X-Message-Number: 32253
Date: Thu, 24 Dec 2009 20:10:08 -0800 (PST)
From:
Subject: Cucujus clavipes has mastered vitrification
[In addition to antifreeze proteins, the red flat bark beetle (Cucujus
clavipes) owes its ability to survive liquid nitrogen vapour temperatures
(-150 C) to 10 mol glycerol. Obviously this animal has developed a
complete and comprehensive solution to cryoprotectant toxicity. Note that,
unlike most other cryoprotectants, glycerol does not denature proteins,
so this mode of toxicity is not an issue. Glycerol's known modes of
toxicity include iron catalyzed free radical formation, and osmotic
shock. This animal achieves 10 mol glycerol by a process of gradual
partial dehydration, which could plausibly eliminate osmotic
shock. Presumably Cucujus clavipes also sequesters iron in some fashion
similar to that of quercetin which protects against glycerol induced
renal failure in rats. After evolving ultimate cold hardiness over
millions of years, Cucujus clavipes has a lot to teach about how to avoid
vitrification solution toxicity.]
http://www.gi.alaska.edu/ScienceForum/ASF18/1877.html
Alaska Science Forum
October 17, 2007
Alaska beetles survive 'unearthly' temperatures
Article #1877
by Ned Rozell
This column is provided as a public service by the Geophysical Institute,
University of Alaska Fairbanks, in cooperation with the UAF research community.
Ned Rozell is a science writer at the institute.
As we pull on winter coats and wool hats to shield our tropical bodies from the
cold, there is a creature in our midst that survives Alaska's coldest
temperatures bare-naked.
The red flat bark beetle lives as far north as there are balsam poplar trees in
Alaska, hunkering down for the winter in the moist area between dead bark and
tree. Scientists like Todd Sformo, from the University of Alaska's Institute of
Arctic Biology, find most of them in the larval stage, where they resemble
segmented worms a bit longer than a grain of rice. He finds a smaller number of
adults that have handsome segmented bodies the color of teak.
The beetles are special among living things in Alaska because they have the
ability to spend the winter above the snow, exposed to the coldest air of
winter. Sformo, a graduate student working in Professor Brian Barnes' lab, has
cooled the beetles to minus 94 degrees Fahrenheit (minus 70 degrees Celsius) in
the lab, and they have not died. Yellowjackets, stinkbugs, and other insects
that survive winter using the same strategy, known as supercooling, perish at
about minus 13 degrees Fahrenheit (minus 25 degrees Celsius).
"They really have to be under that leaf litter and under the snow (for
insulation from the cold air)," Sformo said.
How cold can the bark beetles get? Sformo shipped a few of the beetles to a lab
in California to find out. The owner of the lab lowered the beetles to a
temperature of about 238 degrees below zero (minus 150 degrees Celsius), and
they didn't freeze. The lowest temperature recorded on Earth was minus 129
degrees Fahrenheit recorded in Antarctica in 1983. Alaska's all-time low is
minus 80 degrees Fahrenheit at Prospect Creek, off the Dalton Highway in 1971.
"Under the right conditions, these things don't freeze, even when you get down
to unearthly temperatures," Sformo said.
The beetles survive by being one of the most successful northern practitioners
of supercooling, where they are able to resist freezing by combining a few
unique talents. Beginning in August, they begin to produce antifreeze proteins
that bind to sites where ice might form. Later in the fall, they produce
glycerol that drives their freezing point down as antifreeze does in a car, and,
finally, they begin to lose water in their bodies.
Sformo and colleagues at the University of Notre Dame also have studied another
Alaska beetle that's nearly as good in the cold as the flat bark beetle. The
larger beetle Upis ceramboides, which has no common name, survives the winter in
dry crevices in trees. Unlike the flat bark beetle, the Upis beetle tolerates
freezing rather than avoiding it. Sformo found that the Upis beetles froze at
about 18.5 degrees Fahrenheit (minus 7.5 degrees Celsius) in the lab and
survived temperatures down to about 104 degrees below zero (minus 76 degrees
Celsius).
"Using opposite strategies, we have two beetles that can survive to the minus
70s (Celsius)," he said.
The Upis beetles have the ability to force water outside their cells, where it
freezes without puncturing cell membranes. They seek out dry places to spend the
winter, which makes the contrasting choice of the red flat bark beetles to
crawl under moist bark seem remarkable, Sformo said. The bodies of red flat
beetles often are stuck to ice, the best substance known to trigger freezing.
"These insects, in winter, are in direct contact with ice crystals, for months,"
he said. "You can't get a better nucleator than that."
