Modulation of stress proteins and apoptotic regulators in the anoxia tolerant turtle brain

Freshwater turtles survive prolonged anoxia and reoxygenation without overt brain damage by well-described physiological processes, but little work has been done to investigate the molecular changes associated with anoxic survival. We examined stress proteins and apoptotic regulators in the turtle during early (1 h) and long-term anoxia (4, 24 h) and reoxygenation. Western blot analyses showed changes within the first hour of anoxia; multiple stress proteins (Hsp72, Grp94, Hsp60, Hsp27, and HO-1) increased while apoptotic regulators (Bcl-2 and Bax) decreased. Levels of the ER stress protein Grp78 were unchanged. Stress proteins remained elevated in long-term anoxia while the Bcl-2/Bax ratio was unaltered. No changes in cleaved caspase 3 levels were observed during anoxia while apoptosis inducing factor increased significantly. Furthermore, we found no evidence for the anoxic translocation of Bax from the cytosol to mitochondria, nor movement of apoptosis inducing factor between the mitochondria and nucleus. Reoxygenation did not lead to further increases in stress proteins or apoptotic regulators except for HO-1. The apparent protection against cell damage was corroborated with immunohistochemistry, which indicated no overt damage in the turtle brain subjected to anoxia and reoxygenation. The results suggest that molecular adaptations enhance pro-survival mechanisms and suppress apoptotic pathways to confer anoxia tolerance in freshwater turtles.     

Signal transduction pathways through cytoprotective, apoptotic and hypertrophic stimuli: a comparative study in adult cardiac myocytes

In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis. Multiple signalling pathways have been implicated in these responses and among them, kinases such as mitogen‐activated protein kinases (MAPKs) and Akt. However, the distinction between signalling pathways originally believed to be specific for either hypertrophy, apoptosis or cell survival is fading. The existing data, coming from different experimental systems, often are conflicting. In this study, we sought to compare aspects of intracellular signalling activated by diverse stimuli in a single experimental system, adult rat cardiac myocytes. Furthermore, we assessed the role of these stimuli in eliciting a particular cell phenotype, i.e. whether they promote hypertrophy, cell survival or apoptosis. The results demonstrate that the hypertrophic agonist phenylephrine is the most potent activator of MAPKs/mitogen and stress‐ activated kinase MSK1, although its effect on Akt phosphorylation is relatively minor. The pro‐apoptotic concentration of H₂O₂ activates strongly both MAPKs and PI3K/Akt pathways. Insulin‐like growth factor‐1 has a minimal effect on phosphorylation of MAPKs/MSK1, but it is a potent activator of Akt. In conclusion, hypertrophic, pro‐survival or apoptotic stimuli operate through the same signalling pathways with different time course and amplitude of kinase activation. Thus, to determine the effect of different stimuli on cell fate, it is important to assess signalling pathways as a network and not as a single pathway. Copyright © 2011 John Wiley & Sons, Ltd.     

Cadmium-induced apoptosis in lymphoblastoid cell line: involvement of caspase-dependent and -independent pathways

Cadmium is a widely used heavy metal that causes severe damage to many organs including liver, kidney and lung. Cadmium toxicity has been described as in vitro and in vivo apoptosis but its molecular mechanisms are not fully understood. In this study, we used the human lymphoblastoid cell line Boleth to characterise cadmium-induced apoptosis further, using sub-lethal (10 microM) and lethal (IC50: 350 microM) doses. At lethal concentration, we observed features of apoptosis between 6 and 8 h after treatment: maturation of caspases 3 and 8, poly(ADP-ribose)polymerase (PARP) cleavage and DNA fragmentation. In order to determine the role of the MAPKs in this process, we investigated p38, ERK1/2 and c-Jun NH2-terminal kinases (JNK) phosphorylation: at lethal concentration, all these pathways were rapidly activated, but no decrease in the apoptotic rate was seen on inhibition of these kinases with drugs. Chemical inhibitors of caspases 3 and 8 blocked cleavage of PARP but not cell death, suggesting the existence of a caspase-independent death. We found that cadmium depolarised membrane potential in less than 1 h, as determined with DiOC6 dye. Interestingly, mitochondrial alteration led to the translocation of apoptosis-inducing factor (AIF) to the nucleus, where we observed chromatin condensation and possibly DNA fragmentation. These results suggest that cadmium-induced apoptosis can occur in the Boleth cell line through caspase-dependent and -independent pathways, independently of activation of major MAPKs.     

