X-Message-Number: 32521
Date: Mon, 29 Mar 2010 19:41:21 -0800 (PST)
From:
Subject: vitrification - the little beetle larvae that could
[Glycerol is one of the "miracle" ingredients responsible for an
amazing feat of natural bioengineering (vitrification) by the Alaskan
beetle larvae Cucujus clavipes. Glycerol is interesting because its
toxicity profile does not include protein denaturation, which is a
limiting factor for many cryoprotectants (eg: DMSO), which are commonly
used in human engineered vitrification solutions. Glycerol itself has seen
relatively little use in vitrification solutions, possiblely due to its
higher risk of osmotic toxicity. Also the higher viscosity of glycerol
would limit the concentration that can be used with organ
cryopreservation. As with beetles, partial slow dehydration would be
required to achieve organ vitrification.]
[Note: Authors include Wowk B, and Fahy GM. Beetle survival was low at
-100C, but this was believed to be due to manhandling of the
specimens causing physical damage. Freezing did not occur in specimens
cooled to -150 C, which confirmed vitrification had occurred.]
J Exp Biol. 2010 Feb;213(Pt 3):502-9.
Deep supercooling, vitrification and limited survival to -100{degrees}C in the
Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae.
Sformo T, Walters K, Jeannet K, Wowk B, Fahy GM, Barnes BM, Duman JG. Institute
of Arctic Biology, University of Alaska, Fairbanks, AK 99775, USA.
Larvae of the freeze-avoiding beetle Cucujus clavipes puniceus (Coleoptera:
Cucujidae) in Alaska have mean supercooling points in winter of -35 to -42
degrees C, with the lowest supercooling point recorded for an individual of
-58 degrees C. We previously noted that some larvae did not freeze when
cooled to -80 degrees C, and we speculated that these larvae vitrified. Here
we present evidence through differential scanning calorimetry that C. c.
puniceus larvae transition into a glass-like state at temperatures<-58
degrees C and can avoid freezing to at least -150 degrees C. This novel
finding adds vitrification to the list of insect overwintering strategies.
While overwintering beneath the bark of fallen trees, C. c. puniceus larvae
may experience low ambient temperatures of around -40 degrees C (and lower)
when microhabitat is un-insulated because of low snow cover. Decreasing
temperatures in winter are correlated with loss of body water from summer
high levels near 2.0 to winter lows near 0.4 mg mg(-1) dry mass and
concomitant increases in glycerol concentrations (4-6 mol l(-1)) and thermal
hysteresis. Finally, we provide direct evidence that Cucujus from Wiseman,
Alaska, survive temperatures to -100 degrees C.
PMID: 20086136 [PubMed - in process]
[Glycerol has been successfully incorporated into a few human engineered
vitrification solutions.]
Cryobiology. 2007 Oct;55(2):148-57. Epub 2007 Jul 4.
Cryopreservation of Radopholus similis, a tropical plant-parasitic nematode.
Elsen A, Vallterra SF, Van Wauwe T, Thuy TT, Swennen R, De Waele D, Panis B.
Laboratory of Tropical Crop Improvement, Department of Biosystems, Faculty of
Bioscience Engineering, KU Leuven, Kasteelpark Arenberg 13, 3001, Leuven,
Belgium.
For obligate plant-parasitic nematodes, cryopreservation has advantages over
the usual preservation methods on whole plants or axenic culture systems,
because the latter two are labourious and time and space consuming. In
addition, cross contamination among different isolates can occur easily.
Moreover, specific genetic studies require maintenance of the original
population. The nematode under investigation, Radopholus similis, is a
plant-parasitic nematode from the humid tropics. Therefore, any treatment at
low temperatures is likely to add extra stress to the nematode, making the
development of a cryopreservation protocol extremely difficult. In this
paper, we describe experiments to achieve a successful cryopreservation
protocol for the tropical nematode R. similis using vitrification
solution-based methods based on a well defined mixture of cryoprotectants in
combination with ultra-rapid cooling and thawing rates. A two-step
treatment was used consisting of an incubation in glycerol followed by the
application of a vitrifying mixture of methanol, glycerol and glucose. After
cryopreservation, the pathogenicity of the nematodes was not altered, since
they could infect and reproduce on carrot discs after recovery in the
Ringer solution. The cryopreservation method described can be used for
routine cryopreservation of R. similis lines from different origins.
PMID: 17707790
[A glycerol/methanol combination gave the best results for flounder
embryos. Methanol probably acted to reduce the osmotic toxicity of
glycerol.]
Theriogenology. 2005 Feb;63(3):763-73.
Toxicity and protective efficiency of cryoprotectants to flounder (Paralichthys
olivaceus) embryos.
Zhang YZ, Zhang SC, Liu XZ, Xu YJ, Hu JH, Xu YY, Li J, Chen SL. Department of
Marine Biology, Ocean University of China, Qingdao 266003, PR China.
With the purpose of finding an ideal cryoprotectant or combination of
cryoprotectants in a suitable concentration for flounder (Paralichthys
olivaceus) embryo cryopreservation, we tested the toxicities, at culture
temperature (16 degrees C), of five most commonly used
cryoprotectants-dimethyl sulfoxide (Me2SO), glycerol, methanol (MeOH),
1,2-propylene glycol (PG) and ethylene glycol (EG). In addition,
cryoprotective efficiency to flounder embryos of individual and combined
cryoprotectants were tested at -15 degrees C for 60 min. Five different
concentrations of each of the five cryoprotectants and 20 different
combinations of these cryoprotectants were tested for their protective
efficiency. The results showed that the toxicity to flounder embryos of the
five cryoprotectants are in the following sequence: PG < MeOH < Me2SO <
glycerol < EG (P < 0.05); whereas the protective efficiency of each
cryoprotectant, at -15 degrees C for a period of 60 min, are in the
following sequence: PG > Me2SO approximately MeOH approximately glycerol >
EG (greater symbols mean P < 0.05, and approximate symbols mean P > 0.05).
