Respiratory gas exchange in the airbreathing fish,Synbranchus marmoratus

Synopsis The partitioning of O₂ uptake between aquatic and aerial gas exchange and its dependence on ambient water PO₂ was studied in the facultative air breathing teleost Synbranchus marmoratus, after acclimation to well aerated water and after acute and chronic exposure to hypoxic water. O₂ uptake was also studied following acute air exposure and after prolonged entrapment in soil. Breathing rates during water and air breathing in response to reduced water PO₂ and tidal volume during air breathing were also studied. S. marmoratus satisfies its O₂ requirement by water breathing alone until water PO₂ falls below 30–50 mm Hg (switching PO₂) depending on the acclimation history. Below the switching PO₂, air breathing is adopted while active water breathing stops. The O₂ uptake varied little for all groups when the principal mode of gas exchange changed at the switching PO₂. The highest O₂ uptake prevailed when the fish employed the mode of gas exchange in operation during the acclimation period (i.e. water breathing for normoxia-acclimated, air breathing for hypoxic-acclimated).Acclimation to chronic hypoxia gave a much higher switching PO₂ 55 mm Hg) than for the other groups (about 30 mm Hg). S. marmoratus maintained its O₂ uptake when acutely exposed to air. When entrapped in soil in an aestivating state, the O₂ uptake was reduced to 25% of that in water or during acute air exposure. The overall gas exchange ratio for air breathing was very low (R_E 0.1).Branchial water pumping increased with lowering of water PO₂. The rate of air breathing was independent of water PO₂.The findings are discussed in the light of the ecophysiological conditions confronting S. marmoratus.     

Characterization and application of an optical sensor for quantification of dissolved O2 in shake-flasks

On-line measurement of dissolved O₂ in shake-flasks was realized via immobilized sensor spots containing a fluorophore with an O₂-dependent luminescent decay time. An unaffected sensor signal during 80 autoclaving cycles suggests multi-usage of sensor equipped shake-flasks. The sensor had a response time of 6 s. Quantification of gas-liquid mass transfer revealed maximum k_La values of 150 h^–1, from which maximum O₂ transfer capacity of 33 mM h^–1 was calculated. Liquid volume and shaking frequency have a strong influence on k_La. Exemplified by cultivations of Corynebacterium glutamicum the importance of shaking rate for O₂ supply of bacterial cultures is shown. Sampling of microbial cultures with intermittent shaking of a few minutes can cause O₂ limitation. Based on the results of this work a simple and straightforward tool is now available for accurate O₂ sensing in shake-flasks, which are widely used in microbial cultivations.     

Placental blood flows and oxygen transfer during uterine contractions: A mathematical model

Theoretical calculations have been performed on the effects of changes in intrauterine pressure on maternal and fetal placental blood flows and pressures and transplacental O₂ exchange. Initial conditions and contraction profiles may be “assumed normal” or abnormal. The model predicts that the fetal O₂ deficit is most sensitive to the maximum intensity of the contraction and maternal mean arterial blood pressure, less sensitive to the duration of the contraction and far less sensitive to the value of maternal arterial PO₂. The umbilical venous PO₂ falls to a minimum at the peak of a contraction. Its level is most sensitive to contraction intensity and maternal blood pressure; while the value of maternal arterial PO₂ has less effect and the duration of contraction has essentially no effect. There is interaction between factors so that the effect of simultaneous changes is not necessarily in proportion to the effects of individual changes.     

Chronic hypoxia upregulates adenosine 2a receptor expression in chromaffin cells via hypoxia inducible factor-2α: role in modulating secretion

Catecholamine (CAT) release from chromaffin tissue plays an essential role in the fetus which develops in a low O₂ environment (hypoxia). To address molecular mechanisms regulating CAT secretion in low O₂, we exposed a fetal chromaffin-derived cell line (MAH cells) to chronic hypoxia (CHox; 2% O₂, 24 h) and assessed gene expression using microarrays, quantitative RT-PCR, and western blot. CHox caused a dramatic ∼12× upregulation of adenosine A2a receptor (A2aR) mRNA, an effect critically dependent upon hypoxia-inducible factor (HIF)-2α which bound the promoter of the A2aR gene. In amperometric studies, acute hypoxia and high K⁺ (30 mM) evoked quantal CAT secretion that was enhanced after CHox, and further potentiated during simultaneous A2aR activation by adenosine. A2aR activation also enhanced stimulus-induced rise in intracellular Ca²⁺ in control, but not HIF-2α-deficient, MAH cells. Thus, A2aR, adenosine, and HIF-2α are key contributors to the potentiation of CAT secretion in developing chromaffin cells during chronic hypoxia.     

