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Lung metastasis is a major cause of mortality in patients with osteosarcoma (OS). A better understanding of the molecular mechanism of OS lung metastasis may facilitate development of new therapeutic strategies to prevent the metastasis. We have established high- and low-metastatic sublines (LM8-H and LM8-L, respectively) from Dunn OS cell line LM8 by using in vivo image-guided screening. Among the genes whose expression was significantly increased in LM8-H compared to LM8-L, the transcription factor lymphoid enhancer-binding factor 1 (LEF1) was identified as a factor that promotes LM8-H cell extravasation into the lungs. To identify downstream effectors of LEF1 that are involved in OS lung metastasis, 13 genes were selected based on LM8 microarray data and genomewide meta-analysis of a public database for OS patients. Among them, the cytoglobin (Cygb) gene was identified as a key effector in promoting OS extravasation into the lungs. CYGB overexpression increased the extravasation ability of LM8-L cells, whereas knocking out the Cygb gene in LM8-H cells reduced this ability. Our results showed a novel LEF1-CYGB axis in OS lung metastasis and may provide a new way of developing therapeutic strategies to prevent OS lung metastasis., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Environmental factors or adverse growth conditions that may reduce cell function or viability are considered stress. The cell ability to sense and respond to environmental stresses determine its function and survival destiny. We recently defined Neuroglobin (NGB), a heme-protein, as a compensatory protein in the 17β-Estradiol (E2) anti-apoptotic activity and as a sensor of oxidative stress in both neurons and breast cancer cells. Here, the possibility that NGB levels could represent a pivotal regulator of integrated response of cancer cells to stress has been evaluated. Data obtained in neuroblastoma and in breast cancer cell lines evidence that nutrient deprivation significantly up-regulated NGB levels at different time points. However, the analysis of autophagy activation led to exclude any possible role of stress- or E2-induced NGB in the upstream regulation of general autophagy. However, the over-expression of Flag-NGB in ERα stable transfected HEK-293 cells completely affects nutrient deprivation-induced decrease in cell number. In addition, reported results indicate that modulation of the anti-apoptotic Bcl-2 level may play a key role in the protective NGB function against energetic stress. Overall, these data define a role of NGB as compensatory protein in the cell machinery activated in response to stress and as general stress adaptation marker of cancer cells susceptible to oxidative stress, oxygen and, as demonstrated here for the first time, even to nutrient willingness. Despite the lacking of any direct NGB role on autophagic flux activated by energetic stress, NGB upregulation appears functional in delaying stress-related cell death allowing an appropriate cell response and adaptation to the changing extracellular conditions.

Objective: The role of hemoglobin and myoglobin in the cardiovascular system is well established, yet other globins in this context are poorly characterized. Here, we examined the expression and function of cytoglobin (CYGB) during vascular injury., Approach and Results: We characterized CYGB content in intact vessels and primary vascular smooth muscle (VSM) cells and used 2 different vascular injury models to examine the functional significance of CYGB in vivo. We found that CYGB was strongly expressed in medial arterial VSM and human veins. In vitro and in vivo studies indicated that CYGB was lost after VSM cell dedifferentiation. In the rat balloon angioplasty model, site-targeted delivery of adenovirus encoding shRNA specific for CYGB prevented its reexpression and decreased neointima formation. Similarly, 4 weeks after complete ligation of the left common carotid, Cygb knockout mice displayed little to no evidence of neointimal hyperplasia in contrast to their wild-type littermates. Mechanistic studies in the rat indicated that this was primarily associated with increased medial cell loss, terminal uridine nick-end labeling staining, and caspase-3 activation, all indicative of prolonged apoptosis. In vitro, CYGB could be reexpressed after VSM stimulation with cytokines and hypoxia and loss of CYGB sensitized human and rat aortic VSM cells to apoptosis. This was reversed after antioxidant treatment or NOS2 (nitric oxide synthase 2) inhibition., Conclusions: These results indicate that CYGB is expressed in vessels primarily in differentiated medial VSM cells where it regulates neointima formation and inhibits apoptosis after injury., (© 2017 American Heart Association, Inc.)

Thiosulfate formation and biodegradation processes link aerobic and anaerobic metabolism of cysteine. In these reactions, sulfite formed from thiosulfate is oxidized to sulfate while hydrogen sulfide is transformed into thiosulfate. These processes occurring mostly in mitochondria are described as a canonical hydrogen sulfide oxidation pathway. In this review, we discuss the current state of knowledge on the interactions between hydrogen sulfide and hemoglobin, myoglobin and neuroglobin and postulate that thiosulfate is a metabolically important product of this processes. Hydrogen sulfide oxidation by ferric hemoglobin, myoglobin and neuroglobin has been defined as a non-canonical hydrogen sulfide oxidation pathway. Until recently, it appeared that the goal of thiosulfate production was to delay irreversible oxidation of hydrogen sulfide to sulfate excreted in urine; while thiosulfate itself was only an intermediate, transient metabolite on the hydrogen sulfide oxidation pathway. In the light of data presented in this paper, it seems that thiosulfate is a molecule that plays a prominent role in the human body. Thus, we hope that all these findings will encourage further studies on the role of hemoproteins in the formation of this undoubtedly fascinating molecule and on the mechanisms responsible for its biological activity in the human body., Competing Interests: The authors declare no conflict of interest.

Sensory receptor neurons match their dynamic range to ecologically relevant stimulus intensities. How this tuning is achieved is poorly understood in most receptors. The roundworm Caenorhabditis elegans avoids 21% O 2 and hypoxia and prefers intermediate O 2 concentrations. We show how this O 2 preference is sculpted by the antagonistic action of a neuroglobin and an O 2 -binding soluble guanylate cyclase. These putative molecular O 2 sensors confer a sigmoidal O 2 response curve in the URX neurons that has highest slope between 15 and 19% O 2 and approaches saturation when O 2 reaches 21%. In the absence of the neuroglobin, the response curve is shifted to lower O 2 values and approaches saturation at 14% O 2 In behavioral terms, neuroglobin signaling broadens the O 2 preference of Caenorhabditis elegans while maintaining avoidance of 21% O 2 A computational model of aerotaxis suggests the relationship between GLB-5-modulated URX responses and reversal behavior is sufficient to broaden O 2 preference. In summary, we show that a neuroglobin can shift neural information coding leading to altered behavior. Antagonistically acting molecular sensors may represent a common mechanism to sharpen tuning of sensory neurons., Competing Interests: The authors declare no conflict of interest.

Objective: To investigate the effect of neuroglobin on oxygen-glucose deprivation and reoxygenation (OGD/R) induced autophagy in a human neuroblastoma cell line (SH-SY5Y). Methods: SH-SY5Y cells were transfected with plasmids (or vector) to establish a stable cell line of NGB overexpression (OE). After treated with OGD/R, cells were collected for the analyses of mRNA (Atg5, Atg7, BECN1 and FUNDC1) and protein levels of LC3. Furthermore, mitochondrial and cytosolic fractions were isolated for protein levels of PINK1 and Parkin. Results: Treatment of OGD/R significantly increased the levels of mRNA of Atg5, Atg7, BECN1 and FUNDC1 (peak levels were 4.90±0.71, 6.72±0.75, 2.71±0.39 and 3.96±0.78 fold, all P <0.05). The protein level of Parkin increased in mitochondria and decreased in cytoplasm after the treatment. Compared with the vector group, Ngb OE group showed a significant higher level of FUNDC1 mRNA (3.96±0.78 versus 6.86±0.63 fold, P <0.05), while Atg5, Atg7 and BECN1 mRNA levels showed no significant difference. Moreover, the mitochondrial or cytosolic protein levels of PINK1 or Parkin showed no significant difference between Ngb OE and vector group. Conclusions: Overexpression of Ngb can not affect autophagy or mitohpagy in OGD/R treated SH-SY5Y cells. Overexpression of Ngb can increase the mRNA level of FUNDC1 and the mechanism needs further study.

