Gamma Chain Production Research Articles

Synthesis of hemoglobin F in adult simian erythroid progenitor-derived colonies.

The simian hematopoietic system is known to respond to anemic stress with the production of erythrocytes containing large amounts of fetal hemoglobin. To determine the regulatory mechanism responsible for hemoglobin F (HbF) production in stress erythropoiesis, adult simian bone marrow cells were cultured in plasma clots in the presence or absence of erythropoietin and burst-promoting activities, and the HbF content of progenitor-derived colonies was determined by radioimmunoligand assay. Three classes of erythroid progenitors were detected: BFU-E, CFU-E, and a very mature cohort of dense highly erythropoietin-responsive cells (HERC). These classes varied in inverse proportion to their maturity with respect to their potential for HbF accumulation in the colonies to which they give rise. Both erythropoietin and burst-promoting activity stimulated HbF production, particularly in colonies derived from immature progenitors. For example, under conditions of high erythropoietin stimulation, BFU-E colonies contained 13.7-37.7% HbF, CFU-E colonies contained 2.8-13.5% HbF, and HERC colonies 0-1% HbF. These results suggest that under nonstress conditions simian erythrocytes are derived almost entirely from HERC progenitors and proerythroblasts in which gamma chain synthesis is suppressed. During stress erythropoiesis, augmented HbF accumulation could be explained by the rapid entrance into the marrow of proerythroblasts directly derived from immature progenitors. Gamma chain production in these proerythroblasts is additionally regulated by the changes in environmental erythropoietin and burst-promoting activities.

The British type of non-deletion HPFH: characterization of developmental changes in vivo and erythroid growth in vitro.

Further studies of a unique British family with a rare type of hereditary persistence of fetal haemoglobin (HPFH) have confirmed that this condition appears to result from a regulatory defect affecting globin gene expression. Adult heterozygotes for this disorder have 3.5-10.0% Hb F while homozygotes have 18-21% Hb F and in both cases the Hb F contained predominantly A gamma chains (89-94%). In all other respects these individuals were haematologically normal. Two obligate heterozygotes were studied from birth, at which time both the proportion of Hb F and the G gamma/A gamma ratio were completely normal. There was a delayed decline in Hb F which continued well into childhood and a parallel decline in the proportion of G gamma chains. The growth of erythroid bursts showed no difference in number, size or erythropoietin responsiveness between homozygous HPFH, heterozygous HPFH or normal family members. Haemoglobin synthesis in these bursts showed the normal pattern of asynchrony between gamma and beta chain synthesis but there was an overall increase in gamma chain production in the HPFH cases, particularly the homozygotes, compared to normal. In addition, the abnormal G gamma/A gamma ratio was faithfully reproduced in vitro. This condition appears to result, therefore, from a regulatory abnormality affecting the postnatal repression of gamma chain synthesis.

Synthesis of hemoglobin chains in adult and newborn goats: possible influence of the beta c synthesis on the production of alpha chains.

The rates of in vitro synthesis of hemoglobin alpha and non-alpha chains were determined in adult goats during blood loss anemia and in newborn goats during postnatal development. The reticulocytes were incubated in a medium supporting protein synthesis and containing [14C] leucine. The hemoglobin chains were separated by CM-cellulose chromatography. The adult animals responded to the phlebotomy with a production of nearly 100% beta C chain about 10 days after the start of the experiment. Severe anemia and the production of beta C chain was accompanied by a significant increase in the alpha/non-alpha synthesis ratio. The gamma chain production in the newborns declined rapidly after birth and was completely replaced by that of the beta C chain in 7-10 days. No significant difference in the relative synthesis of the three types of alpha chain was observed during this period. Goats with either a homozygosity for the I alpha or I alpha B chains synthesize the I alpha (or I alpha B) and II alpha chains at a ratio of 3:1. When a heterozygosity for I alpha B chains is present the I alpha:I alpha B:II alpha ratio approximates 2:1:1. Results of kinetic studies suggest that the II alpha chains are synthesized at a slower rate than the II alpha chains. The difference in the alpha/non-alpha ratio during severe anemia may be due to a decreased production of beta C chains. No significant difference in the relative synthesis of the I alpha, I alpha B, and II alpha chains was observed between the anemic adult animal and the newborn goat or the growing kid.

The Gamma Chain Heterogeneity of Fetal Hemoglobin in Black β-Thalassemia and HPFH Heterozygotes

