X-Message-Number: 4732
Date: Mon, 7 Aug 1995 18:51:46 +0200 (MET DST)
From: Eugen Leitl <>
Subject: nature's cryonics (II)

Temperature Adaptation cot'd (10 pages of 14).

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as stated above, terrestrial invertebrates that tolerate ice formation
in their extracellular fluids generally are in a state of months-long
hibernation.

Among aquatic animals the intertidal invertebrates of high latitudes
may display impressive degrees of freezing tolerance. For instance,
Kanwisher (1955) showed that two intertidal molluscs, Mytilus edulis,
and Littorina rudis, withstand freezing of approximately 70 percent of
their total body water at an ambient temperature -20 deg C, a common
winter temperature for the North Atlantic intertidal regions where
these specimens were obtained. All freezing was in the extracellu-
lar space. Although the cells appeared shrunken and distorted under
microscopic observation, they contained no ice crystals (Kanwischer,
1955). In a related study, Murphy and Pierce (1975) examined the
ability of another intertidal mollusc, Modioulus demissus demissus, to
acclimate to low temperatures and, thereby, to increase its tolerance of
freeezing. They were able to demonstrate that a pronounced increase in
the mollusc's capacity to withstand tissue desiccation accompanied low
temperature acclimation. Specimens acclimated to 23 deg C died when 35
percent of the tissue water was removed via extracellular ice formation;
0 deg C-acclimated individuals could tolerate the loss of 41 percent of tis-
sue water. An important conclusion from this study is that low tem-
perature acclimation does not reduce the amount of tissue water lost
during extracellular freezing, but instead leads to a greatly increased
tolerance of partial dehydration. The molecular basis of this acclima-
tory effect is not known, albeit the involvement of macromolecular
antifreezes in certain intertidal molluscs (Theede et al., 1976) raises the
possibility that these molecules may play some role in stabilizing bio-
chemical structures and functions in face of reduced water activities,
a suggestion that has also been made in the case of xerotolerant insects
such as Tenebrio molitor (Patterson and Duman, 1979; Schneppenheim
and Theede, 1980).

/* Mike, whadda you say to this below: */

A final group of compound merits consideration in the context of
freezing tolerance. These are polyhydroxyl alcohols (polyols) like glyc-
erol, sorbitol, and mannitol, which may accumulate to high concentra-
tions in many insects that are either freeze-tolerant or freeze-resistant.
Polyols lower the supercooling point of a solution approximately twice
as much as they reduce the true freezing point, so these compounds
may be of great significance in the prevention of ice formation. The
potential disadvantages of polyols are at least twofold. First, because
they function in a strictly colligative manner, unlike the peptide and
glycopeptide antifreezes discussed later, polyols will sharply increase
the osmotic concentration of the body fluids. If polyols are uniformly


Table 11-11. "Antifreeze" Proteins and Glycoproteins in Fishes and 
Invertebrates.


--------------------------------------------------------------------------------------------
Group of organisms     Type of		Molecular weight  Thermal hys-	  Reference
		       "antifreeze"     (daltons)         teresis deg C


--------------------------------------------------------------------------------------------
Antarctic fishes       Glycoproteins	8 size classes	  1.27		  DeVries (1974)
 Notothenidae				00-33700
 (e.g. Pagothenia
 borchgrevinkii)
									  DeVries (1974)
Zoarcidae	       Glycoproteins			  0.76
 (Rhigophila
 dearborni)

Arctic fishes
 flounder	       Proteins 	3 size classes	  0.62 (winter)   Duman and DeVries

 (Pseoudopleuronectes                                                     (1974,
 1976)
 americanus)
cod		       Glycoproteins	7 size classes	  1.18		  Van Voorhies et al.

 (Gadus ogac)                           same as Antarct.                  (1978)
 					nototheniids
saffron cod	       Glycoproteins			  1.0		  Raymond et al.

 (Eleginus gracilis)                                                      (1975)
 sculpin 	       Protein				  1.4		  Raymond et al.