The proteins created by the red flat bark beetle fascinate scientists because
they are ingredients of an all-natural antifreeze liquid, a version of which
might someday be used to cool human organs to preserve them. Other applications
that might be teased from the secrets of Alaska's beetles are a non-toxic
de-icing solution for aircraft, a concrete that builders can pour in the cold,
or an ingredient that would prevent the re-crystallization of ice cream, which
makes large crystals form inside an open pint.
J Exp Biol. 2005 Dec;208(Pt 23):4467-77.
Comparative overwintering physiology of Alaska and Indiana populations of the
beetle Cucujus clavipes (Fabricius): roles of antifreeze proteins, polyols,
dehydration and diapause.
Bennett VA, Sformo T, Walters K, Toien O, Jeannet K, Hochstrasser R, Pan Q,
Serianni AS, Barnes BM, Duman JG. Department of Biological Sciences, University
of Notre Dame, Notre Dame, IN 46556, USA.
The beetle Cucujus clavipes is found in North America over a broad
latitudinal range from North Carolina (latitude approximately 35 degrees N)
to near tree line in the Brooks Range in Alaska (latitude, approximately 67
degrees 30' N). The cold adaptations of populations from northern Indiana
(approximately 41 degrees 45' N) and Alaska were compared and, as expected,
the supercooling points (the temperatures at which they froze) of these
freeze-avoiding insects were significantly lower in Alaska insects. Both
populations produce glycerol, but the concentrations in Alaska larvae were
much higher than in Indiana insects (approximately 2.2 and 0.5 mol l(-1),
respectively). In addition, both populations produce antifreeze proteins.
Interestingly, in the autumn both populations have the same approximate
level of hemolymph thermal hysteresis, indicative of antifreeze protein
activity, suggesting that they synthesize similar amounts of antifreeze
protein. A major difference is that the Alaska larvae undergo extreme
dehydration in winter wherein water content decreases from 63-65% body water
(1.70-1.85 g H2O g(-1) dry mass) in summer to 28-40% body water (0.40-0.68
g H2O g(-1) dry mass) in winter. These 2.5-4.6-fold reductions in body water
greatly increase the concentrations of antifreeze in the Alaska insects.
Glycerol concentrations would increase to 7-10 mol l(-1) while thermal
hysteresis increased to nearly 13 degrees C (the highest ever measured in
any organism) in concentrated hemolymph. By contrast, Indiana larvae do not
desiccate in winter. The Alaska population also undergoes a diapause while
insects from Indiana do not. The result of these, and likely additional,
adaptations is that while the mean winter supercooling points of Indiana
larvae were approximately -23 degrees C, those of Alaska larvae were -35 to
-42 degrees C, and at certain times Alaska C. clavipes did not freeze when
cooled to -80 degrees C.
PMID: 16339867
[It is not difficult to protect against glycerol toxicity.]
Pharmacology. 2005 Jan;73(1):49-56. Epub 2004 Sep 27.
Reversal of experimental myoglobinuric acute renal failure in rats by quercetin,
a bioflavonoid.
Chander V, Singh D, Chopra K. Division of Pharmacology, University Institute of
Pharmaceutical Sciences, Panjab University, Chandigarh, India.
The occurrence of acute renal failure (ARF) following rhabdomyolysis has
been put at between 10 and 40% of cases, and accounts for between 3 and 15%
of all cases of ARF. Reactive oxygen intermediates have been demonstrated to
play an etiological role in myoglobinuric renal failure. This study was
performed to explore the protective effect of quercetin, a bioflavonoid, in
an experimental model of myoglobinuric ARF in rats. Four groups of rats were
employed in this study: group 1 served as control, group 2 was given 50%
glycerol (8 ml/kg, i.m.), group 3 was given glycerol + quercetin (2 mg/kg,
i.p.), and group 4 was given glycerol + DMSO (the solvent for quercetin, 5
ml/kg, i.p.). Renal injury was assessed by measuring serum creatinine, blood
urea nitrogen, creatinine and urea clearance. The oxidative stress was
measured by renal malondialdehyde levels, reduced glutathione levels and by
enzymatic activity of catalase, glutathione reductase, and superoxide
dismutase. Glycerol administration resulted in a marked renal oxidative
stress, significantly deranged the renal functions as well as renal
cytoarchitecture. All these factors were significantly improved by quercetin
treatment. Because of its radical-scavenging and iron-chelating properties,
quercetin protected the kidney against the glycerol-induced oxidative
stress and resultant renal dysfunction. Based on these results, this study
confirms the role of oxidative stress and demonstrates the renoprotective
potential of quercetin in this rhabdomyolysis-mimicking model. 2005 S.
