Taurine depletion impairs cardiac function and affects tolerance to hypoxia and high temperatures in brook char (Salvelinus fontinalis).

Physiological and environmental stressors can cause osmotic stress in fish hearts, leading to a reduction in intracellular taurine concentration. Taurine is a ß-amino acid known to regulate cardiac function in other animal models but its role in fish has not been well characterized. We generated a model of cardiac taurine deficiency (TD) by feeding brook char (Salvelinus fontinalis) a diet enriched in ß-alanine, which inhibits cardiomyocyte taurine uptake. Cardiac taurine levels were reduced by 21% and stress-induced changes in normal taurine handling were observed in TD brook char. Responses to exhaustive exercise and acute thermal and hypoxia tolerance were then assessed using a combination of in vivo, in vitro and biochemical approaches. Critical thermal maximum was higher in TD brook char despite significant reductions in maximum heart rate. In vivo, TD brook char exhibited a lower resting heart rate, blunted hypoxic bradycardia and a severe reduction in time to loss of equilibrium under hypoxia. In vitro function was similar between control and TD hearts under oxygenated conditions, but stroke volume and cardiac output were severely compromised in TD hearts under severe hypoxia. Aspects of mitochondrial structure and function were also impacted in TD permeabilized cardiomyocytes, but overall effects were modest. High levels of intracellular taurine are required to achieve maximum cardiac function in brook char and cardiac taurine efflux may be necessary to support heart function under stress. Taurine appears to play a vital, previously unrecognized role in supporting cardiovascular function and stress tolerance in fish.

Thermal variation near the thermal optimum does not affect the growth, metabolism or swimming performance in wild Atlantic salmon Salmo salar.

Typically, laboratory studies on the physiological effects of temperature are conducted using stable acclimation temperatures. Nonetheless, information extrapolated from these studies may not accurately represent wild populations living in thermally variable environments. The aim of this study was to compare the growth rate, metabolism and swimming performance of wild Atlantic salmon exposed to cycling temperatures, 16-21°C, and stable acclimation temperatures, 16, 18.5, 21°C. Growth rate, metabolic rate, swimming performance and anaerobic metabolites did not change among acclimation groups, suggesting that within Atlantic salmon's thermal optimum range, temperature variation has no effect on these physiological properties.

Reversion to developmental pathways underlies rapid arm regeneration in juvenile European cuttlefish, Sepia officinalis (Linnaeus 1758).

Coleoid cephalopods, including the European cuttlefish (Sepia officinalis), possess the remarkable ability to fully regenerate an amputated arm with no apparent fibrosis or loss of function. In model organisms, regeneration usually occurs as the induction of proliferation in differentiated cells. In rare circumstances, regeneration can be the product of naïve progenitor cells proliferating and differentiating de novo . In any instance, the immune system is an important factor in the induction of the regenerative response. Although the wound response is well-characterized, little is known about the physiological pathways utilized by cuttlefish to reconstruct a lost arm. In this study, the regenerating arms of juvenile cuttlefish, with or without exposure at the time of injury to sterile bacterial lipopolysaccharide extract to provoke an antipathogenic immune response, were assessed for the transcription of early tissue lineage developmental genes, as well as histological and protein turnover analyses of the resulting regenerative process. The transient upregulation of tissue-specific developmental genes and histological characterization indicated that coleoid arm regeneration is a stepwise process with staged specification of tissues formed de novo, with immune activation potentially affecting the timing but not the result of this process. Together, the data suggest that rather than inducing proliferation of mature cells, developmental pathways are reinstated, and that a pool of naïve progenitors at the blastema site forms the basis for this regeneration.

Diel cycling hypoxia enhances hypoxia tolerance in rainbow trout (Oncorhynchus mykiss): evidence of physiological and metabolic plasticity.

