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Chaperone proteins have crucial roles to play in all animal species and are involved in mediating both the folding of newly synthesized peptides into their mature conformation, the refolding of misfolded proteins, and the trafficking of proteins between subcellular compartments. These highly conserved proteins have particularly important roles to play in dealing with disruptions of the proteome as a result of environmental stress since abiotic factors, including temperature, pressure, oxygen, water availability, and pollutants can readily disrupt the conformation and/or function of all types of proteins, e.g., enzymes, transporters, and structural proteins. The current review provides an update on recent advances in understanding the roles and responses of chaperones in aiding animals to deal with environmental stress, offering new information on chaperone action in supporting survival strategies including torpor, hibernation, anaerobiosis, estivation, and cold/freeze tolerance among both vertebrate and invertebrate species.

The wood frog, Rana sylvatica (aka Lithobates sylvaticus) is the main model for studies of natural freeze tolerance among amphibians living in seasonally cold climates. During freezing, ∼65% of total body water can be converted to extracellular ice and this imposes both dehydration and hypoxia/anoxia stresses on cells. The current study analyzed the responses of the alpha subunit of the hypoxia-inducible transcription factor (HIF-1), a crucial oxygen-sensitive regulator of gene expression, to freezing, anoxia or dehydration stresses, examining six tissues of wood frogs (liver, skeletal muscle, brain, heart, kidney, skin). RT-PCR revealed a rapid elevation hif-1α transcript levels within 2 h of freeze initiation in both liver and brain and elevated levels of both mRNA and protein in liver and muscle after 24 h frozen. However, both transcript and protein levels reverted to control values after thawing except for HIF-1 protein in liver that dropped to ∼60% of control. Independent exposures of wood frogs to anoxia or dehydration stresses (two components of freezing) also triggered upregulation of hif-1α transcripts and/or HIF-1α protein in liver and kidney with variable responses in other tissues. The results show active modulation of HIF-1 in response to freezing, anoxia and dehydration stresses and implicate this transcription factor as a contributor to the regulation of metabolic adaptations needed for long term survival of wood frogs in the ischemic frozen state.

Obesity caused by excessive fat accumulation in adipocytes is a growing global problem and is a major contributing risk factor for many chronic metabolic diseases. There is increasing evidence that oxidative stress plays a crucial role in both obesity progression and obesity-related complications. In recent years, Drosophila models of diet-induced obesity and associated pathologies have been successfully developed through manipulation of carbohydrate or fat concentrations in the food. Obese flies accumulate triacylglycerols in the fat body, an organ homologous to mammalian adipose tissue and exhibit metabolic and physiological complications including hyperglycemia, redox imbalance and shortened longevity; these are all similar to those observed in obese humans. In this review, we summarize current data on the mechanisms of oxidative stress induction in obesity, with emphasis on metabolic switches and the involvement of redox-responsive signaling pathways such as NF-κB and Nfr2. The recent achievements with D. melanogaster model suggest a complicated relationship between obesity, oxidative stress, and longevity but the Drosophila model offers probably the best opportunities to delve further into unraveling these interactions, particularly the roles of antioxidants and of Nrf2-regulated responses, in order to increase our understanding of the obese metabolic phenotype and test and develop anti-obesity pharmaceuticals.

Overweight and obesity became the worldwide epidemic resulting from overeating especially when a so-called Western diet rich in carbohydrates and fats is used. It is widely accepted that limitation of food consumption could help to withstand such state of adult organism, but information about younger groups is contradictory. The present study was undertaken to characterize the effects of intermittent fasting, using an every other day (EOD) fasting/feeding protocol, on hematological parameters and biochemical blood plasma indices in young mice from one to two months old. It was shown that intermittently fasted mice were characterized by a reduced body weight, reduced total number of blood leucocytes, lower glucose and lactate levels and higher activity of alanine aminotransferase and aspartate aminotransferase in blood plasma as compared with the age-matched control mice. To gain the same mass EOD animals needed to eat more food than ad libitum fed animals. These differences may probably be explained by a need to expend certain resources to combat stress induced by intermittent fasting. Our data showed that EOD feeding at a young age may negatively influence young mammals.

The effects of an alpha-ketoglutarate-supplemented diet on lifespan and functional senescence were evaluated in the Canton S strain of Drosophila melanogaster. The results suggest that effects of dietary alpha-ketoglutarate (AKG) are dose- and gender-dependent. In males, diets containing 1-10 mM AKG did not affect mean and maximum lifespans, except that an increased maximum lifespan observed at 10 mM AKG. Diet with 20 mM AKG shortened median lifespan and had no effect on maximum lifespan of males. In females, diets with low concentrations of AKG (1 and 5 mM) did not affect lifespan, whereas diets supplemented with 10 and 20 mM AKG increased both median and maximum lifespans. At a lifespan-prolonging concentration (10 mM), AKG decreased fecundity, increased cold resistance and had no effect on climbing activity or resistance to oxi­dative stress in flies of either gender at middle (24 days) and old (40 days) ages. Moreover, middle-aged AKG-fed females but not males were more resistant to heat stress that was accompanied by higher levels of HSP90 protein as compared with controls. Middle-aged flies on AKG-supplemented diets showed elevated oxidative stress and had higher total protein and triacylglycerol levels as compared with controls. Hence, anti-aging effects of AKG do not seem to be related to preventing oxidative stress development but involve metabolic rearrangement and synthesis of specific protective proteins, which aid to resist destructive processes with age.

Copper is an essential element, but at high concentrations, it is toxic for living organisms. The present study investigated the responses of goldfish, Carassius auratus, to 96 h exposure to 30, 300, or 700 μg L−1 of copper II chloride (Cu2+). The content of protein carbonyls was higher in kidney (by 158%) after exposure to 700 mg L−1 copper, whereas in gills, liver, and brain, we observed lower content of protein carbonyls after exposure to copper compared with control values. Exposure to copper resulted in increased levels of lipid peroxides in gills (76%) and liver (95–110%) after exposure to 300 and 700 μg L−1 Cu2+. Low molecular mass thiols were depleted by 23–40% in liver and by 29–67% in kidney in response to copper treatment and can be used as biomarkers toxicity of copper. The activities of primary antioxidant enzymes, superoxide dismutase and catalase, were increased in liver as a result of Cu2+ exposure, whereas in kidney catalase activity was decreased. The activities of glutathione-related enzymes, glutathione peroxidase, glutathione-S-transferase, and glutathione reductase were decreased as a result of copper exposure, but glutathione reductase activity increased by 25–40% in liver. Taken together, these data show that exposure of fish to Cu2+ ions results in the development of low/high intensity oxidative stress reflected in enhanced activities of antioxidant and associated enzymes in different goldfish tissues.

L-Arginine, a precursor of many amino acids and of nitric oxide, plays multiple important roles in nutrient metabolism and regulation of physiological functions. In this study, the effects of L-arginine-enriched diets on selected physiological responses and metabolic processes were assessed in Drosophila melanogaster. Dietary L-arginine at concentrations 5-20 mM accelerated larval development and increased body mass, and total protein concentrations in third instar larvae, but did not affect these parameters when diets contained 100 mM arginine. Young (2 days old) adult flies of both sexes reared on food supplemented with 20 and 100 mM L-arginine possessed higher total protein concentrations and lower glucose and triacylglycerol concentrations than controls. Additionally, flies fed 20 mM L-arginine had higher proline and uric acid concentrations. L-Arginine concentration in the diet also affected oxidative stress intensity in adult flies. Food with 20 mM L-arginine promoted lower protein thiol concentrations and higher catalase activity in flies of both sexes and higher concentrations of low molecular mass thiols in males. When flies were fed on a diet with 100 mM L-arginine, lower catalase activities and concentrations of protein thiols were found in both sexes as well as lower low molecular mass thiols in females. L-Arginine-fed males demonstrated higher climbing activity, whereas females showed higher cold tolerance and lower fecundity, compared with controls. Food containing 20 mM L-arginine shortened life span in both males and females. The results suggest that dietary L-arginine shows certain beneficial effects at the larval stage and in young adults. However, the long-term consumption of L-arginine-enriched food had unfavorable effects on D. melanogaster due to decreasing fecundity and life span.