Aerobic animals constantly monitor and adapt to changes in O2 levels. The molecular mechanisms involved in sensing O2 are, however, incompletely understood. Previous studies showed that a hexacoordinated globin called GLB-5 tunes the dynamic range of O2-sensing neurons in natural C. elegans isolates, but is defective in the N2 lab reference strain (McGrath et al., 2009; Persson et al., 2009). GLB-5 enables a sharp behavioral switch when O2 changes between 21 and 17%. Here, we show that GLB-5 also confers rapid behavioral and cellular recovery from exposure to hypoxia. Hypoxia reconfigures O2-evoked Ca(2+) responses in the URX O2 sensors, and GLB-5 enables rapid recovery of these responses upon re-oxygenation. Forward genetic screens indicate that GLB-5's effects on O2 sensing require PDL-1, the C. elegans ortholog of mammalian PrBP/PDE6δ protein. In mammals, PDE6δ regulates the traffic and activity of prenylated proteins (Zhang et al., 2004; Norton et al., 2005). PDL-1 promotes localization of GCY-33 and GCY-35, atypical soluble guanylate cyclases that act as O2 sensors, to the dendritic endings of URX and BAG neurons, where they colocalize with GLB-5. Both GCY-33 and GCY-35 are predicted to be prenylated. Dendritic localization is not essential for GCY-35 to function as an O2 sensor, but disrupting pdl-1 alters the URX neuron's O2 response properties. Functional GLB-5 can restore dendritic localization of GCY-33 in pdl-1 mutants, suggesting GCY-33 and GLB-5 are in a complex. Our data suggest GLB-5 and the soluble guanylate cyclases operate in close proximity to sculpt O2 responses., (Copyright © 2014 Gross et al.)

The human pathogenic fungus Aspergillus fumigatus normally lives as a soil saprophyte. Its environment includes poorly oxygenated substrates that also occur during tissue invasive growth of the fungus in the human host. Up to now, few cellular factors have been identified that allow the fungus to efficiently adapt its energy metabolism to hypoxia. Here, we cultivated A. fumigatus in an O2 -controlled fermenter and analysed its responses to O2 limitation on a minute timescale. Transcriptome sequencing revealed several genes displaying a rapid and highly dynamic regulation. One of these genes was analysed in detail and found to encode fungoglobin, a previously uncharacterized member of the sensor globin protein family widely conserved in filamentous fungi. Besides low O2 , iron limitation also induced transcription, but regulation was not entirely dependent on the two major transcription factors involved in adaptation to iron starvation and hypoxia, HapX and SrbA respectively. The protein was identified as a functional haemoglobin, as binding of this cofactor was detected for the recombinant protein. Gene deletion in A. fumigatus confirmed that haem-binding fungoglobins are important for growth in microaerobic environments with O2 levels far lower than in hypoxic human tissue., (© 2014 John Wiley & Sons Ltd.)

Background: Neuroglobin (Ngb) is a hexacoordinated globin expressed mainly in the central and peripheral nervous system of vertebrates. Although several hypotheses have been put forward regarding the role of neuroglobin, its definite function remains uncertain. Ngb appears to have a neuro-protective role enhancing cell viability under hypoxia and other types of oxidative stress. Ngb is phylogenetically ancient and has a substitution rate nearly four times lower than that of other vertebrate globins, e.g. hemoglobin. Despite its high sequence conservation among vertebrates Ngb seems to be elusive in invertebrates., Principal Findings: We determined candidate orthologs in invertebrates and identified a globin of the placozoan Trichoplax adhaerens that is most likely orthologous to vertebrate Ngb and confirmed the orthologous relationship of the polymeric globin of the sea urchin Strongylocentrotus purpuratus to Ngb. The putative orthologous globin genes are located next to genes orthologous to vertebrate POMT2 similarly to localization of vertebrate Ngb. The shared syntenic position of the globins from Trichoplax, the sea urchin and of vertebrate Ngb strongly suggests that they are orthologous. A search for conserved transcription factor binding sites (TFBSs) in the promoter regions of the Ngb genes of different vertebrates via phylogenetic footprinting revealed several TFBSs, which may contribute to the specific expression of Ngb, whereas a comparative analysis with myoglobin revealed several common TFBSs, suggestive of regulatory mechanisms common to globin genes., Significance: Identification of the placozoan and echinoderm genes orthologous to vertebrate neuroglobin strongly supports the hypothesis of the early evolutionary origin of this globin, as it shows that neuroglobin was already present in the placozoan-bilaterian last common ancestor. Computational determination of the transcription factor binding sites repertoire provides on the one hand a set of transcriptional factors that are responsible for the specific expression of the Ngb genes and on the other hand a set of factors potentially controlling expression of a couple of different globin genes.

Brain neurons and tissues respond to sublethal injury by activating endogenous protective pathways. Recently, following the failure of a large number of clinical trials for protective strategies against stroke that aim to inhibit a specific ischemia response pathway, endogenous neuroprotection has emerged as a more promising and hopeful strategy for development of therapeutics against stroke and neurodegenerative disorders. Neuroglobin (Ngb) is an oxygen-binding globin protein that is highly and specifically expressed in brain neurons. Accumulating evidence have clearly demonstrated that Ngb is an endogenous neuroprotective molecule against hypoxic/ischemic and oxidative stress-related insults in cultured neurons and animals, as well as neurodegenerative disorders such as Alzheimer's disease, thus any pharmacological strategy that can up-regulate endogenous Ngb expression may lead to novel therapeutics against these brain disorders. In this review, we summarize recent studies about the biological function, regulation of gene expression, and neuroprotective mechanisms of Ngb. Furthermore, strategies for identification of chemical compounds that can up-regulate endogenous Ngb expression for neuroprotection against stroke and neurodegenerative disorders are discussed.

Neuroglobin (Ngb), a neuron-specific oxygen-binding globin with an unknown function, has been proposed to play a key role in neuronal survival. We have previously shown Ngb to be highly expressed in the rat suprachiasmatic nucleus (SCN). The present study addresses the effect of Ngb deficiency on circadian behavior. Ngb-deficient and wild-type (wt) mice were placed in running wheels and their activity rhythms, endogenous period and response to light stimuli were investigated. The effect of Ngb deficiency on the expression of Period1 (Per1) and the immediate early gene Fos was determined after light stimulation at night and the neurochemical phenotype of Ngb expressing neurons in wt mice was characterized. Loss of Ngb function had no effect on overall circadian entrainment, but resulted in a significantly larger phase delay of circadian rhythm upon light stimulation at early night. A light-induced increase in Per1, but not Fos, gene expression was observed in Ngb-deficient mice. Ngb expressing neurons which co-stored Gastrin Releasing Peptide (GRP) and were innervated from the eye and the geniculo-hypothalamic tract expressed FOS after light stimulation. No PER1 expression was observed in Ngb-positive neurons. The present study demonstrates for the first time that the genetic elimination of Ngb does not affect core clock function but evokes an increased behavioural response to light concomitant with increased Per1 gene expression in the SCN at early night.

