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First published online September 5, 2008
Journal of Experimental Biology 211, 2899-2900 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012476
JEB Classics |
TREHALOSE AND ANHYDROBIOSIS: THE EARLY WORK OF J. S. CLEGG
University of California, Davis
jhcrowe{at}ucdavis.edu
|
), at which water
contents metabolism as we understand it ceases
(Clegg, 1973). The dry cysts
persist in this state, known as `anhydrobiosis' for decades (reviewed in
Clegg and Conte, 1980), but
when they are rehydrated they rapidly imbibe water and resume active
metabolism and development. Following further development, they then emerge
from the cysts as free-swimming nauplii. Many of the important papers on these
interesting animals have appeared in The Journal of Experimental
Biology, including this JEB Classic from my friend and colleague Jim
Clegg, in which he studied the metabolic events occurring during the
resumption of development, implicating trehalose as a key molecule involved in
the survival of desiccation, which eventually sparked a revolution in the
preservation of biological systems.
It was already known that Artemia cysts are capable of undergoing
development at a variety of salinities in nature. Clegg found under carefully
controlled laboratory conditions that development proceeded normally under a
wide range of external osmotic pressures; at pressures up to 30 atm (1 atm is
101 kPa) the only effect on development was to decrease the rate, but not
the final success at emergence. Some cysts were seen to complete development
at pressures as high as 65 atm. This surprising discovery clearly meant that
when the cysts were exposed to elevated external osmotic pressures they most
likely balanced the osmotic gradient by synthesis of solutes, so he next
studied the metabolic fates of the predominant osmotically active solutes
– glycerol and trehalose – during development. He found that
trehalose was utilized as a metabolite and at low external osmotic pressures
was converted to glycogen. However, at high external osmotic pressures the
trehalose was converted to glycerol, and under all hyperosmotic conditions a
large net synthesis of glycerol was observed prior to emergence. So why was
all this glycerol synthesized? Clegg suggested that not only was the glycerol
used as an osmotic effector, but also it was accumulated near the end of
development, thus increasing internal osmolarity to the point where the cysts
swelled and ruptured, permitting emergence of the nauplii
(Clegg, 1964).
In a closely related paper published the following year he studied
metabolism in Artemia embryos of two types: one enters a dormant
stage, encysts, and can survive dehydration; the other undergoes direct
development without a dormant stage and does not survive drying
(Clegg, 1965). He discovered in
this technically difficult study that the pre-dormant cysts synthesized large
quantities of trehalose (in fact, they converted as much as 15% of their dry
mass to this sugar), while the non-dormant ones contained essentially no
trehalose. The dormant and non-dormant cysts also contained glycerol, but the
dormant ones had about twice as much as the non-dormant ones. He speculated at
the time that the trehalose was an energy source and was utilized mainly
because of its high stability, but he offered no explanation for the presence
of the massive amounts of glycerol other than for osmotic rupturing of the
cyst at the completion of development. However, the clear correlation between
the presence of glycerol and trehalose in the dormant cysts and survival in
the dry state suggested in retrospect that these molecules might be related to
the cysts' ability to survive drying. At the time, the thinking of all of us
interested in the phenomenon of anhydrobiosis was influenced by the well-known
discovery in the field of cryobiology that glycerol is a potent
cryoprotectant, so we assumed – incorrectly, as it turned out –
that glycerol would be the important molecule in stabilizing biological
structures during anhydrobiosis.
Where did Clegg go from there? He continued to work with Artemia,
but mostly as a model for investigations on the status of intracellular water,
profoundly influencing our understanding of metabolic complexes and the
channeling of metabolism (see Clegg,
1991a; Clegg,
1991b). His work from the 1960s
(Clegg, 1964;
Clegg, 1965), which gave us an
interesting metabolic and developmental picture in its own right, might well
have been forgotten, except that it turned out to have more general
significance than he realized at the time.
A decade passed before much more progress was made in sorting out the roles
of glycerol and trehalose. In the 1970s we discovered that biological
membranes can be stabilized during drying in the presence of trehalose, but
not glycerol; in fact, trehalose was clearly superior to every other sugar
tested [but see some addenda to these findings in Crowe et al.
(Crowe et al., 2001)]. We and
others then promptly found that liposomes and proteins can be preserved by
drying with trehalose, both of which findings led to many commercial products,
particularly in the pharmaceutical industry. For instance, Ambisome is a
liposomal based therapeutic agent for treating systemic fungal infections that
is delivered as a dry powder, using technology that came out of our studies on
the stabilization of liposomes by trehalose.
