RESUMEN

The grey tree frog, Dryophytes versicolor, survives whole-body freezing for weeks during cold winter months. Survival in a state devoid of available food, water, or oxygen forces a reliance on metabolic rate depression (MRD) and the reprioritization of bodily functions. This study utilizes next-generation sequencing (NGS) and bioinformatic analyses to characterize changes in the microRNAome of D. versicolor. When comparing control to frozen groups, five microRNAs (miRNA) were found to be differentially regulated (miR-143-3p, miR-30e-3p, miR-10a-5p, miR-140-3p, and miR-148a-3p), suggesting that they play key roles in freeze survival. The KEGG and GO analyses of these changes predicted a significant negative enrichment of terms associated with cell proliferation and active metabolism while simultaneously predicting the upregulation of cell signalling terms. These results suggest a fast-acting regulatory role for miRNA in contributing to the reorganization of gene expression and the limitation of energy-expensive processes during MRD in the hind leg skeletal muscle of the frog.

RESUMEN

Wood frogs are freeze-tolerant vertebrates that can endure weeks to months frozen during the winter without breathing and with as much as 65% of total body water frozen as extracellular ice. Underlying tolerances of anoxia and of cellular dehydration support whole body freezing. One pro-survival mechanism employed by these frogs is epigenetic modifications via DNA hypomethylation processes facilitating transcriptional repression or activation. These processes involve proteins such as DNA Methyltransferases (DNMTs), Methyl Binding Domain proteins (MBDs), Ten-Eleven Translocases (TETs), and Thymine Deglycosylase (TDG). The present study evaluates the responses of these proteins to dehydration and anoxia stresses in wood frog liver. DNMT relative protein expression was reduced in liver, but nuclear DNMT activity did not change significantly under anoxia stress. By contrast, liver DNMTs and nuclear DNMT activity were upregulated under dehydration stress. These stress-specific differences were speculated to arise from Post-Translational Modifications (PTMs). DNMT3A and DNMT3B showed increased relative protein expression during recovery from dehydration and anoxia. Further, MBD1 was elevated during both conditions suggesting transcriptional repression. TET proteins showed varying responses to anoxia likely due to the absence of oxygen, a main substrate required by TETs. Similarly, TDG, an enzyme that corrects DNA damage, was downregulated under anoxia potentially due to lower levels of reactive oxygen species that damage DNA, but levels returned to normal during reperfusion of oxygen. Our results indicate differential stress-specific responses that indicate the need for more research in the DNA hypomethylation mechanisms employed by the wood frog during stress.

Asunto(s)

RESUMEN

Naked mole-rats, Heterocephalus glaber, are champion hypoxia-tolerant rodents that live under low oxygen conditions in their subterranean burrows. Detrimental effects of low oxygen can be mitigated through metabolic rate depression (MRD), metabolic reorganization, and global downregulation of nonessential cellular processes. Recent research has progressively implicated epigenetic modifications - rapid, reversible changes to gene expression that do not alter the DNA sequence itself - as major players in implementing and maintaining MRD. N6-adenosine (m6A) methylation is the most prevalent mammalian RNA modification and is responsible for pre-mRNA processing and mRNA export from the nucleus. Hence, m6A -mediated conformational changes alter the cellular fate of transcripts. The present study investigated the role of m6A RNA methylation responses to 24 h of hypoxia exposure in H. glaber cardiac tissue. Total protein levels of m6A writers/readers/erasers, m6A demethylase activity, and total m6A quantification were measured under normoxic vs. hypoxic conditions in H. glaber heart. While there was no change in either demethylase activity or total m6A content, many proteins of the m6A pathway were downregulated during hypoxia. Overall, m6A may not be a signature hypoxia-responsive characteristic in H. glaber heart, but downregulation of the protein machinery involved in m6A cycling points to an alternate biological involvement. Further research will explore other forms of RNA modifications and other epigenetic mechanisms to determine the controls on hypoxia endurance in this subterranean mammal.

Asunto(s)

RESUMEN

For the breadth of the winter, Dryophytes versicolor can survive full body freezing utilizing a phenomenon known as metabolic rate depression (MRD). Epigenetic transcriptional control on gene expression, such as histone methylation and acetylation, can aid in implementing a balance between permissive and restricted chromatin required to endure this stress. As such, this study explores the interplay between histone lysine methyl and acetyl transferases (HKMTs, HATs), as well as the abundance of various acetyl-lysine and methyl-lysine moieties on histone H3 and H4. Results showing that overexpression of transcriptionally repressive marks, and under expression of active ones, suggest a negative effect on overall gene transcription in skeletal muscle tissue.

