Identification of hemoglobins within individual rhesus monkey (Macaca mulatta) erythrocytes.

Rabbit and goat antibodies to monkey adult and fetal hemoglobin were prepared and purified to apparent monospecificity. After conjugation with fluorescein isothiocyanate, the antibodies were employed to identify the hemoglobin types within individual cells in peripheral erythrocyte smears. The percentage of neonatal monkey erythrocytes containing fetal hemoglobin was found to decrease with time. The existence of adult or fetal hemoglobin in the erythrocytes appeared to be mutually exclusive.     

Heterogeneity in the globin chain composition of a human minor fetal hemoglobin component

The first hemoglobin found to contain an acetyl blocking group was the minor human fetal hemoglobin, Hb FI, present as 10-15% of the total fetal hemoglobin in umbilical cord blood red cells. Acetylation occurs at the amino-terminal glycine of the gamma-globin chain. Assays for the acetyl group by two different methods gave values less than the 2 per tetramer expected for a fully acetylated hemoglobin. We have purified acetylated fetal hemoglobin FIc to homogeneity. The globin chain composition of Hb FIc has been examined by both globin chain separation on CM-cellulose and by tryptic peptide mapping by HPLC. The identities of the gamma globin chains and of the gamma T-1 peptides were confirmed by amino acid analysis. Globin chain separation profiles showed the presence of 22.3 +/- 7.0% of gamma 0 globin (of the total gamma globin) in Hb FIc. Accordingly, the tryptic peptide maps of Hb FIc tetramers also showed the presence of a similar amount of gamma 0T-1 peptide. The gamma 0T-1 peptide was not present in the maps of isolated gamma Ic globin. It is evident that column purified Hb FIc contains a certain percentage of non-acetylated gamma-globin chains, thus indicating a hybrid globin chain composition for this minor fetal hemoglobin component.     

Effect of fibroblast donor cell age and cell cycle on development of bovine nuclear transfer embryos in vitro

The effects of cell cycle stage and the age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages, and the in vitro life span of these cell lines was analyzed. Fibroblasts from both fetuses and calves had similar in vitro life spans of approximately 30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both fetuses and adult animals were cultured as a population, the percentage of cells in G1 increased linearly with time, whereas the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was higher in adult fibroblasts than in fetal fibroblasts. To study the individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate the cell cycle characteristics of both fetal and adult fibroblasts from either 25% or 100% confluent cultures. Irrespective of the age, the mean cell cycle length in isolated cells was shorter (9.6-15.5 h) than that observed for cells cultured as a population. Likewise, the length of the G1 stage in these isolated cells, as indicated by 5-bromo-deoxyuridine labeling, lasted only about 2-3 h. There were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent cultures of fetal or adult cell lines. This study suggests that there are substantial differences in cell cycle characteristics in cells derived from animals of different ages or cultured at different levels of confluence. However, these factors had no effect on in vitro development of nuclear transfer embryos.     

On the kinetics of erythroid cell differentiation in fetal mice. I. Microspectrophotometric determination of the hemoglobin content in erythroid cells during gestation

In a microspectrophotometric study, photographic emulsions and a computer are used for measuring the hemoglobin content of a large number (about 50,000) of erythroid cells in fetal mice. Histograms of the hemoglobin content in erythroid cells illustrate the kinetics of erythropoiesis in yolk sac derived nucleated cells in the fetal peripheral blood, in fetal liver, and in fetal spleen. After the occasional extrusion of their nucleus, yolk sac derived erythrocytes remain as “macrocytes” in fetal circulation two or three days longer than the nucleated yolk sac derived erythrocytes do. Erythrocytes in fetal liver have a constant hemoglobin content of 28 pg ² until day 17 of gestation. During further erythropoiesis in liver and then in the spleen, this amount is gradually adapted to the normal hemoglobin content in red blood cells of 16 pg.     

The transition from HK to LK phenotype in the red cells of newborn genetically LK lambs

Red cells from newborn lambs were separated into different age populations by centrifugation, and cells with fetal hemoglobin (Hb) were distinguished from those with adult Hb by an acid elution technique. Changes were followed during development in rates of K+ transport (active and passive), numbers of Na+/K+ pump sites per cell, cell volumes, and numbers of Lp and L1 antigen sites per cell. These changes were correlated with the percentage of cells with adult hemoglobin. (The Lp and L1 antigens are associated with K+ transport in that specific alloantibody against Lp, anti-Lp, stimulates active transport, and anti-L1 inhibits passive transport.) Active K+ transport decreased during development because of a decline in number of Na+/K+ pumps (from measurements of ouabain binding) and because of an alteration in the affinity of the pumps for intracellular K+ (from kinetic studies in which the intracellular K+ concentration was varied). Cells with fetal Hb had fewer Lp sites and were larger than cells with adult Hb. As transport properties changed, the number of Lp sites increased and continued to increase after all the cells had adult Hb Cells with fetal Hb had as many L1 sites as lamb cells with adult Hb, but the number of L1 sites was less than those found previously for adult sheep. A population of small cells with intermediate K+ concentration and intermediate numbers of Lp sites appeared soon after birth. The various points of evidence suggested that the developmental process leading to cells with adult transport properties was a gradual one and did not coincide precisely with the switch from fetal to adult Hb.     