High pressure liquid chromatography (HPLC) was applied to the HbF isolated from blood of numerous black patients with beta-thalassemia trait or homozygosity, G gamma-delta beta-thalassemia trait, G gamma A-gamma HPFH heterozygosity, or the G gamma-[delta+ beta+]-HPFH condition. The method allowed an accurate evaluation of the relative quantities of three types of gamma-chain (G gamma, A gamma I, A gamma T) in the fetal hemoglobins. The results have shown the following. (A) The incidence of the A gamma T-chain in beta-thal heterozygotes and G gamma A gamma-HPFH heterozygotes is about the same as has been observed in black newborn; about one of five blacks are heterozygous for this A gamma-chain variant. The A gamma T-chain was not detected in the nine G gamma-delta beta-thal heterozygotes nor in the eight G gamma-[delta+ beta+]-HPFH heterozygotes. (B) In most cases, the A gamma T-chain was produced by the A gamma gene in trans to the beta-thal or HPFH determinant. The contribution by the gamma-chain genes in trans to the beta-thal or HPFH determinant is about 15% of the total gamma-chain production in both conditions. (C) Three black beta-thal heterozygotes (and five additional relatives) had the A gamma T gene in cis to the beta-thal determinant. Four of these patients had a low levels of G gamma-chain (the "adult" level), and the contribution by the A gamma gene in cis to the beta-thal determinant was about three times that of the A gamma gene in trans. The four additional patients, all members of one family, had a high level of G gamma-chain (the "newborn" level), and the contribution of the A gamma gene in cis was half of that seen in the previously mentioned four patients while that of the A gamma gene in trans was essentially the same. These limited data suggest that the genetic anomaly causing high high G gamma levels in adult beta-thal heterozygotes is linked to the beta-thal determinant and that one of its primary effects is a decreased synthetic expression of the A gamma gene in cis to the beta-thal determinant.

The gamma chain heterogeneity of fetal hemoglobin in black beta- thalassemia and HPFH heterozygotes

High pressure liquid chromatography (HPLC) was applied to the HbF isolated from blood of numerous black patients with beta-thalassemia trait or homozygosity, G gamma-delta beta-thalassemia trait, G gamma A- gamma HPFH heterozygosity, or the G gamma-[delta+ beta+]-HPFH condition. The method allowed an accurate evaluation of the relative quantities of three types of gamma-chain (G gamma, A gamma I, A gamma T) in the fetal hemoglobins. The results have shown the following. (A) The incidence of the A gamma T-chain in beta-thal heterozygotes and G gamma A gamma-HPFH heterozygotes is about the same as has been observed in black newborn; about one of five blacks are heterozygous for this A gamma-chain variant. The A gamma T-chain was not detected in the nine G gamma-delta beta-thal heterozygotes nor in the eight G gamma-[delta+ beta+]-HPFH heterozygotes. (B) In most cases, the A gamma T-chain was produced by the A gamma gene in trans to the beta-thal or HPFH determinant. The contribution by the gamma-chain genes in trans to the beta-thal or HPFH determinant is about 15% of the total gamma-chain production in both conditions. (C) Three black beta-thal heterozygotes (and five additional relatives) had the A gamma T gene in cis to the beta-thal determinant. Four of these patients had a low levels of G gamma-chain (the “adult” level), and the contribution by the A gamma gene in cis to the beta-thal determinant was about three times that of the A gamma gene in trans. The four additional patients, all members of one family, had a high level of G gamma-chain (the “newborn” level), and the contribution of the A gamma gene in cis was half of that seen in the previously mentioned four patients while that of the A gamma gene in trans was essentially the same. These limited data suggest that the genetic anomaly causing high high G gamma levels in adult beta-thal heterozygotes is linked to the beta-thal determinant and that one of its primary effects is a decreased synthetic expression of the A gamma gene in cis to the beta-thal determinant.

Haemolytic Disease of the Newborn and Chronic Anaemia Induced by γβ Thalassaemia in a Dutch Family

Severe haemolytic anaemia with hyperbilirubinaemia and erythroblastaemia was observed in nine newborn infants belonging to one large family. One infant was still-born, two died shortly after birth, five recorded after receiving one or more exchange transfusions and one improved without transfusional therapy. In four out of six newborns whose bilirubin levels were determined, a mixed hyperbilirubinaemia was found with high concentrations of both free and conjugated bilirubin. At the end of the first year of life the surviving children, although in satisfactory general condition, showed a mild microcytic hypochromic anaemia with decreased red cell osmotic fragility and morphological abnormalities of the erythrocytes consisting of microcytosis, anisopoikilocytosis and target cells. The serum iron levels were normal. The same haematological picture was also present in those parents of the affected children that are members of this family; these adult had normal levels of Hbs A2 and F. Incorporation of [3H]leucine into the globin chains of two adults with this syndrome revealed a reduced beta-chain synthesis (beta/alpha ratio of 0 . 49 and 0 . 53, respectively). In the two infants available for this investigation, reduced gamma-chain production was found shortly after birth. In parallel with the switch from fetal to adult haemoglobin, the deficient gamma-chain production was replaced by a similar reduction of beta-chain synthesis. These results suggested, therefore, a combined deficiency of gamma- and beta-chain production. The normal levels of Hb A2 were compatible with a defective delta-chain synthesis as well. Analysis with restriction enzymes had shown previously a large deletion, comprising gamma and delta genes, in one of the chromosomes of the affected individuals. The same procedure had established that, in spite of the defective beta-chain production, the beta-globin structural gene is intact.

Haemoglobin synthesis in human fetal liver maintained in short-term tissue culture.

