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162. Identification of microRNA (miRNA) expression in hibernating thirteen-lined ground squirrels, Spermophilus tridecemlineatus
Abstracts / Cryobiology 55 (2007) 324–378 CO (1998) 235-268]. For this reason, we examined the post-storage quality of the parental through the F2 generation of G. ashmeadi using the stepwise cooling regime. Our studies indicated that GWSS eggs parasitized by G. ashmeadi can be stored up to 20 days without affecting emergence, survival, development, or progeny quality through the F2 generation. After 30 d of storage, survival of the parental generation was decreased by 24%, the developmental time was extended 4 d, and the fecundity of the F1 females decreased by 32%. After 40 d of storage, only 12% of the wasps emerged from their host eggs, 44% of the females were sterile, and the production of males by the non-sterile females increased by 155%. The F1 female progeny of these stored parents subsequently exhibited a 70% reduction in parasitism of host eggs and a 73% decrease in overall fecundity. Chilling also delayed the peak emergence of the parents but not of either the F1 or F2 generations. Examination of the F1 and F2 populations by demographic analysis also confirmed that the F1 generation was affected by 30 d of chilling of the parents but the damage did not extend to the F2 generation. Expression of reduced fitness by progeny of parents exposed to a stressful environment has been observed in a wide variety of organisms (e.g. maternal effects). Most studies report exposure of an environmental stress to the adult parent and this damage is passed to the progeny. With G. ashmeadi, the exposure to cold stress occurs while the wasps are still immature and within their hosts. Whether the damage is directly imparted to the developing wasps or indirectly through a damaged host has yet to be resolved. ((Conflicts of interest: None declared. Source of funding: USDA-ARS.) doi:10.1016/j.cryobiol.2007.10.163
161. Protein expression patterns and low temperature adaptation in antarctic alga, Chaetoceros neogracile. Koang-Man Choi a, Seul-Ki Park a, EonSeon Jin b, Mi-Young Lee a, a Department of Biotechnology, Soonchunhyang University, Asan, Chungnam, 336-600, Korea; b Department of Life Science, Hanyang University, Seoul 133-743, Korea In order to better understand the survival mechanism of antarctic alga in the low temperature, protein expression patterns in the antarctic alga, Chaetoceros neograciles were examined. Standard 2-dimensional maps for the proteins of Chaetoceros neograciles grown at 4 °C were obtained. Differentially expressed proteins of Chaetoceros neograciles following transient cold-shock or heat-shock were examined by a proteomic technique. Proteomics is a powerful tool to describe the alterations in protein expression. The differentially expressed proteins were identified by MALDI-TOF mass spectrometry. Changes in the total activities and isozyme patterns of various antioxidant enzymes such as superoxide dismutase, glutathione reductase and glutathione transferase were also examined following temperature stress treatment. (Conflicts of interest: None declared. Source of funding: A grant from Korea Science and Engineering Foundation, R01-2006-000-10856-0). doi:10.1016/j.cryobiol.2007.10.164
Hibernation and hypothermia 162. Identification of microRNA (miRNA) expression in hibernating thirteen-lined ground squirrels, Spermophilus tridecemlineatus. Adrian Dubuc, Kenneth B. Storey., Carleton University, Ottawa, ON, Canada MicroRNAs (miRNAs) are known to down-regulate mRNA expression levels, thereby constituting a pivotal role in all major cellular functions, including differentiation and development, metabolism, and insulin secretion. This broad-based regulation led to the hypothesis that miRNA may constitute a vital role in hibernation. Characterization of miRNA expression levels in muscle and liver tissue provided a miRNAsignature for the entry into a state of torpor in Spermophilus tridecemlineatus. The long term objective of this study includes the elucidation of miRNA involvement in the maintenance of low-temperature viability,
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necessary for increased longevity of human organ storage capabilites. Reverse Transcriptase (RT)-PCR examination of miR-15a, miR-21, miR-122a and miR-146 in the liver and miR-143, miR-146 and miR-206 in skeletal muscle of euthermic versus hibernating animals displayed stable expression. However, miR-122a was significantly reduced by 0.76-fold in muscle of hibernating ground squirrels. This change indicates a potential role for miRNA-silencing of hibernator genes. (Conflicts of interest: None declared. Source of funding: NSERC Canada.) doi:10.1016/j.cryobiol.2007.10.165