Many fish naturally encounter a daily cycle of hypoxia, but it is unclear whether this exposure hardens hypoxia-intolerant fish to future hypoxia or leads to accumulated stress and death. The rainbow trout (Oncorhynchus mykiss) is a putatively hypoxia-sensitive species found in rivers and estuaries that may routinely experience hypoxic events. Trout were exposed to one of four 135 h treatments in a swim-tunnel respirometer: (1) air-saturated control (20.7 kPa PO2 ); (2) diel cycling O2 (20.7-4.2 kPa PO2  over 24 h); (3) acute hypoxia (130 h at 20.7 kPa PO2  followed by 5 h at 4.2 kPa PO2 ); and (4) the mean oxygen tension (12.4 kPa PO2 ) experienced by the diel cycled fish. Some responses were similar in diel O2 cycled and mean PO2 -treated fish, but overall, exposure to ecologically representative diel hypoxia cycles improved hypoxia tolerance. Diel hypoxia-induced protective responses included increased inducible HSP70 concentration and mean corpuscular hemoglobin concentration, as well as reduced plasma cortisol. Acclimation to diel hypoxia allowed metabolic rates to decline during hypoxia, reduced oxygen debt following subsequent exposures, and allowed fish to return to an anabolic phenotype. The data demonstrate that acute diel cycling hypoxia improves hypoxia tolerance in previously intolerant fish through the activation of cellular protective mechanisms and a reduction in metabolic O2 requirements.

The importance of incorporating natural thermal variation when evaluating physiological performance in wild species.

Environmental variability in aquatic ecosystems makes the study of ectotherms complex and challenging. Physiologists have historically overcome this hurdle in the laboratory by using 'average' conditions, representative of the natural environment for any given animal. Temperature, in particular, has widespread impact on the physiology of animals, and it is becoming increasingly important to understand these effects as we face future climate challenges. The majority of research to date has focused on the expected global average increase in temperature; however, increases in climate variability are predicted to affect animals as much or more than climate warming. Physiological responses associated with the acclimation to a new stable temperature are distinct from those in thermally variable environments. Our goal is to highlight these physiological differences as they relate to both thermal acclimation and the 'fallacy of the average' or Jensen's inequality using theoretical models and novel empirical data. We encourage the use of more realistic thermal environments in experimental design to advance our understanding of these physiological responses such that we can better predict how aquatic animals will respond to future changes in our climate.

Metabolic rate and rates of protein turnover in food-deprived cuttlefish, Sepia officinalis (Linnaeus 1758).

To determine the metabolic response to food deprivation, cuttlefish (Sepia officinalis) juveniles were either fed, fasted (3 to 5 days food deprivation), or starved (12 days food deprivation). Fasting resulted in a decrease in triglyceride levels in the digestive gland, and after 12 days, these lipid reserves were essentially depleted. Oxygen consumption was decreased to 53% and NH4 excretion to 36% of the fed group following 3-5 days of food deprivation. Oxygen consumption remained low in the starved group, but NH4 excretion returned to the level recorded for fed animals during starvation. The fractional rate of protein synthesis of fasting animals decreased to 25% in both mantle and gill compared with fed animals and remained low in the mantle with the onset of starvation. In gill, however, protein synthesis rate increased to a level that was 45% of the fed group during starvation. In mantle, starvation led to an increase in cathepsin A-, B-, H-, and L-like enzyme activity and a 2.3-fold increase in polyubiquitin mRNA that suggested an increase in ubiquitin-proteasome activity. In gill, there was a transient increase in the polyubiquitin transcript levels in the transition from fed through fasted to the starved state and cathepsin A-, B-, H-, and L-like activity was lower in starved compared with fed animals. The response in gill appears more complex, as they better maintain rates of protein synthesis and show no evidence of enhanced protein breakdown through recognized catabolic processes.

Physiological responses to hypersalinity correspond to nursery ground usage in two inshore shark species (Mustelus antarcticus and Galeorhinus galeus).

Shark nurseries are susceptible to environmental fluctuations in salinity because of their shallow, coastal nature; however, the physiological impacts on resident elasmobranchs are largely unknown. Gummy sharks (Mustelus antarcticus) and school sharks (Galeorhinus galeus) use the same Tasmanian estuary as a nursery ground; however, each species has distinct distribution patterns that are coincident with changes in local environmental conditions, such as increases in salinity. We hypothesized that these differences were directly related to differential physiological tolerances to high salinity. To test this hypothesis, we exposed wild, juvenile school and gummy sharks to an environmentally relevant hypersaline (120% SW) event for 48 h. Metabolic rate decreased 20-35% in both species, and gill Na(+)/K(+)-ATPase activity was maintained in gummy sharks but decreased 37% in school sharks. We measured plasma ions (Na(+), K(+), Cl(-)) and osmolytes [urea and trimethylamine oxide (TMAO)], and observed a 33% increase in plasma Na(+) in gummy sharks with hyperosmotic exposure, while school sharks displayed a typical ureosmotic increase in plasma urea (∼20%). With elevated salinity, gill TMAO concentration increased by 42% in school sharks and by 30% in gummy sharks. Indicators of cellular stress (heat shock proteins HSP70, 90 and 110, and ubiquitin) significantly increased in gill and white muscle in both a species- and a tissue-specific manner. Overall, gummy sharks exhibited greater osmotic perturbation and ionic dysregulation and a larger cellular stress response compared with school sharks. Our findings provide physiological correlates to the observed distribution and movement of these shark species in their critical nursery grounds.