Asunto(s)

RESUMEN

The Arctic is exposed to unprecedented warming, at least three times higher than the global average, which induces significant melting of the cryosphere. Freshwater inputs from melting glaciers will subsequently affect coastal primary production and organic matter quality. However, due to a lack of basic knowledge on the physiology of Arctic organisms, it remains difficult to understand how these future trophic changes will threaten the long-term survival of benthic species in coastal habitats. This study aimed to gain new insights into the seasonal lipid dynamics of four dominant benthic bivalves (Astarte moerchi, Hiatella arctica, Musculus discors, and Mya truncata) collected before and after sea ice break-up in a high-Arctic fjord (Young Sound, NE Greenland). Total lipid content and fatty acid composition of digestive gland neutral lipids were analyzed to assess bivalve energy reserves while the fatty acid composition of gill polar lipids was determined as a biochemical indicator of interspecies variations in metabolic activity and temperature acclimation. Results showed a decrease in lipid reserves between May and August, suggesting that bivalves have only limited access to fresh organic matter until sea ice break-up. The lack of seasonal variation in the fatty acid composition of neutral lipids, especially essential ω3 fatty acids, indicates that no fatty acid transfer from the digestive glands to the gonads occurs between May and August, and therefore, no reproductive investment takes place during this period. Large interspecies differences in gill fatty acid composition were observed, which appear to be related to differences in species life span and metabolic strategies. Such differences in gill fatty acid composition of polar lipids, which generally influence metabolic rates and energy needs, may imply that not all benthic species will be equally sensitive to future changes in primary production and organic matter quality in Arctic coastal habitats.

RESUMEN

The thirteen-lined ground squirrel Ictidomys tridecemlineatus is a rodent that lives throughout the United States and Canada and uses metabolic rate depression to facilitate circannual hibernation which helps it survive the winter. Metabolic rate depression is the reorganization of cellular physiology and molecular biology to facilitate a global downregulation of nonessential genes and processes, which conserves endogenous fuel resources and prevents the buildup of waste byproducts. Facilitating metabolic rate depression requires a complex interplay of regulatory approaches, including post-transcriptional modes such as microRNA. MicroRNA are short, single-stranded RNA species that bind to mRNA transcripts and target them for degradation or translational suppression. Using next-generation sequencing, we analyzed euthermic vs. hibernating cardiac tissue in I. tridecemlineatus to predict seven miRNAs (let-7e-5p, miR-122-5p, miR-2355-3p, miR-6715b-3p, miR-378i, miR-9851-3p, and miR-454-3p) that may be differentially regulated during hibernation. Gene ontology and KEGG pathway analysis suggested that these miRNAs cause a strong activation of ErbB2 signaling which causes downstream effects, including the activation of MAPK and PI3K/Akt signaling and concurrent decreases in p53 signaling and cell cycle-related processes. Taken together, these results predict critical miRNAs that may change during hibernation in the hearts of I. tridecemlineatus and identify key signaling pathways that warrant further study in this species.

RESUMEN

The wood frog, Rana sylvatica endures whole body freezing for weeks/months while overwintering at subzero temperatures. Survival of long-term freezing requires not only cryoprotectants but also strong metabolic rate depression (MRD) and reorganization of essential processes in order to maintain a balance between ATP-producing and ATP-consuming processes. Citrate synthase (CS) (E.C. 2.3.3.1) is an important irreversible enzyme of the tricarboxylic acid (TCA) cycle and forms a crucial checkpoint for many metabolic processes. Present study investigated the regulation of CS from wood frog liver during freezing. CS was purified to homogeneity by a two-step chromatographic process. Kinetic and regulatory parameters of the enzyme were investigated and, notably, demonstrated a significant decrease in the Vmax of the purified form of CS from frozen frogs as compared to controls when assayed at both 22 °C and 5 °C. This was further supported by a decrease in the maximum activity of CS from liver of frozen frogs. Immunoblotting also showed changes in posttranslational modifications with a significant decrease in threonine phosphorylation (by 49 %) for CS from frozen frogs. Taken together, these results suggest that CS is suppressed and TCA flux is inhibited during freezing, likely to support MRD survival of harsh winters.