163. Regulation of AKT in Richardson’s ground squirrels during hibernation. Khalil Abnous, Christopher A. Dieni, Kenneth B. Storey, Carleton University, Ottawa, ON, Canada Hibernation is a winter survival strategy for many small mammals metabolic rate falls by >95%, core body temperature can approach 0 °C, and all physiological functions are suppressed. Energy savings of 90% are achieved compared with the costs of remaining euthermic (37 °C) over the winter. The protein kinase Akt (also known as protein kinase B) plays a central role in coordinating growth and survival responses in cells and we hypothesized that regulation of Akt would be critical in hibernation. Kinetic properties of muscle Akt were compared in euthermic versus hibernating states; total and phosphorylated Akt protein was also quantified by Western blotting. Total Akt protein did not change in hibernation but phospho-Akt (the active form) and measured Akt activity decreased significantly by 40% and 60%, respectively, compared with euthermic controls. S0.5 values for Akt peptide fell by 28% during hibernation whereas S0.5 ATP increased by 330%. Low temperature assay (10 °C) strongly affected S0.5 ATP of euthermic Akt (a 350% increase). Akt activation energy (Ea) and sensitivity to denaturation by urea did not change between euthermia and hibernation. DEAE Sephadex chromatography showed three peaks (isozymes) of Akt in euthermic muscle but only two peaks during hibernation. The results document differential regulation of Akt during hibernation and suggest a key role for Akt in cell survival during torpor. (Conflicts of interest: None declared. Source of funding: NSERC Canada.) doi:10.1016/j.cryobiol.2007.10.166
164. Analysis of mRNA and protein levels of PPARa and H-FABP in heart during hibernation of thirteen-lined ground squirrels. Melanie Bouffard, Tarek Abd El Halim, Kenneth B. Storey, Carleton University, Ottawa, ON, Canada Lipid catabolism is the main source of energy for hibernating mammals. Fatty acid binding proteins (FABPs) are small, highly conserved cytoplasmic proteins that have roles in fatty acid uptake and intracellular lipid transport and metabolism. The peroxisome proliferator-activated receptor alpha (PPARa) is a transcription factor that regulates expression of various genes of fatty acid catabolism. We hypothesized that up-regulation of PPARa and its downstream gene, FABP, are important responses supporting hibernation in the 13-lined ground squirrel, Spermophilus tridecemlineatus. The levels of PPARa and FABP mRNA and protein were quantified in heart at selected timepoints over a hibernation cycle using relative PCR and Western blotting, respectively. PPARa mRNA levels increased slowly and peaked in late hibernation then decreased, whereas PPARa protein levels generally increased late in hibernation. FABP mRNA levels were highest during entry into hibernation and decreased while during torpor but FABP protein increased slowly throughout hibernation. (Conflicts of interest: None declared. Source of funding: NSERC Canada.) doi:10.1016/j.cryobiol.2007.10.167
161. Protein expression patterns and low temperature adaptation in antarctic alga, Chaetoceros neogracile. Koang-Man Choi a, Seul-Ki Park a, EonSeon Jin b, Mi-Young Lee a, a Department of Biotechnology, Soonchunhyang University, Asan, Chungnam, 336-600, Korea; b Department of Life Science, Hanyang University, Seoul 133-743, Korea In order to better understand the survival mechanism of antarctic alga in the low temperature, protein expression patterns in the antarctic alga, Chaetoceros neograciles were examined. Standard 2-dimensional maps for the proteins of Chaetoceros neograciles grown at 4 °C were obtained. Differentially expressed proteins of Chaetoceros neograciles following transient cold-shock or heat-shock were examined by a proteomic technique. Proteomics is a powerful tool to describe the alterations in protein expression. The differentially expressed proteins were identified by MALDI-TOF mass spectrometry. Changes in the total activities and isozyme patterns of various antioxidant enzymes such as superoxide dismutase, glutathione reductase and glutathione transferase were also examined following temperature stress treatment. (Conflicts of interest: None declared. Source of funding: A grant from Korea Science and Engineering Foundation, R01-2006-000-10856-0). doi:10.1016/j.cryobiol.2007.10.164
