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R L Nagel

Publications and source records attributed to R L Nagel.

At least 19 recordsLinked to original sources
PubMed1999

Circulating cytokines response and the level of erythropoiesis in sickle cell anemia.

A hemoglobin F (HbF) level between eight and nine percent divides sickle cell anemia (SS) patients into two populations, according to the kinetics of circulating burst forming units-erythroid (BFU-E), long term culture-initialing cells (LTC-IC), and cytokine plasma concentrations. The SS patients with HbF levels lower than 8-9% are more anemic (LFSS patients) than those with HbF levels higher than 8-9% who have less severe anemia (HFSS patients). We report here that the level of erythropoiesis [evaluated by the levels of soluble transferin receptors (sTfR)] is not identical in these two patient populations, supporting the idea that a different set of regulatory mechanisms might be required to maintain the two levels of increased hematopoiesis. The plasma sTfR concentration was increased in all SS samples compared with controls (P < 0.002) and sTfR levels were negatively correlated with peripheral HbF%. (r = -0.574, P < 0.002). Furthermore, sTfR levels were higher in LFSS than in HFSS patients. Erythropoietin (Epo) levels were increased in the plasma of LFSS individuals (range = 34-215 ml U/ml), while the values in HFSS patients were in the normal range (3-20 ml U/ml). Furthermore, we identify here stem cell factor (SCF) and transforming growth factor-beta (TGF-beta) as regulatory factors specifically affected by the presence of SS genotype and its level of severity. The plasma concentrations of SCF and TGF-beta were increased compared with normal controls and high levels of SCF (up to 7,000 pg/ml) were detected in LFSS patients. The latter also showed increased proportion of SCF+ CD34 enriched circulating cells (49%). Low SCF in HFSS patients is associated with elevated TGF-beta, suggesting a regulatory role of the latter on either SCF release or c-kit expression in progenitor cells. Occasional elevation of granulocyte macrophage-colony stimulating factor (G-CSF), interleukin (IL)-7, and macrophage inflammatory protein (MIP)-1alpha in plasma of SS patients is not specific because no relation to HbF could be demonstrated. All plasma tested for leukemia inhibitory factor (LIF) were negative. Data presented here, complementing previously published information, supports a model in which HFSS patients achieve a balance between inhibitory (TGF-beta) and stimulatory (SCF, IL-3) factors, resulting in moderate erythropoietic response. In contrast, in LFSS patients, low levels of TGF-beta and the increased release of GM-CSF and SCF maintain the intense erythropoiesis in response to higher erythropoietic stress, in these more severe patients.

Adult↗
PubMed1999

Plasmodium falciparum: enhanced gametocyte formation in vitro in reticulocyte-rich blood.

Concentrates of late stage parasites of the gametocyte-forming clone HB-3 were mixed with blood rich in reticulocytes from anemic patients, or with normal control blood, and kept under culture conditions for 4 days. Significantly more gametocytes were always formed in the reticulocyte-rich blood than in the control. This was true whether the anemic blood supported a larger asexual parasitemia than the control, or a lower one, or the same and without regard to the cause of the anemia. Gametocytes as a percentage of asexual forms were up to 10 times higher in reticulocyte-rich blood than in normal blood.

Anemia↗
PubMed1999

Will the genetic individualization of disease force a new paradigm?

Disease, through the ages, has been considered as a 'visitation' from afar to the body or as 'disequilibrium' of an internal order. In practical terms, these two concepts of disease have generated a nosology, that is a classification of disease states. Genetics, in its dazzling recent advances, has opened another conceptual framework: disease is an individual event, inasmuch as our growing understanding of the genetic 'terrain' makes all subjects true individuals. Hence, it is time to abandon the old conceptual dichotomy for a Hegelian synthesis: there are no diseases (either as visitation or disequilibrium) only diseased individuals.

Disease↗
PubMed1999

Accuracy of pulse oximetry in sickle cell disease.

