Structure and reactivity of hexacoordinate hemoglobins

The heme prosthetic group in hemoglobins is most often attached to the globin through coordination of either one or two histidine side chains. Those proteins with one histidine coordinating the heme iron are called "pentacoordinate" hemoglobins, a group represented by red blood cell hemoglobin and most other oxygen transporters. Those with two histidines are called "hexacoordinate hemoglobins", which have broad representation among eukaryotes. Coordination of the second histidine in hexacoordinate Hbs is reversible, allowing for binding of exogenous ligands like oxygen, carbon monoxide, and nitric oxide. Research over the past several years has produced a fairly detailed picture of the structure and biochemistry of hexacoordinate hemoglobins from several species including neuroglobin and cytoglobin in animals, and the nonsymbiotic hemoglobins in plants. However, a clear understanding of the physiological functions of these proteins remains an elusive goal.     

Presence and possible function of root effect hemoglobins in fishes lacking functional swim bladders

The adult hemoglobins of 15 species of teleost and the midgestation fetal hemoglobin of the seaperch Embiotoca lateralis show a pronounced decrease in oxygen-carrying capacity at low pH, a Root effect. All of these fishes lack a functional swim bladder, which is generally thought to be the likely site of Root effect hemoglobin function. All of the teleosts examined, including the fetal sea perch, however, have a choroid rete, a structure that is proposed to be involved in oxygen secretion to the eye. The data are inconsistent with the generalization that only fishes with swim bladders possess Root effect hemoglobins, and that the only function of Root effect hemoglobins is in the secretion of oxygen to a swim bladder. The data for the fishes examined in this study suggests that a better correlation may exist between Root effect hemoglobins and the presence of a choroid rete. This is consistent with the hypothesis that Root effect hemoglobins may be involved in the physiology of the eye in many fishes.     

The effect of experimental anaemia on red cell glutathione reductase

The effect of experimental anaemia on red cell glutathione reductase (GR) was investigated in rabbits, guinea-pigs and cattle. The ratio of active and inactive forms of GR did not change during the experimental period in either the rabbits or the cattle. However, there was a marked rise in the ratio of inactive form in the guinea-pigs during anaemia. It appears that, in addition to the most important regulator of GR activity, namely the flavin content of the red blood cells, there are other mechanisms which are operating in the red blood cells of anaemic animals and that these mechanisms vary among different species of animals.     

Hemoglobin of the Antarctic fishes Trematomus bernacchii and Trematomus newnesi: structural basis for the increased stability of the liganded tetramer relative to human hemoglobin

Hemoglobins extracted from fishes that live in temperate waters show little or no dissociation even in the liganded form, unlike human hemoglobin (HbA). To establish whether cold adaptation influences the tendency to dissociate, the dimer-tetramer association constants (L(2,4)) of the carbonmonoxy derivatives of representative hemoglobins from two Antarctic fishes, Trematomus newnesi (Hb1Tn) and Trematomus bernacchii (Hb1Tb), were determined by analytical ultracentrifugation as a function of pH in the range 6.0-8.6 and compared to HbA. HbA is more dissociated than fish hemoglobins at all pH values and in particular at pH 6.0. In contrast, both fish hemoglobins are mostly tetrameric over the whole pH range studied. The extent of hydrophobic surface area buried at the alpha(1)beta(2) interface upon association of dimers into tetramers and the number of hydrogen bonds formed are currently thought to play a major role in the stabilization of the hemoglobin tetramer. These contributions were derived from the X-ray structures of the three hemoglobins under study and found to be in good agreement with the experimentally determined L(2,4) values. pH affects oxygen binding of T. bernacchii and T. newnesi hemoglobins in a different fashion. The lack of a pH effect on the dissociation of the liganded proteins supports the proposal that the structural basis of such effects resides in the T (unliganded) structure rather than in the R (liganded) one.     

Plants,humans and hemoglobins

New developments have forced a re-evaluation of our understanding of the structure and function of hemoglobins. Leghemoglobins regulate oxygen affinity through a mechanism different from that of myoglobin using a novel combination of heme pocket amino acids that lower the oxygen affinity. The hexacoordinate hemoglobins are characterized by intramolecular coordination of the ligand binding site at the heme iron, and were first identified in plants as the 'non-symbiotic plant hemoglobins'. They are now known to be present in animals and bacteria. Many of these proteins are upregulated in both plants and animals during hypoxia or similar stresses. Therefore, there might be a common physiological function for hexacoordinate hemoglobins in plants and animals.     