Asunto(s)

RESUMEN

Malagasy tenrecs are placental hibernating mammals that seal the entrances to their burrows and hibernate either singly or in groups for 8-9 months, which is likely to create a hypoxic and hypercapnic burrow environment. Therefore, we hypothesized that tenrecs are tolerant to environmental hypoxia and hypercapnia. Many hypoxia- and hypercapnia-tolerant fossorial mammals respond to hypoxia by decreasing metabolic rate and thermogenesis, and have blunted ventilatory responses to both environmental hypoxia and hypercapnia. However, tenrecs exhibit extreme metabolic and thermoregulatory plasticity, which exceeds that of most heterothermic mammals and approaches that of ectothermic reptiles. Thus, we predicted that tenrecs would have abnormal physiological responses to hypoxia and hypercapnia relative to other fossorial mammals. To test this, we exposed common tenrecs (Tenrec ecaudatus) to moderate and severe hypoxia (9 and 4% O2) or hypercapnia (5 and 10% CO2) in either 28 or 16°C while non-invasively measuring metabolic rate, thermogenesis and ventilation. We found that tenrecs exhibit robust metabolic decreases in both hypoxia and hypercapnia. Furthermore, tenrecs have blunted ventilatory responses to both hypoxia and hypercapnia, and these responses are highly temperature sensitive such that they are reduced or absent in 16°C. Thermoregulation was highly variable in 16°C but constrained in 28°C across all treatment conditions and was not impacted by hypoxia or hypercapnia, unlike in other heterothermic mammals. Taken together, our results indicate that physiological responses to hypoxia and hypercapnia in tenrecs are highly dependent on environmental temperature and differ from those of other mammalian heterotherms.

Asunto(s)

RESUMEN

The rapid and reversible nature of microRNA (miRNA) transcriptional regulation is ideal for implementing global changes to cellular processes and metabolism, a necessary asset for the freeze-tolerant gray tree frog (Dryophytes versicolor). D. versicolor can freeze up to 42% of its total body water during the winter and then thaw completely upon more favorable conditions of spring. Herein, we examined the freeze-specific miRNA responses in the gray tree frog using RBiomirGS, a bioinformatic tool designed for the analysis of miRNA-seq transcriptomics in non-genome sequenced organisms. We identified 11 miRNAs differentially regulated during freezing (miR-140-3p, miR-181a-5p, miR-206-3p, miR-451a, miR-19a-3p, miR-101-3p, miR-30e-5p, miR-142-3p and -5p, miR-21-5p, and miR-34a-5p). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis suggests these miRNAs play roles in downregulating signaling pathways, apoptosis, and nuclear processes while enhancing ribosomal biogenesis. Overall, these findings point towards miRNA inducing a state of energy conservation by downregulating energy-expensive pathways, while ribosomal biogenesis may lead to prioritization of critical processes for freeze-tolerance survival.

Asunto(s)

RESUMEN

Couch's spadefoot toad (Scaphiopus couchii) spends most of the year underground in a hypometabolic state known as estivation. During this time, they overcome significant dehydration and lack of food through many mechanisms including employing metabolic rate depression (MRD), increasing urea concentration, switching to lipid oxidation as the primary energy source, and decreasing their breathing and heart rate. MicroRNA (miRNA) are known to regulate translation by targeting messenger RNA (mRNA) for degradation or temporary storage, with several studies having reported that miRNA is differentially expressed during MRD, including estivation. Thus, we hypothesized that miRNA would be involved in gene regulation during estivation in S. couchii heart. Next-generation sequencing and bioinformatic analyses were used to assess changes in miRNA expression in response to two-month estivation and to predict the downstream effects of this expression. KEGG and GO analyses indicated that ribosome and cardiac muscle contraction are among the pathways predicted to be upregulated, whereas cell signaling and fatty acid metabolism were predicted to be downregulated. Together these results suggest that miRNAs contribute to the regulation of gene expression related to cardiac muscle physiology and energy metabolism during estivation.