Freeze tolerance is an amazing winter survival strategy used by various amphibians and reptiles living in seasonally cold environments. These animals may spend weeks or months with up to ∼65% of their total body water frozen as extracellular ice and no physiological vital signs, and yet after thawing they return to normal life within a few hours. Two main principles of animal freeze tolerance have received much attention: the production of high concentrations of organic osmolytes (glucose, glycerol, urea among amphibians) that protect the intracellular environment, and the control of ice within the body (the first putative ice-binding protein in a frog was recently identified), but many other strategies of biochemical adaptation also contribute to freezing survival. Discussed herein are recent advances in our understanding of amphibian and reptile freeze tolerance with a focus on cell preservation strategies (chaperones, antioxidants, damage defense mechanisms), membrane transporters for water and cryoprotectants, energy metabolism, gene/protein adaptations, and the regulatory control of freeze-responsive hypometabolism at multiple levels (epigenetic regulation of DNA, microRNA action, cell signaling and transcription factor regulation, cell cycle control, and anti-apoptosis). All are providing a much more complete picture of life in the frozen state.

Alpha-ketoglutarate (AKG) is involved in multiple metabolic and regulatory pathways. In this work, the effects of AKG-supplemented diets on selected physiological responses and metabolic processes, including metabolism of reactive oxygen species, was assessed in larvae and adult (both 2 and 24 days old) Drosophila melanogaster . Dietary supplementation with AKG resulted in dose-dependent effects on larval development, body composition and antioxidant status of third instar larvae. Larvae and young (2 days post-eclosion) adult females fed on AKG shared similar metabolic changes such as higher total protein levels, lower triacylglyceride levels and higher values for oxidative stress indices, namely lipid peroxides and low molecular mass thiols. The latter indicated the development of oxidative stress which, in turn, may induce adaptive responses that can explain the higher resistance of AKG-fed young females to heat shock and hydrogen peroxide exposure. In contrast to young flies, middle-aged females (24 days) on AKG-containing diet possessed higher total protein, glucose and triacylglyceride levels, whereas oxidative stress parameters were virtually the same as compared with control females of the same age. In parallel, females fed an AKG-supplemented diet showed lower fecundity, higher heat shock resistance but no change in oxidative stress resistance at middle age which in combination with levels of protein, glucose, and triacylglycerides can be considered as potentially beneficial AKG effects for aging organisms. To our best knowledge, this is the first study on age-matched AKG influence on animals' organism which shows that Drosophila may be used as a model for previous quick study in cost-efficient manner age-related AKG effects in mammals and humans.

Winter survival for many animal species depends freeze tolerance, a capacity to endure the conversion of as much as 65-70% of total body water into extracellular ice while reorganizing metabolism to provide cells with cryoprotection against insults that include prolonged ischemia and hyperosmotic stress. Natural freeze tolerance involves not just de novo preservation mechanisms such as synthesis of high levels of cryoprotectants or novel proteins that manage ice formation, but also requires attention to and co-ordination of many cellular processes. The present review examines recent studies of the freeze-tolerant wood frog (Rana sylvatica) that probed previously unexplored areas of metabolic adaptation for freezing survival, with a particular emphasis on mitochondria. Post-translational controls on enzyme function play a prominent role in resculpting metabolic responses of the wood frog to freezing including reversible phosphorylation control over fuel processing at the pyruvate dehydrogenase locus and modulation of antioxidant defense enzymes (Mn-SOD, catalase). Enzymes involved in mitochondrial nitrogen metabolism (glutamate dehydrogenase, carbamoyl phosphate synthetase) are also differentially regulated during freezing but by different post-translational modifications including ADP-ribosylation, lysine acetylation or glutarylation. The action of microRNAs in mediating post-translational controls on gene expression aid the suppression of energy-expensive (cell cycle) or destructive (apoptosis) processes in the frozen state while also providing storage of transcripts that will be immediately available for repair or reactivation of metabolic processes after thawing. The effects of low temperature in strengthening mRNA-microRNA interactions can also provide a passive mechanism of metabolic suppression in the frozen state.

Freezing, dehydration, salinity variations, hypoxia or anoxia are some of the environmental constraints that many organisms must frequently endure. Organisms adapted to these stressors often reduce their metabolic rates to maximize their chances of survival. However, upon recovery of environmental conditions and basal metabolic rates, cells are affected by an oxidative burst that, if uncontrolled, leads to (oxidative) cell damage and eventually death. Thus, a number of adapted organisms are able to increase their antioxidant defenses during an environmental/functional hypoxic transgression; a strategy that was interpreted in the 1990s as a “preparation for oxidative stress” (POS). Since that time, POS mechanisms have been identified in at least 83 animal species representing different phyla including Cnidaria, Nematoda, Annelida, Tardigrada, Echinodermata, Arthropoda, Mollusca and Chordata. Coinciding with the 20th anniversary of the postulation of the POS hypothesis, we compiled this review where we analyze a selection of examples of species showing POS-mechanisms and review the most recent advances in understanding the underlying molecular mechanisms behind those strategies that allow animals to survive in harsh environments. Fil: Giraud Billoud, Maximiliano German. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Rivera Ingraham, Georgina A.. Laboratoire Environnement de Petit Saut, Hydreco-Guyane; Guayana Francesa Fil: Moreira, Daniel C.. Universidade do Brasília; Brasil Fil: Burmester, Thorsten. Universitat Hamburg; Alemania Fil: Castro Vazquez, Alfredo Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Carvajalino Fernández, Juan M.. Universidade do Brasília; Brasil Fil: Dafre, Alcir. Universidade Federal de Santa Catarina; Brasil Fil: Niu, Cuijuan. Beijing Normal University; China Fil: Tremblay, Nelly. Alfred Wegener Institute for Polar and Marine Research, Helgoland; Alemania Fil: Paital, Biswaranjan. University of Agriculture and Technology; India Fil: Rosa, Rui. Universidade de Lisboa, Lisboa; Portugal Fil: Storey, Janet M.. Carleton University; Canadá Fil: Vega, Israel Aníbal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Zhang, Wenyi. Beijing Normal University; China Fil: Yepiz Plascencia, Gloria. Centro de Investigacion En Alimentacion y Desarrollo; México Fil: Zenteno Savin, Tania. Centro de Investigaciones Biológicas del Noroeste; México Fil: Storey, Kenneth B.. Carleton University; Canadá Fil: Hermes Lima, Marcelo. Universidade do Brasília; Brasil

The protective effects of dietary alpha-ketoglutarate (AKG) are described that aid fruit flies, Drosophila melanogaster, to resist sodium nitroprusside (SNP) and hydrogen peroxide toxicity. Food supplementation with 10mM AKG alleviated toxic effects of 1mM SNP added to food and improved fly development. Dietary AKG also prevented the increase in levels of oxidative stress markers seen in SNP-reared adult flies. In vitro AKG did not affect the rate of SNP decomposition and did not bind iron and nitrite ions released in this process. Alpha-ketoglutarate also displayed high H2O2-scavenging activity in vitro and efficiently protected adult flies against this compound in combined treatments. Based on the observed antioxidant activity of AKG, it may be suggested that the antioxidant mode of AKG action (apart from its cyanide-binding capability) may be used to prevent the toxic effects of SNP and improve general physiological state of D. melanogaster and other animals and humans.

