X-Message-Number: 4747
Date: Wed, 9 Aug 1995 11:34:01 +0200 (MET DST)
From: Eugen Leitl <>
Subject: nature's cryonics (III)

Hochachka's Temperature Adaptation excerpt cot'd (14 pages of
14), yet to be rounded up with a fragment on the role of polyols
in anhydrobiosis, which seems to be at least a significant part
of the mechanism of polyol-linked cryoprotection in
vitrification cryopreservation.

The bibliography can be supplied on demand. (Read: I'm too damn
lazy to hack in text most people will skip anyway). A short
discussion whether and what we can learn from nature's cryonics
philosophy might be constructive. (But please wait for "polyols
in anhydrobiosis" part, first).

-- Eugene

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only one type of antifreeze compound in terms of basic chemical struc-
ture (but not molecular size), some insects contain a battery of com-
pounds that all may be involved in establishing freezing resistance or
freezing tolerance: freezing-point depressing peptide antifreezes, ice nu-
cleating agents, and cryoprotective substances like glycerol. How do
these multiple components interact to provide insects with the abilities
either to avoid ice formation or withstand freezing of the extra-
cellular fluids?

Duman (1980) has summarized the different roles likely to be played
by these three classes of molecules. It is fairly clear that the nucleating
agents have a single role, that of preventing significant supercooling
wich could lead to spontaneous ice formation in the intracellular
space. The full roles of polyols remain controversial despite years of
study. They may aid in supercooling, they may be cryoprotectants, and
they may aid in desiccation resistance. The contributions played by
antifreeze peptides may also be multifaceted. Prior to the acquisition of
freezing tolerance late in the fall by the beetle Dendroides canadensis,
the antifreeze peptides present in its hemolymph may adequately pro-
tect against ice formation. The antifreeze peptides may also act as
supercooling agents (Duman et al., 1982). Once the beetles acquire
freezing tolerance, it is not clear what role the antifreeze pepides can
play. Nonetheless, these peptides reach their maximum concentrations
in this beetle when freezing tolerance is greatest. Duman (1980) con-
jectures that these proteins may serve as cryoprotectants under these
latter condition; this idea awaits experimental test.

Regulation of Antifreeze Synthesis and Degradation. Antifreeze
peptides and glycoproteins may be present at relatively high concen-
trations in the body fluids (approximately 3 percent weight/volume in
the serum of Antarctic fishes; DeVries, 1980), and their biosynthesis
probably involves a substantial amount of metabolic energy. It is not
unexpected, therefore, that antifreeze synthesis in many species occurs
on a seasonal basis, beginning in the autumn and ceasing in the spring.
Antarctic fishes are an exception, of course, as they continously ex-
perience subzero temperatures and must maintain antifreeze titres
during their entire lives.

A number of recent studies have addressed he control mechanisms
involved in triggering changes in antifreeze synthesis/degradation rates.
Much as antifreeze molecules differ in structure among animals, so are
the control mechanisms varied, at least in terms of the environmental
cues which are utilized to sense the needs for antifreeze production.
Duman and DeVries (1974) found that a "fail-safe" control mechanism
was operative in governing antifreeze synthesis and degradation in
some northern fishes. Although the synthesis of antifreeze was triggered
by holding fish at low temperatures, the disappearance of anifreezes
required a combination of long photoperiod and warm temperatures.
Thus, removal of antifreezes in response to an unusual warm spell in
late winter or early spring would not occur due to the overriding in-
fluence of photoperiod. The kinetics of antifreeze synthesis required
three to six weeks for peak antifreeze titres to be built up, and loss of
antifreeze occured over a similiar time course.

The hormonal and molecular-genetic bases of antifreeze turnover
have also been studied in some fishes. In the winter flounder, Pseudo-
pleuronectes americanus, pituitary regulation of antifreeze degradation
was shown by Hew and Fletcher (1979). Hypophysectomized fish con-
tinued to synthesize antifreeze in summer, while control fish did not.
The levels of control involved in regulating antifreeze synthesis appear
to be complex. Lin (1979) and Lin and Long (1980) have shown that
the specific messenger RNA (mRNA) for antifreeze in the winter floun-
der appears well before antifreeze synthesis begins, and disappers
about a month prior to the disappearance of antifreeze in the spring.
Thus, both transcriptional and translational control of antifreeze syn-
thesis in fish liver appear to occur.

In terrestrial invertebrates, a variety of regulatory schemes exist. Not
all species studied exhibit the "fail-safe" degradation scheme noted for
certain fishes. Thus, while the darkling beetle, Merancantha contracta,
requires a combination of high temperature and long photoperiod to
induce loss of antifreeze (Duman, 1977), the spider Philodromus sp.
lost antifreeze only in response to elevation in temperature. In the
beetle Dendroides canadensis, Duman (1980) showed that a long photo-
period was necessary to trigger loss of antifreeze; high temperatures and
short photoperiod were not effective in this regard. The neural and
hormonal mechanisms effecting these responses to environmental cues
remain to be elucidated.