By the mid-1980s we had worked out a possible mechanism for these
stabilizing effects, which is still being tested experimentally and by
molecular dynamics (reviewed in Crowe,
2007). Based on experimental biophysical data, we proposed that
trehalose serves as a water replacement in the dry state, conferring on
membranes and proteins physical properties that resemble those seen in the
fully hydrated state. Further, we proposed that this effect involves direct
interaction between the trehalose and polar groups on membrane lipids and
proteins by hydrogen bonding, a suggestion that came to be known as the water
replacement hypothesis. Experimental data have shown that higher order
structures in membranes such as rafts can be stabilized by drying with
trehalose, a phenomenon that can be explained by the water replacement
hypothesis (Leidy et al.,
2004).
In fact, the level of complexity that can be preserved in the dry state is
startling; we have applied these findings to human blood cells, particularly
platelets. We found a way of introducing trehalose into platelets and
discovered that the trehalose-loaded platelets survive freeze drying, with a
greatly extended shelf life. The rehydrated platelets show complex responses
such as Ca2+ and H+ transport in response to agonists
and clot formation (reviewed in Crowe et
al., 2001; Crowe,
2007). These higher order responses require that several receptor
and signaling processes be maintained intact. Numerous laboratories around the
world are involved in developing this technology for the preservation of a
variety of cells of interest in biology, agriculture and medicine, and several
trehalose-stabilized freeze-dried products are currently in various stages of
development and clinical trials for human use.
In recent years, Clegg returned to the field of cellular preservation, and
his discovery and purification of certain stress proteins in Artemia
(e.g. Clegg, 2005) are
beginning to play a role in the stabilization of eukaryotic cells in the
presence of trehalose (Ma et al.,
2005). One word of caution: trehalose is being applied in many
areas where it is inappropriate; for example, the cosmetics industry is adding
trehalose to their products, often with no clear rationale for doing so. Also,
while trehalose does have some special properties that make it useful
(reviewed in Crowe et al.,
2001; Crowe, 2007),
the same result can be achieved with other sugars under ideal conditions.
Clegg's papers from the early 1960s pointed the way to a new field of inquiry that is currently remarkably active, with implications as far ranging as ecology and human medicine. His 1964 paper is a classic in many realms, which will continue to inspire.
Footnotes
John Crowe discusses Jim Clegg's 1964 paper: The control of emergence and metabolism by external osmotic pressure and the role of free glycerol in developing cysts of Artemia salina. A copy of the paper can be obtained from http://jeb.biologists.org/cgi/reprint/41/4/879.
References
Clegg, J. S. (1964). The control of emergence
and metabolism by external osmotic pressure and the role of free glycerol in
developing cysts of Artemia salina. J. Exp. Biol.
41,879
-892.
Clegg, J. S. (1965). The origin of trehalose and its significance during the formation of encysted dormant embryos of Artemia salina. Comp. Biochem. Physiol. 14,135 -143.[Medline]
Clegg, J. S. (1973). Do dried cryptobiotes have a metabolism? In Anhydrobiosis (ed. J. H. Crowe and J. S. Clegg), pp. 141-146.
Clegg, J. S. (1978). Residual water content of dried but viable cells. Experientia, 34,734 -735.[CrossRef][Medline]
Clegg, J. S. (1991a). Metabolic organization and ultrastructure of animal cells. Biochem. Soc. Trans. 19,985 -986.[Medline]
Clegg, J. S. (1991b). The physiological significance of metabolite channeling – an idea whose time has come. J. Theor. Biol. 152,63 -64.[CrossRef][Medline]
Clegg, J. S. (2005). Desiccation tolerance in encysted embryos of the animal extremophile, Artemia. Integr. Comp. Biol. 45,715 -724.[CrossRef]
Clegg, J. S. and F. Conte (1980). A review of the cellular and developmental biology of Artemia. In The brine shrimp, Artemia, vol. 2 (ed. G. P. Persoone, P. Sorgeloos, O. Roels and E. Jaspers), pp.11 -54. Wetteren, Belgium: Universa Press.
Crowe, J. H. (2007). Trehalose as a chemical chaperone: fact and fantasy. Adv. Exp. Med. Biol. 595,143 -158.
Crowe, J. H., Crowe, L. M., Oliver, A. E. and Tablin, F. (2001). The trehalose myth revisited: introduction to a symposium on stabilization of cells in the dry state. Cryobiology 43,89 -105.[CrossRef][Medline]
Leidy, C., Gousset, K., Ricker, J., Wolkers, W. F., Tsvetkova, N. M., Tablin, F. and Crowe, J. H. (2004). Lipid phase behavior and stabilization of domains in membranes of platelets. Cell Biochem. Biophys. 40,123 -148.[CrossRef][Medline]
Ma, X., Jamil, K., Macrae, T. H., Clegg, J. S., Russell, J. M., Villeneuse, T. S., Euloth, M., Sun, Y., Crowe, J. H., Tablin, F. and Oliver, A. E. (2005). A small stress protein acts synergistically with trehalose to confer desiccation tolerance on mammalian cells. Cryobiology, 51,15 -28.[CrossRef][Medline]
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