The effects of 96 h exposure to 7.14, 35.7, or 71.4 mg L−1 of Sencor were studied on liver and plasma parameters in goldfish, Carassius auratus L. Goldfish exposure to 71.4 mg L−1 of Sencor for 96 h resulted in a decrease in glucose concentrations in plasma and liver by 55%, but did not affect liver glycogen levels. An increase in the activity of aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase (by 24–27%, 32–72%, and 87–102%, respectively) occurred in plasma of Sencor exposed goldfish, whereas in liver activities of these enzymes decreased (by 15–17%, 19%, and 20%, respectively). Lactate concentration in plasma increased by 22–36% in all treated fish groups, whereas in liver it increased by 64% only after exposure to 35.7 mg L−1 of Sencor. Herbicide exposure enhanced lipid peroxide levels by 49–75% and decreased activities of catalase by 46%, glutathione reductase by 25–48% and glutathione peroxidase by 21–26% suggesting development of oxidative stress in liver. The treatment induced various histological changes in goldfish liver, such as dilated sinusoids, hypertrophy and dystrophy of hepatic cells and detachment of endothelial cytoplasm with diffuse hemorrhage. The data collectively let us propose that mild oxidative stress might be responsible for the hepatotoxicity of Sencor.

There are very few studies that have directly analyzed the effects of dietary intake of slowly digestible starches on metabolic parameters of animals. The present study examined the effects of slowly digestible starch with high amylose content (referred also as amylose starch) either alone, or in combination with metformin on the development, lifespan, and levels of glucose and storage lipids in the fruit fly Drosophila melanogaster. Consumption of amylose starch in concentrations 0.25-10% did not affect D. melanogaster development, whereas 20% starch delayed pupation and reduced the number of larvae that reached the pupal stage. Starch levels in larval food, but not in adult food, determined levels of triacylglycerides in eight-day-old adult flies. Rearing on diet with 20% starch led to shorter lifespan and a higher content of triacylglycerides in the bodies of adult flies as compared with the same parameters in flies fed on 4% starch diet. Food supplementation with 10mM metformin partly attenuated the negative effects of high starch concentrations on larval pupation and decreased triacylglyceride levels in adult flies fed on 20% starch. Long-term consumption of diets supplemented with metformin and starch decreased lifespan of the insects, compared with the diet supplemented with starch only. The data show that in Drosophila high starch consumption may induce a fat fly phenotype and metformin may partially prevent it.

Objectives: The study was conducted to assess the redox status of Drosophila flies upon oral intake of insulin-mimetic salt, sodium molybdate (Na(2)MoO(4)). Methods: Oxidative stress parameters and activities of antioxidant and associated enzymes were analyzed in two-day-old D. melanogaster insects after exposure of larvae and newly eclosed adults to three molybdate levels (0.025, 0.5, or 10 mM) in the food. Results: Molybdate increased content of low molecular mass thiols and activities of catalase, superoxide dismutase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase in males. The activities of these enzymes were not affected in females. Males exposed to molybdate demonstrated lower carbonyl protein levels than the control cohort, whereas females at the same conditions had higher carbonyl protein content and catalase activity than ones in the control cohort. The exposure to 10 mM sodium molybdate decreased the content of protein thiols in adult flies of both sexes. Sodium molybdate did not affect the activities of NADP-dependent malate dehydrogenase and thioredoxin reductase in males or NADP-dependent isocitrate dehydrogenase in either sex at any concentration. Discussion: Enhanced antioxidant capacity in upon Drosophila flies low molybdate levels in the food suggests that molybdate can be potentially useful for the treatment of certain pathologies associated with oxidative stress.

Winter survival for many insect species includes a need to maintain metabolic homeostasis and structural/functional integrity of macromolecules not only over a wide range of cold temperatures but also in response to rapid temperature change. Chaperones are well-known to protect/stabilize protein structure with regard to heat stress but less is known about their potential involvement in long-term protection of the proteome at subzero temperatures. The present study assessed the participation of chaperone proteins in the cold hardiness of larvae of the goldenrod gall moth, Epiblema scudderiana (Clemens) (Lepidoptera, Olethreutidae), monitoring changes in nine proteins over the winter months as well as their responses to laboratory cold acclimation or anoxia exposure. Four heat shock proteins (HSPs: Hsp110, Hsp70, Hsp60, Hsp40), three glucose-regulated proteins (GRPs: Grp78, Grp 94, Grp170) and the tailless complex polypeptide 1 (TCP-1) as well as the heat shock transcription factor (HSF1) were investigated. In general, all were significantly elevated in larvae collected from an outdoor site between October and March, as compared with September values, and chaperone levels were reduced again in April. The October to March interval also includes the period of diapause followed by cold quiescence in the species. Relative expression of Hsp70, Hsp60 and Hsp40 rose by 2-3-fold, GRPs increased 1.5-3-fold, and levels of active (hyperphosphorylated) HSF1 increased by 4-4.8-fold over the midwinter months. Chilling from 15°C to 4°C in the laboratory upregulated Grp78 protein content that remained high as temperature was further reduced to -4°C and then -20°C whereas Hsp110, Hsp70 and HSF1 levels increased when larvae were exposed to -4°C and -20°C. Grp170 (also known as oxygen-regulated protein 150) was the only chaperone that increased significantly in the larvae in response to anoxia exposure at 4°C. The data also indicated that multiple subcellular compartments received enhanced protection for their proteome since upregulation of chaperones included proteins known to occur in cytosolic (Hsp40, Hsp70), mitochondrial (Hsp60) and endoplasmic reticulum (Grp170) locations. Overall, the data indicate that chaperones have a significant role to play in the winter cold hardiness of E. scudderiana and identify declining temperatures (and perhaps also oxygen restriction) as potential modulators of chaperone production. The data add support to a relatively understudied area of insect cold hardiness - the long-term protection and stabilization of the proteome over the winter months.

The toxicity of sodium nitroprusside (SNP) (an inducer of oxidative/nitrosative stress) and the attenuation of SNP effects by 2,4-dinitrophenol (DNP) (that induces mild uncoupling of respiration) were evaluated in the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with 1.0 mM SNP, 0.5 or 1.25 mM DNP, or with mixtures 1.0 mM SNP plus 0.5 or 1.25 mM DNP. Food supplementation with SNP decreased larval viability and pupation height whereas supplementation with DNP substantially reversed these changes. Biochemical analyses of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies emerged from control larvae and larvae fed on food supplemented with SNP, DNP, or SNP/DNP mixtures. Larval exposure to SNP lowered activities of aconitase, while the presence of DNP reduced the negative impact of SNP by raising aconitase activity back to near control levels. Larval treatment with SNP also elevated the contents of carbonyl protein, uric acid and low molecular mass thiols and produced higher activities of superoxide dismutase, glutathione S-transferase, glucose-6-phosphate dehydrogenase and thioredoxin reductase in adult flies. However, the presence of DNP in the food mixtures prevented SNP-induced changes in thioredoxin reductase and glucose-6-phosphate dehydrogenase activities, as well as uric acid and low-molecular-mass thiol content. The potential mechanisms by which DNP exerts protective effects against SNP toxicity are discussed.