Free Radical Scavenging by Myocardial Fatty Acid Binding Protein

Recent investigations have indicated the presence of a fatty acid binding protein (FABP) in mammalian heart. This protein binds free fatty acids and their esters with high affinity, however, its physiological role remains unknown. Since FABP constitutes a significant amount of cystolic protein, it is likely that it would be a target for free radical attack. To test this hypothesis, FABP was examined for scavenging against free radicals such as the superoxide anion (O^?₂,). hydroxyl radical (OH') and hypochlorite radical (OCl') which may be present in an ischemic reperfused heart. Our results suggest that FABP scavenges O^?₂, OH' and OCl' as indicated by the FABP inhibition of O^?₂-dependent reduction of cytochrome c, OH'-dependent hydroxybenzoic acid formation and OCl'-mediated chemiluminescence response. FABP was found to be a more potent scavenger of these free radicals compared to bovine serum albumin. Furthermore, FABP was more effective in scavenging OH' than O^?₂, and inhibited OH' mediated lipid peroxidation process. These results indicate that FABP can scavenge free radicals which may be present in an ischemic/reperfused heart and, thus, may play a significant physiological role in the heart during ischemia and reperfusion.     

Responses of the teleost Hoplias malabaricus to hypoxia

Synopsis Oxygen uptake (VO₂) during graded hypoxia, rate of hypoxia acclimation, breathing frequency (f_R), breath volume (V_{S, R}) and gill ventilation (V_G) were measured in Hoplias malabaricus. Normoxia and hypoxia acclimated fish had similar and constant VO₂ and V_G in a range of water PO₂ from 150 to 25 mmHg. Hypoxia acclimated fish showed significantly higher VO₂ in severe hypoxia (PO₂ <15 mmHg). Normoxia acclimated fish showed symptoms similar to hypoxic coma after 1 h of exposure to water PO₂ of 10 mmHg whereas the same symptoms were observed only at PO₂ of 5 mmHg for fish acclimated to hypoxia. Fish required 14 days to achieve full acclimation to hypoxia (PO₂ ≥25 mmHg). Lowering of water PO₂ from 150 to 25 mmHg resulted in normoxic fish showing a 3–2 fold increase in V_G. The increase was the result of an elevation in V_{S, R} rather than f_R. Among normoxia acclimated specimens, small fish showed a higher V_G per unit weight than the large ones in both normoxia (PO₂ =150 mmHg) and hypoxia (PO₂ = 15 mmHg). A decrease in the ventilatory requirement (V_G/VO₂) with increased body weight was recorded in hypoxia (PO₂ = 15 mmHg).     

Luminescent sensing and imaging of oxygen: Fierce competition to the Clark electrode

Luminescence‐based sensing schemes for oxygen have experienced a fast growth and are in the process of replacing the Clark electrode in many fields. Unlike electrodes, sensing is not limited to point measurements via fiber optic microsensors, but includes additional features such as planar sensing, imaging, and intracellular assays using nanosized sensor particles. In this essay, I review and discuss the essentials of (i) common solid‐state sensor approaches based on the use of luminescent indicator dyes and host polymers; (ii) fiber optic and planar sensing schemes; (iii) nanoparticle‐based intracellular sensing; and (iv) common spectroscopies. Optical sensors are also capable of multiple simultaneous sensing (such as O₂ and temperature). Sensors for O₂ are produced nowadays in large quantities in industry. Fields of application include sensing of O₂ in plant and animal physiology, in clinical chemistry, in marine sciences, in the chemical industry and in process biotechnology.     

Online monitoring of oxygen in spinner flasks

We show the application of a novel optical on-line sensor fixed in spinner flasks for the online monitoring of dissolved O₂ concentrations during mammalian cell growth. Using this sensor that requires only minute changes to the flask to be made, we could determine the volumetric O₂ transfer coefficient as well as O₂ consumption rates. Under normal growth conditions the cells did not undergo O₂ limitation. Also, the transfer of O₂ from the atmosphere to the spinner flasks is influenced by the use of screw caps. The on-line measurement was further applied to determine the O₂ uptake rates which can then be used to monitor the metabolic state of the cells and also for online process monitoring.     

