Long-term Anoxia Research Articles

Visualization of intracellular ATP dynamics in different nephron segments under pathophysiological conditions using the kidney slice culture system

ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here, we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated the ATP synthesis pathway, and analyzed intracellular ATP dynamics using an exvivo IR-mimicking model and a cisplatin nephropathy model. Proximal tubules (PTs) were found to be strongly dependent on oxidative phosphorylation (OXPHOS) using the inhibitor oligomycin A, whereas podocytes relied on both OXPHOS and glycolysis using phloretin an active transport inhibitor of glucose. We also confirmed that an exvivo IR-mimicking model could recapitulate ATP dynamics invivo; ATP recovery in PTs after reoxygenation varied depending on anoxic time length, whereas ATP in distal tubules (DTs) recovered well even after long-term anoxia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor, cimetidine, suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent (Mitochonic Acid 5) delayed the cisplatin-induced ATP decrease in PTs. Thus, our novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease.

Two decades of research on anoxia tolerance - mitochondria, -omics and physiological diversity.

Just over two decades ago, Bob Boutilier published a much-cited Review in this journal on the mechanisms of cell survival in hypoxia and hypothermia. Here, we celebrate this important Review by describing how our knowledge of the mechanisms behind anoxia tolerance have progressed since 2001, including new key roles of mitochondria, something Boutilier had started exploring. Evidence now suggests that, in anoxia-tolerant brains, mitochondria initiate responses aimed at suppressing electrical activity and energy use. These responses are largely dependent on gamma-aminobutyric acid (GABA) release. Animals that survive anoxia must also tolerate reoxygenation - a major challenge that could cause a massive production of damaging reactive oxygen species (ROS). Here, the handling of succinate, which builds up during anoxia, is critical. Interestingly, there are clear species differences in succinate handling among anoxia-tolerant vertebrates (Trachemys and Chrysemys turtles and crucian carp, Carassius carassius). Trachemys turtles suppress succinate build-up during anoxia, presumably to limit ROS production during reoxygenation. By contrast, in crucian carp, reduction of fumarate to succinate during anoxia appears to be essential for keeping their mitochondria charged and viable. Consequently, during anoxia, crucian carp accumulate much more succinate than Trachemys turtles. Moreover, during anoxia, succinate is apparently transported from crucian carp brain and heart to the liver, which handles succinate upon reoxygenation. This is one example of the striking physiological diversity among vertebrates that survive long-term anoxia. More examples are given, and we argue that -omics approaches are, and will be, helpful in providing new insight and moving the field forward.

DNA damage and repair responses to freezing and anoxia stresses in wood frogs, Rana sylvatica

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.

A new conceptual framework for the transformation of groundwater dissolved organic matter

Groundwater comprises 95% of the liquid fresh water on Earth and contains a diverse mix of dissolved organic matter (DOM) molecules which play a significant role in the global carbon cycle. Currently, the storage times and degradation pathways of groundwater DOM are unclear, preventing an accurate estimate of groundwater carbon sources and sinks for global carbon budgets. Here we reveal the transformations of DOM in aging groundwater using ultra-high resolution mass spectrometry combined with radiocarbon dating. Long-term anoxia and a lack of photodegradation leads to the removal of oxidised DOM and a build-up of both reduced photodegradable formulae and aerobically biolabile formulae with a strong microbial signal. This contrasts with the degradation pathway of DOM in oxic marine, river, and lake systems. Our findings suggest that processes such as groundwater extraction and subterranean groundwater discharge to oceans could result in up to 13 Tg of highly photolabile and aerobically biolabile groundwater dissolved organic carbon released to surface environments per year, where it can be rapidly degraded. These findings highlight the importance of considering groundwater DOM in global carbon budgets.