Winter survival for many kinds of animals involves freeze tolerance, the ability to endure the conversion of about 65% of total body water into extracellular ice and the consequences that freezing imposes including interruption of vital processes (e.g., heartbeat and breathing), cell shrinkage, elevated osmolality, anoxia/ischemia, and potential physical damage from ice. Freeze-tolerant animals include various terrestrially hibernating amphibians and reptiles, many species of insects, and numerous other invertebrates inhabiting both terrestrial and intertidal environments. Well-known strategies of freezing survival include accumulation of low molecular mass carbohydrate cryoprotectants (e.g., glycerol), use of ice nucleating agents/proteins for controlled triggering of ice growth and of antifreeze proteins that inhibit ice recrystallization, and good tolerance of anoxia and dehydration. The present article focuses on more recent advances in our knowledge of the genes and proteins that support freeze tolerance and the metabolic regulatory mechanisms involved. Important roles have been identified for aquaporins and transmembrane channels that move cryoprotectants, heat shock proteins and other chaperones, antioxidant defenses, and metabolic rate depression. Genome and proteome screening has revealed many new potential targets that respond to freezing, in particular implicating cytoskeleton remodeling as a necessary facet of low temperature and/or cell volume adaptation. Key regulatory mechanisms include reversible phosphorylation control of metabolic enzymes and microRNA control of gene transcript expression. These help to remodel metabolism to preserve core functions while suppressing energy expensive metabolic activities such as the cell cycle. All of these advances are providing a much more complete picture of life in the frozen state.

Intensive use of pesticides, particularly dithiocarbamates, in agriculture often leads to contamination of freshwater ecosystems. To our knowledge, the mechanisms of toxicity to fish by the carbamate fungicide Tattoo that contains mancozeb [ethylenebis(dithiocarbamate)] have not been studied. The present study aimed to evaluate the effects of Tattoo on goldfish gills and blood, tissues that would have close early contact with the pollutant. Exposure of goldfish Carassius auratus to 3, 5 or 10mgL(-1) of Tattoo for 96h resulted in moderate lymphopenia (by 8 percent) with a concomitant increase in both stab (by 66-88 percent) and segmented (by 166 percent) neutrophils. An increase in the content of protein carbonyl groups in blood (by 137-184 percent) together with decreased levels of protein thiols (by 23 percent) and an enhancement of lipid peroxide concentrations (by 29 percent) in gills after exposure to 10mgL(-1) of Tattoo demonstrated the induction of mild oxidative stress in response to Tattoo exposure. At the same time, the activities of selected antioxidant enzymes were enhanced in gills: superoxide dismutase by 18-25 percent and catalase by 27 percent. A 34 percent increment in low molecular mass thiol concentrations (mainly represented by glutathione) also occurred in gills and could be related to increased activity (by 13-30 percent) of glucose-6-phosphate dehydrogenase. The results indicate that Tattoo exposure perturbs free radical processes, i.e. induces mild oxidative stress and enhances the activity of certain antioxidant and associated enzymes in goldfish gills. It is clear that goldfish respond to the presence of waterborne pesticide by adjusting antioxidant defenses through upregulation of activities of antioxidant and associated enzymes.

Metribuzin belongs to the family of asymmetrical triazine compounds and is an active ingredient in many commercial herbicides including Sencor. Effects on goldfish (Carassius auratus L.) of exposure for 96h to 7.14, 35.7 or 71.4mgL(-1) Sencor 70 WG (corresponding to 5, 25 and 50mgL(-1) of metribuzin) were examined by evaluating oxidative stress markers and activities of antioxidant and associated enzymes in gills. Fish exposed to the lowest Sencor concentration (7.14mgL(-1)) showed a 94% increase in levels of protein carbonyls in gills as well as 45% and 144% increases in the activities of glutathione peroxidase and glutathione-S-transferase. Exposure to the highest Sencor concentration (71.4mgL(-1)) resulted in reduced levels of protein carbonyls by 56% and lipid peroxides by 40%, as compared with controls, but enhanced levels of low and high molecular mass thiols by 71% and 36%, respectively. The activities of superoxide dismutase, glutathione peroxidase and glutathione-S-transferase were increased in gills of goldfish exposed to 71.4mgL(-1) Sencor. At any concentration tested, Sencor did not affect the activities of glutathione reductase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase or acetylcholine esterase in gills. The results of this study indicate that acute exposure of goldfish to Sencor had effect on free radical processes in gills and glutathione-dependent antioxidants effectively protect proteins and lipids from oxidation.

The toxicity of sodium nitroprusside (SNP) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with SNP at concentrations of 0.01-1.5 mM. Food supplementation with SNP caused a developmental delay in flies and reduced adult eclosion. Biochemical analyses such as levels of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies emerged from control and SNP-fed larvae. Larval exposure to SNP resulted in lower activities of aconitase and catalase in adult flies relative to the control cohort. However, larval treatment with SNP led to higher carbonyl protein content and higher activities of superoxide dismutase, glucose-6-phosphate dehydrogenase, thioredoxin reductase, and glutathione-S-transferase in flies. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by SNP. The study also suggests that the toxicity of SNP may arise not just from its direct effects, but also from its decomposition products such as nitric oxide and iron ions.

The toxicity of nickel to mammals is well studied, whereas information on nickel effects on fish is scant. Goldfish exposure to 10–50 mg L −1 of waterborne Ni 2+ for 96 h showed reduced glycogen levels by 27–33% and 37–40% in liver and white muscle, respectively, accompanied by substantial increases in blood glucose levels (by 15–99%). However, indices of oxidative damage to proteins (carbonyl proteins) and lipids (lipid peroxides) were largely unaffected by nickel exposure. In liver, the activities of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx), were not affected by Ni 2+ treatment, while catalase activity was elevated by 26%. In white muscle, however, substantial increases in SOD (by 38–147%) and GPx (by 2.5–5.5-fold) activities appeared to compensate for decreased catalase activity (by 59–69%) in order to resist Ni-induced oxidative perturbations. Both hepatic and muscular glutathione reductase activities were suppressed by 10–30% and 12–21%, respectively, after goldfish exposure to all Ni 2+ concentrations used. However, the activity of glucose-6-phosphate dehydrogenase was remarkably enhanced (by 1.6–5.4-fold) in white muscle of Ni-exposed fish, indicating a strong potential increase in NADPH production under Ni exposure. Thus, the exposure of goldfish to 10–50 mg L −1 of Ni 2+ for 96 h induces glycogenolysis and hyperglycemia, showing some similarities with a hypoxia response, and leads to a substantial activation of defense systems against reactive oxygen species in liver and white muscle in tissue-specific and concentration-dependent manner.

