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1.
A new recombinant, human anti-sickling beta-globin polypeptide designated beta(AS3) (betaGly(16) --> Asp/betaGlu(22) --> Ala/betaThr(87) --> Gln) was designed to increase affinity for alpha-globin. The amino acid substitutions at beta22 and beta87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxy-HbS polymerization. The beta16 substitution confers the recombinant beta-globin subunit (beta(AS3)) with a competitive advantage over beta(S) for interaction with the alpha-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (alpha(2)beta(AS3)(2)) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that beta(AS3) has a distinct advantage over beta(S) for dimerization with alpha-globin. When equal amounts of beta(S)- and beta(AS3)-globin monomers compete for limiting alpha-globin chains up to 82% of the tetramers formed is HbAS3. Knock-out transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knock-out transgenic sickle mice the beta(AS3) polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of beta(AS3)-globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 30% percent of these individuals who coinherit alpha-thalassemia. Therefore, constructs expressing the beta(AS3)-globin gene may be suitable for future clinical trials for sickle cell disease.  相似文献   

2.
Hemoglobin zeta(2)beta(2)(S) is generated by substituting embryonic zeta-globin subunits for the normal alpha-globin components of Hb S (alpha(2)beta(2)(S)). This novel hemoglobin has recently been shown to inhibit polymerization of Hb S in vitro and to normalize the pathological phenotype of mouse models of sickle cell disease in vivo. Despite its promise as a therapeutic tool in human disease, however, the basic O(2)-transport properties of Hb zeta(2)beta(2)(S) have not yet been described. Using human hemoglobins purified from complex transgenic-knockout mice, we show that Hb zeta(2)beta(2)(S) exhibits an O(2) affinity as well as a Hill coefficient, Bohr response, and allosteric properties in vitro that are suboptimally suited for physiological O(2) transport in vivo. These data are substantiated by in situ analyses demonstrating an increase in the O(2) affinity of intact erythrocytes from mice that express Hb zeta(2)beta(2)(S). Surprisingly, though, co-expression of Hb zeta(2)beta(2)(S) leads to a substantial improvement in the tissue oxygenation of mice that model sickle cell disease. These analyses suggest that, in the context of sickle cell disease, the beneficial antisickling effects of Hb zeta(2)beta(2)(S) outweigh its O(2)-transport liabilities. The potential structural bases for the antisickling properties of Hb zeta(2)beta(2)(S) are discussed in the context of these new observations.  相似文献   

3.
We investigated a transgenic mouse model of sickle cell disease, homozygous for deletion of mouse beta-globin and containing transgenes for human beta(S) and beta(S-antilles) globins linked to the transgene for human alpha-globin. In these mice, basal cGMP production in aortic rings is increased, whereas relaxation to an endothelium-dependent vasodilator, A-23187, is impaired. In contrast, aortic expression of endothelial nitric oxide synthase (NOS) is unaltered in sickle mice, whereas expression of inducible NOS is not detected in either group; plasma nitrate/nitrite concentrations and NOS activity are similar in both groups. Increased cGMP may reflect the stimulatory effect of peroxides (an activator of guanylate cyclase), because lipid peroxidation is increased in aortae and in plasma in sickle mice. Despite increased vascular cGMP levels in sickle mice, conscious systolic blood pressure is comparable to that of aged-matched controls; sickle mice, however, evince a greater rise in systolic blood pressure in response to nitro-L-arginine methyl ester, an inhibitor of NOS. Systemic concentrations of the vasoconstrictive oxidative product 8-isoprostane are increased in sickle mice. We conclude that vascular responses are altered in this transgenic sickle mouse and are accompanied by increased lipid peroxidation and production of cGMP; we suggest that oxidant-inducible vasoconstrictor systems such as isoprostanes may oppose nitric oxide-dependent and nitric oxide-independent mechanisms of vasodilatation in this transgenic sickle mouse. Destabilization of the vasoactive balance in the sickle vasculature by clinically relevant states may predispose to vasoocclusive disease.  相似文献   