The Antarctic icefish Chaenocephalus aceratus lacks the globins common to most vertebrates, hemoglobin and myoglobin, but has retained neuroglobin in the brain. This conserved globin has been cloned, over-expressed and purified. To highlight similarities and differences, the structural features of the neuroglobin of this colourless-blooded fish were compared with those of the well characterised human neuroglobin as well as with the neuroglobin from the retina of the red blooded, hemoglobin and myoglobin-containing, closely related Antarctic notothenioid Dissostichus mawsoni. A detailed structural and functional analysis of the two Antarctic fish neuroglobins was carried out by UV-visible and Resonance Raman spectroscopies, molecular dynamics simulations and laser-flash photolysis. Similar to the human protein, Antarctic fish neuroglobins can reversibly bind oxygen and CO in the Fe(2+) form, and show six-coordination by distal His in the absence of exogenous ligands. A very large and structured internal cavity, with discrete docking sites, was identified in the modelled three-dimensional structures of the Antarctic neuroglobins. Estimate of the free-energy barriers from laser-flash photolysis and Implicit Ligand Sampling showed that the cavities are accessible from the solvent in both proteins.Comparison of structural and functional properties suggests that the two Antarctic fish neuroglobins most likely preserved and possibly improved the function recently proposed for human neuroglobin in ligand multichemistry. Despite subtle differences, the adaptation of Antarctic fish neuroglobins does not seem to parallel the dramatic adaptation of the oxygen carrying globins, hemoglobin and myoglobin, in the same organisms.

Neuroglobin (NGB), a newly discovered member of the globin superfamily, may regulate neuronal survival under hypoxia or oxidative stress. Although NGB is greatly expressed in retinal neurons, the biological functions of NGB in retinal diseases remain largely unknown. We investigated the role of NGB in an experimental model of glaucoma, a neurodegenerative disorder that usually involves elevation of intraocular pressure (IOP). Elevated IOP is thought to induce oxidative stress in retinal ganglion cells (RGCs), thereby causing RGC death and, eventually, blindness. We found that NGB plays a critical role in increasing RGC resistance to ocular hypertension and glaucomatous damage. Elevation of IOP stimulated a transient up-regulation of endogenous NGB in RGCs. Constitutive overexpression of NGB in transgenic mice prevented RGC damage induced by glutamate cytotoxicity in vitro and/or by chronic IOP elevation in vivo. Moreover, overexpression of NGB attenuated ocular hypertension-induced superoxide production and the associated decrease in ATP levels in mice, suggesting that NGB acts as an endogenous neuroprotectant to reduce oxidative stress and improve mitochondrial function, thereby promoting RGC survival. Thus, NGB may modulate RGC susceptibility to glaucomatous neural damage. Manipulating the expression and bioactivity of NGB may represent a novel therapeutic strategy for glaucoma., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Neuroglobin (Ngb) is a recently discovered vertebrate globin that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection during oxidative stress that occurs, for example, during ischemia and reperfusion. Recently, we found that zebrafish, but not human, Ngb can translocate into cells. Moreover, we demonstrated that a chimeric ZHHH Ngb protein, in which the module M1 of human Ngb is replaced by the corresponding region of zebrafish Ngb, can penetrate cell membranes and protect cells against oxidative stress-induced cell death, suggesting that module M1 of zebrafish Ngb is important for protein transduction. Furthermore, we recently showed that Lys7, Lys9, Lys21, and Lys23 in module M1 of zebrafish Ngb are crucial for protein transduction activity. In the present study, we have investigated whether module M1 of zebrafish Ngb can be used as a building block to create novel cell-membrane-penetrating folded proteins. First, we engineered a chimeric myoglobin (Mb), in which module M1 of zebrafish Ngb was fused to the N-terminus of full-length human Mb, and investigated its functional and structural properties. Our results showed that this chimeric Mb protein is stable and forms almost the same heme environment and α-helical structure as human wild-type Mb. In addition, we demonstrated that chimeric Mb has a cell-membrane-penetrating activity similar to zebrafish Ngb. Moreover, we found that glycosaminoglycan is crucial for the cell-membrane-penetrating activity of chimeric Mb as well as that of zebrafish Ngb. These results enable us to conclude that such module substitutions will facilitate the design and production of novel functional proteins.

Background: Neuroglobin (Ngb), a neuron-specific globin that binds oxygen in vitro, has been proposed to play a key role in neuronal survival following hypoxic and ischemic insults in the brain. Here we address whether Ngb is required for neuronal survival following acute and prolonged hypoxia in mice genetically Ngb-deficient (Ngb-null). Further, to evaluate whether the lack of Ngb has an effect on hypoxia-dependent gene regulation, we performed a transcriptome-wide analysis of differential gene expression using Affymetrix Mouse Gene 1.0 ST arrays. Differential expression was estimated by a novel data analysis approach, which applies non-parametric statistical inference directly to probe level measurements., Principal Findings: Ngb-null mice were born in expected ratios and were normal in overt appearance, home-cage behavior, reproduction and longevity. Ngb deficiency had no effect on the number of neurons, which stained positive for surrogate markers of endogenous Ngb-expressing neurons in the wild-type (wt) and Ngb-null mice after 48 hours hypoxia. However, an exacerbated hypoxia-dependent increase in the expression of c-FOS protein, an immediate early transcription factor reflecting neuronal activation, and increased expression of Hif1A mRNA were observed in Ngb-null mice. Large-scale gene expression analysis identified differential expression of the glycolytic pathway genes after acute hypoxia in Ngb-null mice, but not in the wts. Extensive hypoxia-dependent regulation of chromatin remodeling, mRNA processing and energy metabolism pathways was apparent in both genotypes., Significance: According to these results, it appears unlikely that the loss of Ngb affects neuronal viability during hypoxia in vivo. Instead, Ngb-deficiency appears to enhance the hypoxia-dependent response of Hif1A and c-FOS protein while also altering the transcriptional regulation of the glycolytic pathway. Bioinformatic analysis of differential gene expression yielded novel predictions suggesting that chromatin remodeling and mRNA metabolism are among the key regulatory mechanisms when adapting to prolonged hypoxia.

