A membrane-bound vertebrate globin.

The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2)-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O(2) binding with a variable affinity (P(50)∼1.3-12.5 torr), depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.

Ontogeny of globin expression in zebrafish (Danio rerio).

Respiratory proteins are responsible for transport and storage of oxygen. It is well established that specific requirements for oxygen during vertebrate ontogeny cause switches of hemoglobin chain expression. Here, we characterize the developmental profiles of zebrafish (Danio rerio) globins by means of quantitative real-time reverse transcription PCR. The total mRNA levels of the hemoglobin chains, including a newly identified embryonic α-chain, as well as myoglobin, neuroglobin, cytoglobin 1 and 2, and globin X were estimated. mRNAs of all globins were detectable in unfertilized eggs, suggesting maternal storage. Embryonic α- and ß-hemoglobin mRNA peaked at hatching and the switch to adult hemoglobin expression occurred 16 dpf. Enhanced myoglobin mRNA levels were detected ~31 h post-fertilization (hpf), coinciding with the heart and the muscle development, while neuroglobin mRNA expression pattern correlates with the formation of the nervous system. Amounts of myoglobin and neuroglobin mRNA were similar within an order of magnitude throughout the ontogeny, tentatively supporting a respiratory role of neuroglobin. Cytoglobin 2 mRNA levels increased gradually, whereas cytoglobin 1 mRNA levels increased strongly after ~31 hpf, which is in agreement with a function in cell proliferation. Globin X mRNA level was highest in oocytes, but low in later stages. Together, these data suggest a specific role for each globin, which are also associated with certain events in fish development.

Oxygen supply from the bird's eye perspective: globin E is a respiratory protein in the chicken retina.

The visual process in the vertebrate eye requires high amounts of metabolic energy and thus oxygen. Oxygen supply of the avian retina is a challenging task because birds have large eyes, thick retinae, and high metabolic rates but neither deep retinal nor superficial capillaries. Respiratory proteins such as myoglobin may enhance oxygen supply to certain tissues, and thus the mammalian retina harbors high amounts of neuroglobin. Globin E (GbE) was recently identified as an eye-specific globin of chicken (Gallus gallus). Orthologous GbE genes were found in zebra finch and turkey genomes but appear to be absent in non-avian vertebrate classes. Analyses of globin phylogeny and gene synteny showed an ancient origin of GbE but did not help to assign it to any specific globin type. We show that the photoreceptor cells of the chicken retina have a high level of GbE protein, which accumulates to ∼10 µM in the total eye. Quantitative real-time RT-PCR revealed an ∼50,000-fold higher level of GbE mRNA in the eye than in the brain. Spectroscopic analysis and ligand binding kinetics of recombinant chicken GbE reveal a penta-coordinated globin with an oxygen affinity of P(50) = 5.8 torrs at 25 °C and 15 torrs at 41 °C. Together these data suggest that GbE helps to sustain oxygen supply to the avian retina.

Changes of globin expression in the Japanese medaka (Oryzias latipes) in response to acute and chronic hypoxia.

Fishes live in an aquatic environment with low or temporally changing O(2) availability. Variations in O(2) levels require many anatomical, behavioral, physiological, and biochemical adaptations that ensure the uptake of an adequate amount of O(2). Some fish species are comparatively well adapted to tolerate low O(2) partial pressure (hypoxia). The Japanese ricefish medaka (Oryzias latipes) is an important model organism for biomedical research that shows remarkable tolerance towards hypoxia. We have investigated the regulation and role of globins under hypoxia. We applied four different regimes of chronic hypoxia (24 and 48 h at PO(2) = 2 or 4 kPa) as well as acute hypoxia (2 h at PO(2) = 0.5 kPa) to adult male medaka. Changes of mRNA levels of seven globin genes (adult hemoglobin α and ß, myoglobin, neuroglobin, cytoglobin 1 and 2, globin X), three hypoxia-response genes (lactate dehydrogenase b, phosphoglycerate kinase, adrenomedullin 1) and two putative reference genes (cyclophilin, acidic ribosomal phosphoprotein P0) were monitored by means of quantitative real-time reverse-transcription PCR. We observed strong upregulation of myoglobin, which is also expressed in the medaka brain, as previously demonstrated for carp, goldfish and zebrafish. The hemoglobin chains were found upregulated, whereas earlier studies found down-regulation of hemoglobin in hypoxic zebrafish. By contrast, neuroglobin mRNA was not affected by hypoxia in medaka, but had been found upregulated in zebrafish. Globin X is induced in medaka brain, but down-regulated in zebrafish. Thus, the patterns of hypoxia response of globins are strikingly different in various fish species, which can be interpreted as indication for different roles of the various globins in hypoxia response and for alternative metabolic strategies of fish species in coping with O(2) deprivation.