Estimates of metabolic rate and major constituents of metabolic demand in fishes under field conditions: Methods, proxies, and new perspectives.

Metabolic costs are central to individual energy budgets, making estimates of metabolic rate vital to understanding how an organism interacts with its environment as well as the role of species in their ecosystem. Despite the ecological and commercial importance of fishes, there are currently no widely adopted means of measuring field metabolic rate in fishes. The lack of recognized methods is in part due to the logistical difficulties of measuring metabolic rates in free swimming fishes. However, further development and refinement of techniques applicable for field-based studies on free swimming animals would greatly enhance the capacity to study fish under environmentally relevant conditions. In an effort to foster discussion in this area, from field ecologists to biochemists alike, we review aspects of energy metabolism and give details on approaches that have been used to estimate energetic parameters in fishes. In some cases, the techniques have been applied to field conditions; while in others, the methods have been primarily used on laboratory held fishes but should be applicable, with validation, to fishes in their natural environment. Limitations, experimental considerations and caveats of these measurements and the study of metabolism in wild fishes in general are also discussed. Potential novel approaches to FMR estimates are also presented for consideration. The innovation of methods for measuring field metabolic rate in free-ranging wild fish would revolutionize the study of physiological ecology.

Multiparametric cytotoxicity assessment: the effect of gold nanoparticle ligand functionalization on SKOV3 ovarian carcinoma cell death.

Gold nanoparticles (AuNP) are promising anti-cancer agents because of their modifiable properties and high biocompatibility. This study used multiple parallel analyses to investigate the cytotoxic properties of 5 nm AuNP conjugated to four different ligands with distinct surface chemistry: polyethylene glycol (PEG), trimethylammonium bromide (TMAB), 4-dimethylaminopyridine (DMAP), and carboxyl (COOH). We used a range of biochemical and high-content microscopy methods to evaluate the metabolic function, oxidative stress, cell health, cell viability, and cell morphology in SKOV3 ovarian cancer cells. Each AuNP displayed a distinct cytotoxicity profile. All AuNP species assessed exhibited signs of dose-dependent cytotoxicity when morphology, clonogenic survival, lysosomal uptake, or cell number were measured as the marker of toxicity. All particles except for AuNP-COOH increased SKOV3 apoptosis. In contrast, AuNP-TMAB was the only particle that did not alter the metabolic function or induce significant signs of oxidative stress. These results demonstrate that AuNP surface chemistry impacts the magnitude and mechanism of SKOV3 cell death. Together, these findings reinforce the important role for multiparametric cytotoxicity characterization when considering the utility of novel particles and surface chemistries.

Contrasting strategies of hypoxic cardiac performance and metabolism in cichlids and armoured catfish.

The heart of tropical fishes is a particularly useful model system in which to investigate mechanisms of hypoxic tolerance. Here we focus on insights gained from two groups of fishes, cichlids and armoured catfishes. Cichlids respond to hypoxia by entering a sustained hypometabolism with decreased heart performance to match whole animal circulatory needs. Heart rate is decreased along with protein turnover to reduce adenosine triphosphate demand. This occurs despite the inherent capacity for high levels of cardiac power development. Although highly hypoxic tolerant at the whole animal level, the heart of cichlids does not have high constitutive activities of glycolytic enzymes compared to other species. Information is conflicting with respect to changes in glycolytic gene expression and enzyme activity following hypoxic exposure with some studies showing increases and others decreases. In contrast to cichlids, species of armoured catfish, that are routinely exposed to water of low oxygen content, do not display hypoxic bradycardia. Under hypoxia there are early changes in glucose trafficking suggestive of activation of glycolysis before lactate accumulation. Thereafter, heart glycogen is mobilized and lactate accumulates in both heart and blood, in some species to very high levels. Heart performance under hypoxia is enhanced by defense of intracellular pH. A functional sarcoplasmic reticulum and binding of hexokinase to the outer mitochondrial membrane may also play a role in cardioprotection. Maintenance of heart performance under hypoxia may relate to a tradeoff between air breathing via a modified stomach and circulatory demands for digestion.