4.
Hb Presbyterian is a variant hemoglobin that carries Lys at Asn-108 of beta-globin. This variant Lys(beta108) residue enhances the stability of Hb in the deoxy-state, conferring the low affinity for oxygen-binding in vitro. In the present study, we generated mutant mice carrying the Presbyterian mutation (Asn(beta108)-->Lys) at the beta-globin locus by a targeted knock-in strategy. Heterozygous mice showed the expression of Hb Presbyterian in 27.7% of total peripheral blood without any hematological abnormalities, which well mimicked human cases. On the other hand, homozygous mice exclusively expressed Hb Presbyterian in 100% of peripheral blood associated with hemolytic anemia, Heinz body formation, and splenomegaly. Hb Presbyterian showed instability in an in vitro precipitation assay. Erythrocytes from homozygous mice showed a shortened life span when transfused into wild-type mice, confirming that the knocked-in mutation of Lys(beta108) caused hemolysis in homozygous mice. This is the first report on the hemolytic anemia of unstable hemoglobin in an animal model. These results confirm the notion that the higher ratio of an unstable variant beta-globin chain in erythrocytes triggers the pathological precipitation and induces hemolysis in abnormal hemoglobinopathies.  相似文献   

5.
A murine model of sickle cell disease was tested by studying the polymerization of hybrid hemoglobin tetramers between alpha mouse and human beta S or beta S Antilles chains were prepared from Hb S Antilles, which was a new sickling hemoglobin inducing a sickle cell syndrome more severe than Hb S. The hybrid molecules did not polymerize in solution, indicating that the mouse alpha chains inhibited fiber formation. Consequently, a mouse model for sickle cell disease requires the transfer and expression of both alpha and beta S or beta S Antilles genes.  相似文献   

6.
Sickle cell disease is a hereditary disorder characterized by erythrocyte deformity due to hemoglobin polymerization. We assessed in vivo the potential curative threshold of fetal hemoglobin in the SAD transgenic mouse model of sickle cell disease using mating with mice expressing the human fetal Agamma-globin gene. With increasing levels of HbF, AgammaSAD mice showed considerable improvement in all hematologic parameters, morphopathologic features and life span/survival. We established the direct therapeutic effect of fetal hemoglobin on sickle cell disease and demonstrated correction by increasing fetal hemoglobin to about 9-16% in this mouse model. This in vivo study emphasizes the potential of the SAD mouse models for quantitative analysis of gene therapy approaches.  相似文献   

7.
Owing to the episodic and unpredictable nature of the sickling crisis, many aspects of the disease sickle cell anemia have resisted in vivo analysis. The lack of an animal model has hindered the pathophysiological investigation of this disease, as well as deterred the development of pharmacological therapies. The transgenic mouse system offers a new means for creating animals that make a specified mutant gene product, and we have used this system to create a series of mice that contain the human beta s-globin gene. These animals express this gene in the appropriate tissues and at the same point in development as the adult mouse globin genes are expressed. We have crossed the human beta s-containing transgenic mice with a beta-thalassemic mouse line and examined the hemoglobins produced by these mice. Their red cells contain 10% mouse alpha/human beta s hybrid hemoglobin, which partially corrects the thalassemic phenotype of the homozygous beta-thalassemic animals. Though the red cells do not sickle, other properties of the human beta s gene in these mice indicate the potential for the eventual development of a transgenic animal model for sickle cell anemia.  相似文献   