 (Myoxocephalus                                                           (1975)
  verruccosus)

insects

 (Tenebrio molitor)    Proteins         several size                      
 Patterson and
					classes 			  Duman (1979)
									  Schnppenheim and
									  Theede (1980)

spider							  2.44 (Feb.)     Duman (1979)
 (Philodromus sp.)     Proteins                           0 (June)
spider							  1.88 (Jan.)     Duman (1979)
 (Clubiona sp.)        Proteins                           0 (warm-accl.)

beetle

 (Dendroides           Proteins                           3.62 (cold-     Duman 
 (1980)
 canadensis)						  accl.)
beetle

 (Merancantha          Proteins                           3.71 (Feb.)     Duman 
 (1977)
 contracta)


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distributed throughout all body fluid compartements, however, this
osmotic effect may not be much of a problem,since redistribution of
water among compartments would not occur. Second, the polyols in-
crease the viscosity of body fluids greatly, a problem that again is
avoided by the peptide and glycopeptide antifreezees, which work in
noncolligative manners.

One important feature of polyols like glycerol is that they may serve
a variety of functions in organisms that experience freezing or desic-
cating conditions. Glycerol functions as a cryoprotectant, as a super-
cooling agent, and has been shown to aid in desiccation resistance in
cysts of Artemia (Clegg, 1962). As noted in Chapter 10, glycerol is a
compatible solute that lacks perturbing effects on proteins. Common
inorganic ions, e.g. K+, Na+, and Cl-, do not exhibit compatibility
at high ionic strengths. Thus, glycerol may be an appropriate solute
to use at low water activities, whether these low activities are asso-
ciated with high external osmotic concentrations, with encystment and
dormancy, or with freezing tolerance. It is necessary to realize, how-
ever, that glycerol is not an ubiquitous component of the body fluids of
freeze-tolerant and freeze-resistant animals (Duman, 1980; Zacharias-
sen, 1980). The full chemical armament of such animals usually con-
tains other compounds whose contributions are critical for survival at
low temperatures. These compounds are the peptide and glycopeptide
antifreeze molecules, wwhich have now been described in a variety of
high latitude fishes as well as in several terrestrial invertebrates.

Freezing Resistance and Macromolecular Antifreezes. One of
the most intriguing stories about molecular evolution to have been
developed during the past two decades involves the macromolecular
antifreezes (peptides and glycopeptides), first discovered in larvae of
the insect, Tenebrio molitor (Ramsay, 1964) and blood sera of Antarctic
fishes (reviewed by DeVries, 1980, 1982). The currently identified family
of antifreezes listed in Table 11-11 reveals that these peptides and
glycopeptides are found in a taxonomically diverse assemblage of ani-
mals, differ substantially in chemical structure and molecular weight,
and can vary in concentrations as a function of acclimation or acclima-
tization. All of these antifreezes share one important common property,
however, which is a diagnostic trait for those molecules: they lower the
freezing point (temperature of ice crystal growth) of a solution more
than the melting point (temperature of shrinkage of ice crystals). This
"thermal hysteresis" is used to detect the presence of antifreezes in
solutions and, moreover, may provide an important clue to the mech-
anism of antifreeze action, as discussed later. We will see, in fact, that
in spite of the diversity of antifreeze primary structures (Table 11-11),
a common mechanism of antifreeze action is likely to apply in all
cases.

The glycopeptide and peptide antifreezes are common in high lati-
tude fishes that inhabit the upper regions of the water column where
ice is present (Table 11-11). Fishes of high latitude seas lacking these
antifrezes must seek out ice-free niches, and many species are likely
to remain in a supercooled state throughout their lives (DeVries, 1980).
The antifreezes present in high latitude fishes are a fascinating example
of convergent evolution at the molecular level. The evolutionary devel-
opment of glycopeptides and peptides capable of causing thermal
hysteresis has occured several times. Distantly related fishes have
independently "discovered" the sane antifreeze primary structures
(Fig. 11-27). The repeat tripeptide unit, "alanyl-alanyl-threonyl" (with
a carbohydratemoiety attached tothe threonyl residue(, is found in
the Antarctic nototheniid fishes, e.g, of the genus Trematomus and
in the Arctic rock cod, Gadus ogac (Van Voorhis et al.l, 1978). The
peptide antifreezes of the Arctic sculpin,Myoxocephalus verrucosus,
and flounder, Pseudeopleuronectes americanus, likeise appear to have
similiar structures. Cysteine-rich antifreezes have been described in both
fish (the sea raven, Hemitrepterus americanus; Slaughter et al., 1981)
and insects (Tenebrio molitor; Schneppenheim and Theede, 1980). Thus,
there appears to be a considerable variety of molecular structures that
can function as antifreezes, and the independant evolution of these
different structures has occured again and again, in a variety of fishes
and invertebrates.