Boron Oxide Nanoparticles Exhibit Minor, Species-Specific Acute Toxicity to North-Temperate and Amazonian Freshwater Fishes.

Boron oxide nanoparticles (nB2O3) are manufactured for structural, propellant, and clinical applications and also form spontaneously through the degradation of bulk boron compounds. Bulk boron is not toxic to vertebrates but the distinctive properties of its nanostructured equivalent may alter its biocompatibility. Few studies have addressed this possibility, thus our goal was to gain an initial understanding of the potential acute toxicity of nB2O3 to freshwater fish and we used a variety of model systems to achieve this. Bioactivity was investigated in rainbow trout (Oncorhynchus mykiss) hepatocytes and at the whole animal level in three other North and South American fish species using indicators of aerobic metabolism, behavior, oxidative stress, neurotoxicity, and ionoregulation. nB2O3 reduced O. mykiss hepatocyte oxygen consumption (MO2) by 35% at high doses but whole animal MO2 was not affected in any species. Spontaneous activity was assessed using MO2 frequency distribution plots from live fish. nB2O3 increased the frequency of high MO2 events in the Amazonian fish Paracheirodon axelrodi, suggesting exposure enhanced spontaneous aerobic activity. MO2 frequency distributions were not affected in the other species examined. Liver lactate accumulation and significant changes in cardiac acetylcholinesterase and gill Na+/K+-ATPase activity were noted in the north-temperate Fundulus diaphanus exposed to nB2O3, but not in the Amazonian Apistogramma agassizii or P. axelrodi. nB2O3 did not induce oxidative stress in any of the species studied. Overall, nB2O3 exhibited modest, species-specific bioactivity but only at doses exceeding predicted environmental relevance. Chronic, low dose exposure studies are required for confirmation, but our data suggest that, like bulk boron, nB2O3 is relatively non-toxic to aquatic vertebrates and thus represents a promising formulation for further development.

Corrigendum: Hypoxic Induced Decrease in Oxygen Consumption in Cuttlefish (Sepia officinalis) Is Associated with Minor Increases in Mantle Octopine but No Changes in Markers of Protein Turnover.

[This corrects the article DOI: 10.3389/fphys.2017.00344.].

Interrelationship Between Contractility, Protein Synthesis and Metabolism in Mantle of Juvenile Cuttlefish (Sepia officinalis).

Young juvenile cuttlefish (Sepia officinalis) can grow at rates as high as 12% body weight per day. How the metabolic demands of such a massive growth rate impacts muscle performance that competes for ATP is unknown. Here, we integrate aspects of contractility, protein synthesis, and energy metabolism in mantle of specimens weighing 1.1 g to lend insight into the processes. Isolated mantle muscle preparations were electrically stimulated and isometric force development monitored. Preparations were forced to contract at 3 Hz for 30 s to simulate a jetting event. We then measured oxygen consumption, glucose uptake and protein synthesis in the hour following the stimulation. Protein synthesis was inhibited with cycloheximide and glycolysis was inhibited with iodoacetic acid in a subset of samples. Inhibition of protein synthesis impaired contractility and decreased oxygen consumption. An intact protein synthesis is required to maintain contractility possibly due to rapidly turning over proteins. At least, 41% of whole animal MO2 is used to support protein synthesis in mantle, while the cost of protein synthesis (50 µmol O2 mg protein-1) in mantle was in the range reported for other aquatic ectotherms. A single jetting challenge stimulated protein synthesis by approximately 25% (2.51-3.12% day-1) over a 1 h post contractile period, a similar response to that which occurs in mammalian skeletal muscle. Aerobic metabolism was not supported by extracellular glucose leading to the contention that at this life stage either glycogen or amino acids are catabolized. Regardless, an intact glycolysis is required to support contractile performance and protein synthesis in resting muscle. It is proposed that glycolysis is needed to maintain intracellular ionic gradients. Intracellular glucose at approximately 3 mmol L-1 was higher than the 1 mmol L-1 glucose in the bathing medium suggesting an active glucose transport mechanism. Octopine did not accumulate during a single physiologically relevant jetting challenge; however, octopine accumulation increased following a stress that is sufficient to lower Arg-P and increase free arginine.