Karger AG, Basel.
PMID: 15452363
[An honourable mention here of the freeze-tolerant Alaskan beetle Upis
ceramboides.]
Biological antifreeze keeps hearty beetle alive through -75C winters
Canwest News Service Published: Friday, December 18, 2009
Canadians who have found themselves caught in a deep freeze by recent
temperatures have a thing or two to learn from an Alaskan beetle, similar to
the one pictured. According to a recent study from the University of Alaska
Fairbanks Institute of ArcticBiology, the Alaska Upis beetle can survive
temperatures as low as -75C. The beetle survives with a unique type of
biological antifreeze that prevents ice crystals from forming and penetrating
its cells. This research could eventually lead to the possibility of freezing
tissues or organs for prolonged storage, according to Brian Barnes, director of
the Institute of Arctic Biology. Unlike other biological antifreezes that have
been discovered, the type found in the beetle has little or no protein.
Instead, it comes from the same fatty acid that cell membranes do.
Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20210-5. Epub 2009 Nov 23.
A nonprotein thermal hysteresis-producing xylomannan antifreeze in the
freeze-tolerant Alaskan beetle Upis ceramboides.
Walters KR Jr, Serianni AS, Sformo T, Barnes BM, Duman JG. Department of
Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA.
Thermal hysteresis (TH), a difference between the melting and freezing
points of a solution that is indicative of the presence of
large-molecular-mass antifreezes (e.g., antifreeze proteins), has been
described in animals, plants, bacteria, and fungi. Although all previously
described TH-producing biomolecules are proteins, most thermal hysteresis
factors (THFs) have not yet been structurally characterized, and none have
been characterized from a freeze-tolerant animal. We isolated a highly
active THF from the freeze-tolerant beetle, Upis ceramboides, by means of
ice affinity. Amino acid chromatographic analysis, polyacrylamide gel
electrophoresis, UV-Vis spectrophotometry, and NMR spectroscopy indicated
that the THF contained little or no protein, yet it produced 3.7 +/- 0.3
degrees C of TH at 5 mg/ml, comparable to that of the most active insect
antifreeze proteins. Compositional and structural analyses indicated that
this antifreeze contains a beta-mannopyranosyl-(1-->4) beta-xylopyranose
backbone and a fatty acid component, although the lipid may not be
covalently linked to the saccharide. Consistent with the proposed structure,
treatment with endo-beta-(1-->4)xylanase ablated TH activity. This
xylomannan is the first TH-producing antifreeze isolated from a
freeze-tolerant animal and the first in a new class of highly active THFs
that contain little or no protein.
PMID: 19934038 [PubMed - in process]
J Biol Chem. 2009 Jun 19;284(25):16822-31. Epub 2009 Apr 29.
Cryoprotectant biosynthesis and the selective accumulation of threitol in the
freeze-tolerant Alaskan beetle, Upis ceramboides.
Walters KR Jr, Pan Q, Serianni AS, Duman JG. Department of Biological Sciences,
University of Notre Dame, Notre Dame, Indiana 46556, USA.
Adult Upis ceramboides do not survive freezing in the summer but tolerate
freezing to -60 degrees C in midwinter. The accumulation of two
cryoprotective polyols, sorbitol and threitol, is integral to the
extraordinary cold-hardiness of this beetle. U. ceramboides are the only
animals known to accumulate high concentrations of threitol; however, the
biosynthetic pathway has not been studied. A series of (13)C-labeled
compounds was employed to investigate this biosynthetic pathway using
(13)C{(1)H} NMR spectroscopy. In vivo metabolism of (13)C-labeled glucose
isotopomers demonstrates that C-3-C-6 of glucose become C-1-C-4 of threitol.
This labeling pattern is expected for 4-carbon saccharides arising from the
pentose phosphate pathway. In vitro experiments show that threitol is
synthesized from erythrose 4-phosphate, a C(4) intermediate in the PPP.
Erythrose 4-phosphate is epimerized and/or isomerized to threose
4-phosphate, which is subsequently reduced by a NADPH-dependent polyol
dehydrogenase and dephosphorylated by a sugar phosphatase to form threitol.
Threitol 4-phosphate appears to be the preferred substrate of the sugar
phosphatase(s), promoting threitol synthesis over that of erythritol. In
contrast, the NADPH-dependent polyol dehydrogenase exhibits broad substrate
specificity. Efficient erythritol catabolism under conditions that promote
threitol synthesis, coupled with preferential threitol biosynthesis, appear
to be responsible for the accumulation of high concentrations of threitol
(250 mm) without concomitant accumulation of erythritol.
PMID: 19403530
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