Lessons from the Study of Temperature Adaptation -
and Some Important, Unanswered Questions

In Chapter 1 we proposed that a major reason for doing comparative
biochemistry, that is, for examining adaptive variations on different
molecular themes, is to discover the crucial aspects of biochemical
design. Through the study of molecular adaptations, one not only may
discover how different organisms are biochemically adapted to their
particular habitats, but also what characteristics of molecular systems
are rigorously conserved in all species. The study of temperature adap-
tation offers an excellent means for elucidating these aspects of bio-
chemical design, because temperature has such a pervasive influence
on biochemical systems, that all of the major chemical components of
organisms - water, proteins, lipids and nucleic acids - will have their
structural and functional properties altered by changes in temperature.

/* this passage only for the sake of completeness and background */

Among the strongly conservative trends we noted in temperature
adaptation was the defense of K_m values and catalytic rate potentials
of enzymes at normal physiological temperatures. Temperature com-
pensatory adjustments in enzyme kinetic properties that lead to the
conservation of K_m and catalytic rate seem an ubiquitous feature of
enzyme evolution. Only small changes in habitat (body) temperature
are necessary to foster these types of evolutionary modifications of
enzymes. Thus, in the case of the barracuda congeners, we found that
differences in average body temperature of only a few degrees Celsius
have been sufficient to favor selection for temperature-adapted M_4-
LDH variants. The types of amino acid substitutions that effect these
functional adaptations remain to be discovered. Through understand-
ing the structural bases of these kinetic adaptations we may gain pow-
erful new insights into the basic mechanisms of enzyme function; that
is, we may be able to discern more clearly how changes in primary
sequence effect adjustments in binding and catalytic properties.
Through studies of enzymes from differently adapted ectotherms wew
may come ot understand structure-function relationships in enzyme
families, e.g. M_4-LDHs, at the detailed level that structure-function
relationships in respiratory proteins, e.g. vertebrate hemoglobins, are
now understood. Likewise, study of the structures of subunit inter-
action sites in multimeric proteins like filamentous actins may provide
us with key insights into the types of bondings that stabilize the re-
veresible assembly of many different classes of proteins.

In many ways the adaptations noted in lipid-based systems parallel
those found in proteins. In both cases the need for reversible structural
transformations necessitates a "semistability" of structure (Alexandrov,
1977). To achieve a correct level of structural flexibility for the given
temperature of function, amino acid composition and lipid composi-
tion are found to be adjusted. Like the adaptations in enzyme struc-
tural and functional properties, the adaptations in lipid systems leading
to homeoviscosity appear to be ubiquitous among different types of
organisms. Even though a variety of different regulatory mechanisms
for modifying the fatty acid and head group compositions of membrane
lipids exist in procaryotes and eucaryotes, the end result of these regu-
latory events is the same in all cases.

Adaptive changes in the structures of large molecules, proteins,
lipids and nucleic acids, are complemented by adaptive ajustments in
the microenvironment in which the large molecular ensembles function.
In particular, regulation of pH according ot the alphastat scheme was
shown to be critical for the retention of correct kinetic properties, e.g.,
the K_m of pyruvate for M_4-LDH, and assembly states of multimeric
proteins, e.g. of PFK. The importance of histidine imidazole groups
in temperature adaptation was strongly emphasized. How different
organisms and different tissues within an organism regulate pH merits
much additional study, for although we are beginning to appreciate
the significance of alphastat regulation, the mechanisms employed to
achieve the correct pH_i for particular body temperature are not that
clearly understood.

The study of temperature adaptation provides us with a good means
for comparing the results of different time courses of molecular adap-
tation. We found that long-term, evolutionary changes often were simi-
lar to short-term acclimatory changes. Lipid adaptations offer a good
case in point; homoviscous adjustments were seen to be comparable
in differently adapted species and in differently acclimated populations
of a single species. Alphastat regulation also is noted on inter- and
intraspecific bases. The role of protein variants in temperature acclima-
tion remains unclear. The adaptive trends noted in interspecific com-
parisons, e.g., temperature adaptive modifications in K_m and k_{cat}
values, are typically not observed in comparisons of differently accli-
mateted (-acclimatized) populations of a single species. Nonetheless, there
are enough good examples of acclimation or acclimatization effects,
e.g., the acetylcholine esterase system of Salmo gairdneri and the H_4-
LDH system of Fundulus heteroclitus, to encourage more studies of
these changes.

Finally, our increasing understanding of the end-results of tempera-
ture adaptation processes is not yet matched by an appreciation of the
kinetics of these processes. How rapidly do evolutionary changes in
proteins occur? Do acclimatory adjustments in protein and lipid sys-
tems follow similiar time courses? What regulatory mechanisms are
rate governing in acclimation processes? Questions such as these may
serve as useful focal points in our future attempts to appreciate how
the diverse biochemical systems of organisms are adaptively modified
over evolutionary time and during short-term acclimatory periods for
function under diverse thermal regimes.

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(to be continued with a short fragment on crucial role polyols
play in desiccation resistance in anhydrobiosis)

-- Eugene


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