The effect of progesterone on hemoglobin synthesis in suspension cultures of fetal erythroid cells from calf liver

When fetal calf liver erythroid cells were incubated in the presence of small amounts of progesterone (10(-7)-10(-8) M), the hemoglobin synthesis in these cells was significantly increased. The increase in the amount of radioactivity in de novo synthesized hemoglobins could be demonstrated when techniques such as isoelectric focusing, chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100 were used to isolate the hemoglobin fraction. Using the latter technique, it was shown that the synthesis of cytoplasmic non-hemoglobin proteins in erythroid-cell lysates was also stimulated by progesterone. The presence of hepatocytes in culture nullified the hormone action. It was necessary that progesterone was present during the first hours of culture. Delayed addition of the steroid to the cells had no effect on hemoglobin synthesis. Erythropoietin was necessary to obtain stimulation by progesterone. These results suggest that the target cell of the hormone is an erythropoietin-sensitive cell. High concentrations of progesterone (10(-4) M) strongly inhibited hemoglobin synthesis in fetal calf erythroid cells. Culture of cells under this condition, however, gives rise to a cell population that preferentially synthesizes adult hemoglobin. Our results suggest that in the erythropoietic calf liver, high concentrations of progesterone may preferentially stimulate adult hemoglobin synthesis, or that those cells which have a high capacity to synthesize adult hemoglobins are less sensitive to toxic concentrations of the hormone. The effects of stimulation of hemoglobin synthesis in fetal calf erythroid cells occur at hormone concentrations that suggest a possible physiological role of progesterone in fetal, and eventually also in maternal, erythropoiesis.     

Detection of hemoglobin variants in erythrocytes by flow cytometry

BACKGROUND: With the emergence of fetal hemoglobin (Hb F)stimulating agents as potential treatments for sickle-cell disease and thalassemias, procedures to monitor the effect of these agents on Hb F levels in individuals will be needed. We developed a rapid procedure that detects fetal hemoglobin in erythrocytes (F cells) using a fluorescein isothiocyanate (FITC) conjugated monoclonal antibody against Hb F. METHODS: Ten microliters of washed blood was fixed in formaldehyde and glutaraldehyde, then permeabilized in a Triton X-100/PBS solution containing a FITC-labeled monoclonal antibody to Hb F. The blood was analyzed by flow cytometry to determine the percentage of F cells. RESULTS: Nearly 200 Hb F-containing samples were analyzed by this protocol and demonstrated good correlation to percent Hb F results determined by high pressure liquid chromatography (HPLC). In addition, a number of samples were fixed and permeabilized using this method as well as a previously-described method that uses dimethyl 3,3'dithiobispropionimadate (DTBP) as a fixative as well as a different anti-Hb F monoclonal. Good correlation (r = 0.96, r2 = 0.93, P<0.001) was observed between the two protocols. CONCLUSIONS: This procedure is easy, reproducible, and gives accurate F cell results. It can be used to measure a wide range of F cell percentages and may also be used to dual-stain Hb F along with other hemoglobin variants and erythrocyte surface antigens.     

Differential control of the synthesis of two hemoglobin beta chains in normal mice

A fetal-to-adult switch in the proportion of the mouse minor hemoglobin is described. Although mice have no fetal hemoglobin per se, the timing of this switch in the mouse suggests that the mechanism of its control may directly parallel that of the human switch from fetal to adult hemoglobin expression. The mouse minor hemoglobin is expressed only in strains with the "diffuse" allele for the beta chain complex locus. Fetal liver cells of these mice synthesize a much greater proportion of the betaminor globin chain that do adult hematopoietic cells. Consequently, circulating fetal erythrocytes carry a high level of the minor hemoglobin containing it. By the time of birth, a lowered proportion of betaminor is synthesized in the liver. This low proportion continues to be expressed during early erythroid maturation in the adult. The fetal-to-adult switch is the first indication that in normal mice, the two beta chain loci can be expressed noncoordinately. The similarity between the patterns of the decline of the minor hemoglobin in mice and of the disappearance of fetal hemoglobin in humans suggests that the minor hemoglobin in the "diffuse" mouse may function to some degree as a fetal hemoglobin in the period between the disappearance of the embryonic hemoglobins and the time of birth.     