Cobalt ions can enhance the generation of reactive oxygen species (ROS), which may be the reason for cobalt toxicity. This study aimed to determine whether Co(2+) toxicity in goldfish is related to induced oxidative stress in gills, heart and spleen, and to assess responses of antioxidant systems. Exposure of goldfish to 50, 100 and 150 mg L(-1) of Co(2+) for 96 h elevated total hemoglobin in blood by 23, 44 and 78%, respectively. In gills, cobalt exposure enhanced lipid peroxide levels and activities of primary antioxidant enzymes; superoxide dismutase (SOD) rose by 125% and glutathione peroxidase (GPx) increased by 53-296%. Glutathione-S-transferase (GST) activity also increased by 117-157% and glucose-6-phosphate dehydrogenase (G6PDH) enhanced by 46-96%. Heart showed limited effects of fish exposure to 50 or 100 mg L(-1) of Co(2+), but the exposure to 150 mg L(-1) of Co(2+) elevated concentrations of lipid peroxides by 123% and activities of GPx by 98% and SOD by 208%. The most substantial effects of goldfish exposure to Co(2+) were observed in spleen: a decrease in total protein concentration by 44-60% and high molecular mass thiols by 59-82%, reduced activities of catalase by 24-58% and GR by 25-68%, whereas the level of low molecular mass thiols increased by 153-279% and activities of GPx, GST, G6PDH were enhanced by 114-120%, 192-769%, and 256-581%, respectively. The data show that fish exposure to 50-150 mg L(-1) of Co(2+) elevates blood hemoglobin level, mimicking effects of hypoxia, and causes the activation of defense systems against ROS.

Cobalt is an essential element, but at high concentrations it is toxic. In addition to its well-known function as an integral part of cobalamin (vitamin B₁₂), cobalt has recently been shown to be a mimetic of hypoxia and a stimulator of the production of reactive oxygen species. The present study investigated the responses of goldfish, Carassius auratus, to 96 h exposure to 50, 100 or 150 mg L⁻¹ Co²⁺ in aquarium water (administered as CoCl₂). The concentrations of cobalt in aquaria did not change during fish exposure. Exposure to cobalt resulted in increased levels of lipid peroxides in brain (a 111% increase after exposure to 150 mg L⁻¹ Co²⁺) and liver (30-66% increases after exposure to 50-150 mg L⁻¹ Co²⁺), whereas the content of protein carbonyls rose only in kidney (by 112%) after exposure to 150 mg L⁻¹ cobalt. Low molecular mass thiols were depleted by 24-41% in brain in response to cobalt treatment. The activities of primary antioxidant enzymes, superoxide dismutase (SOD) and catalase, were substantially suppressed in brain and liver as a result of Co²⁺ exposure, whereas in kidney catalase activity was unchanged and SOD activity increased. The activities of glutathione-related enzymes, glutathione peroxidase and glutathione-S-transferase, did not change as a result of cobalt exposure, but glutathione reductase activity increased by ∼40% and ∼70% in brain and kidney, respectively. Taken together, these data show that exposure of fish to Co²⁺ ions results in the development of oxidative stress and the activation of defense systems in different goldfish tissues.

As a result of screening Bacillus sp. strains isolated from different natural substrates, strain BKL20 was identified as a producer of a thermostable alkaline α-amylase. Maximum production of this α-amylase was achieved by optimizing culture conditions. Production of α-amylase seemed to be independent of the presence of starch in the culture medium and was stimulated by the presence of peptone (0.3%, m/v) and yeast extract (0.2%, m/v). The enzyme was thermostable and retained amylolytic activity after 30 min of incubation at 60 and 70 °C. High activity was maintained over a broad pH range, from 6.0 to 11.0, and the enzyme remained active after alkaline incubation for 24 h. Bacillus sp. BKL20 α-amylase was not stimulated by Ca2+or other bivalent metal cations and was not inhibited by EGTA or EDTA at 1–10 mmol/L, suggesting that this α-amylase is a Ca2+-independent enzyme. It also showed good resistance to both oxidizing (H2O2) and denaturating (urea) agents.

In the environment chromium is found mainly in two valence states-hexavalent chromium (Cr6+) and trivalent chromium (Cr3+). The present study evaluates the effects of Cr3+ exposure on goldfish by analyzing parameters of oxidative stress and antioxidant defense in liver and kidney of fish given 96 h exposures to Cr3+ concentrations of 1, 2.5, 5 or 10 mg/l in aquarium water. Cr3+ exposure did not alter two parameters of oxidative stress-protein carbonyl content and lipid peroxide concentrations in either organ. However, Cr3+ exposure did decrease total glutathione concentration in liver by 34-69% and in kidney to 36-49% of the respective control values. Oxidized GSSG content fell by similar percentages so that the ratio [GSSG]/[total glutathione] remained constant at all Cr3+ exposure levels except in liver under the highest, 10 mg/l, exposure level. In liver, exposure to 1-5 mg/l Cr3+ led to a decrease in the activity of superoxide dismutase (SOD) by 29-36%, and at 10 mg/l Cr3+ the reduction was 54%, whereas in kidney approximately 30% reductions in SOD activity were seen at concentrations 1 and 10 mg/l Cr3+. Catalase activity was not significantly affected by 1-5 mg/l Cr3+, but was reduced by 57 and 42% in liver and kidney, respectively. Chromium exposure also reduced the activity of glutathione-S-transferase in both organs by 17-50% but did not affect glutathione reductase or glucose-6-phosphate dehydrogenase activities. A comparison of Cr3+ effects on goldfish liver and kidney metabolism indicates that the trivalent ion induces stronger oxidative stress than Cr6+ at the same concentrations.

AMP-deaminase was partially purified from white skeletal muscle of goldfish, Carassius auratus. The enzyme was highly stable, showing virtually no change in activity at 1 month following the purification process when stored in 1 M KCl at 2–4°C. The specific activity of the purified enzyme was 130–150 U/mg protein, with a pH optimum of about pH 6.5. AMP-aminohydrolase (AMPD) showed non-Michaelis–Menten kinetics, with a S0.5 (half saturation by the substrate) for AMP of 0.73 ± 0.03 mM, a Hill coefficient of 2.01 ± 0.26, and a V max (maximum velocity) of 176 ± 46 U/mg protein. Both sodium and potassium ions activated goldfish AMPD at low concentrations, with maximal activation at about 80 mM of each chloride salt, whereas higher concentrations became inhibitory. Magnesium and calcium ions also inhibited goldfish muscle AMPD, as did phosphate and fluoride; at a concentration of 8 mM, each anion reduced activity by about 66%. ADP and ATP were strong activators and both demonstrated concentration-dependent activation, with maximal effects at 0.5–1.5 mM. Fish exposure to a high concentration of oxygen (18–20 mg/l against 5–6 mg/l in the control) and recovery to the initial level induced a redistribution of AMPD between free and bound forms in goldfish white muscle and brain in a tissue-dependent manner. A spatial–temporal redistribution may be among the mechanisms regulating enzyme operation in vivo. Possible regulatory mechanisms of AMP-deaminase function in fish muscle are discussed.

Metabolic rate depression is an important survival strategy for many animal species and a common element of hibernation, torpor, aestivation, anaerobiosis, diapause, and anhydrobiosis. Studies of the biochemical mechanisms that regulate reversible transitions to and from hypometabolic states are identifying principles of regulatory control that are conserved across phylogenetic lines and that are broadly applied to the control of multiple cell functions. One such mechanism is reversible protein phosphorylation which is now known to contribute to the regulation of fuel metabolism, to ion channel arrest, and to the suppression of protein synthesis during hypometabolism. The present review focuses on two new areas of research in hypometabolism: (1) the role of differential gene expression in supplying protein products that adjust metabolism or protect cell functions for long-term survival, and (2) the mechanisms of protein life extension in hypometabolism involving inhibitory controls of transcription, translation and protein degradation. Control of translation examines reversible phosphorylation regulation of ribosomal initiation and elongation factors, the dissociation of polysomes and storage of mRNA transcripts during hypometabolism, and control over the translation of different mRNA types by differential sequestering of mRNA into polysome versus monosome fractions. The analysis draws primarily from current research on two animal models, hibernating mammals and anoxia-tolerant molluscs, with selected examples from multiple other sources.

