Occurrence of root effect hemoglobinsin Amazonian fishes

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1. 1.The Root effect was measured in hemolysates from representatives of 56 genera of Amazonianfishes.
2. 2.Hemolysates from several species of air-breathing fishes were found to have Root effects, contraryto published hypotheses.
3. 3.Hemolysates from Potamotrygon, a freshwater ray, exhibit a Root effect under our experimental conditions.
4. 4.The pattern of Root effect distribution correlates positively with the distribution of choroid retiamirabile and swimbladders, but not with the distribution of swimbladder retia mirabile; it is proposed that the former is the more primitive structure which is associated with the origin of Root effect hemoglobins
5. 5.Some of the fish hemoglobins differ spectrally from one another. The positions of the absorptionmaxima of the deoxyhemoglobins range from 553 nm (Lepidosiren paradoxa and Potamotrygon sp.) to 560 nm (Plagioscion)
6. 6.Occurrence of Root effects is not correlated with the complexity of the hemoglobin electrophoreticpattern, although several species are found to have multiple hemoglobin systems in which the Root effect is restricted to certain components.

     

Presence and possible function of Zn in the hyaline cartilage.

Using histochemical and autoradiographical techniques the location of Zn in the hyaline cartilage of the Wistar rat was studied. A triple location was observed in the territorial matrix; in the chondrocyte; and in the perichondrio (cellular area). We believe that the molecules which were observed carrying Zn could be alkaline phosphatase, timidin-kinase and chondroitin-sulphuric acid.     

Presence and possible functional role of nerve growth factor in the thymus.

We have previously reported that the nerve growth factor (NGF), a polypeptide known for its neurotrophic activities, is also involved in proliferation, growth and survival of cells of the immune system. Working with animal models, we found that NGF and NGF-receptors (NGF-r) are present in the cells of the medullary layer of the thymus, a lymphoid gland involved in the production and differentiation of T-lymphocytes. Using immunohistochemical and biochemical approaches, we also showed that the expression of NGF in the thymus is high during late prenatal life and decreases later in postnatal life. A significant alteration of NGF levels was also found during pregnancy and aging, two events characterized by thymic involution. The aim of this study is to investigate whether NGF and NGF-r expression in the thymus are influenced by immuno- and neuro-pathological events. These observations will be presented and discussed.     

Molecular and functional adaptations in deep-sea hemoglobins

The past 20 years have witnessed the publication of numerous studies on hemoglobins (Hbs) from deep-sea animals. Most of the animals studied were collected at deep-sea hydrothermal vents and cold seeps, both being environments where the physical-chemical conditions may be severely challenging for metazoans. These environments may be characterized by deep, chronic hypoxia and high concentrations of toxic compounds such as sulfide and heavy metals. Many species from these environments express Hbs, even though they belong to taxa that otherwise were characterised by the absence of respiratory pigments. Hbs from vent and seep invertebrates commonly exhibit high affinities for oxygen when compared to related species from normoxic, shallow-water environments, and marked pH-dependence. These high affinities permit uptake of oxygen from hypoxic waters and the strong Bohr effects favor its release in the metabolizing acidic organs.     

Myoglobin in the heart tissue of fishes lacking hemoglobin

Myoglobin has been identified in the heart tissue of three species of antarctic icefish, Chaenocephalus aceratus, Pseudochaenichthys georgianus and Chaenodraco wilsoni. Quantitative analysis shows myoglobin concentrations that are substantially lower than other teleost fish. A simple and accurate method for the direct measurement of myoglobin in tissue is described.     

The origin and evolution of the surfactant system in fish: insights into the evolution of lungs and swim bladders

