Short in-Frame Insertions/Deletions in the Coding Sequence of the α-Globin Gene. Consequences of the 3D Structure and Resulting Phenotypes: Hb Choisy as an Example.

A small group of hemoglobin (Hb) variants result from 'in-frame' deletion/insertion (del/ins). We describe a new variant of this group (Hb Choisy), found on the α1 gene, which is the exact counterpart of a previously published deletional variant, Hb J-Biskra [codons 51-58 (or codons 52-59) (-24 bp) (-TCTGCCCAGGTTAAGGGCCACGGC); HBA1: c.157_180del (or HBA2)]. In Hb J-Biskra, the sequence Ser-Ala-Gln-Val-Lys-Gly-His-Gly located from positions α52(E1) to α59(E8) is deleted, while in Hb Choisy the same sequence (Ser-Ala-Gln-Val-Lys-Gly-His-Gly) is inserted at position α52(E1). The variant carrying the insertion appears to be less damaging than the one with the deletion. A possible explanation could be that the additional sequence is located in the C to E interhelical region, and is less disturbing to the general structure of the globin chain. This insertion/deletion (ins/del) is likely favored by the repetition, at an interval of 16 nucleotides, of an eight nucleotide sequence. Comparison of variants of this group, found in the HbVar database, shows that structural modifications resulting from insertions are frequently less damaging than that caused by deletions.

Dense red blood cell and oxygen desaturation in sickle-cell disease.

Production of abnormal hemoglobin (HbS) in sickle-cell disease (SCD) results in its polymerization in deoxygenated conditions and in sickled-RBC formation. Dense RBCs (DRBCs), defined as density >1.11 and characterized by increased rigidity are absent in normal AA subjects, but present at percentages that vary of a patient to another remaining stable throughout adulthood for each patient. Polymerized HbS has reduced affinity for oxygen, demonstrated by the rightward shift of the oxygen-dissociation curve, leading to disturbances in oxygen transport. Ninety-two SCD patients' total RBCs were separated into LightDRBC (LRBC) (d < 1.11 g/mL) and DRBC fractions. Venous blood partial oxygen pressure and RBC-fraction-deoxygenation and -reoxygenation Hb-oxygen-equilibrium curves were determined. All patients took a 6-minute walking test (6MWT); 10 had results before and after >6 months on hydroxyurea. 6MWT time with SpO2 < 88% (TSpO2 < 88) assessed the physiological impact of exertion. Elevated mean corpuscular hemoglobin (Hb) concentrations, decreased %HbF, and 2,3-bisphosphoglycerates in DRBCs modulated Hb-oxygen affinity. Deoxygenation and reoxygenation Hb-oxygen equilibrium curves differed between normal Hb AA and SS RBCs and between LRBCs and DRBCs, with rightward shifts confirming HbS-polymerization's role in affinity loss. In bivariate analyses, 50% Hb saturation correlated positively with %DRBCs (P < 0.0001, r(2) = 0.34) and negatively with %HbF (P < 0.0001, r(2) = 0.25). The higher the %DRBCs, the longer the TSpO2 88 (P = 0.04). Hydroxyurea was associated with significantly shorter TSpO2 < 88 (P = 0.01). We report that the %DRBCs directly affects SCD patients' SpO2 during exertion; hydroxyurea improves oxygen affinity and lowers the %DRBCs. Am. J. Hematol. 91:1008-1013, 2016. © 2016 Wiley Periodicals, Inc.

Role of α-globin H helix in the building of tetrameric human hemoglobin: interaction with α-hemoglobin stabilizing protein (AHSP) and heme molecule.

Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the ß-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of ß-Hb, the increase in fluorescence indicates the replacement of AHSP by ß-Hb. The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

The crystal structure of wild-type human brain neuroglobin reveals flexibility of the disulfide bond that regulates oxygen affinity.

Neuroglobin plays an important function in the supply of oxygen in nervous tissues. In human neuroglobin, a cysteine at position 46 in the loop connecting the C and D helices of the globin fold is presumed to form an intramolecular disulfide bond with Cys55. Rupture of this disulfide bridge stabilizes bi-histidyl haem hexacoordination, causing an overall decrease in the affinity for oxygen. Here, the first X-ray structure of wild-type human neuroglobin is reported at 1.74 Šresolution. This structure provides a direct observation of two distinct conformations of the CD region containing the intramolecular disulfide link and highlights internal cavities that could be involved in ligand migration and/or are necessary to enable the conformational transition between the low and high oxygen-affinity states following S-S bond formation.

Dynamics of α-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state.