Asunto(s)

RESUMEN

Freeze tolerance is an adaptive strategy that wood frogs (Rana sylvatica) use to survive the subzero temperatures of winter. It is characterized by a variety of metabolic and physiological changes that facilitate successful freezing and anoxia. As both mRNA regulation and posttranslation protein modification have been implicated in freeze tolerance, we hypothesized that posttranslational RNA regulation is also involved in coordinating freeze-thaw cycles and metabolic rate depression. As such, we investigated the most abundant RNA modification, adenosine methylation (N6 -methyladenosine; m6 A) in wood frog brains during 24 h periods of freezing and anoxia. This was followed by an examination of levels of RNA methyltransferases, demethyltransferases, and the readers of RNA methylation. Despite relative levels of methylation on mRNA remaining constant throughout freezing and anoxia, a significant increase in relative abundance of m6 A methyltransferases METTL3 and METTL14 was observed. In addition, we investigated the effect of m6 A RNA methylation on mRNA triaging to stress granules and report a significant increase in stress granule markers TIAR and TIA-1 in both freezing and anoxia. Our findings are the first report of RNA posttranslational regulation during metabolic rate depression in the wood frog brain and suggest that the dynamic RNA methylation observed is not directly linked to mRNA regulation during periods of extreme metabolic reorganization, warranting future investigations.

Asunto(s)

RESUMEN

The North American amphibian, wood frogs, Rana sylvatica are the most studied anuran to comprehend vertebrate freeze tolerance. Multiple adaptations support their survival in frigid temperatures during winters, particularly their ability to produce glucose as natural cryoprotectant. Freezing and its component consequences (anoxia and dehydration) induce multiple stresses on cells. Among these is endoplasmic reticulum (ER) stress, a condition spawned by buildup of unfolded or misfolded proteins in the ER. The ER stress causes the unfolded protein response (UPR) and the ER-associated degradation (ERAD) pathway that potentially could lead to apoptosis. Immunoblotting was used to assess the responses of major proteins of the UPR and ERAD under freezing, anoxia, and dehydration stresses in the liver and skeletal muscle of the wood frogs. Targets analyzed included activating transcription factors (ATF3, ATF4, ATF6), the growth arrest and DNA damage proteins (GADD34, GADD153), and EDEM (ERAD enhancing α-mannosidase-like proteins) and XBP1 (X-box binding protein 1) proteins. UPR signaling was triggered under all three stresses (freezing, anoxia, dehydration) in liver and skeletal muscle of wood frogs with most tissue/stress responses consistent with an upregulation of the primary targets of all three UPR pathways (ATF4, ATF6, and XBP-1) to enhance the protein folding/refolding capacity under these stress conditions. Only frozen muscle showed preference for proteasomal degradation of misfolded proteins via upregulation of EDEM (ERAD). The ERAD response of liver was downregulated across three stresses suggesting preference for more refolding of misfolded/unfolded proteins. Overall, we conclude that wood frog organs activate the UPR as a means of stabilizing and repairing cellular proteins to best survive freezing exposures.

Asunto(s)

RESUMEN

Rana sylvatica (also known as Boreorana sylvatica) is one of the few vertebrates that spend extreme winters showing no physiological signs of life. Up to 70% of the total body water of the wood frog freezes as extracellular ice. Survival in extreme conditions requires regulation at transcriptional and translational levels to activate prosurvival pathways. N6-methyladenosine (m6A) methylation is one of the most common RNA modifications, regulating transcript processing and translation by executing important functions that affect regulatory pathways in stress conditions. In the study, regulation of m6A-related proteins in the liver of R. sylvatica was analyzed during 24 h frozen and 8 h thaw conditions. Decreases in the activity of demethylases of 28.44 ± 0.4% and 24.1 ± 0.9% of control values in frozen and thaw tissues, respectively, were observed. Total protein levels of m6A methyltransferase complex components methyltransferase-like 14 and Wilm's tumor associated protein were increased by 1.28-fold and 1.42-fold, respectively, during freezing. Demethylase fat mass and obesity, however, showed a decreasing trend, with a significant decrease in abundance during recovery from frozen conditions. Levels of mRNA degraders YTHDF2 and YTHDC2 also decreased under stress. Overall, increased levels of m6A methylation complex components, and suppressed levels of readers/erasers, provide evidence for the potential role of RNA methylation in freezing survival and its regulation in a hypometabolic state.