8.
The beta S-globin gene haplotypes were investigated using restriction endonucleases Hinc II and Hind III in 22 sickle cell anaemia patients from the eastern province, 67 sickle cell anaemia patients from the south-western province and 4 sickle cell anaemia patients from north-western province. The beta S was found to be mainly linked to the haplotype + + - + + in the eastern province (50% homozygous and 45.45% heterozygous), and - - - - + haplotypes in the south-western (44.77% homozygous and 43.28% heterozygous) and north-western (100% homozygous) provinces. A comparison of the haematological values and clinical manifestations in patients with the two major haplotypes revealed significant differences, with the disease presenting more severely in the south-western compared to the eastern population. The level of Hb F was not significantly different in the two groups and no association could be demonstrated between the beta-globin gene haplotype and Hb F level. These results have led us to suggest that the haplotype + + - + + is in some way linked to a benign sickle cell anemia, though the exact mechanism leading to a benign disease is not clear.  相似文献   

9.
We report the generation and characterisation of the first transgenic mice exclusively expressing normal human beta-globin ((hu)beta-globin) from a 183 kb genomic fragment. Four independent lines were generated, each containing 2-6 copies of the (hu)beta-globin locus at a single integration site. Steady state levels of (hu)beta-globin protein were dependent on transgene copy number, but independent of the site of integration. Hemizygosity for the transgene on a heterozygous knockout background ((hu)beta(+/0), (mu)beta(th-3/+)) complemented fully the hematological abnormalities associated with the heterozygous knockout mutation in all four lines. Importantly, the rescue of the embryonic lethal phenotype that is characteristic of homozygosity for the knockout mutation was also demonstrated in two transgenic lines that were homozygous for two copies of the (hu)beta-globin locus, and in one transgenic line, which was hemizygous for six copies of the (hu)beta-globin locus. Our results illustrate the importance of transgene copy number determination and of the hemizygosity/homozygosity status in phenotypic complementation studies of transgenic mice containing large heterologous transgenes. Transgenic mouse colonies with 100% (hu)beta-globin production from the intact (hu)beta-globin locus have been established and will be invaluable in comparative and gene therapy studies with mouse models containing specific beta-thalassemia mutations in the (hu)beta-globin locus.  相似文献   

10.
Hemoglobin E (HbE) is caused by a G-->A mutation at codon 26 of the beta-globin gene, which substitutes Glu-->Lys. This mutation gives rise to functional but unstable hemoglobin and activates a cryptic splice site causing mild anemia. HbE reaches a carrier frequency of 60-80% in some Southeast Asian populations. HbE causes serious disease when co-inherited with a beta-thalassemia mutation. In this study, we report the creation and evaluation of humanized transgenic mice containing the beta(E) mutation in the context of the human beta-globin locus. Developmental expression of the human beta(E) locus transgene partially complements the hematological abnormalities in heterozygous knockout mice ((mu)beta(th-3/+)) and rescues the embryonic lethality of homozygous knockout mice ((mu)beta(th-3/th-3)). The phenotype of rescued mice was dependent on the transgene copy number. This mouse model displays hematological abnormalities similar to HbE/beta-thalassemia patients and represent an ideal in vivo model system for pathophysiological studies and evaluation of novel therapies.  相似文献   

11.
12.
All mammals use hemoglobin (Hb) to transport oxygen. Each Hb molecule is a tetramer of two pairs of unlike globin polypeptide chains. Equal amount of subunit globin chains derived from the corresponding alpha- and beta-like genes can always result during development though the two separate gene clusters are located on two different chromosomes and spatially transcribed within different nuclear domains. Disturbance of this balance will result in degradation or precipitation of the excessive globin chains, which is the character of various thalassemic syndromes. In previous studies, we had established two kinds of bacterial artificial chromosome (BAC) mediated transgenic mouse models, which contain respectively the entire human alpha- and beta-globin cluster. Here, we investigated the regulatory relationship between the two clusters by interbreeding these two kinds of transgenic mice. The levels of human alpha- and beta-mRNA in the various hybrid lines reflect the levels in the original transgenic lines that contain either the alpha- or beta-globin cluster alone. The results suggested that there is no apparent cross talk or regulatory interaction between the two human globin clusters in transgenic mice.  相似文献   

