Observation of a rare hemoglobin variant [Hb Lulu island, beta107(G9)Gly-->Asp, GGC-->GAC] co-inherited with a beta+-thalassemia mutation [IVS-I-110 (G-->A)] or in the heterozygous state in a Greek-Albanian family.

We report clinical, hematological, biochemical, functional and molecular studies carried out on two first cousins from a Greek-Albanian family who have clinical and hematological findings consistent with the diagnosis of thalassemia intermedia. DNA studies determined that they had co-inherited a common Mediterranean beta-thalassemia (thal) mutation, IVS-I-110 (G-->A), in trans to a beta-globin gene mutation at codon 107 (GGC-->GAC), predicted to give rise to a rare unstable beta chain variant Hb Lulu Island or beta107(G9)Gly-->Asp.

The homozygous state for Hb Crete [beta129 (H7) Ala-->Pro] is associated with a complex phenotype including erythrocytosis and functional anemia.

Hb Crete, an electrophoretically neutral, unstable, high oxygen affinity variant, was characterized by protein and DNA analyses in the homozygous state in a 32-year-old woman from Crete, with erythrocytosis and microcytosis. The proband and members of her family over 3 generations, including 5 carriers of Hb Crete, were subject to clinical, hematological and biochemical investigations, and DNA, RNA and protein studies were carried out. The proband demonstrated features associated with disturbed hemoglobin (Hb) structure and function, including erythrocytosis and additionally a state of functional anemia, the latter reflected by increased erythropoetin levels and cardiac output. In addition, all the carriers surprisingly had hematological and biosynthetic findings more usually associated with thalassemia trait. The structural change in Hb Crete only partly explains all the pathological manifestations of this variant, and other mechanisms are discussed.

Hb Montfermeil [beta 130(H8) Tyr-->Cys]: suggests a key role for the interaction between helix A and H in oxygen affinity of the hemoglobin molecule.

Hb Montfermeil [beta130(H8) Tyr-->Cys] is a high oxygen affinity variant causing erythrocytosis. The cysteine replacement is buried in the inside of the beta chain where it alters the interactions between helix A and H, with a further effect on helix E. This position has already been proposed to contribute to the difference in oxygen affinity between human and bovine hemoglobins. Three dimensional structural considerations and comparison of the functional behavior of other variants suggest that this region is an important determinant of the intrinsic oxygen affinity of the hemoglobin molecule.

Inhaled nitric oxide protects transgenic SAD mice from sickle cell disease-specific lung injury induced by hypoxia/reoxygenation.

Central to the pathophysiology of sickle cell disease are the vaso-occlusive events that lead to tissue damages and life-threatening complications. Lungs are particularly vulnerable to vaso-occlusion because of their specific vasculature. We developed a mouse model of hypoxia/reoxygenation lung injury closely mimicking the lung pathology of patients with sickle cell disease. This model involves the exposure of transgenic sickle cell (SAD) mice to hypoxia (8% oxygen) for 4, 10, and 46 hours followed by 2 hours of reoxygenation. Gene expression profiling of SAD lung tissue pointed to the specific induction of genes involved in the response to ischemic stress and microcirculation remodeling: Hspcb, Hsp86-1, Nfe2l2, Ace, and Fgf7. Hypoxia/reoxygenation also induced a marked increase in bronchoalveolar (BAL) total leukocyte and neutrophil counts, BAL total protein content, and BAL tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), IL-1alpha, and macrophage inflammatory protein 2 (MIP-2) levels, all indicators of enhanced inflammatory response as compared with control mice. Nitric oxide (NO) was administered to SAD mice. NO (40 ppm) inhalation protected SAD mice from the histopathologic lesions of ischemic/reperfusion lung injury with corresponding normalization and/or modulation of tissue gene expression profiles. Inhaled NO (1) significantly reduced the increase in BAL total protein content, BAL total leukocyte, and neutrophil counts; (2) modulated BAL cytokine network; and (3) did not affect hemoglobin and methemoglobin levels. The present study provides evidences for the beneficial effects of inhaled NO in pulmonary injury induced by hypoxia/reoxygenation in a mouse model of sickle cell disease (SCD) and opens new avenues in drug design based on tissue gene expression profiling.

Hemoglobin Görwihl [alpha2beta(2)5(A2)Pro-->Ala], an electrophoretically silent variant with impaired glycation.

BACKGROUND: Some of the genetic variants of hemoglobin (Hb) and their chemically modified species are known to affect the measurement of Hb A(1c). The purpose of this study was to characterize Hb species in the blood sample of a 74-year-old German male with an exceptionally low Hb A(1c) value. METHODS: Hemolysates from the propositus and a healthy individual were analyzed by electrophoresis, cation-exchange HPLC, boronate affinity chromatography, and electrospray ionization-mass spectrometry (ESMS). Genomic DNA was amplified by PCR, and the sequencing was performed on an ABI 310 sequencer. Functional properties of Hb were determined by oxygen equilibrium studies and CO recombination kinetics after flash photodissociation. Glycohemoglobin species were synthesized by incubating hemolysates with glucose. RESULTS: A novel, electrophoretically silent beta chain, beta5(A2)Pro-->Ala or Hb Görwihl, was detected by cation-exchange HPLC. It accounted for approximately 44% of the total Hb and had functional properties similar to those of normal Hb A and a mild degree of heat instability. During incubation with glucose, glycation of the beta chains (assessed by ESMS) in the hemolysate of a healthy volunteer was twice as fast as in hemolysate from the propositus. CONCLUSIONS: The substitution beta5(A2)Pro-->Ala seems to affect neither the functional properties nor the heterotropic interactions of Hb, but slows glycation of the N-terminal valine by an unknown mechanism.