Gene expression of hypoxia-inducible factor (HIF), HIF regulators, and putative HIF targets in ventricle and telencephalon of Trachemys scripta acclimated to 21 °C or 5 °C and exposed to normoxia, anoxia or reoxygenation

In anoxia-sensitive mammals, hypoxia inducible factor (HIF) promotes cellular survival in hypoxia, but also tumorigenesis. By comparison, anoxia-tolerant vertebrates likely need to circumvent a prolonged upregulation of HIF to survive long-term anoxia, making them attractive biomedical models for investigating HIF regulation. To lend insight into the role of HIF in anoxic Trachemys scripta ventricle and telencephalon, 21 °C- and 5 °C-acclimated turtles were exposed to normoxia, anoxia (24 h at 21 °C; 24 h or 14 d at 5 °C) or anoxia + reoxygenation and the gene expression of HIF-1α (hif1a) and HIF-2α (hif2a), two regulators of HIF, and eleven putative downstream targets of HIF quantified by qPCR. Changes in gene expression with anoxia at 21 °C differentially aligned with a circumvention of HIF activity. Whereas hif1a and hif2a expression was unaffected in ventricle and telencephalon, and BCL2 interacting protein 3 gene expression reduced by 30% in telencephalon, gene expression of vascular endothelial growth factor-A increased in ventricle (4.5-fold) and telencephalon (1.5-fold), and hexokinase 1 (2-fold) and hexokinase 2 (3-fold) gene expression increased in ventricle. At 5 °C, the pattern of gene expression in ventricle or telencephalon was unaltered with oxygenation state. However, cold acclimation in normoxia induced downregulation of HIF-1α, HIF-2α, and HIF target gene expression in telencephalon. Overall, the findings lend support to the postulation that prolonged activation of HIF is counterproductive for long-term anoxia survival. Nevertheless, quantification of the effect of anoxia and acclimation temperature on HIF binding activity and regulation at the protein level are needed to provide a strong scientific framework whereby new strategies for oxygen related pathologies can be developed.

Screening of ‘King’ Mandarin Hybrids as Tolerant Citrus Rootstocks to Flooding Stress

This work compares the tolerance to long-term anoxia conditions (35 days) of five new citrus ‘King’ mandarin (Citrus nobilis L. Lour) × Poncirus trifoliata ((L.) Raf.) hybrids (named 0501XX) and Carrizo citrange (CC, Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.), the widely used citrus rootstock in Spain. Growth parameters, chlorophyll concentration, gas exchange and fluorescence parameters, water relations in leaves, abscisic acid (ABA) concentration, and PIP1 and PIP2 gene expressions were assessed. With a waterlogging treatment, the root system biomass of most hybrids went down, and the chlorophyll a and b concentrations substantially dropped. The net CO2 assimilation rates (An) and stomatal conductance (gs) lowered significantly due to flooding, and the transpiration rate (E) closely paralleled the changes in gs. The leaf water and osmotic potentials significantly increased in most 0501 hybrids. As a trend, flooding stress lowered the ABA concentration in roots from most hybrids, but increased in the leaves of CC, 05019 and 050110. Under the control treatment (Ct) conditions, most 0501 hybrids showed higher PIP1 and PIP2 expressions than the control rootstock CC, but were impaired due to the flooding conditions in 05019 and 050110. From this study, we conclude that 0501 genotypes develop some adaptive responses in plants against flooding stress such as (1) stomata closure to prevent water loss likely mediated by ABA levels, and (2) enhanced water and osmotic potentials and the downregulation of those genes regulating aquaporin channels to maintain water relations in plants. Although these traits seemed especially relevant in hybrids 050110 and 050125, further experiments must be done to determine their behavior under field conditions, particularly their influence on commercial varieties and their suitability as flooding-tolerant hybrids for replacing CC, one of the main genotypes that is widely used as a citrus rootstock in Spain, under these conditions.

Cytoskeletal Arrest: An Anoxia Tolerance Mechanism.

Polymerization of actin filaments and microtubules constitutes a ubiquitous demand for cellular adenosine-5′-triphosphate (ATP) and guanosine-5′-triphosphate (GTP). In anoxia-tolerant animals, ATP consumption is minimized during overwintering conditions, but little is known about the role of cell structure in anoxia tolerance. Studies of overwintering mammals have revealed that microtubule stability in neurites is reduced at low temperature, resulting in withdrawal of neurites and reduced abundance of excitatory synapses. Literature for turtles is consistent with a similar downregulation of peripheral cytoskeletal activity in brain and liver during anoxic overwintering. Downregulation of actin dynamics, as well as modification to microtubule organization, may play vital roles in facilitating anoxia tolerance. Mitochondrial calcium release occurs during anoxia in turtle neurons, and subsequent activation of calcium-binding proteins likely regulates cytoskeletal stability. Production of reactive oxygen species (ROS) formation can lead to catastrophic cytoskeletal damage during overwintering and ROS production can be regulated by the dynamics of mitochondrial interconnectivity. Therefore, suppression of ROS formation is likely an important aspect of cytoskeletal arrest. Furthermore, gasotransmitters can regulate ROS levels, as well as cytoskeletal contractility and rearrangement. In this review we will explore the energetic costs of cytoskeletal activity, the cellular mechanisms regulating it, and the potential for cytoskeletal arrest being an important mechanism permitting long-term anoxia survival in anoxia-tolerant species, such as the western painted turtle and goldfish.