Pulmonary complications and hypoxemia are common in sickle cell disease (SCD) and may exacerbate microvascular occlusive phenomena. Thus, detecting hypoxemia is of particular importance in SCD. To assess the accuracy of pulse oximetry in the diagnosis of hypoxemia in SCD, we compared 22 pulse oximetric measurements of arterial oxygen saturation (SpO2) in adult patients with SCD and acute vasoocclusive crisis with simultaneously drawn arterial saturation (SaO2 = oxyhemoglobin divided by oxyhemoglobin plus reduced hemoglobin) measured by co-oximetry. We accepted SpO2 readings only if they were stable and characterized by strong and regular photoplethysmographic waves on the oximeter screen. To assess the position of these patients' oxyhemoglobin dissociation curves, we plotted arterial and venous oxygen saturation (SaO2 and SvO2 ) against oxygen tension. We found right-shifted oxyhemoglobin dissociation curves, with pH-corrected p50s ranging from 28 to 38 mm Hg. Pulse oximetry slightly overestimated oxyhemoglobin percentage (by an average of 3.4 percentage points), but it almost always accurately estimated SaO2 (underestimating on average by 1.1 percentage points). The error in SpO2 was never enough to classify a hypoxemic patient erroneously as normoxemic or a normoxemic patient as hypoxemic. We conclude that, as long as strong and regular photoplethysmographic waves are present, pulse oximeters can be relied upon not to misdiagnose either hypoxemia or normoxemia in SCD.

Acute Disease↗
PubMed1998

Two distinct pathways mediate the formation of intermediate density cells and hyperdense cells from normal density sickle red blood cells.

In sickle cell anemia (SS), some red blood cells dehydrate, forming a hyperdense (HD) cell fraction (>1.114 g/mL; mean corpuscular hemoglobin concentration [MCHC], >46 g/dL) that contains many irreversibly sickled cells (ISCs), whereas other SS red blood cells dehydrate to an intermediate density (ID; 1.090 to 1.114 g/mL; MCHC, 36 to 46 g/dL). This study asks if the potassium-chloride cotransporter (K:Cl) and the calcium-dependent potassium channel [K(Ca2+)] are participants in the formation of one or both types of dense SS red blood cells. We induced sickling by exposing normal density (ND; 1.080 to 1.090 g/mL; MCHC, 32 to 36 g/dL) SS discocytes to repetitive oxygenation-deoxygenation (O-D) cycles in vitro. At physiologic Na+, K+, and Cl-, and 0.5 to 2 mmol/L Ca2+, the appearance of dense cells was time- and pH-dependent. O-D cycling at pH 7.4 in 5% CO2-equilibrated buffer generated only ID cells, whereas O-D cycling at pH 6.8 in 5% CO2-equilibrated buffer generated both ID and HD cells, the latter taking more than 8 hours to form. At 22 hours, 35% +/- 17% of the parent ND cells were recovered in the ID fraction and 18% +/- 11% in the HD fraction. Continuous deoxygenation (N2/5% CO2) at pH 6.8 generated both ID and HD cells, but many of these cells had multiple projections, clearly different from the morphology of endogenous dense cells and ISCs. Continuous oxygenation (air/5% CO2) at pH 6.8 resulted in less than 10% dense cell (ID + HD) formation. ATP depletion substantially increased HD cell formation and moderately decreased ID cell formation. HD cells formed after 22 hours of O-D cycling at pH 6.8 contained fewer F cells than did ID cells, suggesting that HD cell formation is particularly dependent on HbS polymerization. EGTA chelation of buffer Ca2+ inhibited HD but not ID cell formation, and increasing buffer Ca2+ from 0.5 to 2 mmol/L promoted HD but not ID cell formation in some SS patients. Substitution of nitrate for Cl- inhibited ID cell formation, as did inhibitors of the K:Cl cotransporter, okadaic acid, and [(dihydroindenyl) oxy]alkanoic acid (DIOA). Conversely, inhibitors of K(Ca2+), charybdotoxin and clotrimazole, inhibited HD cell formation. The combined use of K(Ca2+) and K:Cl inhibitors nearly eliminated dense cell (ID + HD cell) formation. In summary, dense cells formed by O-D cycling for 22 hours at pH 7.4 cycling are predominately the ID type, whereas dense cells formed by O-D cycling for 22 hours at pH 6.8 are both the ID and HD type, with the latter low in HbF, suggesting that HD cell formation has a greater dependency on HbS polymerization. A combination of K:Cl cotransport and the K(Ca2+) activities account for the majority of dense cells formed, and these pathways can be driven independently. We propose a model in which reversible sickling-induced K+ loss by K:Cl primarily generates ID cells and K+ loss by the K(Ca2+) channel primarily generates HD cells. These results imply that both pathways must be inhibited to completely prevent dense SS cell formation and have potential therapeutic implications.

