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The study of transcription factors that determine specialized neuronal functions has provided invaluable insights into the physiology of the nervous system. Peripheral chemoreceptors are neurone-like electrophysiologically excitable cells that link the oxygen concentration of arterial blood to the neuronal control of breathing. In the adult, this oxygen chemosensitivity is exemplified by type I cells of the carotid body, and recent work has revealed one isoform of the hypoxia- inducible transcription factor (HIF), HIF-2[alpha], as having a nonredundant role in the development and function of that organ. Here, we show that activation of HIF-2[alpha], including isolated overexpression of HIF-2[alpha] but not HIF-1[alpha], is sufficient to induce oxygen chemosensitivity in adult adrenal medulla. This phenotypic change in the adrenal medulla was associated with retention of extra-adrenal paraganglioma-like tissues resembling the fetal organ of Zuckerkandl, which also manifests oxygen chemosensitivity. Acquisition of chemosensitivity was associated with changes in the adrenal medullary expression of gene classes that are ordinarily characteristic of the carotid body, including G protein regulators and atypical subunits of mitochondrial cytochrome oxidase. Overall, the findings suggest that, at least in certain tissues, HIF-2[alpha] acts as a phenotypic driver for cells that display oxygen chemosensitivity, thus linking 2 major oxygen-sensing systems., Introduction One of the most vital, but still incompletely understood, functions in homeostatic physiology is that of the arterial chemoreceptors that control breathing in response to changes in blood oxygen, [...]

How oxygen is sensed by the heart and what mechanisms mediate its sensing remain poorly understood. As recent reports show that low P[O.sub.2] levels are detected by the cardiomyocytes in a few seconds, the rapid and short applications of low levels of oxygen (acute hypoxia), which avoid multiple effects of chronic hypoxia, may be used to probe the oxygen-sensing pathway of the heart. Here, we explored the oxygen-sensing pathway, focusing primarily on cellular surface membrane proteins that were first exposed to low P[O.sub.2]. Such studies suggest that acute hypoxia primarily targets the cardiac calcium channels, where either the channel itself or moieties closely associated with it, for instance heme-oxygenase-2 (HO-2) interacting through kinase phosphorylation, signal the [alpha]-subunit of the channel to the altered levels of P[O.sub.2]. Amino acids 1572-1651, the CaMKII phosphorylation sites (S1487 and S1545), CaM-binding sites (I1624 and Q1625), and Ser1928 of the carboxyl tail of the [alpha]-subunit appear to be critical residues that sense oxygen. Future studies on HO-2 knockout mice or CRISPR/Cas9 gene-edited human-induced pluripotent stem-cell-derived cardiomyocytes that reduce CaM-binding affinity are likely to provide deeper insights into the [O.sub.2]-sensing mechanisms. Key words: acute hypoxia, calcium channel, oxygen sensor, cardiomyocytes, CaMKII, heme-oxygenase-2 On ne comprend pas encore tres bien comment le coeur detecte l'oxygene ni les modes d'action qui y sont lies. Comme de recentes publications montrent que les cardiomyocytes peuvent detecter des diminutions de la P[O.sub.2] en quelques secondes, il est possible que l'administration d'oxygene a de faibles concentrations rapidement et sur de courtes durees (hypoxie aigue), ce qui permet d'eluder lesmultiples effets de l'hypoxie chronique, puisse permettre de sonder les voies de signalisation de la detection de l'oxygene dans le coeur. Dans les presents travaux, nous explorons les voies de signalisation de la detection de l'oxygene, en nous centrant principalement sur les proteines de surface desmembranes cellulaires, premier endroit expose a un abaissement de la P[O.sub.2]. De telles etudes laissent entendre que l'hypoxie aigue ciblerait principalement les canaux calciques cardiaques, soit les canaux eux-memes ou certains fragments qui leur sont etroitement associes. Par exemple, l'heme-oxygenase 2 (HO-2) interagit avec la sous-unite [alpha] des canaux par l'intermediaire de la phosphorylation kinasique pour signaler la modification des taux de P[O.sub.2]. Les acides amines 1572-1651, les sites de phosphorylation de la CaMKII (S1487 et S1545), le site de liaison de la CaM (I1624, Q1625) et la Ser1928 de l'extremite carboxyl de la sous-unite [alpha] semblent constituer des residus cruciaux pour la detection de l'oxygene. De futures etudes a l'aide de souris << knockout >> pour l'HO-2 ou avec les hiPSC-CM dont CRISPR/Cas9 sont modifies ou l'affinite de liaison de la CaM est abaissee sont susceptibles de fournir plus de renseignements quant aux modes d'action de la detection de l'[O.sub.2]. [Traduit par la Redaction] Mots-cles : hypoxie aigue, canal calcique, detecteur d'oxygene, cardiomyocytes, CaMKII, heme-oxygenase-2, 1. Introduction Ischemic heart disease is a leading cause of death worldwide, with its global prevalence expected to exceed 1900 cases per 100000 populations by 2030 (Nowbar et al. 2019). [...]