Laser-Induced Fluorescence Spectroscopy in the Diagnosis of Tissue Hypoxia (Review)

The aim of review is to discuss the results of studies on diagnosis of tissue hypoxia by laser-induced spectroscopy, as well as to identify promising trends and prospects of this technique for its further application in experimental and clinical medicine.The review presents the findings of studies of the fluorescence intensity of endogenous fluorophore molecules (reduced nicotinamide adenine dinucleotide, oxidized flavin adenine dinucleotide) as markers of ischemic injury of internal organs (brain, heart, liver, kidneys, etc.). The principles of fluorescence laser-induced spectroscopy in vivo are discussed. The historical aspects of the subject are also covered. The results of the use of the technique in experimental and clinical studies of tissue hypoxia and ischemia are shown. Difficulties in interpreting the intensity values of autofluorescent signal of the studied molecules are revealed. It was noted that the tissue autofluorescence in a long-term anoxia remains unknown, and there are no structured ideas about the impact of exogenous and endogenous factors on autofluorescence intensity.In conclusion, the use of laser-induced fluorescence spectroscopy to diagnose tissue ischemia is a promising area of experimental and clinical medicine, which still has various unresolved issues, despite a large number of studies in this domain.

Assessment of the behaviour and survival of nematodes under low oxygen concentrations.

Oxygen is required for the completion of almost all known metazoan lifecycles, but many metazoans harbour abilities to withstand varying degrees and periods of hypoxia. Caenorhabditis elegans, one of the most popular model organism is extensively used as a model for the study of hypoxia and anoxia biology and it has been found that this nematode is capable of tolerance to varying degrees of hypoxia. Considering the extremely high diversity of nematodes, the effects of low oxygen concentration and mechanisms of adaptation to oxygen depletion differ among species. In this study, we used a simple assay to examine anoxia tolerance in four nematode species, including three free-living and one plant parasitic nematode. We found that the plant parasitic nematode Bursaphelenchus xylophilus can survive more than 14 days under anoxic conditions. Comparisons of behaviour during anoxia induction and the repertoire of oxygen sensation genes among the tested species suggested the existence of different oxygen sensation systems between B. xylophilus and C. elegans, which quickly introduce suspended animation in response to oxygen depletion to survive long-term anoxia.

Prophylaxis of fungal infections in liver transplantation from donation after citizen's death

As one of the commonest postoperative complications after liver transplantation, fungal infection has a high incidence and mortality. Nowadays, the development of the organ transplantation technique in China has already entered into the era of donation after citizen's death (DCD). Donors of DCD are mainly derived from the patients after brain death, cardiac death or death of both brain and cardiac. These donors usually suffer from long-term hypotension, ischemia, anoxia and secon-dary recessive or dominant infections during emergency treatment. Hence, preventing the fungal infections in donor organs and recipients after transplantation plays an important role in improving the success rate of liver transplantation. This review focuses on the clinical significance of preventing fungal infection in DCD of liver transplantation. Key words: Fungal infection; Liver transplantation; Risk factors; Prophylaxis

Anti-apoptotic response during anoxia and recovery in a freeze-tolerant wood frog (Rana sylvatica).

The common wood frog, Rana sylvatica, utilizes freeze tolerance as a means of winter survival. Concealed beneath a layer of leaf litter and blanketed by snow, these frogs withstand subzero temperatures by allowing approximately 65–70% of total body water to freeze. Freezing is generally considered to be an ischemic event in which the blood oxygen supply is impeded and may lead to low levels of ATP production and exposure to oxidative stress. Therefore, it is as important to selectively upregulate cytoprotective mechanisms such as the heat shock protein (HSP) response and expression of antioxidants as it is to shut down majority of ATP consuming processes in the cell. The objective of this study was to investigate another probable cytoprotective mechanism, anti-apoptosis during oxygen deprivation and recovery in the anoxia tolerant wood frog. In particular, relative protein expression levels of two important apoptotic regulator proteins, Bax and p-p53 (S46), and five anti-apoptotic/pro-survival proteins, Bcl-2, p-Bcl-2 (S70), Bcl-xL, x-IAP, and c-IAP in response to normoxic, 24 Hr anoxic exposure, and 4 Hr recovery stages were assessed in the liver and skeletal muscle using western immunoblotting. The results suggest a tissue-specific regulation of the anti-apoptotic pathway in the wood frog, where both liver and skeletal muscle shows an overall decrease in apoptosis and an increase in cell survival. This type of cytoprotective mechanism could be aimed at preserving the existing cellular components during long-term anoxia and oxygen recovery phases in the wood frog.

