Effect of destabilizing factors on the structural-functional hemoglobin parameters in euryhaline and diadromous fish

A comparative analysis of stability of the hemoglobin structural-functional organization to effect of some experimental factors modeling action of internal medium and habitat was performed in representatives of Chondrostei and Teleostei belonging to euryhaline and diadromous fish. Differentiation of hemoglobins by stability parameters was shown to coincide to division of the studied fish into two main ecological groups: euryhaline fish with unstable hemoglobin and diadromous fishes with stable hemoglobin. In turn, the euryhaline fish by the character of destruction of hemoglobins under effects of destabilizing factors are differentiated into three subgroups: cartilaginous fish, cartilaginous ganoids, and bony fish. Thus, differentiation of hemoglobins by parameters of resistance to destabilizing factors is determined by peculiarities of the fish mode of life and is of adaptive nature. The described in the present study method of determination of critical precipitation concentration (CPC) of hemoglobin may be used as an express-method for estimation of hemoglobin resistance to salt effects, while the CPC values—as one of criteria of fish belonging to various ecological groups (diadromous, euryhaline).     

Effect of nitrite-induced methemoglobinemia on the kinetics of blood deoxygenation

Kinetics of blood deoxygenation was studied during acute hypoxia induced by subcutaneous administration of sodium nitrite using polarographic method. Plasma oxygen tension remained unaltered as the dose of sodium nitrite increased, while the dynamics of oxygen release was dose-dependent. The constant of oxyhemoglobin deoxygenation rate proved to vary with blood deoxygenation. The nitrite-induced deceleration of oxyhemoglobin deoxygenation was due to the inactivation of a fraction of hemoglobin as well as to the increased hemoglobin oxygen affinity and possible changes in the oxygen permeability of erythrocyte membranes during acute methemoglobinemia.     

Phylogenetic Studies of Complete Mitochondrial DNA Molecules Place Cartilaginous Fishes Within the Tree of Bony Fishes

It is commonly acknowledged that cartilaginous fishes, Chondrichthyes, have a basal position among the Gnathostomata (jawed vertebrates). In order to explore this relationship we have sequenced the complete mitochondrial genome of the spiny dogfish, Squalus acanthias, and included it in a phylogenetic analysis together with a number of bony fishes and amniotes. The phylogenetic reconstructions placed the dogfish among the bony fishes. Thus, and contrary to the common view, the analyses have shown that the position of the sharks is not basal among the gnathostomes. The presently recognized phylogenetic position of the dogfish was identified irrespective of the outgroup used, echinoderms or agnathan fishes. The lungfish was the most basal gnathostome fish, while the teleosteans had an apical position in the piscine tree. A basal position of the dogfish among the gnathostomes was statistically rejected, but the phylogenetic relationship among the coelacanth, spiny dogfish, and teleosts was not conclusively resolved. The findings challenge the current theory that sharks and other chondrichthyans, if monophyletic, are the sister group to all other extant gnathostomes. The results open to question the status of several morphological characters commonly used in piscine phylogenetic reconstruction, most notably the presence versus absence of endochondral bone in the endoskeleton, the macromeric versus micromeric structure of the exoskeleton, and the presence/absence of swimbladder and/or lung. The study also confirmed recent findings demonstrating that the origin of the amniotes is deeper than the diversification of extant bony fishes. Received: 12 March 1998 / Accepted: 12 June 1998     

Visible Raman excitation laser induced power and exposure dependent effects in red blood cells

We present results of Raman spectroscopic studies carried out on optically trapped red blood cells with Raman excitation wavelength in Q‐band region of the hemoglobin (Hb) absorption spectrum. The results obtained suggest that when exposed to the Raman excitation laser the RBCs get deoxygenated due to photo‐dissociation of oxygen from hemoglobin. For smaller exposure durations (5 s) the level of deoxygenation increases with an increase in power. However, for longer exposure durations the deoxygenated hemoglobin in the cells gets irreversibly oxidized to form a low spin ferric derivative of hemoglobin. The rate of oxidation depends upon the initial level of deoxygenation; higher the initial level of deoxygenation, higher is the rate of oxidation. However, the RBCs deoxygenated via oxygen deprivation (i.e. N₂ purging) were found to be very stable against any laser induced effect. These observations suggests that in case of laser induced deoxygenation of RBCs the free oxygen generated by photo‐dissociation acts as the oxidizing agent and leads to oxidative damage of the RBCs.