Several times throughout their radiation fish have evolved either lungs or swim bladders as gas-holding structures. Lungs and swim bladders have different ontogenetic origins and can be used either for buoyancy or as an accessory respiratory organ. Therefore, the presence of air-filled bladders or lungs in different groups of fishes is an example of convergent evolution. We propose that air breathing could not occur without the presence of a surfactant system and suggest that this system may have originated in epithelial cells lining the pharynx. Here we present new data on the surfactant system in swim bladders of three teleost fish (the air-breathing pirarucu Arapaima gigas and tarpon Megalops cyprinoides and the non-air-breathing New Zealand snapper Pagrus auratus). We determined the presence of surfactant using biochemical, biophysical, and morphological analyses and determined homology using immunohistochemical analysis of the surfactant proteins (SPs). We relate the presence and structure of the surfactant system to those previously described in the swim bladders of another teleost, the goldfish, and those of the air-breathing organs of the other members of the Osteichthyes, the more primitive air-breathing Actinopterygii and the Sarcopterygii. Snapper and tarpon swim bladders are lined with squamous and cuboidal epithelial cells, respectively, containing membrane-bound lamellar bodies. Phosphatidylcholine dominates the phospholipid (PL) profile of lavage material from all fish analyzed to date. The presence of the characteristic surfactant lipids in pirarucu and tarpon, lamellar bodies in tarpon and snapper, SP-B in tarpon and pirarucu lavage, and SPs (A, B, and D) in swim bladder tissue of the tarpon provide strong evidence that the surfactant system of teleosts is homologous with that of other fish and of tetrapods. This study is the first demonstration of the presence of SP-D in the air-breathing organs of nonmammalian species and SP-B in actinopterygian fishes. The extremely high cholesterol/disaturated PL and cholesterol/PL ratios of surfactant extracted from tarpon and pirarucu bladders and the poor surface activity of tarpon surfactant are characteristics of the surfactant system in other fishes. Despite the paraphyletic phylogeny of the Osteichthyes, their surfactant is uniform in composition and may represent the vertebrate protosurfactant.     

Structural and functional properties of class 1 plant hemoglobins

Nonsymbiotic class 1 plant hemoglobins are induced under hypoxia. Structurally they are protein dimers consisting of two identical subunits, each containing heme iron in a weak hexacoordinate state. The weak hexacoordination of heme-iron binding to the distal histidine results in an extremely high avidity to oxygen, with a dissociation constant in the nanomolar range. This low dissociation constant is due to rapid oxygen binding resulting in protein conformational changes that slow dissociation from the heme site. Class 1 hemoglobins are characterized by an increased rate of Fe3(+) reduction which is likely mediated by cysteine residue. This cysteine can form a reversible covalent bond between two monomers as shown by mass spectrometry analysis and, in addition to its structural role, prevents the molecule from autoxidation. The structural properties of class 1 hemoglobins allow them to serve as soluble electron transport proteins in the enzymatic system scavenging nitric oxide produced in low oxygen via reduction of nitrite. During oxygenation of nitric oxide to nitrate, oxidized ferric hemoglobin is formed (methemoglobin), which can be reduced by an associated reductase. The identified candidate for this reduction is monodehydroascorbate reductase. It is suggested that hemoglobin functions as a terminal electron acceptor during the hypoxic turnover of nitrogen, the process aided by its extremely high affinity for oxygen.     

The structure and function of plant hemoglobins.

Plants, like humans, contain hemoglobin. Three distinct types of hemoglobin exist in plants: symbiotic, non-symbiotic, and truncated hemoglobins. Crystal structures and other structural and biophysical techniques have revealed important knowledge about ligand binding and conformational stabilization in all three types. In symbiotic hemoglobins (leghemoglobins), ligand binding regulatory mechanisms have been shown to differ dramatically from myoglobin and red blood cell hemoglobin. In the non-symbiotic hemoglobins found in all plants, crystal structures and vibrational spectroscopy have revealed the nature of the structural transition between the hexacoordinate and ligand-bound states. In truncated hemoglobins, the abbreviated globin is porous, providing tunnels that may assist in ligand binding, and the bound ligand is stabilized by more than one distal pocket residue. Research has implicated these plant hemoglobins in a number of possible functions differing among hemoglobin types, and possibly between plant species.     

Comparative structural and functional studies of avian and mammalian hemoglobins

Thermal stabilities of chicken, grey lag goose (Anser anser), turkey as avian hemoglobins (Hbs); and human, bovine, sheep and horse as mammalian Hbs in hemolysate form were investigated and compared with oxygen affinities taken from literature. The thermal stability was obtained from thermal profiles using temperature scanning spectrophotometry. The buffer conditions were 50 mM Tris, pH 7.2, and 1 mM EDTA. The average of the inverse temperature transitions, average hydrophobicity, total van der Waals volume, partial molal volume and hydration potential were calculated by computational methods. The hemolysed avian Hbs have a lower oxygen affinity, higher thermal stability and higher self association than the mammalian Hbs. These observations are based on amino-acid composition, influence of ionic effectors, and the presence of Hb D in several avian Hbs. The results indicate that the avian Hbs have a more tense (T) conformation than the mammalian Hbs.     

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.     

Variation in protein structure and function: Primate hemoglobins

Summary Variation in structure among primate hemoglobins is associated with variation in function. This supports the hypothesis that most substitutions observed among homologous proteins in different species have been fixed by natural selection because they contribute to the fitness of the genotype. It does not support the concept that most substitutions result from the fixation of neutral alleles by genetic drift.     