Animals have evolved homeostatic responses to changes in oxygen availability that act on different timescales. Although the hypoxia-inducible factor (HIF) transcriptional pathway that controls long-term responses to low oxygen (hypoxia) [...]

We used a mathematical model of [O.sub.2] transport and the urine concentrating mechanism of the outer medulla of the rat kidney to study the effects of blood pH and medullary blood flow on [O.sub.2] availability and [Na.sup.+] reabsorption. The model predicts that in vivo paracellular [Na.sup.+] fluxes across medullary thick ascending limbs (toTALs) are small relative to transcellular [Na.sup.+] fluxes and that paracellular fluxes favor [Na.sup.+] reabsorption from the lumen along most of the mTAL segments. In addition, model results suggest that blood pH has a significant impact on [O.sub.2] transport and Nat reabsorption owing to the Bohr effect, according to which a lower pH reduces the binding affinity of hemoglobin for [O.sub.2]. Thus our model predicts that the presumed greater acidity of blood in the interbundle regions, where mTALs are located, relative to that in the vascular bundles, facilitates the delivery of [O.sub.2] to support the high metabolic requirements of the mTALs and raises the concentrating capability of the outer medulla. Model results also suggest that increases in vascular and tubular flow rates result in disproportional, smaller increases in active [O.sub.2] consumption and mTAL active [Na.sup.+] transport, despite the higher delivery of [O.sub.2] and [Na.sup.+]. That is, at a sufficiently high medullary [O.sub.2] supply, [O.sub.2] demand in the outer medulla does not adjust precisely to changes in [O.sub.2] delivery. mathematical model; thick ascending limb; [T.sub.Na]/[Qo.sub.2] ratio; paracellular; transcellular doi: 10.1152/ajprenal.00572.2009.

The purpose of this experiment was to investigate the effects of winter growing program on organ mass, composition, and oxygen consumption in beef steers. A total of 46 steers were used for the experiment. Four steers were randomly selected as an initial slaughter group. Remaining steers were randomly allotted to 1 of 4 treatment groups: 1) fed a high-concentrate diet for ad libitum intake (CF); 2) grazed on wheat pasture (WP); 3) fed a sorghum silage-based growing diet (SF); or 4) program fed a high-concentrate diet (PF). Steers in the WP, SF, and PF groups were managed to achieve approximately equal rates of BW gain during the growing phase. After the growing phase (112 d), steers in the WP, SF, and PF treatments were adapted to a high-concentrate diet for finishing. Steers from all treatments were slaughtered at a fat thickness of 1.27 cm as estimated by ultrasound. In addition, 6 steers from each treatment were randomly selected fox' slaughter at the end of the growing phase. Weights of all individual organs were measured and tissue samples of duodenum and liver collected. At the end of the growing phase, WP steers had greater (P < 0.05) small intestine, liver, and kidney mass than SF and PF steers. In contrast, mesenteric fat mass and total visceral fat content were greatest (P < 0.01) for PF, intermediate for SF, and least for WP steers. Mass of total viscera and total splanchnic tissues (TST) did not differ (P > 0.10) among treatments. At final slaughter, mass of mesenteric fat, total viscera, and TST were similar among treatments, but liver weights remained greatest (g/kg of empty BW; P < 0.01) for WP steers. There were no differences in oxygen consumption of duodenum or liver tissue on an equal weight basis ([micro]L-[min.sup.-1][g.sup.-1]) at the end of either period. Growing program affected mass of components of the TST at the end of the growing phase, which contributed to differences in rate of splanchnic organ growth during finishing. We conclude that program feeding a high-concentrate diet during the growing phase may result in greater ADG and G:F during the subsequent finishing period compared with forage-based diets due to less accretion of visceral organ mass resulting in reduced maintenance energy requirements during finishing. Key words: beef cattle, growth, organ mass, oxygen consumption, visceral organ doi: 10.2527/jas.2009-2415

Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydA1 from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain ([2Fe.sub.H]) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydA1 which binds to an Fe atom of the [2Fe.sub.H] domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain--a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site. EXAFS | H-cluster | protein film electrochemistry | biological hydrogen production | green algae doi/ 10.1073/pnas.0905343106

Determining the transient chemical properties of the intracellular environment can elucidate the paths through which a biological system adapts to changes in its environment, for example, the mechanisms that enable some obligate anaerobic bacteria to survive a sudden exposure to oxygen. Here we used high-resolution Fourier transform infrared (FTIR) spectromicroscopy to continuously follow cellular chemistry within living obligate anaerobes by monitoring hydrogen bond structures in their cellular water. We observed a sequence of well orchestrated molecular events that correspond to changes in cellular processes in those cells that survive, but only accumulation of radicals in those that do not. We thereby can interpret the adaptive response in terms of transient intracellular chemistry and link it to oxygen stress and survival. This ability to monitor chemical changes at the molecular level can yield important insights into a wide range of adaptive responses. Desulfovibrio | hydrogen bond | synchrotron FTIR spectromicroscopy | oxygen stress | cellular water