BACKGROUND: AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7). METHODS: Recombinant AHSP, WT α-Hb:AHSP and α-Hb(HE7Q):AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex were measured by time-resolved absorption spectroscopy. RESULTS: AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with ß-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer. CONCLUSION: α-Hb reduction could trigger its release from AHSP toward its final Hb ß-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis. GENERAL SIGNIFICANCE: The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia.

Ligand-rebinding kinetics of 2/2 hemoglobin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Kinetic studies were performed on ligand rebinding to a cold-adapted globin of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (Ph-2/2HbO). This 2/2 hemoglobin displays a rapid spectroscopic phase that is independent of CO concentration, followed by the standard bimolecular recombination. While the geminate recombination usually occurs on a ns timescale, Ph-2/2HbO displays a component of about 1µs that accounts for half of the geminate phase at 8°C, indicative of a relatively slow internal ligand binding. The O2 binding kinetics were measured in competition with CO to allow a short-time exposure of the deoxy hemes to O2 before CO replacement. Indeed Ph-2/2HbO is readily oxidised in the presence of O2, probably due to a superoxide character of the FeO2 bond induced by of a hydrogen-bond donor amino-acid residue. Upon O2 release or iron oxidation a distal residue (probably Tyr) is able to reversibly bind to the heme and as such to compete for binding with an external ligand. The transient hexacoordinated ferrous His-Fe-Tyr conformation after O2 dissociation could initiate the electron transfer from the iron toward its final acceptor, molecular O2 under our conditions. The hexacoordination via the distal Tyr is only partial, indicating a weak interaction between Tyr and the heme under atmospheric pressure. Hydrostatic high pressure enhances the hexacoordination indicating a flexible globin that allows structural changes. The O2 binding affinity for Ph-2/2HbO, poorly affected by the competition with Tyr, is about 1Torr at 8°C, pH7.0, which is compatible for an in vivo O2 binding function; however, this globin is more likely involved in a redox reaction associating diatomic ligands and their derived oxidative species. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Neuroglobins, pivotal proteins associated with emerging neural systems and precursors of metazoan globin diversity.

Neuroglobins, previously thought to be restricted to vertebrate neurons, were detected in the brain of a photosymbiotic acoel, Symsagittifera roscoffensis, and in neurosensory cells of the jellyfish Clytia hemisphaerica. For the neuroglobin of S. roscoffensis, a member of a lineage that originated either at the base of the bilateria or of the deuterostome clade, we report the ligand binding properties, crystal structure at 2.3 Å, and brain immunocytochemical pattern. We also describe in situ hybridizations of two neuroglobins specifically expressed in differentiating nematocytes (neurosensory cells) and in statocytes (ciliated mechanosensory cells) of C. hemisphaerica, a member of the early branching animal phylum cnidaria. In silico searches using these neuroglobins as queries revealed the presence of previously unidentified neuroglobin-like sequences in most metazoan lineages. Because neural systems are almost ubiquitous in metazoa, the constitutive expression of neuroglobin-like proteins strongly supports the notion of an intimate association of neuroglobins with the evolution of animal neural systems and hints at the preservation of a vitally important function. Neuroglobins were probably recruited in the first protoneurons in early metazoans from globin precursors. Neuroglobins were identified in choanoflagellates, sponges, and placozoans and were conserved during nervous system evolution. Because the origin of neuroglobins predates the other metazoan globins, it is likely that neuroglobin gene duplication followed by co-option and subfunctionalization led to the emergence of globin families in protostomes and deuterostomes (i.e. convergent evolution).

Neuroglobin involvement in respiratory chain function and retinal ganglion cell integrity.

Neuroglobin is a member of the globin superfamily expressed in vertebrate brain and retina. The protein is thought to be involved in neuronal protection from hypoxia or oxidative stress and could represent a key element of Alzheimer disease pathogenesis. Our aim was to determine whether neuroglobin could be directly associated with mitochondrial metabolism and integrity. We identified three different forms of neuroglobin in the retina, varying in their apparent molecular masses; all forms are abundant in mitochondrial fractions. This indicates that a significant fraction of the protein localizes within the organelle either in the matrix or in the matrix side of the inner membrane. Since neuroglobin was especially abundant in the ganglion cell layer, we transduced retinal ganglion cells with an anti-neuroglobin short hairpin RNA using in vivo electroporation. Neuroglobin knockdown leads to reduced activities of respiratory chain complexes I and III, degeneration of retinal ganglion cells, and impairment of visual function. The deleterious effect on cell survival was confirmed in primary retinal ganglion cells subjected to inhibition of neuroglobin expression. Hence, neuroglobin should be considered as a novel mitochondrial protein involved in respiratory chain function which is essential for retinal ganglion cell integrity.