Isolation of glycosylated hemoglobins by a combination of ion-exchange and gel-filtration chromatographies: a pilot study

The techniques of ion exchange and gel filtration have been combined in a single chromatographic column which allows the simultaneous isolation of hemoglobins glycosylated at their beta-amino termini from other hemoglobin species as well as from molecules differing in size from the hemoglobins. This method is unique because it makes possible isolation of preparative quantities of glycosylated hemoglobins within approximately 15 min. The method works most efficiently with a dry weight-to-weight ratio of Biorex 70 to Sephadex G25 of 1.4 to 1.0. The technique was applied to the determination of the apparent first-order rate constant for the deglycosylation of the labile form of hemoglobin AIc.     

Glycosylated hemoglobins in a diabetic patient with sickle cell anemia

Results of analysis of blood samples from a diabetic sickle cell anemia (SS) patient and 4 nondiabetic SS patients for glycosylated hemoglobins by Bio-Rex 70 chromatography, high-pressure liquid chromatography, and affinity chromatography are presented. Glycosylated components of Hb S and Hb A2 and total glycosylated hemoglobins were quantitated in this manner. The levels of the various glycosylated hemoglobins were increased twofold in the diabetic patient compared to nondiabetic SS patients. The glycosylated hemoglobin levels in the diabetic SS patient and in the nondiabetic SS patients, however, were significantly lower than the levels normally seen in nonsickle diabetics and normal adults, respectively. In contrast to a previously reported diabetic SS patient, the present case appears to be not severely affected by sickle cell disease.     

A comparative characteristic of effector innervation of cerebral arteries in mammals and humans.

The neural fibres of cerebral arteries in humans, rats, guinea-pigs, rabbits, cats, dogs, swine, cows and horses have been studied using the methods of Flack-Hillarp and Koelle. The large arteries of the carotid and vertebral systems bear dense cholinergic and adrenergic plexuses formed by transversal and longitudial neural fibres, which are located in the superficial and deep adventitial layers. The highest density of cholinergic and adrenergic fibres was observed on the arteries of humans and large mammals. Depending on the density of cholinergic and adrenergic fibres in the said arteries, the animals may be listed in the following order: rats, guinea-pigs, rabbits, cats, dogs, swine, cows and horses. Basic differences in the structure of neural plexuses of humans and animals were not observed.     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 1. Purification and characterisation

Antarctic fishes live at a constant temperature of -1.8 degrees C, in an oxygen-rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, 'stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.     

氟骨症不同动物模型的建立与评价

目的 为选择合适的实验动物并建立氟骨症动物模型,研究不同动物对氟化物致骨骼损伤的敏感性.方法 分别选取离乳新西兰兔、Hartley豚鼠、Wistar大鼠、KM小鼠各16只,每种动物随机分为两组,每组8只,即对照组(C):饲喂标准饲料,饮用去离子水;模型组(M):饲喂标准饲料,饮用150 mg/L氟化钠(NaF)去离子水.在饲喂60 d后取股骨制作组织切片观察组织形态变化,并测定股骨骨氟、骨钙、骨磷的含量及血清碱性磷酸酶(ALP)活性.结果 与对照组相比,各种实验动物模型组生长发育受到显著的抑制,股骨形态破坏明显,骨矿化显著减少,骨氟含量显著升高,血清ALP活性显著增强,但股骨钙和磷含量差异无显著性(P＞0.05).结论 高氟可不同程度的损害豚鼠、家兔、小鼠、大鼠的生长发育及骨骼系统,导致产生氟骨症,但综合考察,以豚鼠、家兔较为敏感,其次为小鼠、大鼠.     