Myoglobin is a globular protein involved in oxygen storage and transport. No consensus yet exists on the atomic level mechanism by which oxygen and other small nonpolar ligands move between the myoglobin's buried heme, which is the ligand binding site, and surrounding solvent. This study uses room temperature molecular dynamics simulations to provide a complete atomic level picture of ligand migration in myoglobin. Multiple trajectories--providing a cumulative total of 7/[micro]s of simulation--are analyzed. Our simulation results are consistent with and tie together previous experimental findings. Specifically, we characterize: (i) Explicit full trajectories in which the CO ligand shuttles between the internal binding site and the solvent and (ii) pattern and structural origins of transient voids available for ligand migration. The computations are performed both in sperm whale myoglobin wild-type and in sperm whale V68F myoglobin mutant, which is experimentally known to slow ligand-binding kinetics. On the basis of these independent, but mutually consistent ligand migration and transient void computations, we find that there are two discrete dynamical pathways for ligand migration in myoglobin. Trajectory hops between these pathways are limited to two bottleneck regions. Ligand enters and exits the protein matrix in common identifiable portals on the protein surface. The pathways are located in the 'softer' regions of the protein matrix and go between its helices and in its loop regions. Localized structural fluctuations are the primary physical origin of the simulated CO migration pathways inside the protein. proteins | molecular dynamics | kinetics | structure-function relationships

Oxygen profoundly affects the composition of oral biofilms. Recently, we showed that exposure of Streptococcus mutans to oxygen strongly inhibits biofilm formation and alters cell surface biogenesis. To begin to dissect the underlying mechanisms by which oxygen affects known virulence traits of S. mutans, transcription profiling was used to show that roughly 5% of the genes of this organism are differentially expressed in response to aeration. Among the most profoundly upregulated genes were autolysis-related genes and those that encode bacteriocins, the ClpB protease chaperone subunit, pyruvate dehydrogenase, the tricarboxylic acid cycle enzymes, NADH oxidase enzymes, and certain carbohydrate transporters and catabolic pathways. Consistent with our observation that the ability of S. mutans to form biofilms was severely impaired by oxygen exposure, transcription of the gtfB gene, which encodes one of the primary enzymes involved in the production of water-insoluble, adhesive glucan exopolysaccharides, was down-regulated in cells growing aerobically. Further investigation revealed that transcription of gtfB, but not gtfC, was responsive to oxygen and that aeration causes major changes in the amount and degree of cell association of the Gtf enzymes. Moreover, inactivation of the VicK sensor kinase affected the expression and localization the GtfB and GtfC enzymes. This study provides novel insights into the complex transcriptional and posttranscriptional regulatory networks used by S. mutans to modulate virulence gene expression and exopolysaccharide production in response to changes in oxygen availability.

A fundamental challenge in the redox biology of Mycobacterium tuberculosis (Mtb) is to understand the mechanisms involved in sensing redox signals such as oxygen ([O.sub.2]), nitric oxide (NO), and nutrient depletion, which are thought to play a crucial role in persistence. Here we show that Mtb WhiB3 responds to the dormancy signals NO and [O.sub.2] through its iron-sulfur (Fe-S) cluster. To functionally assemble the WhiB3 Fe-S cluster, we identified and characterized the Mtb cysteine desulfurase (IscS; Rv3025c) and developed a native enzymatic reconstitution system for assembling Fe-S clusters in Mtb. EPR and UV-visible spectroscopy analysis of reduced WhiB3 is consistent with a one-electron reduction of EPR silent [[4Fe-4S].sup.2+] to EPR visible [[4Fe-4S].sup.+]. Atmospheric [O.sub.2] gradually degrades the WhiB3 [[4Fe-4S].sup.2+] cluster to generate a [[3Fe-4S].sup.+] intermediate. Furthermore, EPR analysis demonstrates that NO forms a protein-bound dinitrosyl-iron-dithiol complex with the Fe-S cluster, indicating that NO specifically targets the WhiB3 Fe-S cluster. Our data suggest that the mechanism of WhiB3 4Fe-4S cluster degradation is similar to that of fumarate nitrate regulator. Importantly, Mtb [DELTA]whiB3 shows enhanced growth on acetate medium, but a growth defect on media containing glucose, pyruvate, succinate, or fumarate as the sole carbon source. Our results implicate WhiB3 in metabolic switching and in sensing the physiologically relevant host signaling molecules NO and [O.sub.2] through its [4Fe-4S] cluster. Taken together, our results suggest that WhiB3 is an intracellular redox sensor that integrates environmental redox signals with core intermediary metabolism. dormancy | redox | metabolism | iron-sulfur