Characterization of the hemoglobin of the backswimmer Anisops deanei (Hemiptera).

While O(2)-binding hemoglobin-like proteins are present in many insects, prominent amounts of hemoglobin have only been found in a few species. Backswimmers of the genera Anisops and Buenoa (Notonectidae) have high concentrations of hemoglobin in the large tracheal cells of the abdomen. Oxygen from the hemoglobin is delivered to a gas bubble and controls the buoyant density, which enables the bugs to maintain their position without swimming and to remain stationary in the mid-water zone where they hunt for prey. We have obtained the cDNA sequences of three Anisops deanei hemoglobin chains by RT-PCR and RACE techniques. The deduced amino acid sequences show an unusual insertion of a single amino acid in the conserved helix E, but this does not affect protein stability or ligand binding kinetics. Recombinant A. deanei hemoglobin has an oxygen affinity of P(50) = 2.4 kPa (18 torr) and reveals the presence of a dimeric fraction or two different conformations. The absorption spectra demonstrate that the Anisops hemoglobin is a typical pentacoordinate globin. Phylogenetic analyses show that the backswimmer hemoglobins evolved within Heteroptera and most likely originated from an intracellular hemoglobin with divergent function.

Interaction of haptoglobin with hemoglobin octamers based on the mutation αAsn78Cys or ßGly83Cys.

Octameric hemoglobins have been developed by the introduction of surface cysteines in either the alpha or beta chain. Originally designed as a blood substitute, we report here the structure and ligand binding function; in addition the interaction with haptoglobin was studied. The recombinant Hbs (rHbs) with mutations alpha Asn78Cys or beta Gly83Cys spontaneously form octamers under conditions where the cysteines are oxidized. Oxygen binding curves and CO kinetic studies indicate a correct allosteric transition of the tetramers within the octamer. Crystallographic studies of the two rHbs show two disulfide bonds per octamer. Reducing agents may provoke dissociation to tetramers, but the octamers are stable when mixed with fresh human plasma, indicating that the reduction by plasma is slower than the oxidation by the dissolved oxygen, consistent with an enhanced stability. The octameric rHbs were also mixed with a solution of haptoglobin (Hp), which binds the dimers of Hb: there was little interaction for incubation times of 15 min; however, on longer timescales a complex was formed. Dynamic light scattering was used to follow the interaction of Hp with the alpha Asn78Cys octamer during 24 hours; a transition from a simple complex of 15 nm to a final size of 60 nm was observed. The results indicate a specific orientation of the αß dimers may be of importance for the binding to haptoglobin.

Ligand- and proton-linked conformational changes of the ferrous 2/2 hemoglobin of Pseudoalteromonas haloplanktis TAC125.

The spectroscopic and ligand-binding properties of a 2/2 globin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 have been studied in the ferrous state. It displays two major conformations characterized by CO-association rates that differ by a factor of 20, with relative fractions that depend on pH. A dynamic equilibrium is found between the two conformations, as indicated by an enhanced slower phase when lower CO levels were used to allow a longer time to facilitate the transition. The deoxy form, in the absence of external ligands, is a mixture of a predominant six-coordinate low spin form and a five-coordinate high-spin state; the proportion of low spin increasing at alkaline pH. In addition, at temperatures above the physiological temperature of 1 °C, an enhanced tendency of the protein to oxidize is observed.

Electron transfer function versus oxygen delivery: a comparative study for several hexacoordinated globins across the animal kingdom.

Caenorhabditis elegans globin GLB-26 (expressed from gene T22C1.2) has been studied in comparison with human neuroglobin (Ngb) and cytoglobin (Cygb) for its electron transfer properties. GLB-26 exhibits no reversible binding for O(2) and a relatively low CO affinity compared to myoglobin-like globins. These differences arise from its mechanism of gaseous ligand binding since the heme iron of GLB-26 is strongly hexacoordinated in the absence of external ligands; the replacement of this internal ligand, probably the E7 distal histidine, is required before binding of CO or O(2) as for Ngb and Cygb. Interestingly the ferrous bis-histidyl GLB-26 and Ngb, another strongly hexacoordinated globin, can transfer an electron to cytochrome c (Cyt-c) at a high bimolecular rate, comparable to those of inter-protein electron transfer in mitochondria. In addition, GLB-26 displays an unexpectedly rapid oxidation of the ferrous His-Fe-His complex without O(2) actually binding to the iron atom, since the heme is oxidized by O(2) faster than the time for distal histidine dissociation. These efficient mechanisms for electron transfer could indicate a family of hexacoordinated globin which are functionally different from that of pentacoordinated globins.