Italian wall lizards (Podarcis sicula campestris) were introduced to Long Island, New York, in 1967 and have subsequently spread through many urban and suburban communities. Their ability to spread further may be limited by their ability to tolerate the relatively cold winters of New York. We found that these lizards were able to survive cold temperatures by supercooling if they were kept dry. However, if freezing was initiated as would be expected under shallow hibernation conditions, these lizards froze and died rapidly. We speculate that Podarcis sicula must hibernate below soil freezing depth, estimated at greater than 24 cm deep, to survive.

The effects of food supplementation with sodium chromate at concentrations of 1–500 μM on development of Drosophila melanogaster larvae and food intake, carbohydrate and lipid pools in adult fruit flies were investigated. Food supplementation with hexavalent chromium (Na2CrO4) at high concentrations delayed larval development and decreased the percentage of larvae that pupated which indicated a relatively low toxicity. The supplement decreased glucose levels in fly hemolymph, but at concentrations of 5–25 μM increased fly carbohydrate reserves: hemolymph trehalose and whole body trehalose and glycogen. The data on parameters of carbohydrate metabolism show that chromate possesses some insulin-mimetic properties. The changes in metabolism of carbohydrates under chromate exposure were also accompanied by an increase in total lipid levels and in the portion of triacylglycerides among all lipids. Chromate addition to fly food did not affect male or female body mass, but reduced food consumption by females at all concentrations used, whereas in males only 500 μM chromate decreased food consumption. The data show that: (1) Cr6 + has many of the same effects as Cr3 + suggesting that it might be just as effective to treat diabetic states, likely as a result of intracellular reduction of Cr6 + ions, and (2) the Drosophila model can be used to develop new approaches to investigate the molecular mechanisms of chromium as an insulin-mimetic. Although it is usually believed that hexavalent chromium possesses higher toxicity than the trivalent ion, due to its easier penetration into the cell, application of hexavalent chromium may substantially decrease the chromium doses needed to get the desired effects.

Molybdenum-containing salts have been found to attenuate diabetes complications in mammals by affecting processes normally regulated by insulin and thus were believed to mimic insulin activity. In this study, we used a fruit fly model to test sodium molybdate, Na2MoO4, action in relation to insulin-promoted processes and toxicity. We studied how larval food supplementation with sodium molybdate affected levels of body carbohydrates and lipids in two-day old adult Drosophila melanogaster. Molybdate salt, in the concentrations used (0.025, 0.05, 0.5, 5, and 10 mM), showed low toxicity to fly larvae and slightly influenced development and the percentage of pupated animals. Additionally, sodium molybdate decreased the level of hemolymph glucose in males by 30%, and increased the level of hemolymph trehalose in flies of both sexes. These changes were accompanied by an increase in whole body trehalose and glycogen of about 30–90%. Although total lipid levels in flies of both sexes were depleted by 25%, an increased amount of triacylglycerides among total lipids was observed. These effects were not related to changes in food intake. Taken together, the present data let us suggest that sodium molybdate may at least partly mimic insulin-related effects in Drosophila.

Molecular mechanisms of toxicity by the metribuzin-containing herbicide Sencor to living organisms, particularly fish, have not yet been extensively investigated. In the present work, we studied the effects of 96 h exposure to 7.14, 35.7, or 71.4 mg L(-1) of Sencor (corresponding to 5, 25, or 50 mg L(-1) of its herbicidal component metribuzin) on goldfish (Carassius auratus L.), examining the histology, levels of oxidative stress markers, and activities of antioxidant and related enzymes in kidney as well as hematological parameters and leukocyte profiles in blood. The treatment induced various histopathological changes in goldfish kidney, such as hypertrophy of intertubular hematopoietic tissue, small and multiple hemorrhages, glomerular shrinkage, a decrease in space between glomerulus and Bowman's capsule, degeneration and necrosis of the tubular epithelium. Sencor exposure also decreased activities of selected enzymes in kidney; activities of catalase decreased by 31-34%, glutathione peroxidase by 14-33%, glutathione reductase by 17-25%, and acetylcholinesterase by 31%. However, glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities increased by 25-30% and 22% in kidney after treatment with 7.14 or 35.7 mg L(-1) and 71.4 mg L(-1) Sencor, respectively. Kidney levels of protein carbonyls increased by 177% after exposure to 35.7 mg L(-1) of Sencor indicating extensive damage to proteins. Lipid peroxide concentrations also increased by 25% after exposure to 7.14 mg L(-1) of Sencor, but levels were reduced by 42% in the 71.4 mg L(-1) exposure group. The data indicate that induction of oxidative stress is one of the mechanisms responsible for Sencor toxicity to fish.

The maximal activities of 28 enzymes, representing multiple pathways of intermediary metabolism, were quantified in the brain, liver and skeletal muscle of spadefoot toads Scaphiopus couchii, comparing control toads with animals that had estivated for 2 months. Estivation-induced changes in brain enzyme activities were consistent with suppressed glycolysis and increased ketone body and amino acid catabolism. In liver, estivation resulted in reduced activities of eight enzymes representing carbohydrate, amino acid, ketone body and phosphagen metabolism, but the maximal activity of malic enzyme increased by 2.4-fold. Estivation led to a large-scale reorganization of skeletal muscle affecting most of the enzymes analyzed. Activities of enzymes of carbohydrate catabolism were generally elevated except for glycogen phosphorylase and hexokinase, whereas those of enzymes of fatty acid synthesis and ketone body metabolism were reduced. Increased glutamate dehydrogenase activities in both brain and muscle, as well as activities of other amino-acid-catabolizing enzymes in muscle, correlated with specific changes in the free amino acids pools in those tissues (reduced glutamine activity, increased glutamate, alanine and valine activities) that appear to be related to protein catabolism, for the purposes of elevating urea levels. The effects of estivation on signal transduction systems were also assessed. Total activities of protein kinases A and C (PKA and PKC) were largely unaltered in toad tissues during estivation (except for a 57% reduction in liver total PKC), but in seven organs there were strong reductions in the percentage of PKA present as the active catalytic subunit in estivating animals, and three contained a much lower percentage of membrane-bound active PKC during estivation. Activities of protein phosphatase types 1, 2A, 2B, and 2C were also frequently reduced during estivation. Overall, these results suggest that anuran estivation involves metabolic reorganization, including changing the maximal activities of key enzymes of intermediary metabolism as well as depressing the metabolic rate by suppressing signal transducing enzymes.