Neuroglobin, cytoglobin, and myoglobin contribute to hypoxia adaptation of the subterranean mole rat Spalax.

The subterranean mole rat Spalax is an excellent model for studying adaptation of a mammal toward chronic environmental hypoxia. Neuroglobin (Ngb) and cytoglobin (Cygb) are O(2)-binding respiratory proteins and thus candidates for being involved in molecular hypoxia adaptations of Spalax. Ngb is expressed primarily in vertebrate nerves, whereas Cygb is found in extracellular matrix-producing cells and in some neurons. The physiological functions of both proteins are not fully understood but discussed with regard to O(2) supply, the detoxification of reactive oxygen or nitrogen species, and apoptosis protection. Spalax Ngb and Cygb coding sequences are strongly conserved. However, mRNA and protein levels of Ngb in Spalax brain are 3-fold higher than in Rattus norvegicus under normoxia. Importantly, Spalax expresses Ngb in neurons and additionally in glia, whereas in hypoxia-sensitive rodents Ngb expression is limited to neurons. Hypoxia causes an approximately 2-fold down-regulation of Ngb mRNA in brain of rat and mole rat. A parallel regulatory response was found for myoglobin (Mb) in Spalax and rat muscle, suggesting similar functions of Mb and Ngb. Cygb also revealed an augmented normoxic expression in Spalax vs. rat brain, but not in heart or liver, indicating distinct tissue-specific functions. Hypoxia induced Cygb transcription in heart and liver of both mammals, with the most prominent mRNA up-regulation (12-fold) in Spalax heart. Our data suggest that tissue globins contribute to the remarkable tolerance of Spalax toward environmental hypoxia. This is consistent with the proposed cytoprotective effect of Ngb and Cygb under pathological hypoxic/ischemic conditions in mammals.

Cerebral expression of neuroglobin and cytoglobin after deep hypothermic circulatory arrest in neonatal piglets.

INTRODUCTION: Deep hypothermic circulatory arrest (DHCA) is used in corrective cardiac surgery for complex congenital heart disease. Endogenous protective mechanisms may be responsible for the prevention of brain damage after hypothermic ischemia. Neuroglobin and cytoglobin are expressed in brain cells and appear to modulate hypoxic-ischemic brain injury. However, their neuroprotective potency is still not understood. Thus the aim of this study was to detect the influence exerted by DHCA on their expression. METHODS: The effects of DHCA were analyzed in a neonatal piglet model with cardiopulmonary bypass, DHCA of 60 and 120 min and subsequent reperfusion of 6h. Complete histological analysis and changes in the mRNA expression of neuroglobin and cytoglobin were measured in the brain. RESULTS: In comparison to animals without DHCA, neuroglobin expression was stable after 60 min DHCA and neuronal cell necrosis in the cortex was mild (< 10 %). Neuroglobin expression was significantly reduced after 120 min DHCA, which was accompanied by substantial neuronal cell necrosis (> 50 %). Cytoglobin expression did not differ significantly between animals with neuronal necrosis vs. sham. CONCLUSION: Constitutive expression levels of neuroglobin may explain the mild neuronal injury after 60 min DHCA. Significant neuronal cell death correlates with reduced neuroglobin expression and might reflect a limited capacity to compensate for ischemic injury. Both respiratory cell proteins may constitute attractive targets for therapeutic modulation of gene regulation, but further studies are necessary.

Seasonal influenza risk in hospital healthcare workers is more strongly associated with household than occupational exposures: results from a prospective cohort study in Berlin, Germany, 2006/07.