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.

Kinetics inside the protein: shape of the geminate kinetics in myoglobin.

Synchronized kinetics of ligand binding to a buried active site offers a look inside the protein. Photodissociated ligands are initially alongside their original binding site, so the recombination kinetics describes the trajectory for direct (geminate) rebinding or escape from the protein for the subsequent (bimolecular) rebinding phase. In the model case of myoglobin in water, most of the ligands escape; to better observe the geminate phase, high viscosity cosolvents were used: the kinetics were characterized by multiple barriers and a distribution of rates. An alternative method to enhance the fraction of geminate phase is the application of high pressure which closes the ligand migration channel; in this case of low viscosity without cosolvents, the geminate phase is closer to a simple exponential behavior. Samples with glycerol display the extended geminate kinetics, while samples in water under pressure do not.

Evaluation of the free α-hemoglobin pool in red blood cells: a new test providing a scale of ß-thalassemia severity.

ß-Thalassemias are characterized by an imbalance of globin chains with an excess of α-chains which precipitates in erythroid precursors and red blood cells (RBCs) leading to inefficient erythropoiesis. The severity of the disease correlates with the amount of unpaired α-chains.Our goal was to develop a simple test for evaluation of the free α-hemoglobin pool present in RBC lysates. Alpha-Hemoglobin Stabilizing Protein (AHSP), the chaperone of α-Hb, was used to trap excess a-Hb. A recombinant GST-AHSP fusion protein was bound to an affinity micro-column and then incubated with hemolysates of patients. After washing, the α-Hb was quantified by spectrophotometry in the elution fraction. This assay was applied to 54 patients: 28 without apparent Hb disorder, 20 ß-thalassemic and 6 α-thalassemic. The average value of free α-Hb pool was 93 ± 21 ppm (ng of free α-Hb per mg of Hb subunits)in patients without Hb disorder, while it varies from 119 to 1,756 ppm, in ß-thalassemic patients and correlated with genotype. In contrast,the value of the free α-Hb pool was decreased in α-thalassemic patients (65 ± 26 ppm). This assay may help to characterize ß-thalassemia phenotypes and to follow the evolution of the globin chain imbalance(α/ß+γ ratio) in response to treatment.

A membrane-bound hemoglobin from gills of the green shore crab Carcinus maenas.

Most hemoglobins serve for the transport or storage of O(2). Although hemoglobins are widespread in "entomostracan" Crustacea, malacostracans harbor the copper-containing hemocyanin in their hemolymph. Usually, only one type of respiratory protein occurs within a single species. Here, we report the identification of a hemoglobin of the shore crab Carcinus maenas (Malacostraca, Brachyura). In contrast to the dodecameric hemocyanin of this species, C. maenas hemoglobin does not reside in the hemolymph but is restricted to the gills. Immunofluorescence studies and cell fractioning showed that C. maenas hemoglobin resides in the membrane of the chief cells of the gill. To the best of our knowledge, this is the first time that a membrane-bound hemoglobin has been identified in eukaryotes. Bioinformatic evaluation suggests that C. maenas hemoglobin is anchored in the membrane by N-myristoylation. Recombinant C. maenas hemoglobin has a hexacoordinate binding scheme at the Fe(2+) and an oxygen affinity of P(50) = 0.5 Torr. A rapid autoxidation rate precludes a function as oxygen carrier. We rather speculate that, analogous to prokaryotic membrane-globins, C. maenas hemoglobin carries out enzymatic functions to protect the lipids in cell membrane from reactive oxygen species. Sequence comparisons and phylogenetic studies suggested that the ancestral arthropod hemoglobin was most likely an N-myristoylated protein that did not have an O(2) supply function. True respiratory hemoglobins of arthropods, however, evolved independently in chironomid midges and branchiopod crustaceans.

The globin gene family of the cephalochordate amphioxus: implications for chordate globin evolution.