We studied the metabolic rate, cellular energetic state, hypoxia-inducible factor-1 (HIF-1) activation, and expression of enzymes involved in energy metabolism using rainbow trout (Oncorhynchus mykiss) hepatocytes over the oxygen range from 21 to 1 kPa. Oxygen dependence of these factors was assessed by gradually reducing oxygen supply to cells from 21 kPa to 10, 5, 2, and 1 kPa. Moreover, time course experiments for up to 20 h at oxygen tensions of 1 and 2 kPa were carried out. Reduction of oxygen from 21 kPa to 10, 5, 2, and 1 kPa decreased metabolic rate of the cells by 14, 24, 37, and 46%, respectively. This response was instantaneous and fully reversible upon reoxygenation. Cellular ATP content and the expression of all mRNAs studied decreased when oxygen was reduced from 21 to 5 and 2 kPa. The lowest ATP levels, ~43% of the initial value, were measured at 5 kPa of oxygen, whereas the reduction in mRNA amounts was most pronounced at 2 kPa. At 1 kPa oxygen tension, both ATP content and mRNA amounts returned to normoxic (21 kPa) levels with a concomitant activation of HIF-1, indicating reorganization of energy metabolism in adaptation of cells to low oxygen supply. These results show that oxygen has a direct regulatory effect on metabolism of trout hepatocyte cultures, supporting the view that oxygen has a profound role in metabolic regulation in cells. metabolic regulation; energy metabolism; hypoxia-inducible factor-1; hypoxia; teleost

The oxygen transport capacity of phospholipid vesicles encapsulating purified Hb (HbV) produced with a [Po.sub.2] at which Hb is 50% saturated ([P.sub.50]) of 8 (Hb[V.sub.8]) and 29 mmHg (Hb[V.sub.29]) was investigated in the hamster chamber window model by using microvascular measurements to determine oxygen delivery during extreme hemodilution. Two isovolemic hemodilution steps were performed with 5% recombinant albumin (rHSA) until Hct was 35% of baseline. Isovolemic exchange was continued using HbV suspended in rHSA solution to a total [Hb] of 5.7 g/dl in blood. [P.sub.50] was modified by coencapsulating pyridoxal 5'-phosphate. Final Hct was 11% for the HbV groups, with a plasma [Hb] of 2.1 [+ or -] 0.1 g/dl after exchange with Hb[V.sub.8] or Hb[V.sub.29]. A reference group was hemodiluted to Hct 11% with only rHSA. All groups showed stable blood pressure and heart rate. Arterial oxygen tensions were significantly higher than baseline for the HbV groups and the rHSA group and significantly lower for the HbV groups compared with the rHSA group. Blood pressure was significantly higher for the Hb[V.sub.8] group compared with the Hb[V.sub.29] group. Arteriolar and venular blood flows were significantly higher than baseline for the HbV groups. Microvascular oxygen delivery and extraction were similar for the HbV groups but lower for the rHSA group (P < 0.05). Venular and tissue [Po.sub.2] were statistically higher for the Hb[V.sub.8] vs. the Hb[V.sub.29] and rHSA groups (P < 0.05). Improved tissue [Po.sub.2] is obtained when red blood cells deliver oxygen in combination with a high- rather than low-affinity oxygen carrier. oxygen-carrying capacity; blood substitutes; tissue oxygen; hemoglobin oxygen affinity

The microvascular distribution of oxygen was studied in the arterioles and venules of the awake hamster window chamber preparation to determine the contribution of vascular smooth muscle relaxation to oxygen consumption of the microvascular wall during verapamil-induced vasodilatation. Verapamil HCl delivered in a 0.1 mg/kg bolus injection followed by a continuous infusion of 0.01 mg x [kg.sup.-1] x [min.sup.-1] caused significant arteriolar dilatation, increased microvascular flow and functional capillary density, and decreased arteriolar vessel wall transmural [Po.sub.2] difference. Verapamil caused tissue [Po.sub.2] to increase from 25.5 [+ or -] 4.1 mmHg under control condition to 32.0 [+ or -] 3.7 mmHg during verapamil treatment. Total oxygen released by the microcirculation to the tissue remained the same as at baseline. Maintenance of the same level of oxygen release to the tissue, increased tissue [Po.sub.2], and decreased wall oxygen concentration gradient are compatible if vasodilatation significantly lowers vessel wall oxygen consumption, which in this model appears to constitute an important oxygen-consuming compartment. These findings show that treatment with verapamil, which increases oxygen supply through vasodilatation. may further improve tissue oxygenation by lowering oxygen consumption of the microcirculation. vasoactivity: oxygen gradients; tissue oxygenation; microvessel metabolism