Hibernation and hypothermia 162. Identification of microRNA (miRNA) expression in hibernating thirteen-lined ground squirrels, Spermophilus tridecemlineatus. Adrian Dubuc, Kenneth B. Storey., Carleton University, Ottawa, ON, Canada MicroRNAs (miRNAs) are known to down-regulate mRNA expression levels, thereby constituting a pivotal role in all major cellular functions, including differentiation and development, metabolism, and insulin secretion. This broad-based regulation led to the hypothesis that miRNA may constitute a vital role in hibernation. Characterization of miRNA expression levels in muscle and liver tissue provided a miRNAsignature for the entry into a state of torpor in Spermophilus tridecemlineatus. The long term objective of this study includes the elucidation of miRNA involvement in the maintenance of low-temperature viability,
377
necessary for increased longevity of human organ storage capabilites. Reverse Transcriptase (RT)-PCR examination of miR-15a, miR-21, miR-122a and miR-146 in the liver and miR-143, miR-146 and miR-206 in skeletal muscle of euthermic versus hibernating animals displayed stable expression. However, miR-122a was significantly reduced by 0.76-fold in muscle of hibernating ground squirrels. This change indicates a potential role for miRNA-silencing of hibernator genes. (Conflicts of interest: None declared. Source of funding: NSERC Canada.) doi:10.1016/j.cryobiol.2007.10.165
163. Regulation of AKT in Richardson’s ground squirrels during hibernation. Khalil Abnous, Christopher A. Dieni, Kenneth B. Storey, Carleton University, Ottawa, ON, Canada Hibernation is a winter survival strategy for many small mammals metabolic rate falls by >95%, core body temperature can approach 0 °C, and all physiological functions are suppressed. Energy savings of 90% are achieved compared with the costs of remaining euthermic (37 °C) over the winter. The protein kinase Akt (also known as protein kinase B) plays a central role in coordinating growth and survival responses in cells and we hypothesized that regulation of Akt would be critical in hibernation. Kinetic properties of muscle Akt were compared in euthermic versus hibernating states; total and phosphorylated Akt protein was also quantified by Western blotting. Total Akt protein did not change in hibernation but phospho-Akt (the active form) and measured Akt activity decreased significantly by 40% and 60%, respectively, compared with euthermic controls. S0.5 values for Akt peptide fell by 28% during hibernation whereas S0.5 ATP increased by 330%. Low temperature assay (10 °C) strongly affected S0.5 ATP of euthermic Akt (a 350% increase). Akt activation energy (Ea) and sensitivity to denaturation by urea did not change between euthermia and hibernation. DEAE Sephadex chromatography showed three peaks (isozymes) of Akt in euthermic muscle but only two peaks during hibernation. The results document differential regulation of Akt during hibernation and suggest a key role for Akt in cell survival during torpor. (Conflicts of interest: None declared. Source of funding: NSERC Canada.) doi:10.1016/j.cryobiol.2007.10.166
164. Analysis of mRNA and protein levels of PPARa and H-FABP in heart during hibernation of thirteen-lined ground squirrels. Melanie Bouffard, Tarek Abd El Halim, Kenneth B. Storey, Carleton University, Ottawa, ON, Canada Lipid catabolism is the main source of energy for hibernating mammals. Fatty acid binding proteins (FABPs) are small, highly conserved cytoplasmic proteins that have roles in fatty acid uptake and intracellular lipid transport and metabolism. The peroxisome proliferator-activated receptor alpha (PPARa) is a transcription factor that regulates expression of various genes of fatty acid catabolism. We hypothesized that up-regulation of PPARa and its downstream gene, FABP, are important responses supporting hibernation in the 13-lined ground squirrel, Spermophilus tridecemlineatus. The levels of PPARa and FABP mRNA and protein were quantified in heart at selected timepoints over a hibernation cycle using relative PCR and Western blotting, respectively. PPARa mRNA levels increased slowly and peaked in late hibernation then decreased, whereas PPARa protein levels generally increased late in hibernation. FABP mRNA levels were highest during entry into hibernation and decreased while during torpor but FABP protein increased slowly throughout hibernation. (Conflicts of interest: None declared. Source of funding: NSERC Canada.) doi:10.1016/j.cryobiol.2007.10.167