Thomas G. Gabuzda

Metabolic and molecular heterogeneity of marrow ferritin

Abstract Two isotopes of iron were utilized to label erythroblast and reticuloendothelial cell ferritin in rabbit marrow in vivo . 59 Fe was administered by way of the transferrin pathway and 55 Fe as heat-damaged labeled reticulocytes. Following clearance of these isotopes from the circulation, the marrow ferritin was isolated and submitted to analytical procedures. The results indicate that erythroblast ferritin, identified by the 59 Fe label, can be distinguished from reticuloendothelial-cell ferritin tagged with 55 Fe. In comparison with reticuloendothelial-cell ferritin, erythroblast ferritin has (1) a more electronegative character by electrophoresis and ion-exchange chromatography, (2) larger molecular size by gel filtration and (3) lesser iron content by density-gradient centrifugation.

Erythropoietic kinetics in sheep studied by means of induced changes in hemoglobin phenotype

This investigation is concerned with the kinetics of the reciprocal relationship between sheep hemoglobin (Hb) A and Hb C formation in response to anemia. The relative synthesis of the hemoglobin types was assessed at various times in bone marrow erythroid cells incubated in vitro with (59)Fe. The changeover from Hb A to Hb C formation lagged by about 3 days behind the development of anemia and was complete within about 11 days. After recovery from anemia the reciprocal change back to preanemic conditions proceeded at a much slower rate, Hb C formation gradually declining to unmeasurable levels over about 25 days. Infusions of plasma with high erythropoietin titre induced the formation of relatively large quantities of Hb C in erythroid cells of nonanemic sheep, demonstrating the central importance of a humoral mechanism in the change of expression of the hemoglobin genes. THE FOLLOWING CONCLUSIONS WERE DRAWN: hemoglobin phenotype is determined at a stem cell level. Erythroid stem cells appear to undergo gradual renewal. The identity of the plasma factor which induces Hb C formation is not yet known; it is not present in plasma from nonanemic sheep, and its production is not dependent upon hemoglobin genotype. If the plasma factor turns out to be erythropoietin, then this hormone must have an important influence on the pool of erythroid stem cells.

The Metabolism of the Individual C14-labeled Hemoglobins in Patients with H-Thalassemia, with Observations on Radiochromate Binding to the Hemoglobins during Red Cell Survival

Patients with thalassemia may have abnormal alterations in the proportions of the structurally normal hemoglobins F and A2, or, in more rare circumstances, new hemoglobins of abnormal structure may appear. Such abnormal hemoglobins have not been shown to have substitutions of single amino acids in the polypeptide chain, as exemplified by hemoglobins S and C. Rather, they occur as unusual combinations of subunits of the hemoglobin molecule (a2/32). Thus, tetramer formation with normal polypeptide units is seen. Examples of tetramer formation in thalassemiaare hemoglobins H (,14) (1), Barts (y4) (2), and possibly a4 (3). There is also evidence that a delta chain tetramer may be present in very small quantities (4). Fusion of pieces of the beta and delta polypeptide chains into one polypeptide unit occurs in hemoglobin Lepore (5). Investigations in this laboratory have been concerned with the manner in which the various hemoglobins are distributed among the red cells in thalassemia. Previous reports have dealt with the behavior of hemoglobins A, F, and A2 (6, 7). The present study describes the distribution of

Inhibition of sickling by methyl acetimidate.

A computer generated model recently proposed for the alignment of hemoglobin S molecules in the microtubules which form within deoxygenated sickled cells, has implicated several Lys side chains in the intermolecular contact regions [1 ]. If this aspect of the model is correct, chemical modification of these amino groups would be expected to inhibit sickling. Imidoesters would appear to be appropriate reagents for this purpose since they have been shown to react selectively with protein amino groups [2,3]. MAI* was employed for the initial in vitro studies because: (1) modification of protein amino groups reportedly does not alter the charge of the protein at physiological pH [4] ; (2) reaction of hemoglobin A with MAI does not appreciably affect co-operativity or the Bohr effect [5] ; (3) DMA, a bifunctional imidoester, has been shown to inhibit sickling [6] ; (4) MAI is the smallest imidoester and therefore should introduce less steric obstruction than any other imidoester.

THE CONTROL OF HEMOGLOBIN PHENOTYPE IN CALVES AND SHEEP

The genes for nonalpha globin chain formation undergo sequential changes in activation during development. In man, the 0-chain of adult life ultimately replaces the tand y-chains of earlier developmental stages. Sickle cell anemia and Cooley’s anemia, the two most severe hemoglobinopathies, are both diseases of the phenotypic expression of the abnormal adult gene for beta globin chain expression. The possibility that gene expression may be altered in such a way as to suppress the defective gene while reactivating a normally silent but nondefective gene is considered in the studies that follow. These studies were conducted in sheep in which a normally silent gene for adult nonalpha globin chain production becomes active during anemia. Newborn calves were also used as a model for studies of fetal hemoglobin synthesis. Hemoglobin polymorphism is present in sheep. The two prevalent adult hemoglobin types are called A and B. Sheep possessing the gene for H b A predictably undergo a change in gene activation in response to anemia; the H b A is replaced by a new hemoglobin called C. This new hemoglobin differs from that which it replaces only in the @-chain. Thus, there appears to be a reciprocal response of two closely related @-chain genes during anemia. The switchover may be complete within ten days of the onset of severe anemia.’” The analogy between this change and the reciprocal change during development from yto @-chain formation is obvious. In the former instance, however, the change is reversible, whereas in the latter it is irreversible. The results to be presented will show: ( 1 ) that the stimulus of severe anemia produces consequences in the erythroid stem cells which persist for about 25 days beyond the cessation of the stimulus, and (2) that a factor present in plasma taken from anemic sheep is capable of producing the change in hemoglobin synthesis in nonanemic sheep. All the evidence to date is consistent with the hypothesis that this factor is the same as erythropoietin.