Distinctive regulation and function of PI 3K/Akt and MAPKs in doxorubicin-induced apoptosis of human lung adenocarcinoma cells

Regulation and function of PI 3K/Akt and mitogen-activated protein kinases (MAPKs) in doxorubicin-induced cell death were investigated in human lung adenocarcinoma cells. Doxorubicin induced dose-dependent apoptosis of human lung adenocarcinoma NCI-H522 cells. Prior to cell death, both Akt and the MAPK family members (MAPKs: ERK1/2, JNK, and p38) were activated in response to the drug treatment. The kinetics of the inductions for Akt and MAPKs are, however, distinct. The activation of Akt was rapid and transient, activated within 30 min of drug addition, then declined after 3 h, whereas the activations of three MAPKs occurred later, 4 h after addition of the drug and sustained until cell death occurred. Inhibition of PI 3K/Akt activation had no effect on MAPKs' activation, suggesting that the two pathways are independently activated in response to the drug treatment. Inhibition of PI 3K/Akt and p38 accelerated and enhanced doxorubicin-induced cell death. On the contrary, inhibition of ERK1/2 or JNK had no apparent effect on the cell death. Taken together, these results suggest that PI 3K/Akt and MAPKs signaling pathways are all activated, but with distinct mechanisms, in response to doxorubicin treatment. Activation of PI 3K/Akt and p38 modulates apoptotic signal pathways and inhibits doxorubicin-induced cell death. These responses of tumor cells to cancer drug treatment may contribute to their drug resistance. Understanding of the mechanism and function of the responses will be beneficial for the development of novel therapeutic approaches for improvement of drug efficacy and circumvention of drug resistance.     

Activation of mitogen-activated protein kinases during natural freezing and thawing in the wood frog

The responses of mitogen-activated protein kinase (MAPK) family members, including ERK (extracellular signal-regulated kinase), JNK (c-Jun NH₂-terminal kinase), and p38, in the metabolic responses to whole animal freezing (up to 24 h frozen at –2.5°C) and thawing (up to 4 h at 5°C after a 12 h freeze) were examined in four organs (liver, kidney, heart, brain) of the freeze-tolerant wood frog Rana sylvatica. Levels of the active phosphorylated form of p38 increased within 20 min as an early response to freezing in liver and kidney but rose later (after 12 h) in heart. Both JNK and p38 were activated during thawing in liver, kidney and heart with temporally-distinct patterns in each organ. The only MAPK response to freeze/thaw in frog brain was a transient elevation of p38 after 90 min thawing. ERK activity did not respond to freeze/thaw in any organ. The levels of c-Fos increased during freezing in kidney and brain whereas c-Jun was unaffected by freeze/thaw. Organ-specific responses by MAPKs, particularly p38, suggest that these may have roles in regulating metabolic or gene expression responses that may be adaptive in dealing with freezing stress or metabolic recovery during thawing.     

The western painted turtle genome,a model for the evolution of extreme physiological adaptations in a slowly evolving lineage

Background

We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species'' physiological capacities to withstand extreme anoxia and tissue freezing.

Results

Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.

Conclusions

Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle''s extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.     