     

Electron microscopic studies of the intracellular polymerization of sickle hemoglobin

Transmission electron microscopy has been used to study intracellular sickle hemoglobin polymer in unfractionated cells from the arterial and venous blood of patients and after external deoxygenation. We detect polymerized hemoglobin in up to 10% of the cells in the venous circulation, especially in cells that are "cigar-shaped" and appear to be irreversibly sickled. We could not see well-defined polymer in mixed arterial samples; nevertheless, we found electron opaque spots, which could be ferritin granules, hemosiderin, or small aggregates of hemoglobin S. However, upon sequential chemical deoxygenation using 1.0% sodium metabisulphite, polymer formation was seen at oxygen saturation values of 75%-85%. Cells that were physically deoxygenated using gas mixtures containing nitrogen-carbon dioxide-oxygen mixtures were found to contain distinct polymers of deoxyhemoglobin S at oxyhemoglobin saturation values of 50%-75%. As deoxygenation increases, we detect short, randomly arranged polymer in a loose network, with occasional long polymers. Upon further deoxygenation, the length and number of polymer forms increased. Between 0% and 50% saturation, most erythrocytes were full of long, parallel, closely packed polymers that tend to align and run parallel to the cell membrane. In both chemical and physically deoxygenated blood samples, cells were seen at 50%-75% oxyhemoglobin saturation that retained their normal biconcave disc shape, although they contained significant amounts of polymer. The structural changes in sickle erythrocytes seen in vitro due to physical or chemical deoxygenation of cells, may reflect in vivo intracellular changes in the sickle cell patient.     

Hemoglobin-dialdehyde dextran conjugates: Improvement of their oxygen-binding properties with anionic groups

We studied the conjugates formed between hemoglobin and sulfated or unsulfated oxidized dextran. It appears that the presence of sulfated groups favors imino bond formation between the protein and the polymer, as the average molecular size of the conjugates is larger in this case. Under neutral conditions, the oxygen-binding properties of the conjugates depend on the presence or absence of oxygen during the coupling reaction. With unsulfated dextran, oxyhemoglobin leads to conjugates with increased oxygen affinity (P ₅₀/P ₅₀ native hemoglobin 0.5) compared to that of free hemoglobin (P ₅₀=4 mm Hg), whereas deoxyhemoglobin leads to conjugates with decreased oxygen affinity (P ₅₀/P ₅₀ native hemoglobin 3). The use of sulfated dextran reinforces this lowering in oxygen affinity, which indicates that sulfated dextran acts as a permanent macromolecular effector of hemoglobin (P ₅₀/P ₅₀ native hemoglobin 4). Moreover, it can be assumed that some of the linkages involve the 2,3-diphosphoglycerate binding site, as the strong effector inositol hexaphosphate has only a slight effect on the oxygen-binding properties of the conjugate prepared in the deoxy state (P ₅₀/P ₅₀ native hemoglobin close to 4.4 and 6, respectively, for unsulfated and sulfated conjugates). Although dextran substituted with benzenehexacarboxylic acid (BHC) leads to a low-oxygen-affinity conjugate when linked to oxyhemoglobin through amide bonds (P ₅₀/P ₅₀ native hemoglobin 5), oxidized dextran modified with BHC leads, with oxyhemoglobin, to a conjugate whose oxygen affinity is close to that of free hemoglobin (P ₅₀/P ₅₀ native hemoglobin 1.2).     