Importance of helices A and H in oxygen binding differences between bovine and human hemoglobins.

Human and bovine hemoglobins (Hbs) exhibit several functional differences. They have a similar oxygen affinity in the presence of 2,3-diphosphoglycerate (2,3-DPG); however, bovine Hb has a greatly diminished 2,3-DPG effect, which itself is chloride dependent. The question is to determine whether these differences have a common structural origin, or whether they evolved in an independent fashion. The decreased 2,3-DPG effect can be partially reproduced by mutations at the effector binding sites, substituting the betaNA1 valine-NA2 histidine present in human Hb with a methionine. While changes of human Hb at these sites could provoke the bovine characteristic of the lower 2,3-DPG effect, the oxygen affinities of these mutated Hbs were not as low as that of the bovine Hb. Modifications responsible for tertiary structural modifications of helix A in human Hb might help shift the N-terminal methionine position, thereby locking helix A in place. We replaced the residues proline beta5(A2), arginine beta104(G6), and tyrosine beta130(H8) of human Hb by the residues present in bovine beta-globin, namely alanine, lysine, and phenylalanine, respectively. These mutations did not allow us to obtain a low oxygen affinity recombinant Hb (rHb). This indicates that other factors also influence oxygen binding and the effects are only partially coupled.

Hb Groene Hart: a new Pro-->Ser amino acid substitution at position 119 of the alpha1-globin chain is associated with a mild alpha-thalassemia phenotype.

Alpha-Thalassemia (thal) is generally considered to be an expression defect caused mostly by deletions silencing one or more alpha-globin genes. Although nondeletional alpha-thalassemia is considered rare, in our laboratory we frequently observe alpha-thal phenotypes induced by point mutations. We report a new point mutation generating an abnormal hemoglobin (Hb) associated with a mild alpha-thal phenotype in two members of a Moroccan family, who presented with mild but persistent microcytic hypochromic parameters and a balanced beta/alpha synthetic ratio. All attempts to separate an abnormal native or denatured fraction were unsuccessful using electrophoresis, isoelectrofocusing (IEE), ion exchange and reversed phase high performance liquid chromatography (HPLC), denaturing polyacrylamide gel electrophoresis (PAGE), and electrospray mass spectrometry (ES/MS). The anomalous protein was only predictable by DNA analysis. The mutated gene product, not separable with any of the techniques used, could be a monomer unsuitable for tetramer formation, which is proteolyzed at an early stage. Alternatively, this mutation could perhaps lead to an abnormal splicing. The CCTCT sequence generated by the mutant, not found in the translated region of the gene, but normally present at the end of the IVS-II, could induce a possible exon skipping. This mutant could generate a mild or a critical Hb H disease in combination with one of the common alpha0-thal deletion defects.

Synthesis and X-ray studies of chiral allosteric modifiers of hemoglobin.

This study was designed to investigate the effect of chirality on the allosteric activity of a series of Hb allosteric modifiers. The chiral analogues were based on the lead compound (4), JP7, [1-[4-(((3,5-dimethylanilino)carbonyl)methyl)phenoxy]cyclopentanecarboxylic acid] with different D- and L-amino acids conjugated to the JP7 acid moiety. The D-isomers were the most potent in vitro effectors in Hb solutions as well as with whole blood. In general, this study demonstrated that the chirality of extended amino acid side chains in JP7 conjugates plays an important role in observed degree of allosteric activity. The binding site interactions for four analogues were determined by single crystallographic diffraction studies. Conclusions show that the chiral configuration of some of the D-isomers enable the effectors to bind with a greater number of interactions with the protein residues. D- and L-isomers with equivalent or near equivalent allosteric activity did not show any significant differences or interactions between their amino acid side chains and the protein. The most potent effectors, in vitro, were compounds 15 and 19, D-isomers of leucine and phenylalanine, respectively. Compounds 21, 22, 30, and32 were more potent in vitro in Hb solutions than JP7.

Adaptation to high altitude: effects of small changes in the regulatory behavior of the andean chicken hemoglobin

Se ha descubierto en los Andes Peruanos un grupo de gallinas de altura (Gallus gallus) con alta afinidad de la hemoglobina por el oxígeno. Hemos estudiado el posible mecanismo molecular subyacente de esta adaptación a la altura. Se ha postulado pequeños cambios en la concentración intracelular de inositol pentafosfato (IPP), el principal efecto alostérico de la hemoglobina en los eritrocitos de aves. Hemos estudiado la sangre de gallinas andinas y de nivel del mar. Las afinidades de suspensiones frescas de eritrocitos están significativamente incrementadas en gallinas andinas comparadas con las de nivel del mar. Los valores de los coeficientes de Hill a saturación 50 por ciento (n50) son mayores para suspensiones de gallinas que para las de mamíferos. Esto podría sugerir la existencia de un proceso de agregación molecular dentro de las células deoxigenadas, altamente concentradas de ambos tipos de aves. Para las soluciones "desnudas" de hemolizados de ambos tipos de gallinas, las afinidades son idénticas en el buffer libre de fosfato, indicando que las afinidades intrínsecas de las Hbs de gallinas andinas y de nivel del mar son las mismas. Con la adición de inositol hexafosfato (IHP), un fuerte efecto alostérico de hemoglobinas de aves, observamos un incremento pequeño pero significativo (aproximadamente 20 por ciento) en la afinidad de los hemolisados de altura en relación a los de nivel del mar. Nuestros resultados también sugieren que la diferencia en la afinidad entre sangres de altura y de nivel del mar puede deberse a un proceso adaptativo posiblemente relacionado con una ligera disminución en la concentración y/o actividad del principal efector celular IPP, más bien que un anormalidad estructural de la hemoglobina.