Emerging threats and persistent conservation challenges for freshwater biodiversity.

In the 12 years since Dudgeon et al. (2006) reviewed major pressures on freshwater ecosystems, the biodiversity crisis in the world's lakes, reservoirs, rivers, streams and wetlands has deepened. While lakes, reservoirs and rivers cover only 2.3% of the Earth's surface, these ecosystems host at least 9.5% of the Earth's described animal species. Furthermore, using the World Wide Fund for Nature's Living Planet Index, freshwater population declines (83% between 1970 and 2014) continue to outpace contemporaneous declines in marine or terrestrial systems. The Anthropocene has brought multiple new and varied threats that disproportionately impact freshwater systems. We document 12 emerging threats to freshwater biodiversity that are either entirely new since 2006 or have since intensified: (i) changing climates; (ii) e-commerce and invasions; (iii) infectious diseases; (iv) harmful algal blooms; (v) expanding hydropower; (vi) emerging contaminants; (vii) engineered nanomaterials; (viii) microplastic pollution; (ix) light and noise; (x) freshwater salinisation; (xi) declining calcium; and (xii) cumulative stressors. Effects are evidenced for amphibians, fishes, invertebrates, microbes, plants, turtles and waterbirds, with potential for ecosystem-level changes through bottom-up and top-down processes. In our highly uncertain future, the net effects of these threats raise serious concerns for freshwater ecosystems. However, we also highlight opportunities for conservation gains as a result of novel management tools (e.g. environmental flows, environmental DNA) and specific conservation-oriented actions (e.g. dam removal, habitat protection policies, managed relocation of species) that have been met with varying levels of success. Moving forward, we advocate hybrid approaches that manage fresh waters as crucial ecosystems for human life support as well as essential hotspots of biodiversity and ecological function. Efforts to reverse global trends in freshwater degradation now depend on bridging an immense gap between the aspirations of conservation biologists and the accelerating rate of species endangerment.

Taurine protects cardiac contractility in killifish, Fundulus heteroclitus, by enhancing sarcoplasmic reticular Ca2+ cycling.

Intracellular taurine is abundant in many animals and it influences an array of physiological processes, including osmoregulation, metabolism, and cardiac contractility. Taurine is an important osmolyte in teleost hearts, but its role in stress tolerance, cardiac metabolism, and contractility has not been assessed. The goal of this study was to determine if ventricular taurine concentration changes in response to environmental stress and to characterize its influence on contractility. Cardiac taurine concentrations varied in killifish (Fundulus heteroclitus) but were generally maintained following acute environmental challenges. In isometrically contracting ventricular strips, supplemental taurine (40 mmol L-1) protected peak tension development (F max) at high stimulation frequencies, an effect abolished by treatment with ryanodine, a blocker of sarcoplasmic reticulum Ca2+ release. In the presence of ryanodine, taurine-treated preparations were also better able to maintain F max at supraphysiological extracellular Ca2+ levels, but a prior anoxia exposure abolished this effect. Taurine had no impact on basal F max during or after anoxia, but it provided additive protection to high-frequency contractility post-anoxia. Tissue oxygen consumption and extracellular glucose utilization were unaffected by taurine in non-contracting preparations, indicating that it does not impact energy metabolism. Overall, the results suggest that cardiac taurine levels are well maintained on acute time scales in this highly stress-tolerant species. Supplemental taurine has no effect on aerobic metabolism in vitro, but it significantly improved cardiac contractility in a manner dependent upon sarcoplasmic reticulum Ca2+ cycling. The data indicate that taurine likely plays an important role in the regulation of cardiac performance in teleosts.

Inhibition of Transient Receptor Potential Vanilloid 6 channel, elevated in human ovarian cancers, reduces tumour growth in a xenograft model.