13.
Allelic variation in the mouse beta globin gene complex (Hbb) produces structurally different beta globins in different mouse strains. Like humans, mice with HbbS alleles produce a single beta globin with one reactive cysteine (beta Cys93). In contrast, mice with HbbD alleles produce two structurally different beta globins, each containing an additional cysteine (beta Cys13). beta Cys93 forms mixed disulfides with glutathione and plays a pivotal role in the activities of hemoglobin, glutathione, and nitric oxide. Similar roles for mouse beta Cys13 have not been described. We used capillary electrophoresis to compare reduced glutathione (GSH), glutathione disulfide (GSSG), and S-glutathionyl hemoglobin levels in erythrocytes from inbred C57BL/6J (homozygous HbbS/S) and 129S1/SvImJ (homozygous HbbD/D) mice and their homozygous and heterozygous B6129S/F2J hybrid offspring. S-glutathionyl hemoglobin was nearly undetectable in inbred or hybrid mice with only monocysteinyl beta globins (HbbS/S) but represented up to 10% of total hemoglobin in mice with polycysteinyl beta globins (HbbS/D or HbbD/D). The stepwise increase in beta globin sulfhydryl group concentration in HbbS/S, HbbS/D, and HbbD/D F2 mice was associated with increasing hemoglobin-bound glutathione and decreasing free glutathione (GSH + GSSG) concentrations. Total erythrocyte glutathione (GSH + GSSG + hemoglobin-bound) was not significantly different between groups. In vitro studies showed that beta Cys13 in mouse HbbD beta globins was more susceptible to disulfide exchange with GSSG than beta Cys93. We conclude that reactive beta globin sulfhydryl group concentration is genetically determined in mice, and that polycysteinyl beta globins markedly influence intraerythrocyte glutathione distribution between free and hemoglobin-bound compartments. Although Hbb heterozygosity and polycysteinyl beta globins are common in wild mouse populations, all common human beta globins contain only one reactive cysteine, and homozygosity is the norm. These fundamental differences in mouse and human beta globin genetics have important implications for the study of mouse biology and for the use of some mouse strains as models for humans.  相似文献   

14.
15.
16.
Nitric oxide (NO) is a potential new therapeutic agent for sickle cell disease (SCD). We investigated the effects of NO donor on hypoxia-induced acute lung injury that occurs when transgenic sickle cell SAD mice are exposed to chronic hypoxia, a model for lung vasoocclusive sickle cell events. In wild-type and SAD mice, intraperitoneal injection of S-nitrosoalbumin (NO-Alb) produced no significant hematologic changes under room air conditions, whereas it induced mild temporary hypotension and inhibition of platelet aggregation. NO-Alb administration (300 mg/kg ip twice a day, equivalent to 7.5 microM NO) in wild-type and SAD mice exposed to 46 h of hypoxia (8% oxygen) followed by 2 h of normoxia resulted in 1) reduction of the hypoxia-induced increase in blood neutrophil count, 2) prevention of hypoxia-induced increased IL-6 and IL-1beta levels in bronchoalveolar lavage, 3) reduction of the lung injury induced by hypoxia-reoxygenation, 4) prevention of thrombus formation, and 5) prevention of hypoxia-induced increase of lung matrix metalloproteinase-9 gene expression. These effects provide new insights into the possible use of NO-Alb in the treatment of acute lung injury in SCD.  相似文献   

17.
A recombinant double mutant of hemoglobin (Hb), E6V/L88A(beta), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6(beta) of one tetramer and the hydrophobic region between Phe-85(beta) and Leu-88(beta) on an adjacent tetramer. Thus, a construct encoding the donor Val-6(beta) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88(beta) was assembled. The doubly mutated beta-globin gene was expressed in yeast together with the normal human alpha-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant beta-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.  相似文献   

