Biomedical subjects

A Fago

Publications and source records attributed to A Fago.

14 recordsLinked to original sources

PubMed2005

Effects of short-term hypoxia on neuroglobin levels and localization in mouse brain tissues.

Nerve cells are highly susceptible to ischemic and hypoxic injuries. The neuroglobin (Ngb), found in vertebrate nerve cells, has been suggested to protect nerve cells from ischemic episodes by a yet unknown mechanism. However, contradicting reports exist regarding localization and up-regulation of Ngb in response to hypoxia. The aim of the present study was to probe the distribution of Ngb proteins in mouse brain and retina by immunohistochemistry, and to quantify the levels of Ngb mRNA by reverse-transcription-polymerase chain reaction (RT-PCR) after short-term (2 h) exposure to 7.6% oxygen. We found Ngb to be present throughout the neocortex, most abundantly in the perirhinal, entorhinal and temporal cortical areas, the thalamus and hypothalamus, the choroid plexus, the olfactory bulb and the cranial nerve nuclei in the brainstem. Intense staining was observed in the mesencephalic central grey area and the Purkinje cells. Two-hour hypoxic exposure caused no detectable changes in staining intensity or spatial distribution of Ngb neither in the Purkinje cells nor in any other brain areas observed. The RT-PCR data supported the lack of differences in brain Ngb levels between normal and oxygen-deprived animals. In the retina, Ngb localization by immunohistochemistry was confined to the inner segments of the photoreceptors, the plexiform layers and the ganglion cells. Short-termed hypoxia did not change retinal Ngb levels as assessed by both techniques. The lack of Ngb up-regulation in the brain is consistent with results from previous long-term hypoxic experiments, suggesting that Ngb is not regulated by pure hypoxia in vivo.

Animals↗

PubMed2001

Hagfish hemoglobins: structure, function, and oxygen-linked association.

Cyclostomes, hagfishes and lampreys, contain hemoglobins that are monomeric when oxygenated and polymerize to dimers or tetramers when deoxygenated. The three major hemoglobin components (HbI, HbII, and HbIII) from the hagfish Myxine glutinosa have been characterized and compared with lamprey Petromyzon marinus HbV, whose x-ray crystal structure has been solved in the deoxygenated, dimeric state (Heaslet, H. A., and Royer, W. E., Jr. (1999) Structure 7, 517-526). Of these three, HbII bears the highest sequence similarity to P. marinus HbV. In HbI and HbIII the distal histidine is substituted by a glutamine residue and additional substitutions occur in residues located at the deoxy dimer interface of P. marinus HbV. Infrared spectroscopy of the CO derivatives, used to probe the distal pocket fine structure, brings out a correlation between the CO stretching frequencies and the rates of CO combination. Ultracentrifugation studies show that HbI and HbIII are monomeric in both the oxygenated and deoxygenated states under all conditions studied, whereas deoxy HbII forms dimers at acidic pH values, like P. marinus HbV. Accordingly, the oxygen affinities of HbI and HbIII are independent of pH, whereas HbII displays a Bohr effect below pH 7.2. HbII also forms heterodimers with HbIII and heterotetramers with HbI. The functional counterparts of heteropolymer formation are cooperativity in oxygen binding and the oxygen-linked binding of protons and bicarbonate. The observed effects are explained on the basis of the x-ray structure of P. marinus HbV and the association behavior of site-specific mutants (Qiu, Y., Maillett, D. H., Knapp, J., Olson, J. S., and Riggs, A. F. (2000) J. Biol. Chem. 275, 13517-13528).

Amino Acid Sequence↗

PubMed2001

Oxygen binding by single red blood cells from the red-eared turtle Trachemys scripta.

Oxygen-binding properties of single red blood cells from the red-eared turtle Trachemys scripta were measured by microspectrophotometry to describe the variation in oxygen affinity of red blood cells and to gain insight into the distribution of functionally different hemoglobins among red blood cells. Methodologically, this study represents the first report on the cell-to-cell variation in oxygen-binding properties based on oxygen-binding curves of single vertebrate red blood cells. The cells differed significantly with respect to oxygen affinity. Mean oxygen pressure at half saturation of the cells in a blood sample was found to be 20.1 +/- 3.3 (SD) Torr. The distribution of oxygen affinities among red blood cells is unimodal, indicating that the two hemoglobins found in turtle blood are not segregated in distinct cells. Therefore, the functional interaction shown by these hemoglobins in vitro is likely to take place in vivo. The considerable variation in oxygen affinity between individual red blood cells calls for its incorporation in models of tissue oxygenation.

Adenosine Triphosphate↗

PubMed2000

Isohemoglobin differentiation in the bimodal-breathing amazon catfish Hoplosternum littorale.