Background: Transient Receptor Potential Vanilloid 6 (TRPV6), a non-voltage gated calcium channel, is implicated in malignancies and correlates with Gleason scores in prostate cancer and with poor prognosis in breast cancer. Data on the TRPV6 status of ovarian malignancies has not received significant attention. The effect of inhibiting TRPV6 activity on ovarian tumour growth has never been reported. Methods: We quantified TRPV6 mRNA and protein in biopsies of five types of ovarian cancer at different stages and grades by quantitative PCR and immunohistochemistry respectively. We verified the presence of TRPV6 in SKOV-3 cells and xenografts by Western Blotting. NOD/SCID mice bearing xenografted ovarian tumours derived from SKOV-3 were treated daily with TRPV6-antagonistic peptides (SOR-C13 and SOR-C27) at 400, 600 and 800 mg/kg delivered intraperitoneally (i.p.) over 12 days. Data from qPCR and tumour growth experiments were compared with a Student's t-test. Immunohistochemical ranking of staining were compared with Kruskall-Wallace one-way ANOVA and Dunn's Multiple Comparison post-test. Results: TRPV6 mRNA and protein are significantly elevated at all stages and grades of 5 ovarian cancer types over normal tissue. Overall qPCR log2 values (n, mean, ± SEM) for mRNA in tumour (n = 165, 5.06 ± 0.16) were greater (p < 0.05) than normal tissues (n = 26, 0.45 ± 0.41). All stages and grades included in the biopsy arrays were significantly greater than normal tissues. Immunohistochemical staining of TRPV6 was ranked >2 (faint in most cells) in 80.5% of tumours (123) while 92% of normal tissues (23) ranked ≤ 2. Daily i.p. injection with SOR-C13 (400, 600 and 800 mg/kg) over 12 days inhibits tumour growth (59%) at the highest dose compared to non-treated controls. SOR-C27 at 800 mg/kg SOR-C27 inhibited tumour growth 55% after 12 days. Results of daily and intermittent dosing (Days 1, 2, 3 and 8, 9, 10) with SOR-C13 were indistinguishable. Conclusion: TRPV6 mRNA and protein are elevated in biopsies of ovarian cancers compared to normal tissue. Inhibition of TRPV6 activity significantly reduces ovarian tumour growth providing evidence that TRPV6 is a feasible oncology target in ovarian cancers.

Mechanisms of toxic action of copper and copper nanoparticles in two Amazon fish species: Dwarf cichlid (Apistogramma agassizii) and cardinal tetra (Paracheirodon axelrodi).

Copper oxide nanoparticles (nCuO) are widely used in boat antifouling paints and are released into the environment, potentially inducing toxicity to aquatic organisms. The present study aimed to understand the effects of nCuO and dissolved copper (Cu) on two ornamental Amazon fish species: dwarf cichlid (Apistogramma agassizii) and cardinal tetra (Paracheirodon axelrodi). Fish were exposed to 50% of the LC50 for nCuO (dwarf cichlid 58.31µgL-1 and cardinal tetra 69.6µgL-1) and Cu (dwarf cichlid 20µgL-1 and cardinal tetra 22.9µgL-1) for 24, 48, 72 and 96h. Following exposure, aerobic metabolic rate (MO2), gill osmoregulatory physiology and mitochondrial function, oxidative stress markers, and morphological damage were evaluated. Our results revealed species specificity in metabolic stress responses. An increase of MO2 was noted in cardinal tetra exposed to Cu, but not nCuO, whereas MO2 in dwarf cichlid showed little change with either treatment. In contrast, mitochondria from dwarf cichlid exhibited increased proton leak and a resulting decrease in respiratory control ratios in response to nCuO and Cu exposure. This uncoupling was directly related to an increase in reactive oxygen species (ROS) levels. Our findings reveal different metabolic responses between these two species in response to nCuO and Cu, which are probably caused by the differences between species natural histories, indicating that different mechanisms of toxic action of the contaminants are associated to differential osmoregulatory strategies among species.

Intracellular glucose and binding of hexokinase and phosphofructokinase to particulate fractions increase under hypoxia in heart of the amazonian armored catfish (Liposarcus pardalis).