Asunto(s)

RESUMEN

The wood frog (Rana sylvatica) undergoes physiological and metabolic changes to withstand subzero temperatures and whole body freezing during the winter months. Along with metabolic rate depression, high concentrations of glucose are produced as a cryoprotectant by liver and distributed to all other tissues. Pyruvate kinase (PK; EC:2.7.1.40), the final enzyme of glycolysis, plays an important role in the modulation of glucose metabolism and, therefore, overall metabolic regulation. The present study investigated the functional and kinetic properties of purified PK from liver of control (5 °C acclimated) and frozen (-2.5 °C for 24 h) wood frogs. Liver PK was purified to homogeneity by a two-step chromatographic process, followed by analysis of enzyme properties. A significant decrease in the affinity of PK for its substrates, phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) at 22 °C and 5 °C was noted in liver from frozen frogs, as compared with controls. Immunoblotting also revealed freeze-responsive changes in posttranslational modifications with a significant increase in serine and threonine phosphorylation by 1.46-fold and 1.73- fold for PK from frozen frogs as compared with controls. Furthermore, a test of thermal stability showed that PK from liver of frozen wood frogs showed greater stability as compared with PK from control animals. Taken together, these results suggest that PK is negatively regulated, and glycolysis is suppressed, during freezing. This response acts as an important survival strategy for maintaining continuously elevated levels of cryoprotectant in frogs while they remain in a hypometabolic frozen state.

Asunto(s)

RESUMEN

The naked mole-rat (Heterocephalus glaber) regularly endures intermittent periods of hypoxia in its burrows, surviving in part due to metabolic rate depression (MRD)-a strategy of conserving cellular resources by downregulating nonessential gene expression and reorganizing cellular processes. miRNA are short, noncoding RNAs already implicated for their roles in numerous models of extreme environmental stress; given their rapid, reversible nature, they are ideal for implementing MRD. We performed small RNA sequencing on cardiac tissue from normoxic versus 24 h hypoxic naked mole-rats, and used bioinformatics to predict 18 miRNAs which may be differentially regulated during hypoxia. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway mapping further suggest these miRNAs play roles in largely translation-related functions, including RNA processing and catabolism.

Asunto(s)

RESUMEN

Metabolic rate depression during prolonged bouts of torpor is characteristic of mammalian hibernation, reducing energy expenditures over the winter. Cell cycle arrest is observed in quiescent cells during dormancy, partly due to the retinoblastoma (Rb) protein at G1 /S, given cell division and proliferation are metabolic-costly processes. Rb binds to E2F transcription factors and recruits corepressors (e.g., SUV39H1) to E2F target genes, blocking their transcription and cell cycle passage. Phosphorylation by cyclin-CDK complexes at S780 or S795 abolishes Rb-mediated repression, allowing transition into S phase. The present study compares Rb-E2F1 responses between euthermic and torpid states in five organs (brain, heart, kidney, liver, skeletal muscle) of 13-lined ground squirrels (Ictidomys tridecemlineatus). Immunoblotting assessed the expression of Rb, pRb (S780, S795), E2F1, and SUV39H1. Our findings demonstrate multi-tissue upregulation of Rb and SUV39H1 during torpor, with tissue-specific changes to E2F1 and pRb (S780), suggesting Rb-E2F1 contributes to cell cycle control in hibernation.

Asunto(s)

RESUMEN

Transcriptional suppression is characteristic of extreme stress responses, speculated to preserve energetic resources in the maintenance of hypometabolism. In recent years, epigenetic regulation has become heavily implicated in stress adaptation of many animals, including supporting freeze tolerance of the wood frog (Rana sylvatica). However, nervous tissues are frequently lacking in these multi-tissue analyses which warrants investigation. The present study examines the role of DNA methylation, a core epigenetic mechanism, in the response of wood frog brains to freezing. We use immunoblot analysis to track the relative expression of DNA methyltransferases (DNMT), methyl-CpG-binding domain (MBD) proteins and ten-eleven-translocation (TET) demethylases across the freeze-thaw cycle in R. sylvatica brain, including selected comparisons to freeze-associated sub-stresses (anoxia and dehydration). Global methyltransferase activities and 5-hmC content were also assessed. The data show coordinated evidence for DNA hypomethylation in wood frog brains during freeze-recovery through the combined roles of depressed DNMT3A/3L expression driving lowered DNMT activity and increased TET2/3 levels leading to elevated 5-hmC genomic content (p < 0.05). Raised levels of DNMT1 during high dehydration were also noteworthy. The above suggest that alleviation of transcriptionally repressive 5-mC DNA methylation is a necessary component of the wood frog freeze-thaw cycle, potentially facilitating the resumption of a normoxic transcriptional state as frogs thaw and resume normal metabolic activities.