Cell and molecular biology of SAE, a cell line from the spiny dogfish shark, Squalus acanthias

Cartilaginous fish, primarily sharks, rays and skates (elasmobranchs), appeared 450 million years ago. They are the most primitive vertebrates, exhibiting jaws and teeth, adaptive immunity, a pressurized circulatory system, thymus, spleen, and a liver comparable to that of humans. The most used elasmobranch in biomedical research is the spiny dogfish shark, Squalus acanthias. Comparative genomic analysis of the dogfish shark, the little skate (Leucoraja erincea), and other elasmobranchs have yielded insights into conserved functional domains of genes associated with human liver function, multidrug resistance, cystic fibrosis, and other biomedically relevant processes. While genomic information from these animals is informative in an evolutionary framework, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. We have derived the first multipassage, continuously proliferating cell line of a cartilaginous fish. The line was initiated from embryos of the spiny dogfish shark. The cells were maintained in a medium modified for fish species and supplemented with cell type-specific hormones, other proteins and sera, and plated on a collagen substrate. SAE cells have been cultured continuously for three years. These cells can be transfected by plasmids and have been cryopreserved. Expressed Sequence Tags generated from a normalized SAE cDNA library included a number of markers for cartilage and muscle, as well as proteins influencing tissue differentiation and development, suggesting that SAE cells may be of mesenchymal stem cell origin. Examination of SAE EST sequences also revealed a cartilaginous fish-specific repetitive sequence that may be evidence of an ancient mobile genetic element that most likely was introduced into the cartilaginous fish lineage after divergence from the lineage leading to teleosts.     

Oxygenation of Native or UV-Modified Human Hemoglobin in the Presence of Nitric Oxide

The effect of 0.1–10% nitrosohemoglobin (HbNO) on the functional properties of human oxyhemoglobin (HbO₂) was studied before and after UV irradiation at 151–453 J/m². Oxygen binding analysis showed that HbNO intensified the first stage of oxygenation and weakened the cooperative interactions in the tetramer, decreasing the affinity of hemoglobin for oxygen at physiologically important oxygen partial pressures (40–100 mm Hg). Mixtures of HbO₂ and HbNO were highly resistant to therapeutic doses of UV irradiation. Since the functional activity of hemoglobin depended nonlinearly on the concentration of HbNO in the mixture, it was assumed that sophisticated interactions of HbO₂ and HbNO yielded a new product differing in properties from the initial components.     

Unfolding and release of heme from human hemoglobins A,S and F

• 1.1. Analysis of the Soret spectra of hemoglobins A, S and F has been used to determine the extent of heme exposure and release from these hemoglobins in the presence of several solvent perturbants.
• 2.2. Oxyhemoglobin S unfolding in the presence of either urea or propyl urea resulted in greater heme exposure and release than either oxyhemoglobins A or F.
• 3.3. Methemoglobin formation resulted in lower denaturation midpoints for each hemoglobin compared to the reduced oxyhemoglobin state; methemoglobin F had the lowest denaturation midpoint under isothermal denaturing conditions.
• 4.4. Rate of heme exposure was greater for oxyhemoglobin S than oxyhemoglobin A in the presence of 200 μM the anionic detergent sodium dodecyl sulfate.
• 5.5. Evidence for increased levels of heme release in hemoglobin S may be related to the greater tendency of sickled red cell membranes to undergo lipid oxidation.

     

Modification of hemoglobin A with dimethyl adipimidate. Contribution of individual reacted subunits to changes in oxygen affinity.