The bimodal gill(water)/gut(air)-breathing Amazonian catfish Hoplosternum littorale that frequents hypoxic habitats uses "mammalian" 2,3-diphosphoglycerate (DPG) in addition to "piscine" ATP and GTP as erythrocytic O(2) affinity modulators. Its electrophoretically distinct anodic and cathodic hemoglobins (Hb(An) and Hb(Ca)) were isolated for functional and molecular characterization. In contrast to Hb(An), phosphate-free Hb(Ca) exhibits a pronounced reverse Bohr effect (increased O(2) affinity with decreasing pH) that is obliterated by ATP, and opposite pH dependences of K(T) (O(2) association constant of low affinity, tense state) and the overall heat of oxygenation. Dose-response curves indicate small chloride effects and pronounced and differentiated phosphate effects, DPG < ATP < GTP < IHP. Hb(Ca)-O(2) equilibria analyzed in terms of the Monod-Wyman-Changeux model show that small T state bond energy differences underlie the differentiated phosphate effects. Synthetic peptides, corresponding to N-terminal fragment of the cytoplasmic domain of trout band 3 protein, undergo oxygenation-linked binding to Hb(Ca), suggesting a metabolic regulatory role for this hemoglobin. The amino acid sequences for the alpha and beta chains of Hb(Ca) obtained by Edman degradation and cDNA sequencing show unusual substitutions at the phosphate-binding site that are discussed in terms of its reverse Bohr effect and anion sensitivities.

2,3-Diphosphoglycerate↗

PubMed2000

Respiratory responses to short term hypoxia in the snapping turtle, Chelydra serpentina.

Among vertebrates, turtles are able to tolerate exceptionally low oxygen tensions. We have investigated the compensatory mechanisms that regulate respiration and blood oxygen transport in snapping turtles during short exposure to hypoxia. Snapping turtles started to hyperventilate when oxygen levels dropped below 10% O(2). Total ventilation increased 1.75-fold, essentially related to an increase in respiration frequency. During normoxia, respiration occurred in bouts of four to five breaths, whereas at 5% O(2), the ventilation pattern was more regular with breathing bouts consisting of a single breath. The increase in the heart rate between breaths during hypoxia suggests that a high pulmonary blood flow may be maintained during non-ventilatory periods to improve arterial blood oxygenation. After 4 days of hypoxia at 5% O(2), hematocrit, hemoglobin concentration and multiplicity and intraerythrocytic organic phosphate concentration remained unaltered. Accordingly, oxygen binding curves at constant P(CO(2)) showed no changes in oxygen affinity and cooperativity. However, blood pH increased significantly from 7.50+/-0.05 under normoxia to 7.72+/-0.03 under hypoxia. The respiratory alkalosis will produce a pronounced in vivo left-shift of the blood oxygen dissociation curve due to the large Bohr effect and this is shown to be critical for arterial oxygen saturation.

Adaptation, Physiological↗

PubMed1999

Bicarbonate binding to hemoglobin links oxygen and carbon dioxide transport in hagfish.

Hagfish are unusual among vertebrates in having red cells that almost completely lack the anion-exchanger membrane protein band III and hemoglobins that are highly sensitive to CO2 but only weakly affected by protons. This suggests a different linkage between oxygen and CO2 transport from that of the majority of vertebrates, which is characterized by oxygen-linked proton binding to hemoglobin and chloride-bicarbonate exchange across band III protein. We here report that the hemoglobin of the hagfish Myxtne glutinosa shows oxygen-linked binding of bicarbonate, which decreases the oxygen affinity and thereby enhances oxygen unloading to the tissues. Bicarbonate binding to the hemoglobin moreover facilitates the hydration of CO2 and its transport as intraerythrocytic bicarbonate, and may compensate at least in part for the virtual absence of band III protein. This represents a unique linkage between oxygen and CO2 transport among vertebrates, where the physiological role of protons in the Bohr and Haldane effects is played by bicarbonate ions.

Acetazolamide↗

PubMed1997

The anodic hemoglobin of Anguilla anguilla. Molecular basis for allosteric effects in a root-effect hemoglobin.