Armored catfish (Liposarcus pardalis), indigenous to the Amazon basin, have hearts that are extremely tolerant of oxygen limitation. Here we test the hypothesis that resistance to hypoxia is associated with increases in binding of selected glycolytic enzymes to subcellular fractions. Preparations of isolated ventricular sheets were subjected to 2 h of either oxygenated or hypoxic (via nitrogen gassing) treatment during which time the muscle was stimulated to contract. The bathing medium contained 5 mM glucose and was maintained at 25 degrees C. Initial experiments revealed increases in anaerobic metabolism. There was no measurable decrease in glycogen level; however, hypoxic treatment led to a twofold increase in heart glucose and a 10-fold increase in lactate content. It is suggested that the increase in heart glucose content is a result of an enhanced rate of facilitated glucose transport that exceeds the rate of phosphorylation of glucose. Further experiments assessed activities of metabolic enzymes in crude homogenates and subsequently tracked the degree of enzyme binding associated with subcellular fractions. Total maximal activities of glycolytic enzymes (hexokinase [HK], phosphofructokinase [PFK], aldolase, pyruvate kinase, lactate dehydrogenase), and a mitochondrial marker, citrate synthase, were not altered with the hypoxic treatment. A substantial portion (>/=50%) of HK is permanently bound to mitochondria, and this level increases under hypoxia. The amount of HK that is bound to the mitochondrial fraction is at least fourfold higher in hearts of L. pardalis than in rat hearts. Hypoxia also resulted in increased binding of PFK to a particulate fraction, and the degree of binding is higher in hypoxia-tolerant fish than in hypoxia-sensitive mammalian hearts. Such binding may be associated with increased glycolytic flux rates through modulation of enzyme-specific kinetics. The binding of HK and PFK occurs before any significant decrease in glycogen level.

Gold nanoparticles partition to and increase the activity of glucose-6-phosphatase in a synthetic phospholipid membrane system.

Engineered nanomaterials can alter the structure and/or function of biological membranes and membrane proteins but the underlying mechanisms remain unclear. We addressed this using a Langmuir phospholipid monolayer containing an active transmembrane protein, glucose-6-phosphatase (G6Pase). Gold nanoparticles (nAu) with varying ligand shell composition and hydrophobicity were synthesized, and their partitioning in the membrane and effects on protein activity characterized. nAu incorporation did not alter the macroscopic properties of the membrane. Atomic force microscopy showed that when co-spread with other components prior to membrane compression, nAu preferentially interacted with G6Pase and each other in a functional group-dependent manner. Under these conditions, all nAu formulations reduced G6Pase aggregation in the membrane, enhancing catalytic activity 5-6 fold. When injected into the subphase beneath pre-compressed monolayers, nAu did not affect G6Pase activity over 60 minutes, implying they were unable to interact with the protein under these conditions. A small but significant quenching of tryptophan fluorescence showed that nAu interacted with G6Pase in aqueous suspension. nAu also significantly reduced the hydrodynamic diameter of G6Pase in aqueous suspension and promoted catalytic activity, likely via a similar mechanism to that observed in co-spread monolayers. Overall, our results show that nAu can incorporate into membranes and associate preferentially with membrane proteins under certain conditions and that partitioning is dependent upon ligand shell chemistry and composition. Once incorporated, nAu can alter the distribution of membrane proteins and indirectly affect their function by improving active site accessibility, or potentially by changing their native structure and distribution in the membrane.

Hypoxic Induced Decrease in Oxygen Consumption in Cuttlefish (Sepia officinalis) Is Associated with Minor Increases in Mantle Octopine but No Changes in Markers of Protein Turnover.

The common cuttlefish (Sepia officinalis), a dominant species in the north-east Atlantic ocean and Mediterranean Sea, is potentially subject to hypoxic conditions due to eutrophication of coastal waters and intensive aquaculture. Here we initiate studies on the biochemical response to an anticipated level of hypoxia. Cuttlefish challenged for 1 h at an oxygen level of 50% dissolved oxygen saturation showed a decrease in oxygen consumption of 37% associated with an 85% increase in ventilation rate. Octopine levels were increased to a small but significant level in mantle, whereas there was no change in gill or heart. There were no changes in mantle free glucose or glycogen levels. Similarly, the hypoxic period did not result in changes in HSP70 or polyubiquinated protein levels in mantle, gill, or heart. As such, it appears that although there was a decrease in metabolic rate there was only a minor increase in anaerobic metabolism as evidenced by octopine accumulation and no biochemical changes that are hallmarks of alterations in protein trafficking. Experiments with isolated preparations of mantle, gill, and heart revealed that pharmacological inhibition of protein synthesis could decrease oxygen consumption by 32 to 42% or Na+/K+ ATPase activity by 24 to 54% dependent upon tissue type. We propose that the decrease in whole animal oxygen consumption was potentially the result of controlled decreases in the energy demanding processes of both protein synthesis and Na+/K+ ATPase activity.