The structures of two types of antifreezes are shown in Figure 11-27.
The glycopeptide antifreezes were the first to be sequenced, and they
characteristically exhibit a simple structure in terms of amino acid
sequence. Alanyl residues contribute a high percentage of total residues,
and threonyl residues serve as the atachment sites for the carbohydrate
components of the antifreezes. The basic repeated unit shown in Figure
11-27 is found in antifreezes of different molecular weights. The smallest
glycopeptide is 00 daltons, and the largest is approximately 33000
daltons (DeVries, 1980). In the smalles class of glycopeptide, some of
the alanyl residues are replaced by prolines.

The partial sequence the peptide antifreeze from the winter floun-
der, P. americanus, is shown in the lower panel of Figure  11-27. Alanyl
residues again constitute a major fraction of the amino acid chain.
Threonyl and aspartyl residues are the other dominant components of
the peptide antifreezes. Like the galactose and galactose-amine residues
of the glycopeptide antifreezes, the polar or charged threonyl and





/* upper panel omitted due to the limitations of ASCII art */

	  Antarctic cod (D. mawsoni)

ALA-ALA-THR-ALA-ALA-THR-ALA-ALA-THR-ALA-
	 |	     |		 |
	Gal-A	    Gal-A	Gal-A
	 |	     |		 |
	Gal	    Gal 	Gal


	  Winter flounder (P. americanus)

ASP-THR-ALA-SER-ASP-ALA-ALA-ALA-ALA-ALA-ALA
 | _ |	     |	 |  _
COO  OH      OH  COO

-LEU-THR-ALA-ASP-ALA-ALA-ALA-ALA-ALA-ALA-
      |       | _
      OH     COO

Fig. 11-27. Structures of the glycopeptide and peptide antifreezes of polar
fishes. Upper panel: the basic repeating structural unit of a glycopeptide with
antifreeze properties. The peptide chain contains only two types of residue
(alanyl and threonyl). To each threonine residue is joined a disaccharide
(beta-D-Galactopyranosyl-(1-3)-2-acetoamido-2-deoxy-alpha-D-galactopyranose.

Lower panel: Primary structures of the glycopeptide antifreezes of the Antarctic
nototheniid fish Dissostichus mawsoni (observe the ALA-ALA-THR- repeat
unit, shown in the upper panel), and the peptide antifreeze of the winter
flounder, Pseudopleuronectes americanus. Figure after DeVries (1980).




aspartyl residues provide a "front" of polar groups which may facilitate
strong adsorption to ice crystals, as discussed below.

The insect antifreezes have not been as well characterized at the
structural level. None of the insect antifreezes has been found to con-
tain carbohydrate components, i.e. all of those antifreezes appear to
be peptides (Duman et al., 1982). The percentage contribution of differ-
ent amino acid residues differs form the fish antifreezes. The insect pep-
tide antifreezes characteristically have low alanyl contents, for example.
The antifreeze peptide from the milkweed bug, Oncopeltus fasciatus,
has a high serine content (30.5 percent; Patterson et al., 1981). Other
insect antifreeze peptides contain high cysteine contents and lose their
thermal hysteresis properties when treated with reagents that reduce
disulfide bridges (Duman et al., 1982). How the secondary structure
established by -S-S- bridges contributes to antifreeze functions is
not presently known.