Adenosine Triphosphate↗
PubMed1998

HbS-oman heterozygote: a new dominant sickle syndrome.

Hemoglobin (Hb) S-Oman has two mutations in the beta-chains. In addition to the classic betaS mutation (beta6 Glu --> Val), it contains a second mutation in the same chain (beta121 Glu --> Lys) identical to that of HbOARAB. We have studied a pedigree of heterozygous carriers of HbS-Oman that segregates into two types of patients: those expressing about 20% HbS-Oman and concomitant -/ thalassemia and those with about 14% of HbS-Oman and concomitant -/- thalassemia. The higher expressors of S-Oman have a sickle cell anemia (SS) clinical syndrome of moderate intensity, while the lower expressors have no clinical syndrome, and are comparable to the solitary case first described in Oman. In addition, the higher expressors exhibit a unique form of irreversibly sickled cell reminiscent of a "yarn and knitting needle" shape, in addition to folded and target cells. The CSAT of S-Oman is identical to that of S-Antilles, another supersickling hemoglobin, whose carriers express the abnormal hemoglobin at 40% to 50%, with a very similar clinical picture to HbS-Oman. Because the level of expression is so different and the clinical picture so similar, and based on the hemolysates CSAT's, we conclude that HbS-Oman produces pathology beyond its sickling tendencies. A clue for this additional pathogenesis is found in the fact that homozygous HbOARAB, which has the same second substitution as S-Oman, has a moderately severe hemolytic anemia; when HbOARAB is combined with HbS, it makes the phenotype of this double heterozygote as severe as SS. Properties of HbS-Oman red blood cells (RBCs) include reticulocytes that are much denser than normal (similar to those of SC and CC disease), a decrease in the Km for Ca2+ needed to activate the Gardos' channel (making this transporter more sensitive to Ca2+), increased association of HbS-Oman with the RBC membrane, the presence of dense cells by isopycnic gradient, the presence of folded cells, and abundant nidus of polymerization under the membrane. Other properties include a clear increase in volume and N-ethylmaleimide-stimulated K:Cl cotransport in RBCs expressing more than 20% HbS-Oman. We conclude that the pathology of heterozygous S-Oman is the product of the sickling properties of the beta6 Val mutation which are enhanced by the second mutation at beta121. In addition, the syndrome is further enhanced by a hemolytic anemia induced by the mutation at beta121. We speculate that this pathology results from the abnormal association of the highly positively charged HbS-Oman (3 charges different from normal hemoglobin) with the RBC membrane.

Adolescent↗
PubMed1998

Transgenic mice expressing human fetal globin are protected from malaria by a novel mechanism.