To determine whether culturing peripheral blood mononuclear cells at atmospheric oxygen levels skews responses in comparison with culturing lymphocytes at physiologic oxygen levels, we cultured peripheral blood mononuclear cells at 5%, 10%, and atmospheric (20%) gas-phase oxygen for 5 days. We found that incubator oxygen levels influenced lymphocyte proliferation stimulated by two commonly used stimuli: Con A and antibodies that crosslink surface CD3 and CD28 to mimic antigen presentation. In both cases, proliferation increased as gas-phase oxygen levels increased. In contrast, oxygen levels did not influence proliferation stimulated by phytohemagglutinin, another commonly used mitogen. Similarly, oxygen levels did not impact cell viability in unstimulated cultures. Thus, we conclude that the influence of oxygen levels on proliferation depends on the stimulus, and, most importantly from the standpoint of immune responses, culturing cells at atmospheric rather than physiologic oxygen levels results in significantly increased proliferation responses to the CD3/CD28 crosslinking, a proliferation stimulus commonly used to mimic T cell antigen receptor signaling. CD3/CD28 | incubator | T cell | tissue culture

Binuclear Cu proteins play vital roles in [O.sub.2] binding and activation in biology and can be classified into coupled and noncoupled binuclear sites based on the magnetic interaction between the two Cu centers. Coupled binuclear Cu proteins include hemocyanin, tyrosinase, and catechol oxidase. These proteins have two Cu centers strongly magnetically coupled through direct bridging ligands that provide a mechanism for the 2-electron reduction of [O.sub.2] to a [mu]-[eta.sup.2]:[eta.sup.2] side-on peroxide bridged [Cu.sup.II.sub.2]([O.sup.2-.sub.2]) species. This side-on bridged peroxo-[Cu.sup.II.sub.2] species is activated for electrophilic attack on the phenolic ring of substrates. Noncoupled binuclear Cu proteins include peptidylglycine [alpha]-hydroxylating monooxygenase and dopamine [beta]-onooxygenase. These proteins have binuclear Cu active sites that are distant, that exhibit no exchange interaction, and that activate [O.sub.2] at a single Cu center to generate a reactive [Cu.sup.II]/[O.sub.2] species for H-atom abstraction from the C-H bond of substrates. [O.sub.2] intermediates in the coupled binuclear Cu enzymes can be trapped and studied spectroscopically. Possible intermediates in noncoupled binuclear Cu proteins can be defined through correlation to mononuclear [Cu.sup.II]/[O.sub.2] model complexes. The different intermediates in these two classes of binuclear Cu proteins exhibit different reactivities that correlate with their different electronic structures and exchange coupling interactions between the binuclear Cu centers. These studies provide insight into the role of exchange coupling between the Cu centers in their reaction mechanisms.

The chlamydospore is a distinctive morphological feature of the fungal pathogen Candida albicans that can be induced to form in oxygen-limited environments and has been reported in clinical specimens. Chlamydospores are not produced by the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, so there is limited understanding of the pathways that govern their development. Here, the results of a forward genetic approach that begins to define the genetic control of chlamydospore formation are described. Six genes-ISW2, MDS3, RIM13, RIM101, SCH9 and SUV3-are required for efficient chlamydospore formation, based on the phenotypes of homozygous insertion mutants and reconstituted strains. Mutations in ISW2, SCH9 and SUV3 completely abolish chlamydospore formation. Mutations in RIM13, RIM101 and MDS3 delay normal chlamydospore formation. The involvement of alkaline pH-response regulators Rim13p and Mds3p in chlamydospore formation is unexpected in view of the fact that chlamydospores in the inducing conditions used here are repressed in alkaline media.

In Paracoccus denitrificans at least three fumarate and nitrate reductase regulator (FNR)-like proteins [FnrP, nitrite and nitric oxide reductases regulator (NNR) and NarR] control the expression of several genes necessary for denitrifying growth. To gain more insight into this regulation, [beta]-galactosidase activity from a plasmid carrying the lacZ gene fused to the Escherichia coli melR promoter with the consensus FNR-binding (FF) site was examined. Strains defective in the fnrP gene produced only very low levels of [beta]-galactosidase, indicating that FnrP is the principal activator of the FF promoter. Anoxic [beta]-galactosidase levels were much higher relative to those under oxic growth and were strongly dependent on the nitrogen electron acceptor used, maximal activity being promoted by [N.sub.2]O. Additions of nitrate or nitroprusside lowered [beta]-galactosidase expression resulting from an oxic to micro-oxic switch. These results suggest that the activity of FnrP is influenced not only by oxygen, but also by other factors, most notably by NO concentration. Observations of nitric oxide reductase (NOR) activity in a nitrite-reductase-deficient strain and in cells treated with haemoglobin provided evidence for dual regulation of the synthesis of this enzyme, partly independent of NO. Both regulatory modes were operative in the FnrP-deficient strain, but not in the NNR-deficient strain, suggesting involvement of the NNR protein. This conclusion was further substantiated by comparing the respective NOR promoter activities.