Cytoglobin (Cygb) is a novel member of the vertebrate globin superfamily. Although it is expressed in splanchnic fibroblasts of various organs, details of its function remain unknown. In the present study, kidney ischemia-reperfusion (I/R) increased the number of Cygb-positive cells per area and up-regulated Cygb mRNA and protein expression in kidney cortex tissues. Similarly, hypoxia up-regulated Cygb expression in cultured rat kidney fibroblasts. The biological function of Cygb in vivo was evaluated in Cygb-overexpressing transgenic rats. Renal dysfunction and histologic damage after renal I/R were ameliorated (mean [SE] serum urea nitrogen concentration after I/R injury, 260.6 [44.9] mg/dL in wild-type rats versus 101.0 [36.0] mg/dL in transgenic rats; P < 0.05) in association with improvement of oxidative stress. Primary cultured fibroblasts from Cygb transgenic rat kidney were resistant to exogenous oxidant stimuli, and treatment of immortalized kidney fibroblasts with Cygb-small interfering RNA (siRNA) enhanced cellular oxidant stress and subsequently decreased cell viability (cell count ratio after exposure to hydrogen peroxide, 35.9% [1.6%] in control-siRNA-treated cells versus 25.5% [2.0%] in Cygb-siRNA-treated cells; P < 0.05). Further, chemical or mutant disruption of heme in Cygb impaired its antioxidant properties, which suggests that the heme of Cygb per se possesses a radical scavenging function. These findings show for the first time, to our knowledge, that Cygb serves as a defensive mechanism against oxidative stress both in vitro and in vivo., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Cytoglobin (Cygb), a novel member of the globin superfamily, is expressed by fibroblasts in various organs. However, its function remains unknown. Because of its localization, we speculated that a biological role of Cygb may be related to fibrogenesis. To clarify the role of Cygb in kidney fibrosis, we employed the remnant kidney model in rats. Immunohistochemical analysis showed an increase in Cygb expression in parallel with disease progression. To investigate the functional consequence of Cygb upregulation, we established transgenic rats overexpressing rat Cygb. Overexpression of Cygb improved histological injury, preserved renal function, and ameliorated fibrosis, as estimated by the accumulation of collagen I and IV as well as Masson trichrome staining. These protective effects of Cygb were associated with a decrease in nitrotyrosine deposition in the kidney and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion as a marker of oxidative stress. We also performed in vitro studies utilizing a rat kidney fibroblast cell line transiently overexpressing Cygb, an inducible kidney cell transfected with Cygb, and primary cultured fibroblasts isolated from the kidneys of the transgenic rats. These different experimental systems consistently showed that Cygb inhibited collagen synthesis. Furthermore, mutant disruption of heme in Cygb that impaired its antioxidant properties led to the loss of antifibrotic effects, suggesting that Cygb reduces fibrosis via a radical scavenging function. In conclusion, we showed that Cygb plays an important role in protection of the kidney against fibrosis via the amelioration of oxidative stress both in vitro and in vivo. Cygb might represent a good therapeutic target in chronic kidney disease.

Cell death associated with mitochondrial dysfunction is common in acute neurological disorders and in neurodegenerative diseases. Neuronal apoptosis is regulated by multiple proteins, including neuroglobin, a small heme protein of ancient origin. Neuroglobin is found in high concentration in some neurons, and its high expression has been shown to promote survival of neurons in vitro and to protect brain from damage by both stroke and Alzheimer's disease in vivo. Early studies suggested this protective role might arise from the protein's capacity to bind oxygen or react with nitric oxide. Recent data, however, suggests that neither of these functions is likely to be of physiological significance. Other studies have shown that neuroglobin reacts very rapidly with cytochrome c released from mitochondria during cell death, thus interfering with the intrinsic pathway of apoptosis. Systems level computational modelling suggests that the physiological role of neuroglobin is to reset the trigger level for the post-mitochondrial execution of apoptosis. An understanding of the mechanism of action of neuroglobin might thus provide a rational basis for the design of new drug targets for inhibiting excessive neuronal cell death.

The superfamily of globins has emerged some 4000 Myr from a common ancestor, which was among the basic protein components required for life. Globins are present in the three kingdoms of life. From a structure point of view, these molecules are defined by the presence of a characteristic protein fold, rich in alpha-helix, surrounding a heme group. Depending on the species or organs, they may be physiologically active as monomers, tetramers or large size polymers. Their function varies from the classical reversible binding of oxygen for transport and storage to cytoprotection against reactive oxygen species, NO scavenging, signaling in oxygen dependent metabolic pathways, or possibly other specific properties involving ligand or electron transfer. All these aspects are discussed in this review.

A correct three-dimensional structure is a prerequisite for protein functionality, and therefore for life. Thus, it is not surprising that our cells are packed with proteins that assist protein folding, the process in which the native three-dimensional structure is formed. In general, plasma membrane and secreted proteins, as well as those residing in compartments along the endocytic and exocytic pathways, fold and oligomerize in the endoplasmic reticulum. The proteins residing in the endoplasmic reticulum are specialized in the folding of this subset of proteins, which renders this compartment a protein-folding factory. This review focuses on protein folding in the endoplasmic reticulum, and discusses the challenge of oligomer formation in the endoplasmic reticulum as well as the cytosol.

We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II, the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34(+) cells that differentiate into more developmentally mature, fetal liver-like erythroblasts, that are smaller, express mostly fetal hemoglobin, and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated, and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary, because the most mature cells that can be produced with current systems express less than 2% adult beta-globin mRNA.

Neuroglobin (Ngb), a vertebrate globin expressed primarily in neurons, is induced by and protects against neuronal hypoxia and cerebral ischemia. To investigate the spectrum and mechanism of Ngb's neuroprotective action, we studied the effect of transgenic overexpression of Ngb on NMDA and beta-amyloid (Abeta) toxicity in murine cortical neuron cultures in vitro and on the phenotype of Alzheimer's disease (AD) transgenic (APP(Sw,Ind)) mice. Compared with cortical neuron cultures from wild-type mice, cultures from Ngb-overexpressing transgenic (Ngb-Tg mice) were resistant to the toxic effects of NMDA and Abeta(25-35), as measured by polarization of cell membrane lipid rafts, mitochondrial aggregation, lactate dehydrogenase release, and nuclear fragmentation. In addition, compared with APP(Sw,Ind) mice, double-transgenic (Ngb-Tg x APP(Sw,Ind)) mice showed reductions in thioflavin-S-stained extracellular Abeta deposits, decreased levels of Abeta(1-40) and Abeta(1-42), and improved behavioral performance in a Y-maze test of spontaneous alternations. These findings suggest that the spectrum of Ngb's neuroprotective action extends beyond hypoxic-ischemic insults. Ngb may protect neurons from NMDA and Abeta toxicity by inhibiting the formation of a death-signaling membrane complex, and interventions that increase Ngb expression could have therapeutic application in AD and other neurodegenerative disorders.

Several studies support the hypothesis that neuroglobin and cytoglobin play a protective role against cell death when cellular oxygen supply is critical. Although the underlying molecular mechanisms are unknown, previous reports suggest that this protection can be realised by the fact that they act as ROS scavengers. In this study, expression of neuroglobin and cytoglobin was evaluated in a human neuroblastoma cell line (SH-SY5Y) under conditions of anoxia or oxygen and glucose deprivation (OGD). The cells could survive prolonged anoxia without significant loss of viability. They became anoxia sensitive when deprived of glucose. OGD induced significant cell death after 16 h resulting in 54% dead cells after 32 h. Necrosis was the main process involved in OGD-induced cell death. After reoxygenation, apoptotic neurons became more abundant. Real-time quantitative PCR and Western blotting revealed that neuroglobin and cytoglobin were upregulated, the former under OGD and the latter under anoxic conditions. Under OGD, cell survival was significantly reduced after inhibiting cytoglobin expression by transfection with antisense ODN. Moreover, cell survival was significantly enhanced by neuroglobin or cytoglobin overexpression. When neuroglobin or cytoglobin protein expression increased or decreased, the H(2)O(2) level was found to be lower or higher, respectively. We conclude that neuroglobin or cytoglobin act as ROS scavengers under ischemic conditions.