BACKGROUND: Influenza immunisation for healthcare workers is encouraged to protect their often vulnerable patients but also due to a perceived higher risk for influenza. We aimed to compare the risk of influenza infection in healthcare workers in acute hospital care with that in non-healthcare workers over the same season. METHODS: We conducted a prospective, multicentre cohort study during the 2006/07 influenza season in Berlin, Germany. Recruited participants gave serum samples before and after the season, and completed questionnaires to determine their relevant exposures and possible confounding factors. The main outcome measure was serologically confirmed influenza infection (SCII), defined as a fourfold or greater rise in haemagglutination inhibition antibody titres to a circulating strain of influenza (with post-season titre at least 1:40).Weekly mobile phone text messages were used to prompt participants to report respiratory illnesses during the influenza season. A logistic regression model was used to assess the influence of potential risk factors. RESULTS: We recruited 250 hospital healthcare workers (mean age 35.7 years) and 486 non-healthcare workers (mean age 39.2 years) from administrative centres, blood donors and colleges.Overall SCII attack rate was 10.6%. Being a healthcare worker was not a risk factor for SCII (relative risk 1.1, p = 0.70). The final multivariate model had three significant factors: living with children (odds ratio [OR] 3.7, p = 0.005), immunization (OR 0.50, p = 0.02), and--among persons living in households without children--ownership of a car (OR 3.0, p = 0.02). Living with three or more children (OR 13.8, p < 0.01) was a greater risk than living with one or two children (OR 5.3, p = 0.02). 30% of participants with SCII reported no respiratory illness. Healthcare workers were at slightly higher risk of reporting any respiratory infection than controls (adjusted OR 1.3, p = 0.04, n = 850). CONCLUSIONS: Our results suggest that healthcare workers in hospitals do not have a higher risk of influenza than non-healthcare workers, although their risk of any respiratory infection is slightly raised. Household contacts seem to be more important than exposure to patients. Car ownership is a surprise finding which needs further exploration. Asymptomatic infections are common, accounting for around a third of serologically confirmed infections.

Genomic response of the rat brain to global ischemia and reperfusion.

To identify genes that are involved in ischemia response of the brain, we have evaluated changes of gene expression in rat cerebrum after 15 min complete global ischemia, followed by reperfusion for 1 h, 6 h or 24 h. The expression profiles of approximately 30,000 transcripts from three subjects in each group (including sham-operated controls) were monitored employing oligonucleotide microarrays. About 20,000 transcripts were detectable in rat brains. The levels of 576 transcripts (approximately 2.9%) were significantly altered in response to experimental ischemia. 419 transcripts were up- and 157 downregulated; 39 transcripts changed after 1 h reperfusion, 174 after 6 h and 462 after 24 h. Results from quantitative real-time reverse transcription PCR of 18 selected genes showed excellent agreement with the microarray data. There is surprisingly little overlap between gene regulation patterns at different reperfusion times (only seven genes displayed significant changes in transcript levels at all reperfusion times. Several genes that were previously unknown to be involved in ischemia-response have been identified. Analyses of gene ontology patterns and the most strongly regulated transcripts showed that the immediate response to an ischemia/reperfusion is mediated by the induction of specific transcription factors and stress genes. Delayed gene expression response is characterised by inflammation and immune-related genes. These results support the hypothesis that the brain's response to ischemia is an active, specific and coordinated process.

Oxygen-induced changes in hemoglobin expression in Drosophila.

The hemoglobin gene 1 (dmeglob1) of the fruit fly Drosophila melanogaster is expressed in the tracheal system and fat body, and has been implicated in hypoxia resistance. Here we investigate the expression levels of dmeglob1 and lactate dehydrogenase (a positive control) in embryos, third instar larvae and adult flies under various regimes of hypoxia and hyperoxia. As expected, mRNA levels of lactate dehydrogenase increased under hypoxia. We show that expression levels of dmeglob1 are decreased under both short- and long-term hypoxia, compared with the normoxic (21% O2) control. By contrast, a hypoxia/reoxygenation regime applied to third instar larvae elevated the level of dmeglob1 mRNA. An excess of O2 (hyperoxia) also triggered an increase in dmeglob1 mRNA. The data suggest that Drosophila hemoglobin may be unlikely to function merely as a myoglobin-like O2 storage protein. Rather, dmeglob1 may protect the fly from an excess of O2, either by buffering the flux of O2 from the tracheoles to the cells or by degrading noxious reactive oxygen species.

Coping with cyclic oxygen availability: evolutionary aspects.