Intraerythrocytic organic phosphates of fetal and adult seaperch (Embiotoca lateralis): Their role in maternal-fetal oxygen transport

Summary The viviparous seaperch,Embiotoca lateralis, has unique fetal and adult hemoglobins. Stripped fetal hemoglobin has a higher oxygen affinity than stripped adult hemoglobin at pH 6.5–7.1. The oxygen affinities of both adult and fetal hemoglobins are lowered allosterically by ATP at pH 7.1. Both fetal and adult seaperch erythrocytes include approximately 82% ATP and 18% GTP of the total nucleotide triphosphates (NTP) with a trace of AMP. No 2,3-diphosphoglycerate or inositol polyphosphate was detected. Mid- and late-gestation erythrocytes contain less NTP/mole hemoglobin tetramer than do adult cells. The effective NTP concentration in adult cells is higher than that of the fetal erythrocytes even when the intracellular concentration of Mg²⁺, which complexes with NTP, is accounted for. The difference in adult and fetal intraerythrocytic NTP concentration should enhance transfer of oxygen from maternal to fetal blood. Thus, the teleostEmbiotoca lateralis may employ a dual mechanism in maternal-fetal oxygen transfer. A difference in fetal and maternal hemoglobin structure and oxygen affinities is enhanced by a difference in their respective intraerythrocytic organic phosphate concentrations.     

Oxygen equilibrium characteristics of adult and fetal hemoglobin of Japanese monkey (Macaca fuscata).

Adult hemoglobin and fetal hemoglobin were obtained from Japanese monkey (Macaca fuscata) and their oxygen equilibrium characteristics were studied. (1) The oxygen affinity of fetal hemoglobin was higher than that of adult hemoglobin both in the presence and absence of 2,3-diphosphoglycerate. The presence of diphosphoglycerate lowers the oxygen affinity of adult hemoglobin much greater than does that of HbF and the diphosphoglycerate levels of red cells of adult and newborn monkeys are about the same. (2) The intensity of the Bohr effect, as expressed by -deltalogP50/deltapH, at pH 7.4 was in the order of fetal hemoglobin-diphosphoglycerate greater than adult hemoglobin-diphosphoglycerate greater than fetal hemoglobin greater than adult hemoglobin.     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.     

Embryonic and fetal hemoglobin synthesis in K562 cell line

K562 cell line was grown in liquid suspension and in plasma clot cultures. Morphological studies revealed the presence of a minority of cells, which were identified as erythroblasts. However, the majority of the cells remained unidentified. Biochemical studies confirmed the synthesis of hemoglobin by K562 cells. The pattern of hemoglobin (Hb) production was of the embryonic type, with the presence of small amount of fetal Hb. The addition of several inducers, like Epo and butyrate, was unable to modify the pattern of Hb production of K562. In contrast, the addition of hemin increased the synthesis of Hb and stimulated the synthesis of fetal Hb and probably adult Hb.     

Variations in the relative activities of erythrocyte membrane ATPase with changes in severity of sickle cell anemia

Red blood cells from 31 patients with sickle cell anemia whose hemoglobins were ascertained as SS were assayed for Mg-, Ca-, Na-, and total ATPase activities. The ATPase activities were correlated with the various stages of severity in each patient as determined by clinical parameters. The results demonstrate that increases in ATPase activities were associated with increases in the percentage severity of sickle cell anemia. Severity correlated inversely with fetal hemoglobin levels in the sickle cell patients. ATPase activities were generally higher in SS genotypes than in AS and AA normal individuals.     

Comparison of the tube leukocyte adherence inhibition test with the lymphocyte stimulation assay in a rat transplantation model.

The tube leukocyte adherence inhibition test (LAI) was analyzed in a heterotopic heart transplantation model in BN and Wag/Rij rats. Seven, fourteen, and twenty-two days after transplantation spleen cells of the recipient rat were tested for recognition of histocompatibility antigens in crude membrane extracts prepared from normal rat tissue. It was found that addition of fetal calf serum affected the cell adherence considerably and that there was a large difference in the percentage of adherent cells from different animals. In a comparative study the lymphocyte stimulation assay proved to be more sensitive and reproducible than the LAI in this rat system.     