Methanol combined with any one of the other cryoprotectants gave the best
protection, while ethylene glycol combined with any one of the other
cryoprotectants gave the poorest protection at -15 degrees C. Toxicity
effect was concentration dependent with the lowest concentration being the
least toxic for all five cryoprotectants at 16 degrees C. For PG, MeOH and
glycerol, 20% solutions gave the best protection at -15 degrees C; whereas a
15% solution of Me2SO, and a 10% solution of EG, gave the best protection
at -15 degrees C.
PMID: 15629795
[Glycerol is never used by itself in vitrification solutions.]
J Reprod Fertil. 1993 Nov;99(2):471-7.
Design of vitrification solutions for the cryopreservation of embryos.
Ali J, Shelton JN. John Curtin School of Medical Research, Australian National
University, Canberra.
A series of experiments was performed to determine the concentrations at
which ten cryoprotectants singly and in pairs would vitrify on plunging into
liquid nitrogen and remain vitreous when warmed by plunging into a water
bath at 25 degrees C. From these tests eight solutions (VS) were selected
for testing of toxicity to mouse morulae in vitro. One of these (VS1) was
modified as a further five VS of which one (VS11) was tested for toxicity to
all stages of mouse embryos and to sheep compacted morulae. The
concentrations at which the cryoprotectants vitrified on cooling were:
butylene glycol, 3.0 mol l-1; propylene glycol, 4.0 mol l-1; dimethyl
sulfoxide (DMSO) and glycerol 5.0 mol l-1; ethylene glycol, 6.5 mol l-1.
None of these, at the highest concentration tested, remained vitreous during
warming. Methanol and the high molecular weight polymers, dextran, Ficoll,
polyethylene glycol and polyvinylpyrrolidone, did not vitrify at the
concentrations tested. Toxicity studies showed the order of increasing
toxicity to be ethylene glycol, methanol, DMSO, glycerol, propylene glycol
and butylene glycol. Of the mixtures composed of two cryoprotectants, those
containing ethylene glycol and glycerol were the least toxic at vitrifying
concentrations. VS11 (6.0 mol ethylene glycol l-1 and 1.8 mol glycerol l-1)
was well tolerated by mouse morulae, less well by eight- and one-cell
embryos and poorly by two-cell embryos. Dilution of the VS11 from mouse
embryos by exposure to 1.0 mol sucrose l-1 for 10 min did not enhance their
survival.(ABSTRACT TRUNCATED AT 250 WORDS)
PMID: 8107029
Vet Med (Praha). 1991 Oct;36(10):585-92.
[Comparison of various cryopreservation media for vitrification of 7-day bovine
embryos]
[Article in Czech]
Liehman P. Fyziologicky ustav CSAV, Praha.
A vitrification medium, attested for cryopreservation of mouse
eight-blastomere embryos (6.85 mol/l glycerol as a cryoprotective agent),
was checked up with respect to preservation of bovine seven-day morulas and
blastocysts. As this medium was not found to be convenient either as to its
technical parameters or the reached embryo survival, its composition was
modified. The glycerol content was complemented or partly replaced by other
cryoprotectives (methanol, saccharose, L-proline). A comparison of technical
parameters and viability of rewarmed embryos shows that our requirements
(technically simple technique of freezing and rewarming and good survival)
are met in the best way by a cryoprotective combination of 4.11 mol/l
glycerol and 1.0 mol/l saccharose. The total of 54.5% embryos developed
in-vitro conditions following vitrification in the medium of this
composition and after rewarming.
PMID: 1807015
[Glycerol induced damage can also be reduced by some antioxidants.]
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
Folia Microbiol (Praha). 2009;54(3):230-2. Epub 2009 Aug 2.
Viability of commercial wine yeasts during freezer storage in glycerol-based
media.
Sidari R, Caridi A. Dipartimento di Scienze e Tecnologie Agro-Forestali e
Ambientali, Universita degli Studi Mediterranea di Reggio Calabria, Reggio
Calabria, Italy.
Glycerol-based medium (BM) with and without the addition of 1 g/L ascorbic
acid (Asc) and/or 100 mg/L (+/-)-catechin (Cat) was tested for the storage
of three commercial wine yeasts at -20 degrees C. The medium supplemented
with Asc was also used to store 706 strains to verify the maintenance of the
liquid state. A decline in survival throughout the storage period was
observed. The media containing Asc maintained viability better than the
other three. The BM caused a loss of viability of 7 orders for one strain
and of 6 orders for the other two. All three strains exhibited a loss of
viability of 4 orders when stored in BM+Asc. Two strains decreased viability
by 5 orders while one strain by 4 orders, when stored in BM+Cat. Two
strains decreased viability by 6 orders while one strain by 5 orders, when
stored in BM+Asc+Cat. Regarding the physical state of the medium tested on
706 yeast strains, three cases were observed: completely liquid (56.5 %),
liquid with only the upper part frozen (40.4 %) without involving the yeast
biomass settled at the bottom, and completely frozen (3.12 %). It is
practicable to prepare a BM that remains liquid at -20 degrees C enhancing
yeast viability when Asc is added as cryoprotectant.
PMID: 19649740
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