18.
Surface hydrophobicity, stability, solubility, and kinetics of polymerization were studied using hemoglobins with four different amino acids at the beta 6 position: Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6). The surface hydrophobicity increased in the order of Hb C, Hb A, Hb Machida, and Hb S, coinciding with the hydrophobicity of the amino acid at the beta 6 position. Solubility of the oxy-form of these hemoglobins decreased in relation to increases in their surface hydrophobicity, suggesting that the solubility is controlled by the strength of hydrophobicity of the amino acid at the beta 6 position. The solubility of the oxy-form of these hemoglobins is always higher than that of the deoxy-form. There is a similar linear relationship between the solubility and surface hydrophobicity among deoxyhemoglobins A, C, and Machida. However, the solubility of deoxy-Hb S deviated significantly from the expected value, indicating that the extremely low solubility of deoxy-Hb S is not directly related to the hydrophobicity of the beta 6 valine. Kinetic studies on the polymerization of deoxy-Hb Machida revealed a distinct delay time prior to polymerization. This confirms our previous hypothesis that beta 6 valine is not responsible for the delay time prior to gelation. The kinetics of the polymerization of 1:1 mixtures of sickle and non-sickle hemoglobins were similar to those of pure Hb S, suggesting that only one of the two beta 6 valines is involved in an intermolecular contact. In mixtures of equal amounts of Hb S and Hb A, Hb C, or Hb Machida, half of the asymmetrical AS, SC, and S-Machida hybrid hemoglobins behaved like Hb S during nucleation, while the other half behaved like the non-sickle hemoglobin.  相似文献   

19.

BACKGROUND:

Hemoglobin (Hb) D Punjab disorder is a congenital hemoglobinopathy described in India. It is a disorder due to defect in beta-globin gene.

MATERIALS AND METHODS:

Here, the author assesses the possibility of U7.623 gene therapy for Hb D Punjab disorder. A standard bioinformatic analysis to study the effect of co-expression between nucleic acid sequence for human Hb D Punjab beta-globin chain and U7.623 was performed.

RESULT:

It can be seen that fully recovery of Hb function and biological process can be derived via gene ontology study.

CONCLUSION:

Here, there is a rationale to use U7 small nuclear ribonucleic acid as a possible tool for gene therapy in Hb D Punjab disorder.  相似文献   

20.
Recent studies have suggested that nitric oxide (NO) binding to hemoglobin (Hb) may lead to the inhibition of sickle cell fiber formation and the dissolution of sickle cell fibers. NO can react with Hb in at least 3 ways: 1) formation of Hb(II)NO, 2) formation of methemoglobin, and 3) formation of S-nitrosohemoglobin, through nitrosylation of the beta93 Cys residue. In this study, the role of beta93 Cys in the mechanism of sickle cell fiber inhibition is investigated through chemical modification with N-ethylmaleimide. UV resonance Raman, FT-IR and electrospray ionization mass spectroscopic methods in conjunction with equilibrium solubility and kinetic studies are used to characterize the effect of beta93 Cys modification on Hb S fiber formation. Both FT-IR spectroscopy and electrospray mass spectrometry results demonstrate that modification can occur at both the beta93 and alpha104 Cys residues under relatively mild reaction conditions. Equilibrium solubility measurements reveal that singly-modified Hb at the beta93 position leads to increased amounts of fiber formation relative to unmodified or doubly-modified Hb S. Kinetic studies confirm that modification of only the beta93 residue leads to a faster onset of polymerization. UV resonance Raman results indicate that modification of the alpha104 residue in addition to the beta93 residue significantly perturbs the alpha(1)beta(2) interface, while modification of only beta93 does not. These results in conjunction with the equilibrium solubility and kinetic measurements are suggestive that modification of the alpha104 Cys residue and not the beta93 Cys residue leads to T-state destabilization and inhibition of fiber formation. These findings have implications for understanding the mechanism of NO binding to Hb and NO inhibition of Hb S fiber formation.  相似文献   

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