Mechanisms of Antifreze Action. How do these diverse glycopep-
tide and peptide antifreezes establish the thermal hysteresis which is
their diagnostic characteristic? Ho can these antifreezes function in a
noncolligative manner, i.e., how are they able to reduce the freezing
point (temperature of ice crystal growth) of a solution more than can
be explained on the grounds of total number of antifreeze particles
in solution? One clue to the mode of function of these antifreezes is
the high concentration of polar or charged groups along the antifreeze
primary structure (Fig. 11-27). In the glycopeptide antifreezes, the
sugars provide a regular front of -OH groups, since all of the hydrox-
yls are thought to orient in one plane along the linear, expanded anti-
freeze molecule (DeVries, 1980). In the peptide antifreezes, which have
high percentages of threonyl and aspartyl residues, a similiar polar front
is created. These polar groups appear appropriately spaced to facilitate
a strong interaction with ice (DeVries, 1980). Thus, the antifreezes have
been proposed to exert their effect by strongly absorbing to ice and
making the addition more water molecules to the growing ice front
less thermodynamically favorable (a more detailed discussion of this
"poisoning" of growth mechanism is given by Raymond and DeVries,
1977).

Evidence in support of this hypothesis has come from several types
of experiments. For example, unlike solutes that obey colligative rela-
tionships, the antifreezes do not freeze out of a solution as ice forma-
tion occurs (Duman and DeVries, 1973). This finding suggests that a
strong interaction takes place between the ice and the antifreeze. Using
scanning electronmicroscopy, Raymond and DeVries (1977) provided
striking visual proof of this interaction. They showed that the anti-
freezes did adsorb to ice crystals, inhibiting their growth and affecting
the crystal form of the ice that propagate. The thermal hysteresis
observed in the freezing-melting behaviour of solutions containing anti-
freezes further suggests an unusual interaction between ice and these
molecules. The reduced freezing point observed in antifreeze-contain-
ing solutions may reflect the inhibition of ice crystal growth that results
when antifreeze molecules adsorb to the growing front of a small ice
crystal. Once ice is formed, however, its melting temperature is that
of normal ice. The dependance of antifreeze action on polar groups has
been demonstrated by chemical modification studies, e.g., by blocking
the hydroxyl groups on the galactose residues with sodium borohy-
dride (DeVries, 1980). The removal of these polar groups may prevent
efficient hydrogen bonding between antifreeze and ice, thereby elimina-
ting the molecules' capacities to bind to ice and to inhibit further crystal
growth.

It is important to point out that the unusual capacities for interac-
tion with ice posessed by the peptide and glycopeptide antifreezes do
not mean that these antifreezes have atypical interactions with liquid
water (see DeVries, 1980). The quantity of water bound to the anti-
freezes is similar to the amount bound by proteins of similiar size which
lack the thermal hysteresis properties of the antifreeze molecules. The
fraction of serum water that could be bound by th antifreezes is only
about 1 percent (DeVries, 1980), and this minute amount of bound
water cannot account significantly for the freezing point depressing
abilities of the glycopeptide and peptide antifreezes.

What is the Relationship Between in Vitro and in Situ Function
of Antifreezes? The elucidation of the mechanism of antifreeze action
has, of course, involved in vitro experimentation. The careful studies
of crystal growth in vitro may mislead us into thinking that a
similiar phenomenon occurs throughout the body fluids of antifreeze-
containing animals. In reality, ice formation per se must be avoided in
most freeze-resistant species, notably the Arctic fishes which live
continuously at -1.86 deg C (DeVries, 1980). Thus, even if the antifreezes
in these fishes were capable of preventing the growth of ice crystals
in the blood or cytosol, there is apparently no way to remove these
crystals once they are formed. That is, the fish never reach temperatures
as high as the melting point of the antifreeze-containing body fluids
(approximately -1 deg C).

These considerations have led ot the hypothesis that the in situ func-
tion of antifreezes is one of inhibiting ice crystal propagation across
integumental surfaces (DeVries, 1980). For example, in fishes the most
likely site of ice propagation is across the gills, whih lack a covering
of protective scales and mucus.  Antifreeze molecules in the gill mem-
branes and circulating fluids may, therefore, be critical in preventing
the passage of minute ice crystals from the seawater into the body
fluids. Schneppenheim and Theede (1979) have observed that the integ-
ument of the Arctic sculpin, Myoxocephalus scorpius, contains anti-
freeze peptides, a finding in support of the hypothesis that one of, if
not the key, function(s) of the antifreezes is a "peripheral defense"
involving the blocking of the ice crystal penetration into the interior
of the organism.

Antifreezes, Nucleating Agents, and Polyols in Insects: What
are Their Roles? Unlike polar fishes in which each species contains

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