A possible new mechanism of oxygen affinity modulation in mammalian hemoglobins

Bovine red cells do not contain appreciable amounts of 2,3-diphosphoglycerate (2,3-DPG). Bovine hemoglobin, however, has a particular sensitivity to chloride ions and as a result it can attain oxygen affinity values lower than those measured for human hemoglobin in the presence of 2,3-DPG. The interaction of bovine hemoglobin with anions is modulated by the hydrophobic characteristics of the protein. Comparison of the hydropathy plots of primate and ruminant hemoglobins indicates constant regions of opposite hydrophobicity, which have fixed amino acid differences. A model is proposed for explaining the regulation of oxygen affinity by chlorides, as an alternative to the classic modulation by 2,3-DPG.     

Allosteric effect of water in fish and human hemoglobins

Prompted by the reported lack of solvation effects on the oxygen affinity of fish (trout I) hemoglobin that questioned allosteric water binding in human hemoglobin A (Bellelli, A., Brancaccio, A., and Brunori, M. (1993) J. Biol. Chem. 268, 4742-4744), we have investigated solvation effects in fish and human hemoglobins by means of the osmotic stress method and allosteric analysis. In contrast to the earlier report, we demonstrate that water potential does affect oxygen affinity of trout hemoglobin I in the presence of inert solutes like betaine. Moreover, we show that upon oxygenation electrophoretically anodic hemoglobin from trout and eel bind a similar number of water molecules as does human hemoglobin A, whereas the cathodic hemoglobins of trout and eel bind smaller, but mutually similar, numbers of water molecules. Addition of cofactors strongly increases the number of water molecules bound to eel hemoglobin A (as in human hemoglobin) but only weakly affects water binding to eel hemoglobin C.     

Presence,localization and morphology of TELOCYTES in developmental gonads of fishes

Telocytes are a new defined type of interstitial cells, considered as a stem cell, with very long and thin cytoplasmic extensions. They are present in the vertebrates, and may participate in tissue remodeling. In fish, during gonadal development, the events that culminate with the germinal epithelium formation are well known. However, the interstitial compartment remains poorly explored, although it may have a great contribution to the morpho-functional changes that occur in the gonad. As in other organisms, in fish, the interstitium consists especially of connective tissue elements. However, until now, there are no reports of the presence and the action of the telocytes in the connective tissue of gonads of fish. Thus, this study aimed to detect the presence, localization and morphology of telocytes during the gonadal development of several species of fish. The gonads were analyzed by light microscopy, transmission electron microscopy and immunohistochemistry for localization of CD34, Vimentin, and metalloproteinases. The presence of two proteins characteristics of mesenchymal cell was detected in cells of the gonads of all species. In addition, they presented a typical morphology of telocytes, showing cellular extensions. Gonadal telocytes also presented positive response to metalloproteinases. In mammals, telocytes can undergo de-differentiation contributing to the reorganization of the extracellular matrix. This role may be performed by the metalloproteinases detected here. The detection of Vimentin and CD34 in the same cellular type, associated with its morphological characteristics, allows us to conclude that some interstitial cells in Teleostei are considered telocytes, identical to the ones already described in mammals and other vertebrates.     

Genetic variation and functional properties of Atlantic cod hemoglobins: introducing a modified tonometric method for studying fragile hemoglobins

Hemoglobin polymorphism in Atlantic cod has been investigated with respect to physiological performance at 10, 15 and 20 degrees C applying a modified tonometric method for O2 equilibrium analysis with full control of the equilibrating gas mixture. The results did not indicate any dissociation of the hemoglobins by a reduction in cooperativity and a parallel increase in affinity during the analytical procedure in contrast to the original tonometric method. With the applied preparation technique, we could store the hemolysate for 70 days at -25 degrees C without any significant changes in the O2 binding properties (P < 0.05) demonstrating the high quality of this procedure for analysing fragile fish hemoglobins. The present investigation demonstrates that the oxygen affinity of the hemoglobins varied between the genotypes. At all temperatures, except 20 degrees C and pH 8.0, Hb-I(2/2) had a higher O2 affinity than Hb-I(1/1). These results conform with previous results (16), suggesting Hb-I(2/2), the genotype which is the dominant allele in northern areas, to be the most efficient O2 carrier at low temperatures. The highest O2 affinity, however, was found for Hb-I(2/2b), supporting the results of Fyhn et al. (9), that this genotype is more restricted to coastal and warmer water and thus a better marker of the coastal population. Our results further suggest a correlation between genotype specific growth rates and oxygen affinities at all temperatures studied, with the highest growth rates observed in those genotypes having the highest O2 affinities. In conclusion, the hemoglobin polymorphism of cod seems to be correlated with physiological performance.