Oxygen levels were monitored within the mantle cavities of three barnacle species (Chthamalus stellatus, Semibalanus balanoides, Elminius modestus), using optode microsensors. Conditions were always hypoxic, even when barnacles were actively using the prosoma and cirri to pump aerated seawater into the mantle cavity. Mantle fluid oxygen concentrations were extremely variable; behaviour and oxygen concentrations were not closely coupled. Ventilation of the mantle cavity depended partially on external water flow, with higher and more stable mantle fluid oxygen concentrations being sustained when the water around barnacles was agitated. During emersion, barnacles initially pumped seawater between the mantle cavity and the cone above the opercular plates to achieve ventilation. As water was lost it was replaced by air bubbles, eventually resulting in an air-filled mantle cavity. In S. balanoides and E. modestus, once the mantle cavity was filled with air, the barnacle usually used up the oxygen within the bubble within 2-3 h and did not regain oxic conditions until the barnacle was reimmersed in seawater. In C. stellatus, the air bubble was repeatedly refreshed for many hours by pneumostome formation. In response to low environmental salinity, all three species closed the opercular plates firmly and rapidly used up oxygen within the mantle fluid.

In addition to their role in bacterial killing by leukocytes, reactive oxygen species (ROS) have been increasingly recognized as important components of signaling and host defense in other cell types. The formation of ROS in both phagocytic- and non-phagocytic cells involves membrane-localized NADPH oxidases (Noxs). Nox proteins show structural homology to the cytochrome [b.sub.558] of leukocytes but, until recently, their regulation has been poorly understood. Here, we describe our current understanding of Nox function, and discuss emerging paradigms for regulation of Nox activity by Rac GTPase and/or other cytosolic components.

Two oxygen-responsive regulatory systems controlling numerous symbiotic genes in Bradyrhizobium japonicum were assayed in free-living cultures for their capacity to activate target genes under different oxygen conditions. NifA- and FixLJ-controlled target genes showed disparate relative expression patterns. Induction of NifA-dependent genes was observed only at oxygen concentrations below 2% in the gas phase, whereas that of FixLJ-controlled targets progressively increased when the oxygen concentration was lowered from 21 to 5, 2, or 0.5%. We propose that this reflects a response to a gradient of increasing oxygen deprivation as bacteria invade their host during root nodule development.

Glutathione is an abundant and ubiquitous low-molecular-weight thiol that may play a role in many cellular processes, including protection against the deleterious effects of reactive oxygen species. We address here the role of glutathione in protection against hydrogen peroxide ([H.sub.2][O.sub.2]) in Haemophilus influenzae and show that glutathione and catalase provide overlapping defense systems. H. influenzae is naturally glutathione deficient and imports glutathione from the growth medium. Mutant H. influenzae lacking catalase and cultured in glutathione-deficient minimal medium is completely devoid of [H.sub.2][O.sub.2] scavenging activity and, accordingly, substantial amounts of [H.sub.2][O.sub.2] accumulate in the growth medium. H. influenzae generates [H.sub.2][O.sub.2] at rates similar to those reported for Escherichia coil, but the toxicity of this harmful metabolite is averted by glutathione-based [H.sub.2][O.sub.2] removal, which appears to be the primary system for protection against [H.sub.2][O.sub.2] endogenously generated during aerobic respiration. When [H.sub.2][O.sub.2] concentrations exceed low micromolar levels, the hktE gene-encoded catalase becomes the predominant scavenger. The requirement for glutathione in protection against oxidative stress is analogous to that in higher and lower eukaryotes but is unlike the situation in other bacteria in which glutathione is dispensable for aerobic growth during both normal and oxidative stress conditions.

Transcription of the nrdDG operon, which encodes the class III nucleotide reductase, which is only active under anaerobic conditions, was strongly induced after a shift to anaerobiosis. The induction was completely dependent on the transcriptional activator FNR and was independent of the ArcA-ArcB two-component response regulator system. The nrdD transcript start site was mapped to a position immediately downstream of two FNR binding sites. Transcription of the other two nucleotide reductase operons, nrdAB and nrdEF, did not respond to oxygen conditions in a wild-type background, but nrdAB expression was increased in the fnr mutant under anaerobic conditions.

The Aer and Tsr chemoreceptors in Escherichia coli govern tactic responses to oxygen and redox potential that are parts of an overall behaviour known as energy taxis. They are also proposed to mediate responses to rapidly utilized carbon sources, glycerol and succinate, via the energy taxis mechanism. In this study, the Aer and Tsr proteins were individually expressed in an 'all-transducer-knockout' strain of E. coli and taxis was analysed in gradients of various oxidizable carbon sources. In addition to the known response to glycerol and succinate, it was found that Aer directed taxis towards ribose, galactose, maltose, malate, proline and alanine as well as the phosphotransferase system (PTS) carbohydrates glucose, mannitol, mannose, sorbitol and fructose, but not to aspartate, glutamate, glycine and arabinose. Tsr directed taxis towards sugars (including those transported by the PTS), but not to organic acids or amino acids. When a mutated Aer protein unable to bind the FAD cofactor was expressed in the receptor-less strain, chemotaxis was not restored to any substrate. Aer appears to mediate responses to rapidly oxidizable substrates, whether or not they are effective growth substrates, whereas Tsr appears to mediate taxis to substrates that support maximal growth, whether or not they are rapidly oxidizable. This correlates with the hypothesis that Aer and Tsr sense redox and proton motive force, respectively. Taken together, the results demonstrate that Aer and Tsr mediate responses to a broad range of chemicals and their attractant repertoires overlap with those of specialized chemoreceptors, namely Trg (ribose, galactose) and Tar (maltose).