The effect of dimethyl adipimidate, a bifunctional imidoester, on the oxygen affinity of hemoglobin A has been studied. Treatment of human oxyhemoglobin with 5 mM dimethyl adipimidate at pH 8.5, room temperature is accompanied by an increase in oxygen affinity in the presence and absence of 2,3-diphosphoglyceric acid. Circular dichroism measurements in the ultraviolet region indicate that dimethyl adipimidate-treated hemoglobin exhibits a reduced conformational change upon deoxygenation. In order to study the contribution of reacted individual subunits, alpha and beta subunits of dimethyl adipimidate-treated and untreated hemoglobin have been separated and reconstituted to form hybrid tetramers containing either the alpha-treated (alpha t beta c) or the beta-treated subunits (alpha c beta t). Electrophoresis on sodium dodecyl sulfate polyacrylamide gels of isolated alpha and beta globin subunits as well as hybrid tetramers from dimethyl adipimidate-treated hemoglobin reveals that 20% of the globin subunits are cross-linked. In the absence of 2,3-diphosphoglyceric acid, modification of alpha subunits increases the oxygen affinity and reduces the conformational change of the tetramer upon deoxygenation whereas modification of beta subunits has no effect. However, treatment of beta subunits decreases the effect of 2,3-diphosphoglyceric acid on the oxygen affinity of the hybrids and reduces the 2,3-diphosphoglyceric acid-induced spectral changes in oxyhemoglobin. Therefore the interaction of dimethyl adipimidate with both the alpha and beta subunits contributes to regulating the oxygen affinity of human hemoglobin.     

Sulfide-hemoglobin interactions in the sulfide-tolerant salt marsh resident,the California killifish Fundulus parvipinnis

Summary Sulfide can potentially damage hemoglobin or be detoxified by hemoglobin. In the sulfide-tolerant California killifish neither seems to be the case at environmentally realistic (micromolar) and physiologically relevant (millimolar) sulfide concentrations. An 8-h exposure of killifish to 5 and 8 mmol sulfide · 1^-1 results in 50–100% mortality, but not due to sulfhemoglobin (where sulfide covalently binds to the porphyrin) nor ferric hemoglobin (Hb⁺), both dysfunctional hemoglobin derivatives. Killifish hemoglobin converts to sulfhemoglobin in vitro only in the presence of 1–5 mmol sulfide · 1^-1. The amount of sulfhemoglobin formed increases with time and heme concentration but decreases with pH. Hb⁺ binds sulfide as ferric hemoglobin sulfide (Hb⁺S, an unstable complex where sulfide ligates to the iron), and also as sulfhemoglobin. Killifish blood does not catalyze the oxidation of 10–500 mol sulfide · 1^-1 to any appreciable extent. Radiolabeled sulfide incubated with oxyhemoglobin or whole blood disappears at rates greater than in buffers, but only minimal amounts of thiosulfate and no sulfate nor sulfite are formed (elemental sulfur and bound sulfide not quantified). Sulfide disappearance rates increase linearly with initial sulfide concentration. Hb⁺ does catalyze the oxidation of sulfide to thiosulfate in vitro. Similar experiments on another sulfide-tolerant species, the long-jawed mudsucker Gillichthys mirabilis, produced similar results.Abbreviations ANOVA analysis of variance - BV benzyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high-pressure liquid chromatography - RBC red blood cells - SHb sulfhemoglobin     

The ultrastructure of the interstitial cells of the mesonephros in the teleost fishes of the Black Sea

The ultrastructure of cells that form mesonephros tissues in eight species of bony fish of the Black Sea, Gaidropsarus mediterraneus (L.), Lisa aurata (Risso), Trachurus mediterraneus (Staindachner), Diplodus annularis (L.), Spicara flexuosa (Rafinesque), Gobius niger jozo L., Mullus barbatus ponticus (Essipov) and Scorpaena porcus (L.), was studied. It was shown that the ultrastructure of agranulocytes and rodlet cells in the studied species is similar to that of cells in freshwater bony fish. The differences were observed in the number of ionocyte mitochondria, in the vesicle ultrastructure in the cells with radial vesicle array, and in the ultrastructure of the specific secondary granules of neutrophils and eosinophils.     

A compact cartilaginous fish model genome

The genomes of several vertebrates, including six mammals, the chicken, Xenopus and four ray-finned fishes have been sequenced or are currently being sequenced to provide a better understanding of the human genome through comparative analysis. However, this list does not include cartilaginous fishes, which are the most basal living jawed vertebrates [1]. The genomes of the current ‘popular’ cartilaginous fishes such as the nurse shark, dogfish, and horn shark are larger than the human genome (∼3800 Mb to 7000 Mb) [2], and are not attractive for whole-genome sequencing. Here, we report the characterization of the relatively small genome (1200 Mb) of a cartilaginous fish, the elephant fish (Callorhinchus milii), and propose it as a model for whole-genome sequencing.     