Generation of a hypoxia‐sensing mouse model

Oxygen (O₂) homeostasis is essential to the metazoan life. O₂‐sensing or hypoxia‐regulated molecular pathways are intimately involved in a wide range of critical cellular functions and cell survival from embryogenesis to adulthood. In this report, we have designed an innovative hypoxia sensor (O₂CreER) based on the O₂‐dependent degradation domain of the hypoxia‐inducible factor‐1α and Cre recombinase. We have further generated a hypoxia‐sensing mouse model, R26‐O₂CreER, by targeted insertion of the O₂CreER‐coding cassette in the ROSA26 locus. Using the ROSA^mTmG mouse strain as a reporter, we have found that this novel hypoxia‐sensing mouse model can specifically identify hypoxic cells under the pathological condition of hind‐limb ischemia in adult mice. This model can also label embryonic cells including vibrissal follicle cells in E13.5–E15.5 embryos. This novel mouse model offers a valuable genetic tool for the study of hypoxia and O₂ sensing in mammalian systems under both physiological and pathological conditions.     

Special relationship between sterols and oxygen: Were sterols an adaptation to aerobic life?

A fascinating link between sterols and molecular oxygen (O₂) has been a common thread running through the fundamental work of Konrad Bloch, who elucidated the biosynthetic pathway for cholesterol, to recent work supporting a role of sterols in the sensing of O₂. Synthesis of sterols by eukaryotes is an O₂-intensive process. In this review, we argue that increased levels of O₂ in the atmosphere not only made the evolution of sterols possible, but that these sterols may in turn have provided the eukaryote with an early defence mechanism against O₂. The idea that nature crafted sterols as a feedback loop to adapt to, or help protect against, the hazards of O₂ is novel and enticing. We marshal several lines of evidence to support this thesis: (1) coincidence of atmospheric O₂ and sterol evolution; (2) sterols regulate O₂ entry into eukaryotic cells and organelles; (3) sterols act as O₂ sensors across eukaryotic life; (4) sterols serve as a primitive cellular defence against O₂ (including reactive oxygen species). Therefore, sterols may have evolved in eukaryotes partially as an adaptive response to the rise of terrestrial O₂, rather than merely as a consequence of it.     

Regulation of the Tyrosine Kinase Pyk2 by Calcium Is through Production of Reactive Oxygen Species in Cytotoxic T Lymphocytes

Pyk2 was identified as a Ca²⁺-dependent kinase, however, the regulation of Pyk2 by Ca²⁺ in T cells remains controversial. We found that Ca²⁺ mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca²⁺ dependent but relied on the strength of T cell receptor stimulation. Ionomycin-stimulated Pyk2 phosphorylation did not require calmodulin activity, because phosphorylation was not inhibited by the calmodulin inhibitor W7, and we detected no Ca²⁺-regulated association between Pyk2 and calmodulin. Ca²⁺-stimulated Pyk2 phosphorylation was dependent on Src-family kinase activity, even at the Pyk2 autophosphorylation site. We sought to identify a Ca²⁺-regulated pathway that could trigger Pyk2 phosphorylation in T cells and found that ionomycin stimulated the production of reactive oxygen species and an H₂O₂ scavenger inhibited ionomycin-induced Pyk2 phosphorylation. Additionally, H₂O₂ induced strong Erk activation and ionomycin-stimulated Pyk2 phosphorylation was Erk dependent. These data support the conclusion that Ca²⁺ mobilization induces the production of reactive oxygen species, which in turn activate the Erk pathway, leading to Src-family kinase-dependent Pyk2 phosphorylation. Our data demonstrate that Pyk2 is not a Ca²⁺-dependent kinase in T cells but instead, increased intracellular Ca²⁺ induces Pyk2 phosphorylation through production of reactive oxygen species. These findings are consistent with the possibility that Pyk2 acts as an early sensor of numerous extracellular signals that trigger a Ca²⁺ flux and/or reactive oxygen species to amplify tyrosine phosphorylation signaling events.     

Dynamics of Nitric Oxide and Peroxynitrite During Global Brain Ischemia/Reperfusion in Rat Hippocampus: NO-sensor Measurement and Modeling Study

The dynamics of nitric oxide (NO) and peroxynitrite concentration changes during brain ischemia/reperfusion are poorly understood. In this paper, a NO-selective sensor was used to measure NO concentration changes in the rat brain hippocampus during global brain ischemia/reperfusion. Four-vessel occlusion model of transient global brain ischemia was used. Global cerebral ischemia was induced by occluding both common carotid arteries with artery nips (for 20 min) and reperfusion was induced by loosening the artery nips. Results showed that the changes of NO concentration during global brain ischemia/reperfusion could be divided into different stages. Together with the effects of O₂ tension changes and NO synthase (NOS) on nitric oxide levels, we determined five stages in the NO concentration profile: (1) acute O₂-limited decrease stage; (2) O₂-limited steady stage; (3) neuronal NOS activation stage; (4) acute O₂-recovery elevation stage; and (5) O₂-recovery steady stage. In addition, a chemical reaction network model was constructed to simulate the dynamics of peroxynitrite during the reperfusion stage, and the effects of a change in the NO formation rate on the dynamics of peroxynitrite were investigated specifically. Results show the rate of NO formation has a great influence on peroxynitrite dynamics.     

