, Hexagonal ice, 189, 196, 252, 254 254, 257, 260, 269,274, 276Homogeneous ice-nucleation, 67, 68, 287, 289, 291, 293, 312-315 95 Ice nucleation, 67, 112-115, 129, 132Homogenous nucleation, 185,188,197 139, 188, 189, 197, 199, 201, Hydrophobic interaction, 165 204, 205, 207-209, 215, 218, Hydroxyethylstarch, 29, 32, 60, 61, 228, 234, 236, 237, 240-244, 190 247, 253-255, 286, 292, 293, Hyperkalemic, 28, 32, 43, 44, 62 313,315 Hypothermia, 1, 2, 5, 7, 9-18, 20-29, Ice nucleators, 107, 112-117, 127, 32, 37, 39-47, 50-52, 54-64 129, 134-136, 138 Hypothermic cardiac arrest, 26, 27, Intracellular ice, 66, 67, 73, 76, 89, 91, 93-95, 98, 105, 108, 253, 42, 56, 59 Hypothermic circulatory arrest, 5, 9, 257, 260, 269, 276-286, 293, 313-315 13, 15, 18, 27, 46, 51, 52, 54Ionic balance, 32, 43 61,63 Ischemia, 1, 2, 5, 6, 9, 11, 15-17, 27, Hypothermic whole-body washout, 28 29, 45, 46, 48, 50, 52, 54, 56, Hypothermosol, 1, 28, 29, 32, 33, 35, 57, 59-61, 63 38-47, 49, 50, 52, 53, 63 Johnson-Mehl-Avrami-Kolmogoroff theory, 190, 191, 200 Ice, 7, 24, 57, 61, 66-76, 78-80, 82, 84, 88, 89, 91, 93-95, 98, 99, Kinetics of the glass transition, 185, 101-105, 107-122, 124, 126209-211,214 139, 142, 146, 147, 149, 152, 162, 174, 176, 178, 179, 185, Kohlraush-WiUiams-Watts model, 217, 222 186, 188-202, 204, 205, 207209, 215, 218, 228, 229, 231255, 257, 260, 269, 274, 276- Lactate dehydrogenase, 37-39 287,289,291-293,310-315 Lactobionate, 29, 42, 56, 59, 61-63 Lactobionic acid, 30 Ice crystal growth, 109, 116, 120, 122, 127, 174, 186, 188, 192- Late embryogenesis abundant pro196, 198, 199, 204, 218, 237, teins, 158, 159, 162, 170, 241-244, 250, 252, 291-293 174, 175, 179, 180

Glycerol, 151, 180, 194,211,214, 222, 225-228, 246, 249, 250, 252-254,262,271,278,313 Glycine, 54, 151-153, 298-302, 306, 310

321

Index

Lectins, 124, 136, 141, 158, 163-165, 167, 169, 170, 175, 177, 178, 180 Lobelia, 113, 114 Lobelia deckenii, 113 Lobelia telekii, 113 Low-temperature scanning electron microscopy, 65, 71-73, 75, 76, 78-80, 82, 84, 99, 103105, 109, 138 Lucilia cuprina, 308 Lycopodium dendroideum, 119 Malus domestica, 72, 109 Melittin, 160-162, 175, 182 Membrane fluidity, 5, 151, 174, 179, 182 Merocyanine 540, 156, 157, 178, 181 Milkweed bug, 124 Musca domestica, 308 Mutation rates, 307, 308 Narayanaswamy-Tool model, 214 Neurotransmitters, 9, 10 Nitrogen slush, 288, 290, 291, 311 Oncopeltus fasciatus, 124 Opuntia, 113-115, 136 Opuntia ficus-indica, 113 Opuntia humifusa, 113 Opuntia streptacantha, 113 Organic acids, 171-173 Osmolytes, 151, 153 Osmotic behavior, 257, 269 Osmotic stress, 175,177,181,297,314 Ostwald ripening, 196, 207, 250, 254 Oxygen consumption, 5-10, 14, 17, 56, 57, 62 Pathogenesis-related proteins, 124126, 131, 135, 138 Peach, 68, 94, 102, 103, 105, 106, 110, 132, 133, 135, 138, 139

Permeability, 16, 31, 101, 102, 106, 133, 147, 151, 153, 154, 156, 157, 164-167, 170, 174, 177, 182,183,257,261,262,269, 270,277,293,310,313,315 Permeabilization, 257-270, 277, 287, 289,291,293,296,298,305, 309-311,313-315 Phaenicia sericata, 308 Phase separation, 188, 209, 218, 226229,245,251 Photosynthesis, 137, 144, 145, 174 Picea glauca, 119 Picea mariana, 119 Plant freezing tolerance, 142, 143, 158 Plant glycolipids, 153 Plastocyanin, 145-150, 152-155, 160165, 168-170, 176 Poaceae, 131 Poly(ethylene)glycol, 229, 240 Polyalcohol, 190, 192, 194, 195, 247 Polysaccharides, 30, 113, 131, 136, 240, 241, 245, 253, 254 Proline, 151, 152, 175, 180, 182 Propanediol, 194, 197, 199-202, 206208, 221-224, 226-230, 235, 237-239,242,246,251,252 Propylene glycol, 211, 219, 220, 222, 228,246,250,252,262,271, 280 Prunus, 94, 110, 113-115, 136 Prunus persica, 94, 110 Pseudomonas syringae, 114, 197 QIO, 7, 8, 10, 11 Quench freezing, 70 Quercus coccinea, 109 Quercus rubra, 78, 109 Recrystallization inhibition, 121, 129 Red ash, 78, 80, 81, 109 Red oak, 78, 80, 109