Do abiotic and ontogenetic factors influence the diet of a generalist predator? Feeding ecology of the Pacific spiny dogfish (<Emphasis Type="Italic">Squalus suckleyi</Emphasis>) in the northeast Pacific Ocean

Stomach contents from 1221 Pacific spiny dogfish (Squalus suckleyi) were collected from the Gulf of Alaska over 2004–2006 and analyzed to determine predominant prey species. Pacific spiny dogfish in the Gulf of Alaska have a variable diet suggesting that they are generalist feeders. Of the 68.30% of stomachs with prey contents, the most important prey groups based on the percent prey-specific index of relative importance (%PSIRI) were shrimp (27.06%), cephalopods (17.16%), and forage fish (17.11%). Remaining components of the diet were inconsistent. Commercially valuable species, such as salmon, Oncorhynchus spp., and rockfish, Sebastes spp. constituted only 2.37% and 1.51% of the diet of Pacific spiny dogfish, respectively. Diet diversity was not significantly influenced by spatial, temporal or ontogenetic factors; however, trophic level of prey and average prey item weight varied both interannually and with ontogeny. With increasing size, Pacific spiny dogfish incorporate larger prey items into their diet, and diet composition may be driven more by seasonal availability and prey size than any other factors.     

Systematic position of fish species and ganglioside composition and content

The ganglioside content in brain of cartilaginous and bony fishes studied varies from 110 to 581 and from 104 to 595 micrograms sialic acid per g of wet weight respectively. A high degree of alkali lability and the predominance of C18-sphingosine and N-acetylneuraminic acid are typical of fish brain gangliosides. A high content of oligosialogangliosides with four and more residues of sialic acid and the predominance of gangliosides with gangliotetraosyl carbohydrate chain are characteristic for teleost brain. No pronounced difference was revealed in ganglioside composition and content of clupeomorphs and percomorphs. Gangliosides with short (lactosyl and gangliotriaosyl) carbohydrate chain predominate in brain of all cartilaginous fishes studied. A statistically significant difference was found in ganglioside content, relative oligosialoganglioside content and ganglioside fatty acid composition of squalomorphs and rajiformes, on one hand, and dasyatiformes and galeomorphs, on the other hand.     

Demonstration of bone resorbing cells in elasmobranchs: Comparison with osteoclasts

We have previously shown that Elasmobranchs-characterized by a partially calcified cartilaginous endoskeleton-presented a bony vertebral arch containing osteoblasts, osteocytes and resorbing cells. The aim of this study is to test the ability of Elasmobranchs to resorb bone tissue. The subcutaneous implantation in dogfish (Scyliorhinus canicula) of devitalized mineral-containing bone particles, obtained from a bony fish (the eel, Anguilla anguilla) resulted, after 21 2 months, in the formation of mononucleated as well as multinucleated cells around and between the bone fragments. By light microscopy, the multinucleated giant cells presented the general aspect of osteoclastic cells whereas, by transmission electron microscopy they never showed ruffled borders which are considered as the typical features of osteoclasts. Except for this character, the mononucleated and multinucleated cells exhibited the typical ultrastructural aspects leading us to say that these cells are involved in the resorption of the bone fragments. This study shows that Elasmobranchs are able to resorb implanted bone.     

Root effect hemoglobins in marine fish

• 1.1. The Root effect was evaluated in hemolysates from 26 species of bony fish and 20 species of cartilaginous fish found on the Brazilian southeastern coast.
• 2.2. Teleost Root shifts, with a single exception, are correlated with the presence of the choroid rete mirabile but not with its counterpart in the swimbladder.
• 3.3. Five ray species displayed weak and moderate Root effects despite the absence of choroid and swimbladder rete.
• 4.4. The presence and intensity of the Root effect is probably primarily related to the high oxygen demand of the retina and with the importance of visual perception in fish.
• 5.5. In marine teleosts the magnitude of the Root effect seems to be associated with the presence and size of both the choroid rete and the pseudobranch.
• 6.6. An antioxidant protection of the fish eyes can be advocated for the pseudobranch.