Molluscan live and dead assemblages in an anthropogenically stressed shallow-shelf: Levantine margin of Israel

Abstract Mismatches between live and dead molluscan assemblages associated with recent, rapid, anthropogenic changes, can be used to track these changes. The eastern Mediterranean is naturally oligotrophic, but recent urbanization of the Israeli coastal plain has enriched the littoral environment by injection of large amounts of treated wastewater onto the shelf. The largest point source is the Dan region wastewater project (Shafdan). Taxonomy and species rank order of abundance of modern (sediment-top) death assemblages were compared to live-collected mollusk assemblages, from two clean control stations (PL29, PL64) and a polluted site (PL3), near the Shafdan sewage sludge outlet at 36 m water depth. Seasonal variability was captured by dredge and box-core sampling in winter (January), spring (May), summer (July) and fall (November) of 2012. Over 11,000 individuals of bivalves and gastropods were collected and analyzed. Diversity indices and nMDS and cluster analysis showed significant differences between live and dead assemblages from all stations and seasons. However, the rank order of the abundant live and dead species remained similar at each sampling station, resulting in high live–dead agreement of Jaccard-Chao index and Spearman's rho similarity coefficient. Live–dead agreement of molluscan assemblages was preserved in the Shafdan area by the naturally high abundance of species that are tolerant to pollution, and by the annual dispersion of the sludge by winter storms, which prevent development of long-term anoxia. Moreover, the high frequency of storms in the winter of 2012 prevented a substantial amount of sludge from accumulating on the seafloor, explaining the similarity between the live assemblages of the polluted and the control sampling sites. Live–dead agreement is a test that is too conservative to track the impact of the Shafdan sludge on the macrobenthic fauna in the Israeli shelf. Other tests, especially the increase in the abundance of deposit-feeding pollution-tolerant species in the live assemblage, show that there is an ongoing impact of the sludge on the benthic community.

Flooding impairs Fe uptake and distribution in Citrus due to the strong down-regulation of genes involved in Strategy I responses to Fe deficiency in roots.

This work determines the ffects of long-term anoxia conditions—21 days—on Strategy I responses to iron (Fe) deficiency in Citrus and its impact on Fe uptake and distribution. The study was carried out in Citrus aurantium L. seedlings grown under flooding conditions (S) and in both the presence (+Fe) and absence of Fe (-Fe) in nutritive solution. The results revealed a strong down-regulation (more than 65%) of genes HA1 and FRO2 coding for enzymes proton-ATPase and Ferric-Chelate Reductase (FC-R), respectively, in –FeS plants when compared with –Fe ones. H+-extrusion and FC-R activity analyses confirmed the genetic results, indicating that flooding stress markedly repressed acidification and reduction responses to Fe deficiency (3.1- and 2.0-fold, respectively). Waterlogging reduced by half Fe concentration in +FeS roots, which led to 30% up-regulation of Fe transporter IRT1, although this effect was unable to improve Fe absorption. Consequently, flooding inhibited 57Fe uptake in +Fe and –Fe seedlings (29.8 and 66.2%, respectively) and 57Fe distribution to aerial part (30.6 and 72.3%, respectively). This evidences that the synergistic action of both enzymes H+-ATPase and FC-R is the preferential regulator of the Fe acquisition system under flooding conditions and, hence, their inactivation implies a limiting factor of citrus in their Fe-deficiency tolerance in waterlogged soils.