A cDNA library prepared from heart of hibernating golden-mantled ground squirrels, Spermophilus lateralis, was differentially screened to clone genes that were up-regulated during hibernation. Two differentially expressed clones were found after three rounds of screening and were confirmed as up-regulated by Northern blotting. Clone Ang6 encoded a polypeptide with 116 amino acids that was identified as the ventricular isoform of myosin light chain 1 (MLC1v). Clone Ang19 coded for 274 amino acid residues of the mitochondrially encoded protein subunit 2 of NADH‐ ubiquinone oxidoreductase (ND2). Both proteins showed high amino acid sequence identity with their human counterparts, 97.5% for MLC1v and 66% for ND2. Northern blot hybridization revealed differential expression of these genes in multiple organs during hibernation. Transcript levels of both were approximately twofold higher in heart and three- to fourfold higher in skeletal muscle of hibernating, versus euthermic, animals. ND2 was also up-regulated in hibernator liver. Hibernation-induced up-regulation of MLC1v suggests that a restructuring of myosin subunit composition could contribute to changes in muscle contractility needed for hypothermic function, whereas changes in ND subunit composition may affect the function of the electron transport chain during hibernation. © 2000 Academic Press

A cDNA library constructed from liver of 2-month estivating female spadefoot toads, Scaphiopus couchii, was differentially screened to reveal genes that were induced or upregulated during estivation. After two rounds of screening a clone was isolated that showed 60% higher expression in liver of estivating, versus control, toads. The clone possessed a 1.0 kb insert which annealed to a single 0.7 kb band on Northern blots. Sequencing revealed a 1053 nucleotide full-length cDNA; the largest potential open reading frame was 708 nucleotides which encoded a protein of 235 amino acids. A homology search in Genbank indicated that the protein was a riboflavin binding protein (RfBP), a monomeric phosphoglycoprotein produced by the liver of female birds, reptiles, and mammals that functions to bind plasma riboflavin and load the vitamin into eggs or fetus. To our knowledge, this is the first demonstration that RfBP is also present in amphibians. Toad RfBP showed 50% of residues identical with the chicken or turtle liver proteins and many essential structural features were conserved in the toad protein including 18 cysteine residues, two asparagine glycosylation sites, and 6 tryptophan residues. However, a region with eight phosphoserines in the chicken or turtle proteins that functions in RfBP binding to the oocyte membrane contained only three serine residues in toad RfBP, suggesting that recog- nition and binding to oocyte receptors must be different in toads. Northern hybridization showed that toad RfBP was largely liver-specific; no mRNA transcripts were detected in brain, gut, heart, or kidney but low message levels occurred in hind leg skeletal muscle of estivating, but not con- trol, toads. Upregulation of RfBP in liver of estivating toads may be linked with maturation of eggs in preparation for the explosive breeding that occurs immediately upon emergence from esti- vation but might also have a role for the adult in "caching" riboflavin to maintain an endogenous vitamin pool over the 9-10 months of each year that toads are dormant. J. Exp. Zool. 284:325- 333, 1999. © 1999 Wiley-Liss, Inc. The Couch spadefoot toad, Scaphiopus couchii, inhabits arid regions of the American southwest and spends as much as 9-10 months of each year burrowed underground in a state of dormancy, termed estivation (McClanahan, '67). Long-term survival is ensured by strong metabolic rate de- pression, oxygen consumption decreasing to only 20-30% of the resting rate when aroused (Sey- mour, '73), and by adaptations that minimize the loss of body water as the soil dries out (Mc- Clanahan, '67). Metabolism while dormant is fu- eled primarily by the consumption of lipid reserves but as water stress becomes severe toads also in- crease the catabolism of protein (Seymour, '73; Jones, '80). From this point, they accumulate the nitrogenous end product, urea, in amounts up to 300 mM in plasma and other body fluids and this enhances water retention via colligative action (McClanahan, '67; Jones, '80). Whereas considerable attention has been given to energy metabolism and water balance during amphibian estivation (Seymour, '73; Jones, '80; Flanigan et al., '90; Pinder et al., '92; Shoemaker, '92; Cowan and Storey, '98; Cowan et al., '98) little is yet known about the role of gene expression in promoting survival. Presumably, specific up- or downregulation of selected genes is needed in or- der to readjust and optimize numerous metabolic processes to sustain long-term dormancy. Indeed, changes in the levels of various proteins (chiefly enzymes) are known to occur during estivation.

▪ Abstract Natural freeze-tolerance supports the winter survival of many animals including numerous terrestrial insects, many intertidal marine invertebrates, and selected species of terrestrially hibernating amphibians and reptiles. Freeze-tolerant animals typically endure the conversion of 50% or more of total body water into extracellular ice and employ a suite of adaptations that counter the negative consequences of freezing. Specific adaptations control the sites and rate of ice formation to prevent physical damage by ice. Other adaptations regulate cell-volume change: Colligative cryoprotectants minimize cell shrinkage during extracellular ice formation; other protectants stabilize membrane structure; and a high density of membrane transporter proteins ensure rapid cryoprotectant distribution. Cell survival during freezing is also potentiated by anoxia tolerance, mechanisms of metabolic rate depression, and antioxidant defenses. The net result of these protective mechanisms is the ability to reactivate vital functions after days or weeks of continuous freezing. Magnetic resonance imaging has allowed visual examinations of the mode of ice penetration through the body of freeze-tolerant frogs and turtles, and cryomicroscopy has illustrated the effects of freezing on the cellular and microvasculature structure of tissues. Various metabolic adaptations for freezing survival appear to have evolved out of pre-existing physiological capacities of animals, including desiccation-resistance and anoxia-tolerance.

The effects of various catecholamines, α- and β-adrenergic agonists and antagonists, hormones, hormone receptor blockers, and other inhibitors, as well as destruction of the nervous system by pithing, were analyzed for their ability to modulate the freezing-induced cryoprotectant output by liver of the wood frog Rana sylvatica. Of the numerous treatments tried only intraperitoneal injections of glucagon (a glycogenolytic hormone) and propranolol (a blocker of β-adrenergic receptors), both of which modulate hepatic cyclic AMP levels, altered cryoprotectant output. Glucagon significantly enhanced glucose output into the blood during freezing (by 3.5-fold compared with sham-injected frogs), whereas propranolol significantly reduced the hyperglycemia, liver and blood glucose concentrations being reduced by 55 and 42%, respectively, compared with values for untreated frozen frogs. However, treatment with the α-adrenergic blocker phentolamine did not block freezing-induced cryoprotectant output, indicating that Ca 2+ -mediated mechanisms are not involved in the hyperglycemic response by liver in these frogs. The mechanism of propranolol action was to suppress the activation of liver glycogen phosphorylase so that the amount of active phosphorylase a in liver of propranolol-treated frogs after freezing exposure was only 5% of the corresponding activity in liver of untreated frozen frogs. The β-adrenergic nature of catecholamine regulation of blood glucose levels was confirmed by the ability of the β-adrenergic agonist isoproterenol to mimic the hyperglycemia induced by epinephrine or norepinephrine injection in unfrozen frogs at 3°C, whereas the α-adrenergic agonist phenylephrine was ineffective. The data indicate that the cryoprotectant output response during freezing of R. sylvatica is mediated by cAMP-dependent mechanisms within the liver under the control of plasma hyperglycemic hormones.

The gastropod mollusk, Littorina littorea L., is a common inhabitant of the intertidal zone along rocky coastlines of the north Atlantic. This species has well-developed anoxia tolerance and freeze tolerance and is extensively used as a model for exploring the biochemical adaptations that support these tolerances as well as for toxicological studies aimed at identifying effective biomarkers of aquatic pollution. This article highlights our current understanding of the molecular mechanisms involved in anaerobiosis and freezing survival of periwinkles, particularly with respect to anoxia-induced metabolic rate depression. Analysis of foot muscle and hepatopancreas metabolism includes anoxia-responsive changes in enzyme regulation, signal transduction, gene expression, post-transcriptional regulation of mRNA, control of translation, and cytoprotective strategies including chaperones and antioxidant defenses. New studies describe the regulation of glucose-6-phosphate dehydrogenase by reversible protein phosphorylation, the role of microRNAs in suppressing mRNA translation in the hypometabolic state, modulation of glutathione S-transferase isozyme patterns, and the regulation of the unfolded protein response.