The recognition of the important role of the polar habitats in global climate changes has awakened great interest in the evolutionary biology of polar organisms. They are exposed to strong environmental constraints, and it is important to understand how they have adapted to cope with these challenges and to what extent adaptations may be upset by current climate changes. We present an introductory overview of the evolution of the Antarctic fish fauna with emphasis on the dominant perciform sub-order Notothenioidei, as well as some specific comments on the biogeography of the three phyletically basal notothenioid families. The wealth of information on the ecology and biodiversity of the species inhabiting high-Antarctic and sub-Antarctic regions provides a necessary framework for better understanding the origin, evolution and adaptation of this unique group of fish. Notothenioidei offer opportunities for identification of the biochemical characters or the physiological traits responsible for thermal adaptation. The availability of phylogenetically related taxa in a wide range of latitudes has allowed to look into the molecular bases of environmentally driven phenotypic gain and loss of function. In the process of cold adaptation, the evolutionary trend of notothenioids has produced unique specialisations, including modification of hematological characteristics, e.g. decreased amounts and multiplicity of hemoglobins. The Antarctic family Channichthyidae (the notothenioid crown group) is devoid of hemoglobin. This loss is related to a single deletional event removing all globin genes with the exception of the inactive 3' end of adult alpha-globin. In reviewing hemoglobin structure, function and phylogeny, the evolution of the fish Root effect is analysed in detail. Adaptation of the oxygen-transport system in notothenioids seems to be based on evolutionary changes involving levels of biological organisation higher than the structure of hemoglobin.

Objective: Sickle cell anemia is a genetic blood disease resulting from production of mutant beta-globin (beta(S)) and has severe clinical consequences. It is known that a higher cellular gamma-globin level, e.g., higher ratio of cellular gamma-globin to beta(S)-globin (gamma/beta(S) ratio), inhibits sickle hemoglobin (HbS) polymerization tendency. Hence, therapeutic treatment of sickle cell anemia has been focused on introducing gamma-globin gene into red blood cells to increase the cellular gamma/beta(S) ratio. Here, we have introduced ribozymes and small interfering RNAs (siRNAs) against beta(S)-globin mRNA into blood cells as a means to increase the gamma/beta(S) ratio., Materials and Methods: Single and multiribozymes against beta(S)-globin mRNA have been tested in vitro and in human erythroleukemia K562beta(S) cells that stably express exogenous beta(S)-globin gene. Primary human hematopoietic progenitor cells were also transfected with multiribozyme and the gamma/(gamma + beta) ratio determined and compared with cells transfected with long hairpin beta-globin cDNA and synthetic siRNA genes., Results: We have found that the multiribozyme zb21A containing two ribozyme units effectively reduces beta(S)-globin mRNA both in vitro and in K562beta(S) cells. The gamma-globin mRNA to beta(S)-globin mRNA ratio in the multiribozyme transfected cells is about a factor of 2 more than that in the control cells. We have also found that the gamma/(gamma + beta) ratio in the transfected hematopoietic progenitor cells is increased by more than twofold in cells treated with multiribozyme zb21A or siRNA ib5., Conclusion: Our results suggest that introducing multiribozymes or siRNAs into red blood cells is comparable in their effectiveness to increase the ratio of cellular gamma-globin mRNA to beta- or beta(S)-globin mRNA, providing possible strategies to increase the effectiveness of gamma-globin gene transfer as gene therapy for treatment of patients with sickle cell anemia.

Given the unique thermal history of the Antarctic continent, fishes of dominant suborder Notothenioidei offer a remarkable opportunity to study the physiological and biochemical characters gained and, conversely, lost during their evolutionary history and to map this information on the species phylogenetic trees. The availability of phylogenetically related notothenioid taxa living in a wide range of latitudes (in the Antarctic, sub-Antarctic and temperate regions) allows to look into the molecular bases of environmentally driven gene birth and death. This evolutionary perspective has also been supported by comparison of some features of the hemoprotein devoted to the oxygen transport in fish species living in the other polar region, the Arctic. The Arctic and Antarctic marine ichthyofaunas differ by age and isolation. Fishes of the two polar regions have undergone different regional histories which have engineered the physiological diversities, so that Antarctic fish are much more stenothermal than Arctic ones. Understanding the mechanisms of phenotypic response to cold exposure in species living at different latitudes in polar habitats offers fundamental insights into environmental adaptations. This review aims at surveying the current knowledge of molecular structure, functional features, phylogeny and adaptations of the hemoglobin of fish thriving in the Antarctic, sub-Antarctic and Arctic regions (with some excursions in the temperate latitudes). Investigating the evolutionary adaptations of hemoglobins to these environments can provide new insights into adaptation currently studied merely in temperate organisms, and can shed light into the convergent processes that evolved in response to thermal adaptations.

Cytoglobin (Cygb) is a novel tissue hemoprotein relatively similar to myoglobin (Mb). Because Cygb shares several structural features with Mb, we hypothesized that Cygb functions in the modulation of oxygen and nitric oxide metabolism or in scavenging free radicals within a cell. In the present study we examined the spatial and temporal expression pattern of Cygb during murine embryogenesis. Using in situ hybridization, RT-PCR, and Northern blot analyses, limited Cygb expression was observed during embryogenesis compared with Mb expression. Cygb expression was primarily restricted to the central nervous system and neural crest derivatives during the latter stages of development. In the adult mouse, Cygb is expressed in distinct regions of the brain as compared with neuroglobin (Ngb), another globin protein, and these regions are responsive to oxidative stress (i.e., hippocampus, thalamus, and hypothalamus). In contrast to Ngb, Cygb expression in the brain is induced in response to chronic hypoxia (10% oxygen). These results support the hypothesis that Cygb is an oxygen-responsive tissue hemoglobin expressed in distinct regions of thenormoxic and hypoxic brain and may play a key role in the response of the brain to ahypoxic insult.

The visual function of the vertebrate retina relies on sufficient supply with oxygen. Neuroglobin is a respiratory protein thought to play an essential role in oxygen homeostasis of neuronal cells. For further understanding of its function, we compared the distribution of neuroglobin and mitochondria in both vascular and avascular mammalian retinae. In the vascular retinae of mouse and rat, oxygen is supplied by the outer choroidal, deep retinal, and inner capillaries. We show that in this type of retina, mitochondria are concentrated in the inner segments of photoreceptor cells, the outer and the inner plexiform layers, and the ganglion cell layer. These are the same regions in which oxygen consumption takes place and in which neuroglobin is present at high levels. In the avascular retina of guinea pig the deep retinal and inner capillaries are absent. Therefore, only the inner segments of the photoreceptors adjacent to choroidal capillaries display an oxidative metabolism. We demonstrate that in the retina of guinea pigs both neuroglobin and mitochondria are restricted to this layer. Our results clearly demonstrate an association of neuroglobin and mitochondria, thus supporting the hypothesis that neuroglobin is a respiratory protein that supplies oxygen to the respiratory chain.