The functional and structural basis for the Root effect has been investigated in the anodic hemoglobin of the European eel, Anguilla anguilla. This hemoglobin exhibits a large Bohr effect, which is accounted for by oxygen-linked binding of seven to eight protons in the presence of GTP at pH 7.5. Oxygen equilibrium curves show nonlinear lower asymptote of Hill plots, indicating the occurrence of heme-heme interactions within the T state. Analysis of the curves according to the co-operon model (Brunori, M., Coletta, M., and Di Cera, E. (1986) Biophys. Chem. 23, 215-222) reveals that T state cooperativity is positive at high pH and in the stripped hemoglobin (where the T --> R allosteric transition is operative) and negative at low pH and in the presence of organic phosphate (where the molecule is locked in the low affinity structure), indicating site heterogeneity. The complete amino acid sequence of eel anodic hemoglobin has been established and compared with that of other fish hemoglobins. The presence of the Root effect correlates with a specific configuration of the alpha1beta2 switch interface, which at low pH would stabilize subunit ligation in the T state without changing the quaternary structure. We propose that the major groups involved in the binding of oxygen-linked protons in eel anodic hemoglobin are located on the beta chain and comprise His-HC3 at the C terminus, His-FG4 at the switch interface, and Lys-EF6 and the N terminus at the phosphate-binding site.

Amino Acid Sequence↗

PubMed1997

Oxygen binding and aggregation of hemoglobin from the common European frog, Rana temporaria.

The hemoglobin from the European frog, Rana temporaria, consists of one major and three minor components. The tetramers aggregate upon deoxygenation notably at pH 7:3. Aggregation due to formation of disulphide bridges, as occurs in related species, was observed only in polyacrylamide gels. The hemolysate showed a pronounced Bohr effect. Oxygen affinity decreased with increasing hemoglobin concentration, indicating that aggregation affects the functional properties of the hemolysate. Oxygen binding equilibria of unfractionated hemolysate are insensitive to chloride and show low sensitivity to ATP. Analysis of oxygen equilibrium curves in terms of the two-state allosteric model (MWC) showed that pH change exerted a greater effect on the association constant of the oxygenated state (KR) than that of the deoxy state (KT). The number of interacting binding sites (q) increased with hemoglobin concentration. Cooperativity of oxygen binding, evaluated as Hill coefficient n, never exceeded the value of 3.0. Earlier studies on hemoglobin and blood from this and related species, report significantly higher n values at high O2 saturation. Molecular adaptive aspects are discussed.

Animals↗

PubMed1996

The unique hemoglobin system of Pleuragramma antarcticum, an antarctic migratory teleost. Structure and function of the three components.

Pleuragramma antarcticum (suborder Notothenioidei, family Nototheniidae) is the most abundant fish in the antarctic shelf. This pelagic species has a circum-antarctic distribution and is characterized by spawning migration. This species displays the highest multiplicity of major hemoglobins (three); the other notothenioids have a single one (except one species, having two) with relatively low oxygen affinity regulated by pH and organophosphates. The hemoglobins of P. antarcticum display strong Bohr and Root effects; however, they reveal important functional differences in subunit cooperativity and organophosphate regulation and, above all, in the response of oxygenation to temperature. Despite the substitution ValbetaE11 --> Ile found in Hb 2, which decreases the affinity in human mutants, the hemoglobins have similar oxygen affinity, higher than that of the other notothenioids. Hb 1 has the alpha chain in common with Hb 2 and the beta in common with Hb 3. The amino acid sequence of all four chains has been established. Thus the hematological features of P. antarcticum differ remarkably from those of antarctic notothenioids. This unique and sophisticated oxygen transport system may adequately meet the requirements of the unusual mode of life of this fish.

Allosteric Regulation↗

PubMed1995

The cathodic hemoglobin of Anguilla anguilla. Amino acid sequence and oxygen equilibria of a reverse Bohr effect hemoglobin with high oxygen affinity and high phosphate sensitivity.

As in other fish, the cathodic hemoglobin of the eel Anguilla anguilla is considered to play an important role in oxygen transport under hypoxic and acidotic conditions. In the absence of phosphates this hemoglobin shows a reverse Bohr effect and high oxygen affinity, which is strongly modulated over a side pH range by GTP (whose concentration in the red blood cells varies with ambient oxygen availability). GTP obliterates the reverse Bohr effects in the cathodic hemoglobin. The molecular basis for the reverse Bohr effect in fish hemoglobins has remained obscure due to the lack of structural data. We have determined the complete amino acid sequence of the alpha and beta chains of the cathodic hemoglobins of A. anguilla and relate it to the oxygen equilibrium characteristics. Several substitutions in crucial positions are observed compared with other hemoglobins, such as the replacement of the C-terminal His of the beta chain of Phe (that suppresses the alkaline Bohr effect) and of residues at the switch region between alpha and beta subunits (that may alter the allosteric equilibrium, thus causing the high intrinsic oxygen affinity and low cooperativity). The residues binding organic phosphate in the beta cleft of fish hemoglobins are conserved, which explains the strong effect of GTP on oxygen affinity and suggests that these residues contribute to the reverse Bohr effect in the absence of alkaline Bohr groups. Moreover, His beta 143 that is considered to be responsible for the reverse Bohr effect in human and tadpole Hbs is replaced by Lys.