The ecology of overwintering among turtles: where turtles overwinter and its consequences

Turtles are a small taxon that has nevertheless attracted much attention from biologists for centuries. However, a major portion of their life cycle has received relatively little attention until recently - namely what turtles are doing, and how they are doing it, during the winter. In the northern parts of their ranges in North America, turtles may spend more than half of their lives in an overwintering state. In this review, I emphasise the ecological aspects of overwintering among turtles, and consider how overwintering stresses affect the physiology, behaviour, distributions, and life histories of various species.Sea turtles are the only group of turtles that migrate extensively, and can therefore avoid northern winters. Nevertheless, each year a number of turtles, largely juveniles, are killed when trapped by cold fronts before they move to safer waters. Evidently this risk is an acceptable trade-off for the benefits to a population of inhabiting northern developmental habitats during the summer.Terrestrial turtles pass the winter underground, either in burrows that they excavate or that are preformed. These refugia must provide protection against desiccation and lethal freezing levels. Some burrows are extensive (tortoise genus Gopherus), while others are shallow, or the turtles may simply dig into the ground to a safe depth (turtle genus Terrapene). In the latter genus, freeze tolerance may play an adaptive role.Most non-marine aquatic turtles overwinter underwater, although Clemmys (Actinemys) marmorata routinely overwinters on land when it occurs in riverine habitats, Kinosternon subrubrum often overwinters on land, and several others may overwinter terrestrially on occasion, especially in more southern climates. For northern species that overwinter underwater, there are two physiological groupings, those that are anoxia-tolerant and those that are relatively anoxia-intolerant. All species fare well physiologically in water with a high partial pressure of oxygen (PO2). A lack of anoxia tolerance limits the types of habitats that a freshwater turtle may live in, since unlike sea turtles, they cannot travel long distances to hibernate.Hatchlings of some species of turtles spend their first winter in or below the nest cavity, while hatchlings of other species in the same area, including northern areas, emerge in the autumn and presumably hibernate underwater. All hatchlings are relatively anoxia-intolerant, and there are no studies to date of where hatchling turtles that do not overwinter in or below the nest cavity spend their first winter. Equally little is known of the ontogeny of anoxia tolerance, other than that adults of all species are more anoxia-tolerant than their hatchlings, probably because of their better ossified shells, which provide adults with more buffer reserves and a larger site in which to sequester lactate. The northern limits of turtles are most likely determined by reproductive limitations (time for egg-laying, incubation, and hatching) than by the rigors of hibernation.Mortality is typically lower in turtle populations during hibernation than it is during their active periods. However, episodic mortality events do occur during hibernation, due to freezing, prolonged anoxia, or predation.     

Rapid transmission of oxidative and nitrosative stress signals from roots to shoots in Arabidopsis.

Protein kinases play a central role in signal transduction pathways in eukaryotes. A highly conserved group of kinases, termed mitogen-activated-protein kinases (MAPKs) was shown to mediate many diverse stress responses. In plants, MAPKs were shown to function in resistance responses to many biotic and abiotic stresses. Here, we show that exposure of Arabidopsis roots to hydrogen peroxide or to nitric oxide resulted in rapid activation of protein kinases in the shoots that exhibited MAPK properties. The same pattern of kinases was induced by direct injection of these compounds into leaves, indicating accurate long-distance transmission of H2O2 and NO signals. These results are important for the understanding of redox signal transmission from the rhizosphere throughout the plant.     

In the cellular garden of forking paths: how p38 MAPKs signal for downstream assistance

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes which connect cell-surface receptors to regulatory targets within cells and convert receptor signals into various outputs. In mammalian cells, four distinct MAPKs have been identified: the extracellular signal-related kinases (ERK)-1/2, the c-jun N-terminal kinases or stress-activated protein kinases 1 (JNK1/2/3, or SAPK1s), the p38 MAPKs (p38 alpha/beta/gamma/delta, or SAPK2s), and the ERK5 or big MAP kinase 1 (BMK1). The p38 MAPK cascade is activated by stress or cytokines and leads to phosphorylation of its central elements, the p38 MAPKs. Downstream of p38 MAPKs there is a diversification and extensive branching of signalling pathways. For that reason, we will focus in this review on the different signalling events that are triggered by p38 activity, and analyse how these events contribute to specific gene expression and cellular responses.