Studies in vitro by Pasvol et al (Nature, 270:171, 1977) have indicated that the growth of Plasmodium falciparum in cells containing fetal hemoglobin (HbF = alpha2gamma2) is retarded, but invasion is increased, at least in newborn cells. Normal neonates switch from about 80% HbF at birth to a few percent at the end of the first year of life. Carriers of beta-thalassemia trait exhibit a delay in the normal HbF switch-off, which might partially explain the protection observed in populations with this gene. To study this hypothesis in vivo, we used transgenic (gamma) mice expressing human Agamma and Ggamma chains resulting in 40% to 60% alpha2Mgamma2 hemoglobin, infected with rodent malaria. Two species of rodent malaria were studied. P chabaudi adami causes a nonlethal infection, mainly in mature red blood cells (RBC). P yoelii 17XNL is a nonlethal infection, invading primarily reticulocytes, whereas P yoelii 17XL is a lethal variant of P yoelii 17XNL and causes death of mice in approximately 1 to 2 weeks. Data indicate that this strain may cause a syndrome resembling cerebral malaria caused by P falciparum (Am J Trop Med Hyg, 50:512, 1994). In gamma transgenic mice infected with P chabaudi adami, the parasitemia rose more quickly (in agreement with Pasvol) than in control mice, but was cleared more rapidly. In mice infected with P yoelii 17XNL, a clear reduction in parasitemia was observed. Interestingly, splenectomy before this infection, did not reverse protection. The most striking effect was in lethal P yoelii 17XL infection. Control mice died between 11 to 13 days, whereas gamma mice cleared the infection by day 22 and survived, a phenomenon also observed in splenectomized animals. These results suggest that HbF does indeed have a protective effect in vivo, which is not mediated by the spleen. In terms of mechanisms, light microscopy showed that intraerythrocytic parasites develop slowly in HbF erythrocytes, and electron microscopy showed that hemozoin formation was defective in transgenic mice. Finally, digestion studies of HbF by recombinant plasmepsin II demonstrated that HbF is digested only half as well as hemoglobin A (HbA). We conclude that HbF provides protection from P falciparum malaria by the retardation of parasite growth. The mechanism involves resistance to digestion by malarial hemoglobinases based on the data presented and with the well-known properties of HbF as a super stable tetramer. In addition, the resistance of normal neonates for malaria can now be explained by a double mechanism: increased malaria invasion rates, reported in neonatal RBC, will direct parasites to fetal cells, as well as F cells, and less to the approximately 20% of HbA containing RBC, amplifying the antimalarial effects of HbF.

Animals↗
PubMed1998

Hematological effects of atypical and Cameroon beta-globin gene haplotypes in adult sickle cell anemia.

To examine the effects of unusual or atypical beta-globin gene cluster haplotypes on the hematological features and Hb F levels of sickle cell anemia, we studied African Americans who had an atypical or Cameroon haplotype chromosome in association with a typical haplotype. We identified over 20 atypical haplotypes. The distribution of 5' sub-haplotypes of the atypical chromosomes mirrored the distribution of common haplotypes in African Americans with sickle cell anemia. Neither 5' nor 3' subhaplotypes of the atypical chromosomes affected Hb F levels, packed cell volume, or mean corpuscular volume in individuals with a Benin chromosome. That the 5' subhaplotype is unaffected might be a consequence of the small numbers of Senegal 5' subhaplotypes in our sample, the need for linkage of both 5' and 3' subhaplotypes of any haplotype for an effect on Hb F to be present, or the likelihood that a normal beta-globin gene contributed the 5' subhaplotypes of some atypical haplotypes.

Adult↗
PubMed1998

Molecular identification and expression of erythroid K:Cl cotransporter in human and mouse erythroleukemic cells.

A major pathway for K+ efflux in human reticulocytes and young RBCs is K:Cl cotransport (K:Cl-CT). The activity of K:Cl-CT is increased in pathologic RBCs containing hemoglobins S and C and may contribute to the abnormal dehydration state of these cells. Human K:Cl-CT (gene product KCC1) has been recently sequenced from human (hKCC1), rabbit and rat tissue by Gillen et al. (J Biol Chem 271:16237, 1996). We report here the sequence of KCC1 from human and mouse erythroleukemic cells (K562 and MEL cells, respectively). The cDNA for human erythroid-KCC1 is 100% identical to hKCC1 and the cDNA for mouse erythroid-KCC1 shares 89% identity with hKCC1, which translates to 96% identity at the amino acid level. Mammalian KCC1 is strongly conserved with >95% identity between human, rabbit, rat, and mouse KCC1 proteins. We did not detect any full-length mRNA transcripts of human erythroid-KCC1 in circulating reticulocytes. We detected two mRNA isoforms of human erythroid-KCC1 that resulted in C-terminal truncated proteins (73 amino acid and 17 amino acids, respectively). Human and mouse erythroidKCC1 differed at several consensus sites including a predicted PKC phosphorylation site at 108threonine and a predicted CK2 phosphorylation site at 51serine, within the predicted cytoplasmic N-terminal, that are present in human but not mouse erythroid-KCC1. Expression of MEL-KCC1 mRNA increases substantially upon DMSO-induced differentiation opening the possibility that erythroid-KCC1 plays a role in early erythroid maturation events. The molecular identification of erythroid-KCC1 is an important step towards understanding the physiologic role mediated by this protein in young and pathologic RBCs and during erythropoiesis, as well as providing a new tool for the elucidation of pathways and signals involved in RBC volume regulation.