322

Red osier dogwood, 68, 78-80, 82, 84, 85, 87-89, 92, 96, 98-101, 104, 105, 109 Resuscitation, 1, 37, 41, 44-47, 50, 58,61,63 Rhizoplaca chrysoleuca, 113 Rhododendron, 115, 137 Ribes nigrum, 110, 139 Rubbery state, 222 Salix babylonica, 78, 109 Salix matsudana, 78 Salix matsudana f. tortuosa, 109 Salt stress, 145, 150-153, 177, 180 Saxifraga caespitosa, 114, 138 Scarlet oak, 109 Secale cereale, 104, 109, 128, 130, 135, 137, 179 Secondary nucleation, 107, 112, 129, 132 Signal sequences, 159 Simplified stroma medium, 148 Solanum dulcamara, 119, 136 Solute loading, 146, 147, 150, 151, 153, 154, 157, 164, 170, 174 Solute permeability, 151, 154, 156, 157, 164-167, 170, 177, 183 Sorbitol, 247, 299-303, 305 Spinach, 143-146, 148, 149, 153, 155, 156, 160, 161, 163, 165, 167, 169, 174-181, 183 Spinodal decomposition, 188, 226, 228 Suberization, 133 Sucrose, 29, 56, 63, 147-149, 153156, 168, 169, 171, 172, 176178, 180,233,236,241,247, 261-263,300-305,310,311 Sugar acids, 157, 177 Supercooled water, 66, 67, 73, 91, 104, 110, 112

INDEX

Supercooling, 65-70, 78, 80-82, 84, 88, 89, 91, 93-96, 101-106, 108-110, 115, 116, 119, 127, 133-136, 138, 139, 174, 187, 190, 192, 193, 196, 197, 202, 205,257,259,280-282,315 Tenebrio molitor, 124, 129 Thaumatin-like proteins, 126 Thermal hysteresis, 116, 119-122, 126, 127, 131, 136, 137, 139, 161, 162 Thermal hysteresis proteins, 119, 136, 137, 139, 161, 162, 175 Thylakoid membranes, 137, 138, 141, 144, 151, 152, 156, 157, 160, 162, 167, 169, 176, 177,180, 183 Tipula trivittata, 114 Total body hypothermic protection, 2,45 Trees, 66, 70, 72, 77, 78, 87, 88, 98, 103, 109, 132, 133, 135, 142 Trehalose, 153-156, 158, 175, 177, 179, 180, 183, 241, 247, 249 Triticum aestivum, 109, 179, 182 Tsuga canadensis, 119 TTT-curves, 199, 243 Ultraprofound hypothermia, 1, 5, 17, 20, 22, 23, 25-28, 37, 39, 40, 45, 47, 50, 52, 54, 56, 63 Universal tissue preservation solutions, 28 Vascular differentiation, 132, 134, 135 Vascular segmentation, 132, 133 Viscosity, 14-17, 21, 27, 32, 187, 189, 190, 193, 195, 196, 218, 219, 226, 229, 235, 252, 262

Index

Vitrification, 70, 94, 137, 185-188, 190, 193, 197, 199, 205, 208, 209, 229, 232, 233, 235, 238243, 245-254, 257-260, 263, 270, 273, 276, 286-293, 295307,311-315 Vogel-Fulcher-Tamman law, 210, 215,219 Warming rate, 191, 196, 206, 208, 214, 217, 218, 225, 244, 259, 291-293,314 Water permeability, 257, 261, 269, 270,313 Weeping willow, 78, 80, 81, 102, 109

323

Wheat, 105, 109, 137, 138, 142, 145, 151, 152, 174-179, 182 Winter rye, 105, 109, 114, 115, 117, 119-131, 133-138, 176, 179, 181,315 Wood, 65, 66, 68-73, 76-78, 80-82, 84-86, 88, 89, 91, 94-96, 99, 101-103, 105, 106, 113, 136, 139 Xylem, 65-67, 73, 76-78, 80-96, 98106,109, 110,112, 129, 132, 133, 135, 139 Xylem ray parenchyma, 105, 110, 129, 133

J A I P R E S S

Advances in Low-Temperature Biology Edited by Peter L. Steponkus, Department of Soil, Crop and Atmospheric Sciences, Cornell University