Human globin knock-in mice complete fetal-to-adult hemoglobin switching in postnatal development

Elevated levels of fetal γ-globin can cure disorders caused by mutations in the adult β-globin gene. This clinical finding has motivated studies to improve our understanding of hemoglobin switching. Unlike humans, mice do not express a distinct fetal globin. Transgenic mice that contain the human β-globin locus complete their fetal-to-adult hemoglobin switch prior to birth, with human γ-globin predominantly restricted to primitive erythroid cells. We established humanized (100% human hemoglobin) knock-in mice that demonstrate a distinct fetal hemoglobin (HbF) stage, where γ-globin is the dominant globin chain produced during mid- to late gestation. Human γ- and β-globin gene competition is evident around the time of birth, and γ-globin chain production diminishes in postnatal life, with transient production of HbF reticulocytes. Following completion of the γ- to-β-globin switch, adult erythroid cells synthesize low levels of HbF. We conclude that the knock-in globin genes are expressed in a pattern strikingly similar to that in human development, most notably with postnatal resolution of the fetal-to-adult hemoglobin switch. Our findings are consistent with the importance of BCL11A in hemoglobin switching, since removal of intergenic binding sites for BCL11A results in human γ-globin expression in mouse definitive erythroid cells.     

Decline in lung liquid volume and secretion rate during oligohydramnios in fetal sheep

Reduced amniotic fluid volume often results in fetal lung hypoplasia. Our aim was to examine the effects of prolonged drainage of amniotic and allantoic fluids on lung liquid volume (Vl), secretion rate (Vs), and tracheal flow rate (Vtr) in fetal sheep. In five experimental animals, amniotic and allantoic fluids were drained from 107 to 135 days of gestation. The volume of fluid drained from the experimental animals was 411.8 +/- 24.4 ml/day (n = 140). In six control animals, amniotic fluid volume was 747.7 +/- 89.7 ml (n = 15). Wet and dry lung weights were 20-25% lower in experimental fetuses than in control fetuses. Fetal hemoglobin, O2 saturation, arterial PO2, pH, and hematocrit were unchanged by drainage. During the drainage period, Vl was up to 65% lower, Vs was up to 35% lower, and Vtr was up to 40% lower in experimental fetuses than in control fetuses. We conclude that prolonged drainage of amniotic and allantoic fluids decreases Vl, Vs, and Vtr in fetal sheep. These findings indicate that fetal lung hypoplasia associated with oligohydramnios may be the result of a prolonged reduction in Vl.     

Prostaglandin E2 mediated effects on the synthesis of fetal and adult hemoglobin in blood erythroid bursts

The effects of prostaglandin E2 (PGE2) in association with erythropoietin on the synthesis of fetal and adult hemoglobin in peripheral blood-derived erythroid burst colonies from normal adults and from patients with sickle cell anemia were investigated. The synthesized hemoglobin at the end of 8, 14 or 18 days in culture was separated by DEAE-cellulose chromatography of 35S-methionine labelled hemoglobin. Quantitative estimation of the synthesized hemoglobin phenotypes, for the three indicated culture periods, showed preferential synthesis of Hb F in addition to an overall increase in hemoglobin synthesis in PGE2 treated colonies. Furthermore, the reactivation of fetal hemoglobin production by PGE2 was more pronounced when the adherent cells were included in the culture dishes. These results indicate that the addition of PGE2 to culture dishes presumably constitutes an environmental change to promote the functional changes seen in the blood erythroid bursts in terms of Hb synthesis and switching.     

Mouse fetal hemoglobin.

Using isoelectric focusing, a fetal hemoglobin was found in the peripheral blood of C57BL/6 fetal mouse during the 14 to 20 days gestational period. In acid-urea polyacrylamide gel the pattern of this fetal hemoglobin was different from that of the adult hemoglobin. The mouse fetal hemoglobin was differentiated from the mouse adult hemoglobin by immunodiffusion reaction. It suggests that there is a transient fetal hemoglobin in the C57BL/6 mouse during gestational age.     

Iodination-Coupled Tetrazonium and Ferric Ferricya-Nide Reduction Stains for Differentiating Red Blood Cells Containing Fetal or Adult Hemoglobin

Either the iodination-coupled tetrazonium reaction or the ferric ferricyanide reduction procedure can be used to differentiate red blood cells containing fetal hemoglobin (hemoglobin F) from those containing adult hemoglobin (hemoglobin A) in blood smears. Oxalated blood is diluted with 3 parts of physiological saline, and smears are made on slides. The air-dried slides are treated with absolute ethanol for 2 min, dried, and placed in phosphate-citrate buffer of pH 3.2-3.6 for 1 min at 37°C. They are then rinsed in distilled water, and dried for storage or stained at once by either the iodination-coupled tetrazonium or the ferric ferricyanide reduction procedure. Adult hemoglobin is extracted by the buffer, so that red blood cells containing fetal hemoglobin give a much darker stain than those containing adult hemoglobin. The hemoglobin S of patients with sickle-cell anemia behaves like adult hemoglobin.