Characterization of hematopoiesis/erythropoiesis in thalassemias from multipotent primitive cells to mature erythrocytes is of fundamental importance and clinical relevance. We investigated this process in alpha- and beta-globin hemizygous mice, lacking the two adult tandemly organized genes from either the alpha- or beta-globin locus. Although both mice backcrossed on a homogeneous background exhibited similar reduced red blood cell (RBC) survival, beta-globin hemizygous mice had less severe reticulocyte loss and globin chain imbalance, suggesting an apparently milder thalassemia than for alpha-globin hemizygous mice. In contrast, however, beta-globin hemizygous mice displayed a more marked perturbation of hematologic parameters. Quantification of erythroid precursor subpopulations in marrow and spleen of beta-globin hemizygous mice showed more severely impaired maturation from the basophilic to orthochromatophilic erythroblasts and substantial loss of these late precursors probably as a consequence of a greater susceptibility to an excess of free alpha-chain than beta-chain. Hence, only erythroid precursors exhibiting stochastically moderate chain imbalance would escape death and mature to reticulocyte/RBC stage, leading to survival and minimal loss of reticulocytes in the beta-globin hemizygous mice. Furthermore, in response to the ineffective erythropoiesis in beta-globin hemizygous mice, a dynamic compensatory hematopoiesis was observed at earlier differentiation stage as evidenced by a significant increase of erythroid progenitors (erythroid colony-forming units approximately 100-fold) as well as of multipotent primitive cells (day 12 spleen colony-forming units approximately 7-fold). This early compensatory mechanism was less pronounced in alpha-globin hemizygous mice. The expansion of multipotent primitive and potentially stem cell populations, taken together with ineffective erythropoiesis and increased reticulocyte/RBC destruction could confer major cumulative advantage for gene targeting/bone marrow transplantation. Therefore, this study not only corroborated the strong potential effectiveness of transplantation for thalassemic hematopoietic therapy but also demonstrated the existence of a differential regulatory response for alpha- and beta-thalassemia.

Gene replacement or gene reactivation therapies for sickle-cell disease (SCD) typically target the mutant beta(S)-globin subunits of hemoglobin-S (alpha(2)beta(S)(2)) for substitution by nonpathological beta-like globins. Here we show, in vitro and in vivo in a transgenic mouse model of SCD, that the adverse properties of hemoglobin-S can be reversed by exchanging its normal alpha-globin subunits for zeta-globin, an endogenous, developmentally silenced, non-beta-like globin.

Neuroglobin (Ngb) is a newly discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. It has been reported that Ngb expression levels increase in response to oxygen deprivation and that it protects neurons from hypoxia in vitro and in vivo. However, the mechanism of this neuroprotection remains unclear. In the present study, we tried to clarify the neuroprotective role of Ngb under oxidative stress in vitro. By surface plasmon resonance, we found that ferric Ngb, which is generated spontaneously as a result of the rapid autoxidation, binds exclusively to the GDP-bound form of the alpha subunit of heterotrimeric G protein (Galphai). In GDP dissociation assays or guanosine 5'-O-(3-thio)triphosphate binding assays, ferric Ngb behaved as a guanine nucleotide dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP. The interaction of GDP-bound Galphai with ferric Ngb will liberate Gbetagamma, leading to protection against neuronal death. In contrast, ferrous ligand-bound Ngb under normoxia did not have GDI activities. Taken together, we propose that human Ngb may be a novel oxidative stress-responsive sensor for signal transduction in the brain.

Neuroglobin (Ngb) is an O(2)-binding protein localized to cerebral neurons of vertebrates, including humans. Its physiological role is unknown but, like hemoglobin, myoglobin, and cytoglobin/histoglobin, it may transport O(2), detoxify reactive oxygen species, or serve as a hypoxia sensor. We reported recently that hypoxia stimulates transcriptional activation of Ngb in cultured cortical neurons and that antisense inhibition of Ngb expression increases hypoxic neuronal injury, whereas overexpression of Ngb confers resistance to hypoxia. These findings are consistent with a role for Ngb in promoting neuronal survival after hypoxic insults in vitro. Here we report that in rats, intracerebroventricular administration of an Ngb antisense, but not sense, oligodeoxynucleotide increases infarct volume and worsens functional neurological outcome, whereas intracerebral administration of a Ngb-expressing adeno-associated virus vector reduces infarct size and improves functional outcome, after focal cerebral ischemia induced by occlusion of the middle cerebral artery. We conclude that Ngb acts as an endogenous neuroprotective factor in focal cerebral ischemia and may therefore represent a target for the development of new treatments for stroke.

Visual performance of the vertebrate eye requires large amounts of oxygen, and thus the retina is one of the highest oxygen-consuming tissues of the body. Here we show that neuroglobin, a neuron-specific respiratory protein distantly related to hemoglobin and myoglobin, is present at high amounts in the mouse retina (approximately 100 microm). The estimated concentration of neuroglobin in the retina is thus about 100-fold higher than in the brain and is in the same range as that of myoglobin in the muscle. Neuroglobin is expressed in all neurons of the retina but not in the retinal pigment epithelium. Neuroglobin mRNA was detected in the perikarya of the nuclear and ganglion layers of the neuronal retina, whereas the protein was present mainly in the plexiform layers and in the ellipsoid region of photoreceptor inner segment. The distribution of neuroglobin correlates with the subcellular localization of mitochondria and with the relative oxygen demands, as the plexiform layers and the inner segment consume most of the retinal oxygen. These findings suggest that neuroglobin supplies oxygen to the retina, similar to myoglobin in the myocardium and the skeletal muscle.

Neuroglobin and cytoglobin are two recently discovered members of the vertebrate globin family. Both are intracellular proteins endowed with hexacoordinated heme-Fe atoms, in their ferrous and ferric forms, and display O2 affinities comparable with that of myoglobin. Neuroglobin, which is predominantly expressed in nerve cells, is thought to protect neurons from hypoxic-ischemic injury. It is of ancient evolutionary origin, and is homologous to nerve globins of invertebrates. Cytoglobin is expressed in many different tissues, although at varying levels. It shares common ancestry with myoglobin, and can be traced to early vertebrate evolution. The physiological roles of neuroglobin and cytoglobin are not completely understood. Although supplying cells with O2 is the likely function, it is also possible that both globins act as O2-consuming enzymes or as O2 sensors. Here, we review what is currently known about neuroglobin and cytoglobin in terms of their function, tissue distribution and relatedness to the well-known hemoglobin and myoglobin. Strikingly, the data reveal that O2 metabolism in cells is more complicated than was thought before, requiring unexpected O2-binding proteins with potentially novel functional features.