Helicobacter pylori whole cells showed high rates of oxygen uptake with L-serine and L-proline as respiratory substrates, and somewhat lower rates with D-alanine and D-proline. These respiratory activities were inhibited by rotenone and antimycin A at low concentrations. Since pyruvate was produced from L-serine and D- and L-alanine in whole cells, the respiratory activities with these amino acids as substrates occurred via pyruvate. Whole cells showed 2,6-dichlorophenolindophenol (DCIP)-reducing activities with D- and L-proline and D-alanine as substrates, suggesting that hydrogen removed from these amino acids also participated in oxygen uptake by the whole cells. High amounts of L-proline, D- and L-alanine, and L-serine were present in H. pylori cells, and these amino acids also predominated in samples of human gastric juice. H. pylori seems to utilize D- and L-proline, D-alanine and L-serine as important energy sources in its habitat of the mucous layer of the stomach.

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) enters intestinal epithelial cells of cattle and other ruminants via a mechanism that remains to be fully elucidated. This study showed that a gene encoding the M. paratuberculosis 35 kDa major membrane protein (MMP) is expressed at a higher level in low-oxygen and high-osmolarity conditions that are similar to the environment of the intestine. In addition, cattle with Johne's disease produced antibodies against MMP, suggesting that the protein is present during infection. The gene encoding MMP was cloned and expressed as a fusion protein with the maltose-binding protein (MBP-MMP) in Escherichia coli. Rabbit antisera were raised against a M. paratuberculosis whole-cell sonicate and MMP-specific antibodies were purified from these sera by affinity chromatography. MMP was localized to the surface of M. paratuberculosis by immunoelectron microscopy and by immunoblot analysis of fractionated protein lysates. Both anti-MMP antibodies and MBP-MMP protein inhibited M. paratuberculosis invasion of cultured Madin-Darby bovine kidney cells by 30 %. In similar invasion experiments with M. paratuberculosis incubated in low oxygen tension, these antibodies and protein decreased invasion by 60%. Collectively, these data show that the 35 kDa MMP is a surface exposed protein that plays a role in invasion of epithelial cells. The authors suggest that the MMP is a virulence factor of M. paratuberculosis that may be important in the initiation of infection in vivo.

Wild-type Escherichia coli cells grow normally in 95% [O.sub.2]/5% C[O.sub.2]. In contrast, cells that cannot make polyamines because of mutations in the biosynthetic pathway are rapidly killed by incubation in 95% [O.sub.2]/5% C[O.sub.2]. Addition of polyamines prevents the toxic effect of oxygen, permitting cell survival and optimal growth. Oxygen toxicity can also be prevented if the growth medium contains an amino acid mixture or if the polyamine-deficient cells contain a manganese-superoxide dismutase (Mn-SOD) plasmid. Partial protection is afforded by the addition of 0.4 M sucrose or 0.4 M sorbitol to the growth medium. We also report that concentrations of [H.sub.2][O.sub.2] that are nontoxic to wild-type cells or to mutant cells pretreated with polyamines kill polyamine-deficient cells. These results show that polyamines are important in protecting cells from the toxic effects of oxygen.

The ascites syndrome in broiler chickens is attributed to metabolic burdening, which results from intensive genetic selection for rapid growth coupled with exposure to extreme environmental conditions, such as low ambient temperature. These conditions impose on the broilers difficulties in fulfilling tissue demands for oxygen, and the birds exhibit a decrease in blood oxygen saturation and high hematocrit values. It is unknown whether the increase in hematocrit results from alteration in erythropoiesis or from fluid exudation out of the blood system to the abdominal cavity. The present study was conducted to examine the association between abnormal stress response and erythropoiesis process in ascitic broilers. Ascitic chickens revealed a uniquely continuous stress response: expressing an increase (P [less than or equal to] 0.05) in plasma corticosterone concentration 2 to 3 wk before death. At 5 wk of age, ascitic broilers exhibited an increase (P < 0.05) in hematocrit, blood cell count, and packed cells and blood volumes, with no significant change in plasma volume. These results confirm an accelerated erythropoiesis process in ascitic birds. Increased blood cell production in ascitic birds was matched by an increase (P < 0.05) in the proportion of immature red blood cells (23%) in comparison with broilers that remained healthy (7%), and by decreased (P < 0.05) hemoglobin content relative to red blood cells. We conclude that continually increased corticosterone concentrations, as an inducer of erythropoiesis proliferation and differentiation arrest, in ascitic chickens, resulted in increased production of red blood cells (partially immature) with decreased hemoglobin content; this decrease in hemoglobin might have contributed to enhanced development of hypoxemia and to aggravation of the syndrome. Key Words: Ascites, Corticosterone, Erythropoiesis, Hemoglobin

Research has been conducted on tetranuclear manganese cluster of the oxygen-evolving complex. The reactivity of nitric oxide (super). with the oxygen-evolving complex in Synechococcus elongatus and spinach has been investigated via the use of flash-induced oxygen evolution measurements and electron paramagnetic resonance spectroscopy, and the results are reported.