The toxicity of sodium nitroprusside (SNP) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with SNP at concentrations of 0.01-1.5 mM. Food supplementation with SNP caused a developmental delay in flies and reduced adult eclosion. Biochemical analyses such as levels of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies emerged from control and SNP-fed larvae. Larval exposure to SNP resulted in lower activities of aconitase and catalase in adult flies relative to the control cohort. However, larval treatment with SNP led to higher carbonyl protein content and higher activities of superoxide dismutase, glucose-6-phosphate dehydrogenase, thioredoxin reductase, and glutathione-S-transferase in flies. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by SNP. The study also suggests that the toxicity of SNP may arise not just from its direct effects, but also from its decomposition products such as nitric oxide and iron ions.

The toxicity of the nitric oxide donor S-nitrosoglutathione (GSNO) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with GSNO at concentrations of 1.0, 1.5 or 4.0 mM. Food supplementation with GSNO caused a developmental delay in the flies. Biochemical analyses of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies that emerged from control larvae and larvae fed on food supplemented with GSNO. Larval exposure to GSNO resulted in lower activities of aconitase in both sexes and also lower activities of catalase and isocitrate dehydrogenase in adult males relative to the control cohort. Larval treatment with GSNO resulted in higher carbonyl protein content and higher activities of glucose-6-phosphate dehydrogenase in males and higher activities of superoxide dismutase and glutathione-S-transferase in both sexes. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by GSNO.

Summary Aestivation is a survival strategy used by many vertebrates and invertebrates to endure arid environmental conditions. Key features of aestivation include strong metabolic rate suppression, strategies to retain body water, conservation of energy and body fuel reserves, altered nitrogen metabolism, and mechanisms to preserve and stabilize organs, cells and macromolecules over many weeks or months of dormancy. Cell signaling is crucial to achieving both a hypometabolic state and reorganizing multiple metabolic pathways to optimize long-term viability during aestivation. This commentary examines the current knowledge about cell signaling pathways that participate in regulating aestivation, including signaling cascades mediated by the AMP-activated kinase, Akt, ERK, and FoxO1.

Winter survival for many insects depends on cold hardiness adaptations as well as entry into a hypometabolic diapause state that minimizes energy expenditure. We investigated whether AMP-activated protein kinase (AMPK) could be involved in this adaptation in larvae of two cold-hardy insects, Eurosta solidaginis that is freeze tolerant and Epiblema scudderiana that uses a freeze avoidance strategy. AMPK activity was almost 2-fold higher in winter larvae (February) compared with animals collected in September. Immunoblotting revealed that phosphorylation of AMPK in the activation loop and phosphorylation of acetyl-CoA carboxylase (ACC), a key target of AMPK, were higher in Epiblema during midwinter whereas no seasonal change was seen in Eurosta. Immunoblotting also revealed a significant increase in ribosomal protein S6 phosphorylation in overwintering Epiblema larvae, and in both Eurosta and Epiblema, phosphorylation of eukaryotic initiation factor 4E-binding protein-1 dramatically increased in the winter. Pyruvate dehydrogenase (PDH) E1α subunit site 1 phosphorylation was 2-fold higher in extracts of Eurosta larvae collected in February versus September while PDH activity decreased by about 50% in Eurosta and 80% in February Eurosta larvae compared with animals collected in September. Glycogen phosphorylase phosphorylation was 3-fold higher in Epiblema larvae collected in February compared with September and also in these animals, triglyceride lipase activity increased by 70% during winter. Overall, our study suggests a re-sculpting of metabolism during insect diapause, which shifted to a more catabolic poise in freeze-avoiding overwintering Epiblema larvae, possibly involving AMPK.

During winter hibernation, small mammals fall into long periods of deep cold torpor where metabolic rate is suppressed by90% and core body temperature can fall to near 0 degrees C. Studies with hibernators illustrate the molecular regulatory mechanisms that coordinate the suppression of metabolic functions during torpor, reprioritize energy use, and preserve/stabilize macromolecules to support long-term viability during cold torpor. This review explores mechanisms including posttranslational modification of proteins, differential regulation of enzymes, global suppression of transcription and translation including a role for microRNA, torpor-responsive gene expression, signal transduction, and regulation of transcription factors. The molecular basis of natural torpor in hibernating mammals offers models and applications that are relevant to issues in clinical science including hypothermia and ischemia resistance, inducible torpor, organ preservation, and atrophy resistance.

The role of chaperone proteins in the winter survival of insects was evaluated in freeze tolerant gall fly larvae, Eurosta solidaginis. Levels of four heat shock proteins (Hsp110, Hsp70, Hsp60, Hsp40), two glucose-regulated proteins (Grp75, Grp78) and three others (tailless complex polypeptide 1 [TCP-1], αA-crystallin, αB-crystallin) were tracked in outdoor larvae from September to April and, in addition, laboratory experiments assessed chilling, freezing, and anoxia effects on these proteins. Gall fly larvae showed consistent elevation of Hsp110, Hsp70, Hsp40, Grp78 and αB-crystallin over the late autumn and winter months, generally 1.5-2.0-fold higher than September values. This suggests that these proteins contribute to cell preservation over the winter months via protection and stabilization of macromolecules. By contrast, levels of the mitochondrial Hsp60 fell to just 40% of September values by midwinter, paralleling the responses by numerous mitochondrial enzymes and consistent with a reduction in total mitochondria numbers over the winter. None of the proteins were altered when 15°C acclimated larvae were chilled to 3°C for 24h but Hsp70, Hsp40 and Grp75 increased during freezing at -16°C for 24h whereas others (Hsp110, TCP-1 and both crystallins) increased significantly after larvae thawed at 3°C. Anoxia exposure (24h under N2 gas at 15°C) elevated levels of Hsp70, Grp78 and the two crystallins. Levels of active hyperphosphorylated heat shock transcription factor (HSF1) were also analyzed, giving an indication of the state of hsp gene transcription in the larvae. HSF1 was high in September and October but fell to less than 40% of September values in midwinter consistent with suppression of gene transcription in diapause larvae. HSF1 levels responded positively to freezing and increased robustly by 4.9-fold under anoxia. Overall, the data provide strong evidence for the importance of protein chaperones as a mechanism of cell preservation in freeze tolerant insects.

Oxygen is critical to insect life. Oxygen-based respiration offers a tremendous advantage for cellular energetics by supporting high-efficiency production of ATP from the complete oxidation of substrates to CO 2 and H 2 O and is an absolute requirement for powering insect flight muscles. However, once tied to aerobic respiration, organisms have to deal with various consequences of an oxygen-based life, including oxygen limitation (hypoxia, anoxia), oxygen overabundance (hyperoxia) and oxygen in its many reactive forms (e.g. superoxide, hydroperoxides, hydroxyl radical, etc.). The last cause oxidative stress, one of the most pervasive forms of cellular stress, since reactive oxygen species (ROS) can attack and damage most types of cellular macromolecules. This chapter looks at insect cold-hardiness from the point of view of the oxygen-related issues that are important for winter survival. We examine hypoxia/anoxia, freeze-induced ischemia, mitochondria, metabolic-rate depression, oxidative stress and antioxidants as they relate to the low-temperature biology of insects. Oxygen challenges in winter Although there are some unique insect solutions for dealing with winter (e.g. migration of monarch butterflies to Mexico or shivering thermogenesis to provide central heating for honey bee colonies), terrestrial insects generally employ three basic options (sometimes in combination) to survive the cold winter months – underground hibernation below the frostline (in burrows or by digging), freeze-avoidance by supercooling, or freeze-tolerance.