Both the gradual rise in atmospheric oxygen over the Proterozoic Eon as well as episodic fluctuations in oxygen over several million-year time spans during the Phanerozoic Era, have arguably exerted strong selective forces on cellular and organismic respiratory specialization and evolution. The rise in atmospheric oxygen, some 2 billion years after the origin of life, dramatically altered cell biology and set the stage for the appearance of multicelluar life forms in the Vendian (Ediacaran) Period of the Neoproterozoic Era. Over much of the Paleozoic, the level of oxygen in the atmosphere was near the present atmospheric level (21%). In the Late Paleozoic, however, there were extended times during which the level of atmospheric oxygen was either markedly lower or markedly higher than 21%. That these Paleozoic shifts in atmospheric oxygen affected the biota is suggested by the correlations between: (1) Reduced oxygen and the occurrences of extinctions, a lowered biodiversity and shifts in phyletic succession, and (2) During hyperoxia, the corresponding occurrence of phenomena such as arthropod gigantism, the origin of insect flight, and the evolution of vertebrate terrestriality. Basic similarities in features of adaptation to hyopoxia, manifest in living organisms at levels ranging from genetic and cellular to physiological and behavioral, suggest the common and early origin of a suite of adaptive mechanisms responsive to fluctuations in ambient oxygen. Comparative integrative approaches addressing the molecular bases of phenotypic adjustments to cyclic oxygen fluctuation provide broad insight into the incremental steps leading to the early evolution of homeostatic respiratory mechanisms and to the specialization of organismic respiratory function.

Regulation and role of neuroglobin and cytoglobin under hypoxia.

Neuroglobin (Ngb) and cytoglobin (Cygb) are two novel members of the globin superfamily that are ubiquitously present in vertebrates. Their exact physiological roles are still uncertain. Here we review the expression of Ngb and Cygb, with particular emphasis on their regulation and potential role under hypoxia. Ngb expression is confined to neurons and some endocrine tissues. At the subcellular level, Ngb is associated with the presence of mitochondria and thus linked to the oxidative metabolism. Hypoxia or ischemic insults most likely do not strongly increase Ngb levels in the rodent brain. This might be explained by the fact that most mammals are not adapted to low oxygen levels. In zebrafish and turtle, however, which live in an environment with naturally changing oxygen conditions, hypoxia dramatically increases Ngb expression in the brains. We also found that hypoxia-tolerant species (e.g. the mole rat Spalax and goldfish) express more Ngb in their brains than their oxygen-deprivation sensitive relatives. These data suggest that Ngb may have a myoglobin-like role and supplies oxygen to the respiratory chain of the metabolically highly active neurons, or protect them from reactive oxygen species. Cygb is predominantly expressed in fibroblasts and related cell types, but also in distinct nerve cell populations. Cygb levels are significantly elevated at low oxygen levels in the fibroblast cell lineage. Cell culture data suggest that in fibroblasts Cygb is involved in cell proliferation, possibly in collagen synthesis. In neurons, there is evidence for an additional role of Cygb related to nitric oxide metabolism.

Duplicated cytoglobin genes in teleost fishes.

Cytoglobin is a recently discovered myoglobin-related O2-binding protein of vertebrates with uncertain function. It occurs as single-copy gene in mammals. Here, we demonstrate the presence of two paralogous cytoglobin genes (Cygb-1 and Cygb-2) in the teleost fishes Danio rerio, Oryzias latipes, Tetraodon nigroviridis, and Takifugu rubripes. The globin-typical introns at positions B12.2 and G7.0 are conserved in both genes, whereas the C-terminal exon found in mammalian cytoglobin is absent in the fish genes. Phylogenetic analyses show that the two cytoglobin genes diverged early in teleost evolution. This is confirmed by gene synteny analyses, which suggest a large-scale duplication event. Although both cytoglobin genes are highly conserved and have evolved under purifying selection, substitution rates are significantly higher in Cygb-1 than in Cygb-2. Similar to their mammalian ortholog, both fish cytoglobins are expressed in a broad range of tissues. However, Cygb-2 is more than 250-fold stronger expressed in neuronal tissues, suggesting a subfunctionalization of the two cytoglobin paralogs after gene duplication.

Neuroglobin and cytoglobin in search of their role in the vertebrate globin family.