A biomechanically derived minimum work model of the fish gill lamellar system exhibits its exquisite morphological arrangement and perfusate regulation for oxygen uptake from water

To evaluate the efficiency of oxygen (O₂) uptake from water through the fish gill lamellar system, a cost function (CF) representing mechanical power expenditure for water ventilation and blood circulation through the gill was formulated, by applying steady-state fluid mechanics to a homogeneous lamellar-channel model. This approach allowed us to express CF as the function of inter-lamellar water channel width (w) and to derive an analytical solution of the width (w_min) at the minimum CF. Morphometric and physiological data for rainbow trout in the literature were referred to calculate CF(w) curves and their w_min values at five intensity stages of swimming exercise. Obtained w_min values were evenly distributed around the standard measure of the width (w_s = 24 μm) in this fish. Individual levels of CF(w_min) were also fairly close to the corresponding CF(w_s) values within a 10% deviation, suggesting the reliability of approximating [CF(w_min) = CF(w_s)]. The cost-performance of O₂ uptake through the gill (η_g) was then assessed from reported data of total O₂ uptake/CF(w_s) at each intensity stage. The η_g levels at any swimming stage exceeded 95% of the theoretical maximum value, implying that O₂ uptake is nearly optimally performed in the lamellar-channel system at all swimming speeds. Further analyses of O₂ transport in this fresh water fish revealed that the water ventilation by the buccal/opercular pumping evokes a critical limit of swimming velocity, due to confined O₂ supply to the peripheral skeletal muscles, which is avoided in ram ventilators such as tuna.     

Sensitive and selective system of benzene detection based on a cataluminescence sensor

Au/La₂O₃ nanomaterials were prepared through calcining Au‐modified La(OH)₃ precursors. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X‐ray diffractometry (XRD) were employed to characterize the as‐prepared samples. Benzene, a common volatile organic compound, was selected as a model to investigate the cataluminescence (CTL)‐sensing properties of the Au/La₂O₃ nanomaterials. Results indicated that the as‐prepared Au/La₂O₃ exhibited outstanding CTL properties such as stable intensity, high signal‐to‐noise values, and short response and recovery times. Under optimized conditions, the benzene assay exhibited a broad linear range of 1–4000 ppm, with a limit of detection of 0.7 ppm, which was below the standard permitted concentrations. Furthermore, the gas sensor system showed outstanding selectivity for benzene compared with seven other types of common volatile organic compounds (VOCs). The proposed gas sensor showed good characteristics with high selectivity, fast response time and long lifetime, which suggested the promising application of the Au/La₂O₃ nanomaterials as a novel highly efficient CTL‐sensing material. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural dynamics of proximal heme pocket in HemAT-Bs associated with oxygen dissociation

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-containing O₂ sensor protein that acts as a chemotactic signal transducer. Binding of O₂ to the heme in the sensor domain of HemAT-Bs induces a conformational change in the protein matrix, and this is transmitted to a signaling domain. To characterize the specific mechanism of O₂-dependent conformational changes in HemAT-Bs, we investigated time-resolved resonance Raman spectra of the truncated sensor domain and the full-length HemAT-Bs upon O₂ and CO dissociation. A comparison between the O₂ and CO complexes provides insights on O₂/CO discrimination in HemAT-Bs. While no spectral changes upon CO dissociation were observed in our experimental time window between 10 ns and 100 μs, the band position of the stretching mode between the heme iron and the proximal histidine, ν(Fe–His), for the O₂-dissociated HemAT-Bs was lower than that for the deoxy form on time-resolved resonance Raman spectra. This spectral change specific to O₂ dissociation would be associated with the O₂/CO discrimination in HemAT-Bs. We also compared the results obtained for the truncated sensor domain and the full-length HemAT-Bs, which showed that the structural dynamics related to O₂ dissociation for the full-length HemAT-Bs are faster than those for the sensor domain HemAT-Bs. This indicates that the heme proximal structural dynamics upon O₂ dissociation are coupled with signal transduction in HemAT-Bs.