Amino Acid Sequence↗
PubMed1998

Peroxynitrite formation and apoptosis in transgenic sickle cell mouse kidneys.

BACKGROUND: In a previous study, nitric oxide synthases (NOS) were found to be strongly expressed in the tubular epithelium of kidneys of a transgenic mouse model of sickle cell disease (alphaHbetaS[betaMDD]). Because NOS activity is often associated with peroxynitrite formation when superoxide radical (.O-2) is present in abundance, we examined the kidneys of sickle cell mice for nitrotyrosine, considered to be a footprint of ONOO-. METHODS: Western blot and immunohistochemistry for nitrotyrosine was carried out. Since peroxynitrite and other reactive oxygen radicals are capable of causing apoptosis, we also performed agarose gel electrophoresis of kidney DNA and TUNEL staining of nuclei, indicators of apoptosis. RESULTS: Nitration of tyrosine residues of three proteins (kD 66, 57 and 22) was found on Western blot of kidney protein extracts of the sickle cell mice. The degree of tyrosine nitration of the 66 kD protein was not significantly different in the control versus transgenic mice, whereas tyrosine nitration of the 57 and 22 kD proteins was clearly increased in transgenic mice. Strong immunostaining for nitrotyrosine was seen in tubular epithelial cells of the sickle cell mice, in close proximity to positive immunostaining of iNOS. Neither iNOS nor nitrotyrosine was expressed in the control mice. DNA "laddering" was found localized to the same zones of the kidney as nitrotyrosine and iNOS immunostaining. TUNEL assay on mouse kidney tissue sections showed minimal tubular cell apoptosis in normal mouse with hypoxia, mild tubular cell apoptosis in sickle cell mouse in room air, and moderate tubular cell apoptosis in sickle cell mouse with hypoxia. CONCLUSIONS: The observations suggest that ONOO- and perhaps other reactive oxygen species are being produced in the sickle cell kidney. The mechanism may be ischemia/reperfusion due to intermittent vascular occlusion by sickle cells. The resulting hypoxia could result in iNOS activation, superoxide radical and peroxynitrite formation. Two consequences of these reactions appear to be nitration of tyrosine residues of some renal proteins and enhanced apoptosis.

Anemia, Sickle Cell↗
PubMed1998

Nonperfusion of retina and choroid in transgenic mouse models of sickle cell disease.

PURPOSE: To determine if vascular occlusion and nonperfusion is associated with the outer retinal atrophy, retinopathy, and choroidopathy (chorioretinopathy) that occurs in the alpha H beta S[beta MDD] and alpha H beta S [alpha MD beta MDD] transgenic mouse models of sickle cell disease. METHODS: Mice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] transgenic mouse lines that express high levels of human beta S globin were anesthetized and administered horseradish peroxidase (HRP) intracardially. After 1 min, the animals were sacrificed, and the retina from one eye was excised, fixed, and developed in diaminobenzidine (DAB). The contralateral eye was fixed, embedded whole in glycol methacrylate, and HRP developed in 2.5 microns sections. RESULTS: HRP reaction product (HRP-RP) and stained erythrocytes (RBCs) (due to endogenous peroxidase) were diffusely distributed within all vascular lumens in flatmount retinas from control animals (littermates homozygous for the mouse Beta Major deletion not expressing the beta S transgene). In 42.5% of the transgenic mice expressing beta S without any proliferative retinopathy, many blood vessels contained RBC plugs and lacked lumenal HRP-RP. In addition to packed RBCs, fibrin was sometimes present at sites of occlusion. In sections from whole eyes of the same animals, foci of photoreceptor degeneration were associated with areas of choriocapillaris nonperfusion (lumen that lacked HRP-PR). In areas with normal photoreceptors, the choriocapillaris appeared perfused (HRP-RP was present). In animals with proliferative chorioretinopathy, some neovascular formations lacked luminal HRP-RP, suggesting autoinfarction. CONCLUSIONS: Nonperfused retinal and choroidal vessels were observed in mice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] lines without retinal and choroidal neovascularization, whereas, all mice with neovascularization had nonperfused areas. Furthermore, small foci of PR loss were associated with areas of nonperfused choriocapillaris. These results suggest that sickle cell-mediated vaso-occlusions are an initial event in the chorioretinopathy and outer retinal atrophy that occurs in these models.