Neuroglobin and cytoglobin are two recent additions to the family of heme-containing respiratory proteins of man and other vertebrates. Here, we review the present state of knowledge of the structures, ligand binding kinetics, evolution and expression patterns of these two proteins. These data provide a first glimpse into the possible physiological roles of these globins in the animal's metabolism. Both, neuroglobin and cytoglobin are structurally similar to myoglobin, although they contain distinct cavities that may be instrumental in ligand binding. Kinetic and structural studies show that neuroglobin and cytoglobin belong to the class of hexa-coordinated globins with a biphasic ligand-binding kinetics. Nevertheless, their oxygen affinities resemble that of myoglobin. While neuroglobin is evolutionarily related to the invertebrate nerve-globins, cytoglobin shares a more recent common ancestry with myoglobin. Neuroglobin expression is confined mainly to brain and a few other tissues, with the highest expression observed in the retina. Present evidence points to an important role of neuroglobin in neuronal oxygen homeostasis and hypoxia protection, though other functions are still conceivable. Cytoglobin is predominantly expressed in fibroblasts and related cell types, but also in distinct nerve cell populations. Much less is known about its function, although in fibroblasts it might be involved in collagen synthesis.

Cytoglobin is a respiratory protein in connective tissue and neurons, which is up-regulated by hypoxia.

Cytoglobin is a recently discovered vertebrate globin distantly related to myoglobin, and its function is unknown. Here we present the first detailed analysis of the distribution and expression of cytoglobin. Northern and Western blotting experiments show the presence of cytoglobin mRNA and protein in a broad range of tissues. Quantitative PCR demonstrates an up-regulation of cytoglobin mRNA levels in rat heart and liver under hypoxic conditions (22 and 44 h of 9% oxygen). Immunofluorescence studies with three antibodies directed against different epitopes of the protein consistently show cytoglobin in connective tissue fibroblasts as well as in hepatic stellate cells. Cytoglobin is also present in chondroblasts and osteoblasts and shows a decreased level of expression upon differentiation to chondrocytes and osteocytes. Cytoglobin is located in the cytoplasm of these cell types. Evidence against an exclusively nuclear localization of cytoglobin, as recently proposed, is also provided by transfection assays with green fluorescent protein fusion constructs, which demonstrates the absence of an active nuclear import. The differential expression of cytoglobin argues against a general respiratory function of this molecule, but rather indicates a connective tissue-specific function. We hypothesize that cytoglobin may be involved in collagen synthesis. Cytoglobin expression was also observed in some neuronal subpopulations of the central and the peripheral nervous systems. Surprisingly, cytoglobin is localized in both the cytoplasm and nucleus of neurons, indicating a possible additional role of this protein in neuronal tissues.

Neuroglobin and cytoglobin: genes, proteins and evolution.

Hemoglobin and myoglobin are oxygen transport and storage proteins of most vertebrates. Neuroglobin (Ngb) and cytoglobin (Cygb)--two recent additions to the vertebrate globin superfamily--have still disputed functions. Combining the data from all available resources, we investigate the evolution of these novel globins. Both Ngb and Cygb show little sequence variation in vertebrate evolution, suggesting conserved structures and functions, and an important role in the animal's metabolism. Exon-intron patterns remained unchanged in Ngb and Cygb, with the exception of the addition of a 3' exon to Cygb early in mammalian evolution. In phylogenetic analyses, Ngb forms a common branch with globin X, another recently identified globin with undefined function in lower vertebrates, and with some invertebrate nerve globins. This shows an early divergence of this branch in animal evolution. Cygb is related to myoglobin, and associated with an eye-specific globin from birds. The pattern of globin evolution shows that proteins with clear respiratory roles evolved independently from intracellular globins with uncertain functions. This result suggests either multiple independent functional changes or a yet undefined respiratory role of tissue globins like Ngb and Cygb.

The cellular and subcellular localization of neuroglobin and cytoglobin -- a clue to their function?

Neuroglobin and cytoglobin are recently discovered respiratory proteins of vertebrates with yet ill-defined physiological functions. Neuroglobin is widely expressed in neurons, but not glia, in the vertebrate central and peripheral nervous systems. Other major expression sites are the retina and endocrine tissues. This distribution is indicative of a function of neuroglobin in metabolically most active, oxygen-consuming cell types, but does not yet allow to safely distinguish between different cellular roles, such as oxygen homeostasis, scavenging of reactive oxygen species or sustaining energy metabolism. Cytoglobin is predominantly expressed in connective tissue fibroblasts and related cell types in the body organs. Its main function may therefore be related to the specific amounts of extracellular matrix. Cytoglobin may hypothetically be involved in the oxygen-consuming maturation of collagen proteins. Cytoglobin is also expressed in distinct cell types of brain and retina. Its distribution strikingly differs from neuroglobin, suggesting an independent, yet unknown function.