Human fetal liver maintained in tissue culture has been examined as a model system for studying the factors involved in the switch from fetal to adult haemoglobin production. In erythropoietin-treated cultures alpha-, beta- and gamma-chain synthesis remains active for up to 12 days. Globin-chain synthesis is balanced and the alpha/beta + gamma-chain production ratio remains constant up to about 8 days; in longer cultures gamma-chain production declines before that of beta chains. It is concluded that the system may be useful for examining factors involved in the switch from gamma- to beta-chain production provided that these are acting on cells already committed to the erythroid compartment.

Inhibition of Synthesis of Fetal Hemoglobin by an Isoleucine Analogue*

Reticulocytes from newborn infants with Rh isoimmune hemolytic disease actively incorporated radioactive amino acids in vitro into hemoglobins F and A. Approximately 50% of the reticulocytes appeared capable of synthesis of both of these hemoglobins within the same cell, as demonstrated by the selective elution technique of Betke and Kleihauer. An isoleucine analogue, L-O-methylthreonine, inhibited the incorporation of a variety of amino acids into hemoglobin F, without significantly affecting the synthesis of hemoglobin A. The inhibition was prevented upon concomitant addition of L-isoleucine to the medium. These observations suggest that an independent biosynthetic apparatus is present in the cell for the synthesis of each of these two hemoglobins. Because isoleucine is present only in the gamma chains of hemoglobin F, the inhibitory effect of the analogue on the synthesis of this hemoglobin must represent a selective effect on the production of gamma chains.

Alpha, Beta and Gamma Hemoglobin Polypeptide Chains During the Neonatal Period with Description of a Fetal Form of Hemoglobin DαSt. Louis

Abstract Five hundred and thirty-nine randomly selected umbilical cord blood samples (449 Negroes and 90 Caucasians) were studied. Observations were made on 15 babies with abnormal hemoglobins until 3 to 19 months of age. Hgb D was found in high concentration, approximately 27 per cent, in the cord blood of two newborn infants. The mobility of the fetal Hgb D by starch block and paper electrophoresis (pH 8.6) was slower than that of the Hgb D found in the parents. The cord blood D had many characteristics of a fetal hemoglobin and even though the abnormality was in the alpha chain, the hemoglobin behaved similarly to normal fetal hemoglobin in respect to alkali denaturation, mobility on agar electrophoresis (pH 6.2) and on resin column chromatography. It appears that gamma chains influence the mobility of the fetal form of Hgb D α on these two supporting media. The N-terminal amino acid was glycine following one hour of acid hydrolysis; valine was present in the 18 hour acid hydrolysate. Observations made on "fingerprints" of tryptic digests of hemolysates of Hgb D indicated that peptide 3, contained in the alpha chain, had moved from the neutral core to overshadow peptide 9. One-dimensional electrophoresis of tryptic digests of Hgb D displayed a peptide between the neutral zone and peptide 10 which was not seen in digests of Hgb A. The peptide gave a strong staining reaction for histidine. Peptides 3 and 9 are α chain peptides; their involvement provides strong evidence for an alpha chain abnormality in Hgb D αSt. Louis. Forty-seven (10.1 per cent) Negro babies had Hgb S in their bloods. The amount ranged from 1.7 to 8.5 per cent of the total hemoglobin and 0.9 to 13.0 per cent of the erythrocytes sickled after treatment with metabisulfite. Five newborns with sickle cell trait were regularly observed. The amount of Hgb S gradually increased to adult levels by 3 to 8 months of age. Nine newborn infants (2.0 per cent) had Hgb C in their cord blood. The concentration of Hgb C approached the maximum value by 6 months of age in one infant observed over a period of one year. The amount of Hgb F in 143 unselected cord bloods ranged from 60 to 96.7 per cent (mean 77.8); the mean value for 134 full term babies was 77.1 and for 9 prematures 81.7 per cent. No correlation was found between the amount of Hgb F and the birth weight, between boys and girls, between Caucasian and Negro, nor between those with normal and those with abnormal hemoglobins. The characteristics of a "fast" Hgb, Hgb γ4 ("Bart’s"), found in cord blood were described. The concentration of Hgb γ4 in 10 bloods quantitated ranged between 2.3 and 12.4 per cent of the total hemoglobin. Of nine infants who were routinely examined, three had other abnormalities, Hgb S and Hgb D, and hereditary elliptocytosis, and six appeared to have no associated blood dyscrasia (except for possible iron deficiency detected at one year in two) and were normal and healthy. Hgb A2 in 90 random cord blood specimens ranged from 0 to 1.8 per cent of the total hemoglobin. There was no correlation between the amount of Hgb A2 and Hgb A1, Hgb F and birth weight. Other minor components of hemoglobins were also described. The conclusions made from the observations were: (1) at birth the rate of synthesis of alpha chains appears to be already at maximum, (2) the synthesis of beta chains was low, and (3) the gamma chain production was active as indicated by polymerization in some apparently normal infants. However, slow synthesis of alpha chains could not be ruled out as a cause for gamma chain tetramers.