The principle that developmentally silenced globin genes can be reactivated in adults with defects in beta-globin gene expression has been well established both in vitro and in vivo. In practice, levels of developmental stage-discordant fetal gamma globin that can be achieved by using currently approved therapies are generally insufficient to fully resolve typical clincopathological features of sickle cell disease. The therapeutic potential of another developmentally silenced globin--embryonic epsilon globin--has been difficult to evaluate in the absence of a convenient expression system or an appropriate experimental model. The current work analyzes the antisickling properties of an epsilon -globin-containing heterotetramer (Hb Gower-2) both in vitro as well as in vivo in a well-established mouse model of sickle cell anemia. These animals, expressing 100% human Hb S, display a chronic hemolytic anemia with compensatory marrow and extramedullary erythropoiesis, abundant circulating sickled erythrocytes, and chronic tissue damage evidenced by parallel histopathological and functional deficits. By comparison, related mice that coexpress Hb S as well as Hb Gower-2 exhibit normal physiological, morphological, histological, and functional attributes. Subsequent in vitro analyses substantiate results from whole-animal studies, indicating that the polymerization of deoxygenated Hb S can be significantly slowed by relatively small quantities of Hb Gower-2. Together, the in vivo and in vitro analyses suggest that reactivation of epsilon-globin gene expression would be therapeutically beneficial to adults with sickle phenotypes, and provide a rationale for detailed investigations into the molecular basis for its developmental silencing.

As alterations in gene expression underlie a considerable proportion of human diseases, correcting such aberrant transcription in vivo is expected to provide therapeutic benefit to the patient. Attempts to control endogenous mammalian genes, however, face a significant obstacle in the form of chromatin. Aberrant gene repression can be alleviated by using small-molecule inhibitors that exert nucleus-wide effects on chromatin-based repressors. Genome-wide chromatin remodeling also occurs during cloning via nuclear transfer, and causes the deregulation of epigenetically controlled genes. Regulation of genes in vivo can be accomplished via the use of designed transcription factors - these result from a fusion of a designed DNA-binding domain based on the zinc finger protein motif to a functional domain of choice.

In the current study, we show that bone morphogenetic proteins (BMPs) play a role in hematopoiesis that is independent of their function in specifying ventral mesodermal fate. When BMP activity is upregulated or inhibited in Xenopus embryos hematopoietic precursors are specified properly but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of BMP activity induces erythroid precursors to undergo apoptotic cell death, whereas constitutive activation of BMPs causes an increase in commitment of hematopoietic progenitors to myeloid differentiation and a concomitant decrease in erythrocytes that is not due to enhanced apoptosis. These blood defects are observed even when BMP activity is misregulated solely in non-hematopoietic (ectodermal) cells, demonstrating that BMPs generate extrinsic signals that regulate hematopoiesis independent of mesodermal patterning. Further analysis revealed that endogenous calmodulin-dependent protein kinase IV (CaM KIV) is required to negatively modulate hematopoietic functions of BMPs downstream of receptor activation. Our data are consistent with a model in which CaM KIV inhibits BMP signals by activating a substrate, possibly cAMP-response element-binding protein (CREB), that recruits limiting amounts of CREB binding protein (CBP) away from transcriptional complexes functioning downstream of BMPs.

Globins are oxygen-binding heme proteins present in bacteria, protists, fungi, plants, and animals. Their functions have diverged widely in evolution, and include binding, transport, scavenging, detoxification, and sensing of gases like oxygen, nitric oxide, and carbon monoxide. Neuroglobin (Ngb) is a recently discovered monomeric globin with high affinity for oxygen and preferential localization to vertebrate brain. No function for Ngb is known, but its affinity for oxygen and its expression in cerebral neurons suggest a role in neuronal responses to hypoxia or ischemia. Here we report that Ngb expression is increased by neuronal hypoxia in vitro and focal cerebral ischemia in vivo, and that neuronal survival after hypoxia is reduced by inhibiting Ngb expression with an antisense oligodeoxynucleotide and enhanced by Ngb overexpression. Both induction of Ngb and its protective effect show specificity for hypoxia over other stressors. We conclude that hypoxia-inducible Ngb expression helps promote neuronal survival from hypoxic-ischemic insults.

Background and Objectives: The degree of globin chain imbalance and tissue hypoxia are important determinants of clinical severity in thalassemia syndromes. Thus phenotypic expression may be modified by interaction of alpha- and beta-thalassemia defects, level and type of hemoglobin synthesized and oxygen release to the tissues. We evaluated hematology, erythroid marrow activity and functional anemia in patients with the rare interaction of a single a-globin gene and heterozygous beta-thalassemia (HbH/beta-thal trait)., Design and Methods: In 7 patients characterized by DNA analysis to have HbH disease genotypes with beta-thalassemia trait, we assessed hematologic findings, serum transferrin receptor (sTfR), serum erythropoietin (Epo), red cell 2,3-disphosphoglycerate (2,3-DPG) and whole blood oxygen releasing capability., Results: Patients with HbH/beta-thal trait had moderate anemia, marked hypochromasia and microcytosis, normal or raised HbA2, and no electrophoretically/chromatographically detectable HbH. Epo and sTfR levels were significantly higher than in beta-thalassemia heterozygotes, but lower than in patients with HbH disease; 2,3-DPG levels were highest in HbH/beta-thal trait. Oxygen binding studies and simulations showed reduced oxygen affinity (P50) in HbH/beta-thal trait, resulting in increased oxygen release (O2R)., Interpretation and Conclusions: Hematologic findings and bone marrow activity in patients with HbH/b-thal trait were consistent with the modified globin chain imbalance and hemoglobin synthesis expected from interaction of HbH disease with heterozygous b-thalassemia, although this rare complex genotype may elude diagnosis based on hematology alone. Significantly higher red cell 2,3-DPG levels were an unexpected finding, and the consequent increase in oxygen release capability resulted in a compensated functional anemia relative to hemoglobin levels.

Variability profiles measured over a set of aligned sequences can be used to estimate evolutionary freedom to vary. Differences in variability profiles between clades can be used to identify shifts in function at the molecular level. We demonstrate such a shift between the alpha and beta subunits of hemoglobin. We also show that the variability profiles for myoglobin are different between whales and primates and speculate that the differences between the two clades may reflect a shift associated with the novel oxygen storage demands in the lineage leading to whales. We discuss the relationship between sequence variability and "evolutionary opportunity" and explore the utility of Maynard Smith's multidimensional evolutionary opportunity space metaphor for exploring functional constraints, genetic redundancy, and the context dependency of the genotype-phenotype map. This work has implications for quantitatively defining and comparing protein function. Supplementary data is available from bioinfo.mbb.yale. edu/align.

Globin-like oxygen-binding proteins occur in bacteria, yeasts and other fungi, and protozoa. The simplest contain protohaem as sole prosthetic group, but show considerable variation in their similarity to the classical animal globins and plant globins. Flavohaemoglobins comprise a haem domain homologous to classical globins and a ferredoxin-NADP+ reductase (FNR)-like domain that converts the globin into an NAD(P)H-oxidizing protein with diverse reductase activities. In Escherichia coli, the prototype flavohaemoglobin (Hmp) is clearly involved in responses to nitric oxide (NO) and nitrosative stress: (i) the structural gene hmp is upregulated by NO and nitrosating agents; (ii) purified Hmp binds NO avidly, but also converts it to nitrate (aerobically) or nitrous oxide (anaerobically); (iii) hmp mutants are hypersensitive to NO and nitrosative stresses. Here, we review recent advances in E. coli and the growing number of microbes in which globins are known, draw particular attention to the essential chemistry of NO and related reactive species and their interactions with globins, and suggest that microbial globins have additional functions unrelated to 'NO' stresses.