Amino Acid Sequence↗

PubMed1995

The hemoglobin system of the hagfish Myxine glutinosa: aggregation state and functional properties.

Hemoglobin (Hb) from the hagfish Myxine glutinosa is composed of six major monomeric subunits. Some of these subunits aggregate to dimers at low pH, and to tetramers when deoxygenated and at high protein concentration. The aggregation is inhibited by the presence of KCl. Oxygen equilibrium studies show the presence of a small Bohr effect which is strongly reduced by KCl, indicating that it originates from pH-dependent aggregation. ATP and DPG cause a similar decrease in the Bohr effect. O2 affinity is dependent on protein concentration, temperature and presence of CO2. Cooperativity is practically absent. O2 binding properties of the separated aggregating and non-aggregating Hbs purified at low pH cannot account for the functional properties of the composite hemolysate, suggesting the presence of other subunits interactions. The results are discussed in relation to literature data for other cyclostome Hbs and for M. glutinosa Hb, where the presence of three major monomeric Hbs and a possible CO2-dependent aggregation had been reported.

Animals↗

PubMed1994

Hemoglobins from Plasmodium-infected rat erythrocytes: functional and molecular characteristics.

Aiming to evaluate the mechanisms responsible for altered O2-transporting properties in blood of Plasmodium-infected animals, stripped (cofactor-free) hemoglobin (Hb) solutions were prepared from infected erythrocytes (IE) and noninfected erythrocytes (NIE) of rats inoculated with Plasmodium berghei bergei for functional and structural characterization. At normal intraerythrocytic pH (+/- 7.2), Hb from IE showed a higher affinity, a larger Bohr effect, and lower sensitivities to 2,3-diphosphoglycerate (DPG) and to temperature than did NIE Hb. Moreover, as judged from electrophoresis, isoelectric focusing, and gel filtration experiments, Hb from IE show changes in charge and molecular assembly. The results indicate that the higher O2 affinity and greater Bohr factors observed in IE compared with those for NIE are attributable to chemical modification of the Hb that increases its intrinsic O2 affinity and decreases its sensitivity to DPG as well as to changes in the intracellular physicochemical milieu, including reduced DPG levels.

Animals↗

PubMed1993

A polymerising Root-effect fish hemoglobin with high subunit heterogeneity. Correlation with primary structure.

The blood of the teleost Chelodonichthys kumu, living in the temperate waters of New Zealand, contains a single hemoglobin. The complete amino acid sequence of the alpha and beta chain has been established. The presence of a reactive Cys in the external position beta CD8(49) causes polymerisation through intermolecular disulfide bridges between beta chains, with no alteration of functional features. C. kumu Root-effect hemoglobin displays very low or no subunit co-operativity in the physiological pH range. Kinetic experiments on the oxygen dissociation and binding of carbon monoxide show a marked, pH-dependent functional heterogeneity of the two chains, which contributes to the observed reduction of co-operativity. In contrast, kinetic heterogeneity was not observed in the process of CO dissociation, indicating that functional differences between the subunits are detectable only for the dynamic ligand association pathway. The allosteric effector, ATP, seems to increase the pKa of the proton-linked effect on the slow-reacting subunit, affecting the quaternary equilibrium through stabilisation of the T state at lower pH, rather than enhancing the functional heterogeneity itself. In position E11 of both chains, Val (usually present at the distal side of the heme), is substituted by Ile. Although this residue has been shown not to significantly alter ligand binding to the alpha chain, to some extent it can perturb the access of oxygen to the beta chain. Thus, this substitution may be the main reason for subunit functional heterogeneity.

Adenosine Triphosphate↗

PubMed1992

The hemoglobins of Notothenia angustata, a temperate fish belonging to a family largely endemic to the Antarctic Ocean.

The blood of the teleost Notothenia angustata contains a major hemoglobin (Hb 1, over 95% of the total), accompanied by a minor component (Hb 2). The two hemoglobins have identical beta chains and differ in their alpha chains. The primary structure of both hemoglobins has been established through the elucidation of the complete amino acid sequence of the three chains. The study of the oxygen-binding properties shows that Hb 1 displays the Bohr and Root effects and has high affinity for organic phosphates. N. angustata belongs to the family Nototheniidae, suborder Notothenioidei. Unlike the vast majority of nototheniid species, which live in isolation in the Antarctic Ocean and have developed cold adaptation, N. angustata inhabits the waters of southern New Zealand and is not cold adapted. Although some hematological parameters typically favour oxygen transport in a temperate environment, the hemoglobin multiplicity and structural and functional features closely resemble those of the Antarctic species of the same family and suborder. Thus, N. angustata may be considered as a link between temperate and Antarctic habitats. The hypothetical separation history of N. angustata from the Antarctic species of the same family is discussed in the light of the present findings.

Acclimatization↗