Foraminiferal species responses to in situ, experimentally induced anoxia in the Adriatic Sea

Abstract. Anoxia was successfully induced in four benthic chambers installed at 24 m depth in the northern Adriatic Sea for periods varying from 9 days to 10 months. During the 10-month period, species richness significantly decreased. Although no significant change in Shannon diversity and evenness was observed, the composition of the foraminiferal assemblages changed with time. This change is due to interspecific differences in tolerance to anoxia. Reophax nanus, Textularia agglutinans and Quinqueloculina stelligera all showed a significant decrease with time, strongly suggesting they are sensitive to anoxia. Conversely, Eggerella scabra, Bulimina marginata, Lagenammina atlantica, Hopkinsina pacifica and Bolivina pseudoplicata appeared to be resistant to the experimental conditions. Quinqueloculina seminula was apparently sensitive to anoxia but showed a clear standing stock increase during the first month of the experiment, which we interpret as an opportunistic response to increasing organic matter availability due to the degradation of the dead macrofaunal organisms. None of the anoxia-sensitive species is able to accumulate intracellular nitrates. Nitrate accumulation could be shown for some tested specimens of the dominant anoxia-tolerant species E. scabra and B. marginata. However, tests on the denitrification capacity of these taxa yielded negative results, suggesting that their resistance to long-term anoxia is not due to their ability to denitrify.

Mitochondria from anoxia-tolerant animals reveal common strategies to survive without oxygen

The mitochondrion plays a critical role in the development of Oxygen (O2)-related diseases. While research has predominantly focused on hypoxia-sensitive mammals as surrogates for humans, the use of animals which have naturally evolved anoxia tolerance has been largely ignored. Remarkably, some animals can live in the complete absence of O2 for days, months and even years, but surprisingly little is currently known about mitochondrial function in these species. In contrast to mammals, mitochondrial function in anoxia-tolerant animals is relatively insensitive to in vitro anoxia and reoxygenation, suggesting that anoxia tolerance transcends to the level of the mitochondria. Furthermore, long-term anoxia is associated with marked changes in the intrinsic properties of the mitochondria from these species, which may afford protection against anoxia-related damage. In the present review, we highlight some of the strategies anoxia-tolerant animals possess to preserve mitochondrial function in the absence of O2. Specifically, we review mitochondrial Ca(2+) regulation, proton leak, redox signaling and mitochondrial permeability transition, in phylogenetically diverse groups of anoxia-tolerant animals. From the strategies they employ, these species emerge as model organisms to illuminate novel interventions to mitigate O2-related mitochondrial dysfunction in humans.

Patterns of ubiquitylation and SUMOylation associated with exposure to anoxia in embryos of the annual killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus acquire extreme tolerance to anoxia during embryonic development. These embryos can survive environmental and cellular conditions that would likely result in death in the majority of vertebrate cells, despite experiencing a massive loss of ATP. It is highly likely that the initial response to anoxia must quickly alter cellular physiology to reprogram cell signaling and metabolic pathways to support anaerobiosis. Covalent protein modifications are a mechanism that can quickly act to effect large-scale changes in protein structure and function and have been suggested by others to play a key role in mammalian ischemia tolerance. Using Western blot analysis, we explored patterns of protein ubiquitylation and SUMOylation in embryos of A. limnaeus exposed to anoxia and anoxic preconditioning. Surprisingly, we report stage-specific protein ubiquitylation patterns that suggest different mechanisms for altering protein turnover in dormant and actively developing embryos that both survive long-term anoxia. Anoxic preconditioning does not appear to alter levels of ubiquitin conjugates in a unique manner. Global SUMOylation of proteins does not change in response to anoxia, but there are stage-specific changes in SUMOylation of specific protein bands. Contrary to other systems, global changes in protein SUMOylation may not be required to support long-term tolerance to anoxia in embryos of A. limnaeus. These data lead us to conclude that embryos of A. limnaeus respond to anoxia in a unique manner compared to other vertebrate models of anoxia tolerance and may provide novel mechanisms for engineering vertebrate tissues to survive long-term anoxia.

Avoidance of apoptosis in embryonic cells of the annual killifish Austrofundulus limnaeus exposed to anoxia.