Anemia, Sickle Cell↗
PubMed1998

Microvascular hemodynamics and in vivo evidence for the role of intercellular adhesion molecule-1 in the sequestration of infected red blood cells in a mouse model of lethal malaria.

The cytoadherence of infected red blood cells (IRBCs) to the vascular endothelium is the major cause of IRBC sequestration and vessel blockage in the cerebral form of human malaria. Among the rodent models of malaria, Plasmodium yoelii 17XL-infected mice show many similarities with the human cerebral malaria caused by P. falciparum. In both, the sequestration of IRBCs in the brain vessels is secondary to the cytoadherence of IRBCs to the vascular endothelium. Similar to P. falciparum infection in the human but in contrast to P. berghei ANKA infection in mice, P. yoelii 17XL results in little, if any, accumulation of monocytes in the brain. In vivo microcirculatory studies reported here were designed to further understand the hemodynamic aspects and mechanisms underlying cytoadherence of IRBCs in the P. yoelii model using the easily accessible cremaster muscle vasculature. The results show significant decreases in arteriovenous red blood cell velocities (Vrbc) and wall shear rates in the microcirculation of P. yoelii-infected mice, with a maximal decrease occurring in small-diameter postcapillary venules, the main sites of cytoadherence. This reflects contributions from IRBC cytoadherence as well as from increased rigidity of parasitized red blood cells. No cytoadherence is observed in arterioles of the infected mice despite decreased wall shear rates, indicating that endothelial receptors for cytoadherence are restricted to venules. Infusion of a monoclonal antibody (MAb) against the intercellular adhesion molecule-1 (ICAM-1) resulted in significant increases in both arteriolar and venular Vrbc and wall shear rates, accompanied by detachment of adhered IRBCs at some venular sites. The peripheral blood smears taken after the MAb infusion showed a distinct increase in the percentage of schizonts, again indicating detachment and/or prevention of cytoadherence. An MAb against the vascular cell adhesion molecule-1 (VCAM-1) as well as an irrelevant control antibody had no effect on these parameters. These results provide the first in vivo microcirculatory evidence indicating involvement of ICAM-1, but not of VCAM-1, in the sequestration of IRBCs in a rodent model of cerebral malaria.

Animals↗
PubMed1998

Correlation of increased expression of intercellular adhesion molecule-1, but not high levels of tumor necrosis factor-alpha, with lethality of Plasmodium yoelii 17XL, a rodent model of cerebral malaria.

Previous studies demonstrated that Plasmodium yoelii 17XL, a lethal strain of rodent malaria, causes a syndrome in SW mice that resembles human cerebral malaria. The mouse brain pathology is characterized by cytoadherence of parasitized erythrocytes. Here, the possible mechanisms mediating cerebral malaria in this model were studied and the results were compared with a nonlethal strain of this parasite, P. yoelii 17XNL (nonlethal), which does not cause cerebral malaria. Immunostaining for intercellular adhesion molecule-1 (ICAM-1) revealed an increase in expression of this protein in the small venules and capillaries of the brains of infected mice that increased with time after infection. Staining was more pronounced during the lethal infection than the nonlethal infection. Some staining with monoclonal antibody to vascular cell adhesion molecule-1 was also observed, but it was quantitatively less than ICAM-1 staining and was limited to larger venules. During the lethal infection, levels of tumor necrosis factor-alpha (TNF-alpha) increased rapidly, peaking on day 4. In contrast, mice infected with nonlethal P. yoelii had a slower serum TNF-alpha response that peaked on day 10, prior to the maximum parasitemia. In addition, mice with a targeted disruption of the TNF-alpha gene (TNF-alpha-/- mice) were infected with the lethal and nonlethal strains of P. yoelii 17X. The TNF-alpha-/- mice infected with the nonlethal parasite had significantly higher levels of parasitemia than controls, whereas TNF-alpha-/- mice infected with the lethal strain had slightly higher levels of infected erythrocytes but were equally susceptible to death from this infection. Thus, TNF-alpha does not appear to be essential in mediating death. These results demonstrate that P. yoelii 17XL infection has features in common with human cerebral malaria and suggest that this model may be useful in testing strategies to alleviate this syndrome.