$109.50

Volume 1,1992, 288 pp. ISBN 1-55938-351-8

CONTENTS: Introduction, Peter L. Steponkus. Photosynthetic Acclimation to Light and Low Temperature in Freezing Tolerant Plants and Psychrophilic Microalgae, Norman P.A. Huner and Charles G. Trick. Vitrification and Devitrification in Cryopreservation, Douglas R. MacFarlane, Maria Forsyth and Catherine A. Barton. Protein Stability Under Conditions of Deep Chill, Felix Franks and Ross H.M. Hatley. Biochemical Adaptations for Winter Survivial in Insects, Kenneth B. Storey and Janet M. Storey. Thermodynamics and Intracelluar Ice Formation, Ronald E. Pitt. Vitrification of Plant Tissues, Peter L. Steponkus and Robert Langis. Volume 2, In preparation, Winter 1996 ISBN 1-55938-536-7

Approx. $109.50

CONTENTS: Preface, Peter L Steponkus. Nucleatlon of Ice Crystals in Biological Cells, Mehmet Toner Freeze-Drying of Red Blood Cells, Raymond P. Goodrich and Samuel O. SowemimO'Coker Cellular Adaptations for Freezing Survival by Amphibians and Reptiles, Kenneth B. Storey and Janet M. Storey. Thermal-Hysterisis Proteins, John G.Duman, Ding Wen Wu, Mark T. Olsen, Maria Urrutia, and Donald Tursman. Genes Induced During Cold Acclimation in Higher Plants, Michael F. Thomashow. A Contrast of the Cryostabllity of the Plasma Membrane of Winter Rye and Spring, Oat-Two Species that Widely Differ in their Freezing Tolerance and Plasma Membrane Lipid Composition, Peter L Steponkus, Murray S. Webb, andMatsuo Uemura. Subject Index.

JAI PRESS INC. 55 Old Post Road No. 2 - P.O. Box 1678 Greenwich, Connecticut 06836-1678 Tel: (203) 661 - 7602 Fax: (203) 661 -0792

Advances in Low-Temperature Biology, Volume 3

Advances in Marine Biology, Volume 41 (Advances in Marine Biology)

Advances In Marine Biology, Volume 53 (Advances in Marine Biology)

Advances in Marine Biology, Volume 39 (Advances in Marine Biology)

Advances In Marine Biology, Volume 51 (Advances in Marine Biology)

Advances in Structural Biology, Volume 6 (Advances in Structural Biology)

Advances In Marine Biology, Volume 46 (Advances in Marine Biology)

Advances in Marine Biology, Volume 44 (Advances in Marine Biology)

Advances in Agronomy, Volume 3

Advances in Computers, Volume 3

Advances in Pharmacology Volume 3

Advances in Pharmacology Volume 3

Advances in Immunology Volume 3

Advances in Genetics Volume 3

Advances in Computers, Volume 3

Advances in Marine Biology (v. 3)

Advances in Marine Biology, Volume 15

Advances in Marine Biology, Volume 26

Advances in Marine Biology, Volume 18

Advances in Marine Biology, Volume 14

Advances in Marine Biology, Volume 37

Advances in Marine Biology, Volume 9

Advances in Marine Biology: Volume 25

Advances in Medicine and Biology, Volume 20

Advances In Marine Biology, Volume 50

Advances In Marine Biology, Volume 56

Advances in Marine Biology, Volume 34

Advances in Marine Biology, Volume 15

Advances in Marine Biology, Volume 21

Advances in Marine Biology, Volume 10

Advances in Developmental Biology, Volume 2

Advances in Low-Temperature Biology, Volume 3

Advances in Marine Biology, Volume 41 (Advances in Marine Biology)

Advances In Marine Biology, Volume 53 (Advances in Marine Biology)

Advances in Marine Biology, Volume 39 (Advances in Marine Biology)

Advances In Marine Biology, Volume 51 (Advances in Marine Biology)

Advances in Structural Biology, Volume 6 (Advances in Structural Biology)

Advances In Marine Biology, Volume 46 (Advances in Marine Biology)

Advances in Marine Biology, Volume 44 (Advances in Marine Biology)

Advances in Agronomy, Volume 3

Advances in Computers, Volume 3

Advances in Pharmacology Volume 3

Advances in Pharmacology Volume 3

Advances in Immunology Volume 3

Advances in Genetics Volume 3

Advances in Computers, Volume 3

Advances in Marine Biology (v. 3)

Advances in Marine Biology, Volume 15

Advances in Marine Biology, Volume 26

Advances in Marine Biology, Volume 18

Advances in Marine Biology, Volume 14

Advances in Marine Biology, Volume 37

Advances in Marine Biology, Volume 9

Advances in Marine Biology: Volume 25

Advances in Medicine and Biology, Volume 20

Advances In Marine Biology, Volume 50

Advances In Marine Biology, Volume 56

Advances in Marine Biology, Volume 34

Advances in Marine Biology, Volume 15

Advances in Marine Biology, Volume 21

Advances in Marine Biology, Volume 10

Advances in Developmental Biology, Volume 2

Recommend Documents