To determine if the activation of the (K+Cl-) cotransport system observed in hemoglobin (Hb) S- or C-containing erythrocytes is related either to a global change of isoelectric point of the Hb molecule or to the specific location of these mutations on the position 6 of the beta chain of Hb, we studied the (K+Cl-) cotransport system in erythrocytes containing beta chain variants exhibiting either the Glu----Lys substitution observed in position beta 6 in Hb C (Hb E: beta 26 Glu----Lys; Hb O-Arab: beta 121 Glu----Lys; Hb Siriraj:beta 7 Glu----Lys) or the Glu----neutral residue substitution observed in position beta 6 in Hb S (Hb G-San Jose: beta 7 Glu----Gly; Hb D Punjab or D-Los Angeles: beta 121 Glu----Gln). The K transport mediated by the (K+Cl-) cotransport was increased in AC, AS and A-Siriraj and A-San Jose red blood cells and was similar to AA control in the other variants. These results indicate that an enhanced (K+Cl-) cotransport is not a property of all positively charged Hb variants, but it is mainly associated with mutations occurring at the beta 6 or beta 7 residues. An interaction of Hb with the cell membrane mediated by the disappearance of one of the negative charged residues (Glu) at this site of the A helix of the beta chain is the most likely candidate for the persistent activation of the (K+Cl-) cotransport system in these Hb variants.

Damaged or old erythrocytes are cleared rapidly from circulation. Because several common biochemical lesions can induce the clustering of integral membrane proteins, we have proposed that formation of microscopic protein aggregates in the membrane might constitute a cell surface marker that promotes removal of the defective/senescent cells. We demonstrate here that treatments that cluster integral membrane proteins in erythrocytes (1 mM ZnCl2, 1 mM acridine orange, and 0.35 microM melittin) induce autologous IgG binding, complement fixation, and phagocytosis by human monocytes in vitro. Removal of the clustering agents prior to incubation in autologous serum or cross-linking of cell surface proteins before addition of clustering agents prohibited the above response, while cross-linking after treatment with the clustering agents preserved the response even if the clustering agents were later removed. Furthermore, subsequent reversal of the chemical cross-link maintaining the clustered distribution also reversed the induction of IgG binding, complement deposition, and phagocytosis. Finally, by deleting or inactivating different steps in the phagocytosis pathway, the chronology of steps was shown to be: (i) integral protein clustering, (ii) IgG binding, (iii) complement deposition, and (iv) phagocytosis.

A novel beta-chain, beta 126(H4)Val----Gly, electrophoretically silent, was detected by reverse-phase high performance liquid chromatography in three unrelated families from Naples (Southern Italy) and accounted for about 30% of the total beta-chains. The amino acid substitution was detected by HPLC fingerprint. The eight heterozygous patients showed hematologic and biosynthetic alterations of mild beta-thalassemia type. The hemoglobin variant showed abnormal stability features. It was unstable in the heat stability and isopropanol precipitation tests, but did not cause a hemolytic syndrome in vivo and was stable in a time-course experiment of biosynthesis in vitro. DNA polymerase chain reaction direct sequencing of the mutated gene from 135 nt upstream of the cap site to 106 nt downstream of the polyadenylation site showed only the beta 126 GTG----GGG mutation, which was confirmed in the other patients by allele-specific oligonucleotide hybridization. The mutation was found to be associated with a type II beta-globin framework and restriction fragment length polymorphism haplotype V. The novel variant was named hemoglobin Neapolis.

Patients with beta zero thalassemia arising from premature terminator codon mutations in the gene for beta globin do not produce beta globin protein; these individuals also exhibit a decreased amount of beta globin mRNA in their erythroid cells. The absence of beta globin protein is readily explained by the inability of the beta zero-39 mRNA to be translated. The decrease in beta globin mRNA has been attributed to either decreased cytoplasmic stability of the nontranslatable decreased cytoplasmic stability of the nontranslatable mRNA or to an undefined nuclear lesion. To compare directly the relative stabilities of normal and beta zero-39 thalassemic globin transcripts, we prepared normal and thalassemic beta globin pre-mRNAs and mRNAs using cloned DNA templates and the SP6 promoter-polymerase system. The stability of the transcripts was assessed by incubation in various cell-free extracts. Our results indicate that although the stabilities of the beta globin transcripts varied considerably from one extract to another the stabilities of the beta zero-39 thalassemic pre-mRNAs and mRNAs were equal to those of normal beta globin mRNAs in every extract tested.

We characterized the molecular defect in a Swiss patient with a spontaneous beta-thalassemia mutation. Cloning and DNA sequencing of her beta-globin gene revealed a new frameshift mutation due to a single nucleotide deletion at codon 64 of the beta-globin gene. Restriction site polymorphism showed that the mutation arose on her paternal chromosome. Direct sequencing of a polymerase chain reaction amplified DNA segment showed absence of the lesion in both alleles of her father's beta-globin gene and confirmed the spontaneous nature of this mutation.

The transgenic mouse system provides an in vivo setting in which to examine the effects on mouse red cells of hemoglobin genes that have been genetically introduced into the animals' genome. In this report, we have analyzed the cellular properties of red cells from homozygous beta-thalassemic mice (Hbbth-1/Hbbth-1), homozygous beta-thalassemic transgenic mice containing a human beta-sickle (beta(s)) gene (Hbb(th-1)/Hbb(th-1) + beta(s)), and normal animals. The presence of human beta(s)-globin chains in red cells from the Hbbth-1/Hbb(th-1) + beta(s) transgenic animals was noted to have a significant effect on cellular deformability and density distribution, as well as on the degree of anemia in these animals. We conclude from these studies that red cell deformability and density distribution is a sensitive means for assessing at the cellular level the effects of globin genes genetically introduced into whole organisms. In addition, these studies suggest that small decreases in the amount of excess alpha-globin chains can significantly ameliorate the severity of anemia in the beta-thalassemic mouse.

The theta globin gene is the most recently discovered member of the alpha globin gene family. Its pattern of expression during development is not fully defined, and its encoded protein has not yet been detected in vivo. The detection of theta globin messenger RNA (mRNA) in embryonic and fetal erythroid tissue but not in adults has suggested that theta is an embryonic globin gene. The present study further defines the pattern of theta globin gene expression. We use a modification of the highly sensitive polymerase chain reaction (PCR) technique to assess the levels of theta globin gene expression during development. We confirm the presence of the theta globin mRNA in embryonic and fetal erythroid tissue, and, in addition, we find theta mRNA in the peripheral reticulocytes of normal adults. Furthermore, using the same analytic approach, we detect low but significant levels of the embryonic zeta and epsilon mRNAs in reticulocytes of normal adults. Both zeta and theta gene expression appears erythroid specific in that neither mRNA species is detected in RNA isolated from brain tissue, peripheral blood mononuclear cells, or three nonerythroid cell lines (B-lymphocyte, T-lymphocyte, and hepatoma cell lines). The relative levels of zeta and theta gene expression were assayed during development by a coamplification technique. The results demonstrate the expected developmental regulation of zeta globin mRNA. In contrast, the level of theta globin mRNA fails to demonstrate the significant changes of the magnitude seen in other globin genes and remains low in embryonic, fetal, and adult life. The lack of zeta and epsilon globin proteins in normal adults using highly sensitive immunologic techniques, as reported by others, stands in contrast to these mRNA results and suggests a gap between mRNA and protein expression.