     

Oxygen-derived free radicals and hemolysis during open heart surgery

Reperfusion injury occurs during open-heart surgery after prolonged cardioplegic arrest. Cardiopulmonary bypass also is known to cause hemolysis. Since reperfusion of ischemic myocardium is associated with the generation of oxygen free radicals, and since free radicals can attack a protein molecule, it seems reasonable to assume that hemolysis might be the consequence of free radical attack on hemoglobin protein. The results of this study demonstrated that reperfusion following ischemic arrest caused an increase in free hemoglobin and free heme concentrations, simultaneously releasing free iron and generating hydroxyl radicals. In vitro studies using pure hemoglobin indicated that superoxide anion generated by the action of xanthine oxidase on xanthine could release iron from the heme ring and cause deoxygenation of oxyhemoglobin into ferrihemoglobin. This study further demonstrated that before the release of iron from the heme nucleus, oxyhemoglobin underwent deoxygenation to ferrihemoglobin. The released iron can catalyze the Fenton reaction, leading to the formation of cytotoxic hydroxyl radical (OH·). In fact, the formation of OH. in conjunction with hemolysis occurs during cardiac surgery, and when viewed in the light of the in vitro results, it seems likely that oxygen-derived free radicals may cause hemolysis during cardiopulmonary bypass and simultaneously release iron from the heme ring, which can catalyze the formation of OH·.     

Molecular dynamics simulations indicate that deoxyhemoglobin,oxyhemoglobin, carboxyhemoglobin,and glycated hemoglobin under compression and shear exhibit an anisotropic mechanical behavior

We developed a new mechanical model for determining the compression and shear mechanical behavior of four different hemoglobin structures. Previous studies on hemoglobin structures have focused primarily on overall mechanical behavior; however, this study investigates the mechanical behavior of hemoglobin, a major constituent of red blood cells, using steered molecular dynamics (SMD) simulations to obtain anisotropic mechanical behavior under compression and shear loading conditions. Four different configurations of hemoglobin molecules were considered: deoxyhemoglobin (deoxyHb), oxyhemoglobin (HbO₂), carboxyhemoglobin (HbCO), and glycated hemoglobin (HbA_1C). The SMD simulations were performed on the hemoglobin variants to estimate their unidirectional stiffness and shear stiffness. Although hemoglobin is structurally denoted as a globular protein due to its spherical shape and secondary structure, our simulation results show a significant variation in the mechanical strength in different directions (anisotropy) and also a strength variation among the four different hemoglobin configurations studied. The glycated hemoglobin molecule possesses an overall higher compressive mechanical stiffness and shear stiffness when compared to deoxyhemoglobin, oxyhemoglobin, and carboxyhemoglobin molecules. Further results from the models indicate that the hemoglobin structures studied possess a soft outer shell and a stiff core based on stiffness.     

Spectrophotometry of Hemoglobin: Absorption Spectra of Bovine Oxyhemoglobin, Deoxyhemoglobin, Carboxyhemoglobin, and Methemoglobin

The absorptivity at 540 nm of bovine hemiglobincyanide (cyanmethemoglobin) was determined on the basis of the iron content and found to be equal to the established value for human hemiglobincyanide (11.0 L · mmol⁻¹ · cm⁻¹). On this basis the absorption spectra of the common derivatives were determined for bovine hemoglobin. There proved to be only slight differences in the oxyhemoglobin, deoxyhemoglobin, and carboxyhemoglobin spectra between bovine and human hemoglobin. For comparison of the methemoglobin spectra a new series of measurements was made for human hemoglobin. As also found in the rat, the methemoglobin spectrum of bovine blood differed considerably from that in the human. These differences should be taken into account in multicomponent analysis.