Animals↗
PubMed1997

Inhibition of sickle beta-chain (betaS)-dependent polymerization by nonhuman alpha-chains. A superinhibitory mouse-horse chimeric alpha-chain.

Horse alpha-chain inhibits sickle beta-chain-dependent polymerization; however, its inhibitory potential is not as high as that of mouse alpha-chain. Horse alpha-(1-30) and alpha-(31-141) segments make, respectively, minor and major contributions to the inhibitory potential of horse alpha-chain. The sum of the inhibitory potential of the two segments does not account for the inhibitory potential of the full-length horse alpha-chain. Although the polymerization inhibitory potential of horse alpha-chain is lower than mouse alpha-chain, the inhibitory potential of horse alpha-(31-141) is comparable to that of mouse alpha-(31-141). When mouse alpha-(1-30) is stitched to horse alpha-(31-141), the product is a chimeric alpha-chain with an inhibitory potential greater than mouse alpha-chain. In contrast, the stitching of horse alpha-(1-30) with mouse alpha-(31-141) had no additional inhibitory potential. Molecular modeling studies of HbS containing the mouse-horse chimeric alpha-chain indicate altered side-chain interactions at the alpha1beta1 interface when compared with HbS. In addition, the AB/GH corner perturbations facilitate a different stereochemistry for the interaction of the epsilon-amino group of Lys-16(alpha) with the beta-carboxyl group of Asp-116(alpha), resulting in a decrease in the accessibility of the side chain of Lys-16(alpha) to the solvent. Based on molecular modeling, we speculate that these perturbations by themselves, or in synergy with the altered conformational aspects of the alpha1beta1 interactions, represent the molecular basis of the superinhibitory potential of the mouse-horse chimeric alpha-chains.

Animals↗
PubMed1997

Molecular cloning and expression of a chloride channel-associated protein pICln in human young red blood cells: association with actin.

We report the cloning and sequencing from human reticulocytes of cDNA coding for the Cl- channel-associated protein, pICln. Human reticulocyte pICln (HRpICln) cDNA encodes a protein (predicted molecular mass 26293Da) identical with human non-pigmented ciliary epithelial cell pICln. By using full-length HRpICln cDNA (approx. 1.2 kb) to probe human lymphocyte metaphase-chromosome spreads, the location of the human ICln gene was mapped to 11q13 by fluorescence in situ hybridization analysis. Polyclonal antibodies to recombinant HRpICln detected bands at approx. 43 kDa and approx. 37 kDa in both normal (AA) and sickle (SS) red blood cell (RBC) ghost membranes. In SS ghosts, and in ghosts from a patient with autoimmune haemolytic anaemia with 9.8% reticulocytes, the amount of HRpICln was increased compared with AA ghosts, suggesting that the expression or membrane assembly of HRpICln is cell age-dependent. Laser scanning confocal fluorescent microscopy immunolocalized HRpICln largely to the RBC membrane. The increased staining intensity of HRpICln in a reticulocyte-enriched AA RBC density-separated fraction is consistent with a dependence of HRpICln membrane content on cell age. HRpICln and beta-actin form stable complexes in vivo, demonstrated with the yeast two-hybrid system. Low-ionic-strength extraction of ghost membranes, which results in the extraction of the spectrin-actin cytoskeleton, also results in the extraction of HRpICln, consistent with the possibility for the association of these proteins in RBCs in vivo. The results presented here establish the presence of the Cl- channel-associated protein, pICln, in human RBCs, and raises the possibility that this protein has a role in RBC Cl- transport and volume regulation in young RBCs. Moreover the association of RBC pICln with actin offers a model in which to test interactions between RBC ion channels and the cytoskeleton.