Structural and functional properties of hemoglobins from unicellular organisms as revealed by resonance Raman spectroscopy

Hemoglobins have been discovered in organisms from virtually all kingdoms. Their presence in unicellular organisms suggests that the gene for hemoglobin is very ancient and that the hemoglobins must have functions other than oxygen transport, in view of the fact that O2 delivery is a diffusion-controlled process in these organisms. Based on sequence alignment, three groups of hemoglobins have been characterized in unicellular organisms. The group-one hemoglobins, termed truncated hemoglobins, consist of proteins with 110-140 amino acid residues and a novel two-over-two alpha-helical sandwich motif. The group-two hemoglobins, termed flavohemoglobins, consist of a hemoglobin domain, with a classical three-over-three alpha-helical sandwich motif, and a flavin-containing reductase domain that is covalently attached to it. The group-three hemoglobins consist of myoglobin-like proteins that have high sequence homology and structural similarity to the hemoglobin domain of flavohemoglobins. In this review, recent resonance Raman studies of each group of these proteins are presented. Their implications are discussed in the context of the structural and functional properties of these novel hemoglobins.     

Monosaccharides bound to hemoglobins in normal and diabetic individuals. Evidence for glucose, mannose and galactose as sugars released by methanolysis of the different hemoglobin components

Direct evidence is given for the presence of glucose, mannose and galactose as the products of hydrolysis of hemoglobins A1a1, A1a2, A1b, A1c and A0. The presence of galactose cannot be explained by the earlier hypothesis of Amadori rearrangement and suggests the existence of further complex rearrangements. Monosaccharide content of the different hemoglobin components varies from 0.2-2.0 mol/mol of alpha beta dimer with an increase of 1.5-2.0-times in diabetic components. This increase is not accompanied by net charge differences, suggesting that additionally bound sugars are not responsible for the pI modification of these hemoglobins. The pattern of glucose, mannose and galactose ratio in normal individuals divides these hemoglobins into two classes, hemoglobins A1b, A1c and A0 (ratio 0.60:0.25:0.15) on one hand and hemoglobins A1a1 and A1a2 (ratio 0.40:0.40: 0.20) on the other. These findings suggest that diverse mechanisms for sugar binding might exist between these two classes of glycosylated hemoglobins. This difference disappears in diabetic components suggesting that the non-NH2-terminal sites are glycosylated in all components by a common mechanism. Increase in glucose at the expense of mannose and galactose, as observed in diabetics, could be an indicator of recent glycosylation.     

Preparation of monospecific antisera to IgA in laboratory animals using cascade immunization without preliminary isolation of the antigen

The proposed method of "cascade" immunization for the preparation of monospecific antisera to IgA of the experimental animals (mice, rats, guinea-pigs) requires no isolation and purification of an antigen, since it is based on using precipitation lines containing IgA. The subsequent steps of the method are the following: 1) preparation of a polyspecific antiserum against one of the secretions or IgA-containing serum fraction; 2) preparation of precipitation lines using this antiserum and some other secretions; 3) immunization of other rabbits with these precipitation lines to obtain monospecific anti-IgA antiserum. The antisera obtained do not require any additional absorption except for the removal of anti-IgA antibodies. The method can be used for the preparation of monospecific antisera to any other serum or secretion proteins.     

Central nervous system myelin of teleosts: comparative electrophoretic analysis of its proteins by staining and immunoblotting

CNS myelin was isolated from 24 teleostean fishes and the proteins were analyzed by staining and immunoblotting. All species showed a 36 K protein, two or more glycosylated hydrophobic intermediate protein (IP) components and several myelin basic protein bands (BP). The 36 K protein was specific for teleostean fishes. The IP and BP components displayed substantial variations in their proportions as well as in molecular sizes when comparing the different teleosts. This contrasts with CNS myelin proteins which appear more stable in terrestrial vertebrates.     

Immunological reaction of guinea-pigs following intranasal Mycoplasma pneumoniae infection and immunization with the 168 kDa adherence protein

Humoral responses to Mycoplasma pneumoniae proteins, especially the 168 kDa protein, were demonstrated by Western blotting in sera and bronchial washings of all groups of infected or immunized guinea-pigs. However, infection was not prevented by these local and systemic antibodies. Hilar lymphocytes of infected and immunized guinea-pigs were stimulated in vitro by sonicated M. pneumoniae antigen and by the 168 kDa protein. Stimulation was significantly lower in animals which had been infected twice or had been preimmunized and challenged by infection. Histologically the most severe lesions were seen in the twice-infected group followed by the preimmunized group which was subsequently infected.     