Embryos of the annual killifish Austrofundulus limnaeus have unequalled ability among vertebrates to survive long-term anoxia. Surprisingly, these embryos can survive for months in anoxia despite a large-scale decrease in ATP levels during the initial hours of anoxic exposure. These conditions are known to trigger apoptotic cell death in mammalian cells as a result of ischemia or anoxia. Anoxia-induced induction of apoptosis was investigated in four developmental stages of A . limnaeus that differ in their tolerance of anoxia, and thus may respond to anoxia uniquely. Exposure to staurosporine was used to determine if A . limnaeus cells were competent to enter apoptosis via cues other than anoxia. Apoptotic cells were identified by TUNEL assays and by measuring caspase 3/7 activity. Exposure to 48 hr of anoxia did not induce an increase in TUNEL-positive cells and generally did not lead to an increase in caspase 3/7 activity. However, treatment of anoxic embryos with 10 μM staurosporine resulted in a significant increase in caspase 3/7 activity in both normoxic and anoxic embryos. These results suggest that apoptosis is avoided in embryos of A . limnaeus following exposure to anoxia at least in part by mechanisms that prevent the activation of caspase 3/7 activity. While this mechanism remains unknown, it may be triggered by a protein kinase that can be experimentally inhibited by staurosporine.

Nitric oxide metabolites during anoxia and reoxygenation in the anoxia-tolerant vertebrate, Trachemys scripta

Moderate elevations of nitrite and nitric oxide (NO) protect mammalian tissues against ischemia (anoxia)-reperfusion damage by inhibiting mitochondrial electron transport complexes and reducing the formation of reactive oxygen species (ROS) upon reoxygenation. Crucian carp appear to exploit this mechanism by upregulating nitrite and other nitrite/NO metabolites (S-nitroso and iron-nitrosyl compounds) in several tissues when exposed to anoxia. We investigated whether this is a common strategy amongst anoxia-tolerant vertebrates by evaluating NO metabolites in red-eared slider turtles during long-term (9 days) anoxia and subsequent reoxygenation at low temperature, a situation naturally encountered by turtles in ice-covered ponds. We also measured glutathione in selected tissues and assessed the impact of anoxia on electrolyte status. Anoxia induced major increases in [nitrite] in the heart, pectoral muscle and red blood cells, while [nitrite] was maintained unaltered in brain and liver. Concomitantly, the concentrations of S-nitroso and iron-nitrosyl compounds increased, showing that nitrite was used to produce NO and to S-nitrosate cellular molecules during anoxia. The changes were gradually reversed during reoxygenation (1 h and 24 h), testifying that the processes were reversible. The increased NO bioavailability occurred in the absence of NO synthase activity (due to global anoxia) and may involve mobilization of internal/external nitrite reservoirs. Our data support the theory that anoxic upregulation of nitrite and other NO metabolites could be a general cytoprotective strategy amongst anoxia-tolerant vertebrates. The possible mechanisms of nitrite-derived NO and S-nitrosation in protecting cells from destructive Ca(2+) influx during anoxia and in limiting ROS formation during reoxygenation are discussed.

Bioinformatic analysis of embryo development related small heat shock protein Hsp26 in Artemia species

Artemia embryos can endure extreme temperature, long-term anoxia, desiccation and other wide variety of stressful conditions. How the embryos survive these stresses is a very interesting and unsolved subject. To solve this question we analyzed the nucleotide and deduced protein sequence for Hsp26, a molecular chaperone specific to Artemia embryo development. cDNAs of Hsp26 were sequenced from eight Artemia species and deduced Hsp26 amino acid sequences were analyzed. Computer-assisted analysis indicated that the 5′-untranslated region and all the 3 introns contain many putative cis-acting elements for Hsp26 gene expression during development, including heat shock elements (HSEs), Dfd, dl, CF2-II, Hb and AP-1 binding sites. Secondary structure of the Hsp26 3′-untranslated terminator contains the basic structure basis for transcriptional termination. Hsp26 shares sequence similarity with sHSPs (small heat shock protein) from other organisms. The physico-chemical properties of the deduced protein, such as theoretical molecular weight, protein extinction coefficient, isoelectric point and antigenic sites were also obtained. One seven-peptide nuclear localization signals (NLS) “PFRRRMM” was found, which suggested that the Hsp26 protein was hypothesized to be located inside the nucleus. The numbers of phosphorylation sites of serine, threonine and tyrosine and kinase specific phosphorylation sites are also located in Hsp26 protein sequence. These studies will help us achieve a better understanding of Hsp26 and broad implications for sHSPs function in crustacean embryo development.

Heterogeneity in Pseudomonas aeruginosa Biofilms Includes Expression of Ribosome Hibernation Factors in the Antibiotic-Tolerant Subpopulation and Hypoxia-Induced Stress Response in the Metabolically Active Population

Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the rmf and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a P. aeruginosa Δrmf strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.