Actins↗
PubMed1997

Red blood cells of a transgenic mouse expressing high levels of human hemoglobin S exhibit deoxy-stimulated cation flux.

Deoxy-stimulated cation fluxes have been implicated in the generation of the dense and irreversibly sickled red blood cells (RBCs) in patients homozygous for hemoglobin S (SS). We now report on the effect of short term deoxygenation on K+ and Na+ transport in RBCs from control mice (C57Bl/6J) and a transgenic (alphaHbetaS[betaMDD]) mouse line that expresses high levels of human alphaH and betaS-chains and has a small percent dense cells but does not exhibit anemia. In transgenic mouse RBCs (n = 5) under oxygenated conditions, K+ efflux was 0.22 +/- 0.01 mmol/L cell x min and Na+ influx was 0.17 +/- 0.02 mmol/L cell x min. Both fluxes were stimulated by 10 min deoxygenation in transgenic but not in control mice. The deoxy-stimulated K+ efflux from transgenic mouse RBCs was about 55% inhibited by 5 nm charybdotoxin (CTX), a blocker of the calcium activated K+-channel. To compare the fluxes between human and mouse RBCs, we measured the area of mouse RBCs and normalized values to area per liter of cells. The deoxy-simulated CTX-sensitive K+ efflux was larger than the CTX-sensitive K+ efflux observed in RBCs from SS patients. These results suggest that in transgenic mice, deoxygenation increases cytosolic Ca2+ to levels which open Ca2+-activated K+ channels. The presence of these channels was confirmed in both control and transgenic mice by clamping intracellular Ca2+ at 10 microM with the ionophore A23187 and measuring Ca2+-activated K+ efflux. Both types of mouse had similar maximal rates of CTX-sensitive, Ca2+-activated K+ efflux that were similar to those in human SS cells. The capacity of the mouse red cell membrane to regulate cytosolic Ca2+ levels was examined by measurements of the maximal rate of calmodulin activated Ca2+-ATPase activity. This activity was 3-fold greater than that observed in human RBCs thus indicating that mouse RBC membranes have more capacity to regulate cytosolic Ca2+ levels. In summary, transgenic mouse RBCs exhibit larger values of deoxy-stimulated K+ efflux and Na+ influx when compared to human SS cells. They have a similar Ca2+-activated K+ channel activity to human SS cells while expressing a very high Ca2+ pump activity. These properties may contribute to the smaller percent of very dense cells and to the lack of adult anemia in this animal model.

Adult↗
PubMed1997

Molecular interactions between Hb alpha-G Philadelphia, HbC, and HbS: phenotypic implications for SC alpha-G Philadelphia disease.

We show here that alpha2(G-Phila.) beta2(C) has an increased rate of crystal nucleation compared to alpha2 beta2(C) (HbC). We conclude from this finding that position alpha68, the mutation site of alpha2(G-Phila.) beta2 (HbG(Philadelphia)), is a contact site in the crystal of HbC. In addition, that HbS enhances HbC crystallization (additive to the effect of alpha(G-Phila.) as shown here) and that alpha(G-Phila.) inhibits polymerization of HbS are pathogenically relevant previously known facts. All of these findings help explain the phenotype of an individual simultaneously heterozygous for the betaS, betaC, and the alpha(G-Phila.) genes (SC alpha-G Philadelphia disease). This disease is characterized by a mild clinical course, abundant circulating intraerythrocytic crystals, and increased folded red cells. This phenotype seems to be the result of increased crystallization and decreased polymerization brought about by the opposite effects of the gene product of the alpha(G-Phila.) gene on the betaC and betaS gene products. Some of the intraerythrocytic crystals in this syndrome are unusually long and thin, resembling sugar canes, unlike those seen in SC disease. The mild clinical course associated with increased crystallization implies that, in SC disease, polymerization of HbS is pathogenically more important than the crystallization induced by betaC chains. The SC alpha-G Philadelphia disease is an example of multiple hemoglobin chain interactions (epistatic effect among globin genes) creating a unique phenotype.

Adult↗