The effect of various host species on the feeding performance of immature stages of the tick Hyalomma truncatum (Acari: Ixodidae)

In order to determine the effect of various hosts on the feeding performance of the tick Hyalomma truncatum, we used three mammalian species as hosts. Larvae and nymphs of H. truncatum were fed, under controlled laboratory conditions, on gerbils, guinea-pigs and rabbits. The larvae fed for 4.3±1.4 days on gerbils, 5.6±1.3 days on guinea-pigs and 4.7±1.2 days on rabbits. The mean weights of the larvae which fed on the rabbits, guinea-pigs and gerbils were 0.58± 0.09, 0.46±0.04 and 0.45±0.04 mg. respectively. The feeding periods of the nymphs on gerbils, guinea-pigs and rabbits were 7.9±1.3, 8.6±1.3 and 9.6±2.2 days respectively. The mean weights of the nymphs which fed on the gerbils, guinea-pigs and rabbits were 22.5±2.8, 19.7±1.3 and 15.8±1.4 mg, respectively. Hyalomma truncatum demonstrated a life cycle of a three-host tick on gerbils and guinea-pigs and of a two-host tick on rabbits. The evolutionary advantage of a two-host cycle over a three-host cycle in metastriate ticks is discussed.     

The phylogeny of polar fishes and the structure,function and molecular evolution of hemoglobin

Fishes thriving in polar habitats offer many opportunities for comparative approaches to understanding protein thermal adaptations. Investigations on the remarkable evolutionary adaptations to these environments of basic proteins such as hemoglobin, the oxygen carrier, can provide new insights into the mechanisms studied in temperate organisms and can shed light on convergent processes evolved in response to thermal adaptations. At the molecular level, hemoglobins are one of the most intriguing systems for studying the relationships between environmental conditions and adaptations. This review summarizes the current knowledge on molecular structure, biological function and phylogeny of hemoglobins of fish species living in both polar habitats but having different evolutionary histories. In benthic, non-migratory, cold-adapted fishes, the stability of thermal conditions may have generated no or few variations in selective pressures on globin sequences through evolutionary time, so that sequences retain the species phylogenetic “signal”. In pelagic, migratory, cold-adapted or temperate fishes, variations in selective pressures on globin sequences caused by variations in temperature accompanying the dynamic life style may have disrupted the phylogenetic “signal” in phenetic trees.     

Stability of the microbial populations of the caecum of guinea-pigs and rabbits

The microbial population of the caecum of rabbits and guinea-pigs, respectively, normally exhibits distinctive features which can be tabulated precisely, and which persist when the animals are kept in the same cage and fed on the same diet. A pronounced resistance, therefore, opposes cross-infection and tends to secure the stability of the population whose characteristics afford useful criteria for the microbiological control of in vitro incubation experiments.     

Activation in vitro of some biological systems by extracts of adult worms and microfilariae of Dirofilaria immitis

Extracts of Dirofilaria immitis adults and microfilariae cause the degranulation of mast cells in rat mesentery when applied in vitro and also cause increased vascular permeability in guinea-pigs and rabbits. Adult worms but not microfilariae are able to activate complement in vitro and both stages contain a plasminogen activator. No proteolytic activity or haemolysins could be detected in either stage. The possibility that these factors may be important in the pathogenesis of dirofilariasis and in the adverse drug reactions that may follow treatment of microfilaraemic animals with diethylcarbamazine is discussed.     

Negative cooperativity in Root-effect hemoglobins: role of heterogeneity

In some animals, the oxygen transport capacity of blood decreaseswhen pH is lowered, yielding oxygen binding curves with Hill-coefficientssmaller than unity. This so-called Root effect is observed inseveral fishes and is important for creating large oxygen partialpressures locally, for example in the swim bladder. While thereis general agreement on the physiological advantages of thiseffect, its molecular basis remains ambiguous. Various studiesshow that isoforms of hemoglobins usually are present in thehemolymph, when the Root effect is observed. Here, we show thatin such a case the mixture of these isoforms can exhibit apparentnegative cooperativity, although each component taken separatelycan be described by the MWC model. In other cases, isolatedisoforms exhibit true negative cooperativity. The well establishedMWC model describes many cooperative phenomena of enzymes andrespiratory proteins but is not capable of describing negativecooperativity. In order to model negative cooperativity withina single molecular species a decoupling model might be employed,as pointed out previously. However, simulations show that itis not mandatory to have species with negative cooperativity,in order to obtain the binding curves typically seen for wholeblood. These two aspects of the Root effect will be discussedon the basis of data from the literature.