BACKGROUND: The lancelet amphioxus (Cephalochordata) is a close relative of vertebrates and thus may enhance our understanding of vertebrate gene and genome evolution. In this context, the globins are one of the best studied models for gene family evolution. Previous biochemical studies have demonstrated the presence of an intracellular globin in notochord tissue and myotome of amphioxus, but the corresponding gene has not yet been identified. Genomic resources of Branchiostoma floridae now facilitate the identification, experimental confirmation and molecular evolutionary analysis of its globin gene repertoire. RESULTS: We show that B. floridae harbors at least fifteen paralogous globin genes, all of which reveal evidence of gene expression. The protein sequences of twelve globins display the conserved characteristics of a functional globin fold. In phylogenetic analyses, the amphioxus globin BflGb4 forms a common clade with vertebrate neuroglobins, indicating the presence of this nerve globin in cephalochordates. Orthology is corroborated by conserved syntenic linkage of BflGb4 and flanking genes. The kinetics of ligand binding of recombinantly expressed BflGb4 reveals that this globin is hexacoordinated with a high oxygen association rate, thus strongly resembling vertebrate neuroglobin. In addition, possible amphioxus orthologs of the vertebrate globin X lineage and of the myoglobin/cytoglobin/hemoglobin lineage can be identified, including one gene as a candidate for being expressed in notochord tissue. Genomic analyses identify conserved synteny between amphioxus globin-containing regions and the vertebrate ß-globin locus, possibly arguing against a late transpositional origin of the ß-globin cluster in vertebrates. Some amphioxus globin gene structures exhibit minisatellite-like tandem duplications of intron-exon boundaries ("mirages"), which may serve to explain the creation of novel intron positions within the globin genes. CONCLUSIONS: The identification of putative orthologs of vertebrate globin variants in the B. floridae genome underlines the importance of cephalochordates for elucidating vertebrate genome evolution. The present study facilitates detailed functional studies of the amphioxus globins in order to trace conserved properties and specific adaptations of respiratory proteins at the base of chordate evolution.

Cytoglobin conformations and disulfide bond formation.

The oligomeric state and kinetics of ligand binding were measured for wild-type cytoglobin. Cytoglobin has the classical globin fold, with an extension at each extremity of about 20 residues. The extended length of cytoglobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoglobin behaves hydrodynamically like a tightly packed globin with a greater flexibility of the N- and C-terminal regions. Cytoglobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoglobin conformations, which may affect the observed oxygen affinity by an order of magnitude.

Pulmonary capillary blood volume and membrane conductance in Andeans and lowlanders at high altitude: a cross-sectional study.

Lung carbon monoxide (CO) transfer and pulmonary capillary blood volume (Vc) at high altitudes have been reported as being higher in native highlanders compared to acclimatised lowlanders but large discrepancies appears between the studies. This finding raises the question of whether hypoxia induces pulmonary angiogenesis. Eighteen highlanders living in Bolivia and 16 European lowlander volunteers were studied. The latter were studied both at sea level and after acclimatisation to high altitude. Membrane conductance (Dm(CO)) and Vc, corrected for the haemoglobin concentration (Vc(cor)), were calculated using the NO/CO transfer technique. Pulmonary arterial pressure and left atrial pressures were estimated using echocardiography. Highlanders exhibited significantly higher NO and CO transfer than acclimatised lowlanders, with Vc(cor)/VA and Dm(CO)/VA being 49 and 17% greater (VA: alveolar volume) in highlanders, respectively. In acclimatised lowlanders, Dm(CO) and Dm(CO)/VA values were lower at high altitudes than at sea level. Echocardiographic estimates of cardiac output and pulmonary arterial pressure were significantly elevated at high altitudes as compared to sea level. The decrease in Dm(CO) in lowlanders might be due to altered gas transport in the airways due to the low density of air at high altitudes. The disproportionate increase in Vc in Andeans compared to the change in Dm(CO) suggests that the recruitment of capillaries is associated with a thickening of the blood capillary sheet. Since there was no correlation between the increase in Vc and the slight alterations in haemodynamics, this data suggests that chronic hypoxia might stimulate pulmonary angiogenesis in Andeans who live at high altitudes.

Artificial oxygen carrier based on polysaccharides-poly(alkylcyanoacrylates) nanoparticle templates.

Biomimetic nanoparticles based on polysaccharides-poly(alkylcyanoacrylates) copolymers were initially developed in view of drug delivery. Core-shell nanoparticles covered with a sufficiently long brush of polysaccharides were shown to be very low complement activators and have the potential for long circulation times in the bloodstream. Such nanoparticles bearing haemoglobin were envisaged as potential red cell substitutes. Different core-shell nanoparticles with a brush shell made of dextran, dextran-sulphate, or heparin were prepared and haemoglobin (Hb) could be adsorbed on their surface. Benzene tetracarboxylic acid (BTCA) was used as a coupling agent for Hb to dextran-coated nanoparticles; the Hb loading capacity of the dextran nanoparticles showed a 9.3 fold increased. The coupled Hb maintained the allosteric properties of free Hb. While modification of nanoparticles by BTCA slightly increased complement activation, the further addition of Hb totally reversed this effect providing Hb-loaded nanoparticles with a very low level of complement activation. Such nanoparticles could be a suitable alternative to haemoglobin solutions in the development of a blood substitute.