The experiments were undertaken to measure, for the first time, cardiac output in yellowtail (Seriola quinqueradiata) during exposure to a harmful red tide flagellate (Chattonella marina). The responses were compared with those during exposure to environmental hypoxia to evaluate the significance of the drop of arterial oxygen partial pressure (Pa[O.sub.2]) in the fish-kill mechanisms by C. marina. Pa[O.sub.2] immediately decreased, whereas heart rate (HR) was maintained until shortly before death during exposure to C. marina. Suffocation developing during the exposure resulted from a decrease in blood oxygen content, but not from lowered blood flow to the tissue. Although exposure to both C. marina and hypoxia immediately decreased Pa[O.sub.2], arterial oxygen content (Ca[O.sub.2]) and pH (pHa) were significantly lower, whereas HR and cardiac output (Q) remained significantly higher, for the C. marina-exposed fish than for hypoxia-exposed fish. Although the drop in Pa[O.sub.2] appears to play a pivotal role in the mechanisms of fish death by C. marina, other physiological response(s) should also be considered.

Regulation of active [Na.sup.+] transport across fetal distal lung epithelial cells (FDLE) by corticosterone (CST), corticotropin-releasing hormone (CRH), and oxygen tension may be crucial for postnatal adaptation. FDLE isolated from 19-day rat fetuses (term: 22 days) were grown on permeable supports to confluent monolayers (duration 3 days) in 2.5, 5, 12, or 20% [O.sub.2] with 5% C[O.sub.2]-balance [N.sub.2] and mounted in Ussing chambers for measurement of short-circuit currents ([I.sub.sc]). FDLE monolayers grown in 20% [O.sub.2] had significantly higher levels of total [I.sub.sc] and of their amiloride-sensitive ([I.sub.amil]) and ouabain-sensitive ([I.sub.ouab]) components than hypoxic cells. Values ([micro]A/[cm.sup.2] [+ or -] SE) for 2.5-5% [O.sub.2] and 20% [O.sub.2] were, respectively, [I.sub.sc] 5.3 [+ or -] 0.2 vs. 8.4 [+ or 0] 0.3 (P < 0.001), [I.sub.amil] 3.4 [+ or -] 0.2 VS. 4.3 [+ or -] 0.2 (P < 0.01), and [I.sub.ouab] 3.4 [+ or -] 0.6 vs. 9.1 [+ or -] 0.6 (P < 0.001). Addition of CST but not CRH to the culture medium at any [O.sub.2] concentration increased [I.sub.amil]. FDLE cells grown at 5% [O.sub.2] expressed significantly lower levels of [alpha]-, [beta]-, and [gamma]-epithelial [Na.sup.+] channel (ENaC), and of the [[alpha].sub.1]-[Na.sup.+]-[K.sup.+]-ATPase, as determined by Western blotting. We conclude that higher [O.sub.2] concentrations increased total vectorial [Na.sup.+] transport, and the function of [Na.sup.+]-[K.sup.+]-ATPase and apical amiloride-sensitive [Na.sup.+] conductance, whereas CST only increased ENaC function. Ussing chamber; short-circuit current; sodium-potassium-adenosinetriphosphatase; epithelial sodium channel; oxygen; corticosterone; corticotropin-releasing hormone

To delineate the roles that oxygen and fibroblast growth factors (FGFs) play in the process of angiogenesis from the embryonic aorta, we cultured mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) in a three-dimensional type I collagen gel matrix. During 8 days of culture under 5% [O.sub.2], but not room air, the addition of FGF2 to explants stimulated the formation of Gs-I[B.sub.4-] positive, CD31-positive, and Flk-1-positive microvessels in a concentration-dependent manner. FGF2-stimulated microvessel formation was inhibited by sequestration of FGF2 via addition of soluble FGF receptor (FGFR) chimera protein or anti-FGF2 antibodies. FGFR1 and FGFR2 were present on explants. Levels of FGFR1, but not FGFR2, were increased in embryonic aorta cultured under 5% [O.sub.2] relative to room air. Our data suggest that low oxygen upregulates FGFR1 expression in embryonic aorta in vitro and renders it more responsive to FGF2. angiogenesis; embryogenesis; endothelial cell; organ culture

Research has been conductedc on plasma membrane cholesterol content. The role of this cholesterol content in intracellular oxygen concentration in Chinese hamster ovary cells and their mutants has been investigated via the use of EPR oximetry, and the details are reported.

The authors review the publications on light, oxygen, or voltage (LOV) domain family. The topics of interest include LOV protein classes, their structure and function, and LOV photophysics and photochemistry.