RESUMEN

Wood frogs, Rana sylvatica, endure the freezing of ∼65% of total body water while overwintering in cold climates, enduring not only internal ice formation but also long-term anoxia due to cessation of heartbeat and breathing. Thawing restores perfusion but rapid reoxygenation can increase vulnerability to reactive oxygen species and induce oxidative damage. This study provides a first assessment of antioxidant capacity, DNA damage, and DNA repair responses comparing freeze/thaw and anoxia/reoxygenation in liver and skeletal muscle of wood frogs. Oxidation of guanine resides in DNA did not change under either stress but total antioxidant capacity rose in both tissues under anoxia. Relative expression of eight proteins involved in double-stranded break repair (Mre11, Rad50, phospho-p95, XLF, DNA ligase IV, XRCC4, Ku70, Rad51) were assessed in both tissues. Freezing suppressed Ku70 and Rad51 in liver and Rad51 in muscle but levels rose again after thawing. Anoxia exposure suppressed XLF, Ku70 and Rad51 proteins in muscle. However, in liver, anoxia exposure led to elevated Mre11, Ku70 and DNA ligase IV, the former two belonging to the MRN complex that binds DNA and marks sites of double stranded breaks (DSBs). Large increases in Mre11 and Ku70 expression suggested DSB damage in liver under anoxia but not during freezing, whereas muscle was resistant to DSB damage under both stresses. These data indicate that DNA damage is minimal during whole body freezing due to tissue and stress specific regulation of antioxidant capacity and DNA damage repair to preserve genomic integrity.

Asunto(s)

RESUMEN

Grey tree frogs (Dryophytes versicolor) have the remarkable ability to endure full-body freezing over the winter, with up to 42% of total body water converted into extracellular ice. Survival is aided by metabolic rate depression that greatly reduces tissue energy costs over the winter. Post-transcriptional controls on gene expression which include miRNA regulation of gene transcripts can aid implementation of the reversible changes required for freeze tolerance, since miRNAs are ideal for facilitating the rapid metabolic reorganization needed for this process. The energy cost for synthesizing new miRNAs is low, and miRNAs' ability to target more than one mRNA transcript (and vice versa) allows a wide versatility in their capability for metabolic restructuring. Western immunoblotting was used to examine protein expression levels of members of the miRNA biogenesis pathway in D. versicolor liver, skeletal muscle, and kidney. Four of these proteins (Dicer, Drosha, Trbp, Xpo5) were upregulated in liver of frozen frogs, suggesting enhanced capacity for miRNA biogenesis, whereas expression of four proteins in frozen muscle (Ago1, Ago2, Dgcr8, Xpo5) and six proteins in kidney (Ago1, Ago2, Ago3, Ago4, Dgcr8, Ran-GTP) were downregulated, indicating an opposite trend. Overall, the data show that miRNA biosynthesis is altered during freezing and differentially regulated across tissues. We suggest that miRNAs are central for the freeze tolerance strategy developed by D. versicolor, and future research will expound upon specific miRNAs and their roles in mediating responses to freezing stress.

Asunto(s)

RESUMEN

Tumor suppressing transcription factor p53 regulates multiple pathways including DNA repair, cell survival, apoptosis and autophagy. Here, we studied the stress-induced activation of p53 in anoxic crayfish (Faxonius virilis). Relative levels of target proteins and mRNAs involved in the DNA damage response were measured in normoxic control and anoxic hepatopancreas and tail muscle. Phosphorylation levels of p53 were assessed using immunoblotting at sites known to be phosphorylated (serine 15 and 37) in response to DNA damage or reduced oxygen signaling. The capacity for DNA binding by phosphorylated p53 (p-p53) was also measured, followed by transcript analysis of a potentially pro-apoptotic downstream target, the etoposide induced (ei24) gene. Following this, both inhibitor (MDM2) and activator (p19-ARF) protein levels in response to low-oxygen stress were studied. The results showed an increase in p-p53 levels during anoxia in both hepatopancreas and tail muscle. Increased transcript levels of ei24 support the activation of p53 under anoxic stress. Cytoplasmic accumulation of Ser15 phosphorylated p53 was observed during anoxia when proteins from cytoplasmic and nuclear fractions were measured. Increased cytoplasmic concentration is known to initiate an apoptotic response, which can be assumed as a preparatory step to prevent autophagy. The results suggest that p53 might play a protective role in crayfish defense against low-oxygen stress. Understanding how anoxia-tolerant organisms are able to protect themselves against DNA damage could provide important clues towards survival under metabolic rate depression and preparation for recovery to minimize damage.

Asunto(s)