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During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process.

Objectives: Detection of iron deficiency during pregnancy with hemoglobin (Hb) and serum measurements is insignificant as the measurements may be affected by e.g. hemodilution or accelerated erythropoiesis. This study tests whether cell indices will give a more reliable measure of iron deficiency in pregnant women at term., Methods: The population was 202 pregnant women. Using the ADVIA 120 hematology system, Hb, mean cell volume (MCV), percentage of hypochromic red blood cells (%HYPOm) and reticulocytes (%HYPOr), and cellular hemoglobin in reticulocytes (CHr) were tested. Additionally, transferrin saturation (TfSat), ferritin, and transferrin receptor (TfR) were analyzed. Receiver operating characteristic (ROC) curves and area under the ROC curves (AUC) were used as statistical methods., Results: When TfSat (< or = 11%) was used as the reference test for iron deficiency, %HYPOm and CHr had a sensitivity of 58.1% and 80.7%, while the specificities were 82.6% and 71.3%, respectively. Additionally, the AUC values were %HYPOr 0.80, CHr 0.79, ferritin 0.77, %HYPOm 0.75, TfR 0.67, MCV 0.63 and Hb 0.64. The results provided by the cell indices alone (%HYPOm or CHr) were in good agreement with the results based on the usage of a combination of three commonly used tests (Hb, MCV, ferritin)., Conclusions: This study suggests that the most practical way to diagnose iron deficiency in pregnant women at term is to use cell indices such as CHr and %HYPOm provided by the automated hematological analyzer. Further studies are needed to determine the usefulness of the cell indices in diagnosing iron deficiency longitudinally during the course of pregnancy.

Microcytic erythropoiesis in case of anemia is frequently due to iron deficiency or may be due to alpha- and beta- thalassemia trait as a result of increased activity of erythropoiesis. The aim of the present study was to evaluate alterations with regard to the degree of hemoglobinization in reticulocytes in comparison with mature erythrocytes. Iron availability in subjects with anemia resulting from iron deficiency and alpha- or beta- thalassemia was studied by application of conventional as well hemocytometric parameters that have recently become available. Participants of the study were reference subjects (n=75), subjects with iron deficiency anemia (IDA, n=52) and alpha- (n=26) or beta-thalassemia trait (n=24). If compared with the reference group obviously increased RBC counts together with decreased values for RDW-sd and MCHC were established in case of alpha- and beta- thalassemia subjects. Deviations were demonstrated to be more pronounced in case of beta- thalassemia. Accelerated erythropoiesis in the case of subjects with IDA and beta-thalassemia is manifested by detection of increased results for immature reticulocyte counts. In particular in case of beta- thalassemia, elevated reticulocyte counts combined with slightly increased values for ZPP/heme ratio reflect increased activity of erythropoiesis. In the case of subjects with beta-thalassemia serum transferrin concentrations revealed slightly decreased results, whereas serum ferritin and iron concentrations demonstrated a tendency towards higher values if compared with the group of reference subjects. At a definitive MCV level, the hemoglobin content of reticulocytes is decreased in the case of IDA if compared with the alpha- or beta- thalassemia trait. For the ratio of hemoglobin content of reticulocytes and erythrocytes, obviously decreased results are demonstrated in the case of subjects with iron deficiency anemia (1.02 +/- 0.08, mean +/- SD) and in the case of beta-thalassemia (1.06 +/- 0.04) if compared with the group of reference subjects (1.11 +/- 0.02) and a-thalassemia (1.11 +/- 0.07). Evaluation of the hemoglobinization state should be performed by means of pattern recognition in concordance with characteristic profiles for parameters reflecting the actual iron state. In case of therapy the result of intervention can be appropriately monitored by longitudinal follow-up.

Reticulocyte hemoglobin content (CHr) is considered an index of iron status, helpful in the differential diagnosis of microcytoses. Its potential can be enhanced by comparing CHr dynamic reference values (CHr-e: expected CHr), which are proportional to the MCVr variations occurring in micro- or macrocytosis, with measured CHr values. We demonstrate that the difference between measured CHr and CHr-e (DeltaCHr) is helpful to differentiate the anemic syndromes and, in particular, beta-thalassemia vs. presumable sideropenia. DeltaCHr can also indicate when to interrupt iron supplementation. DeltaCHr allows an insight into the erythropoiesis of thalassemic and sideropenic subjects, pointing out the reduced hemoglobin production and ineffective erythroid activity in these conditions.

A pure population of young reticulocytes was isolated from the blood of healthy blood donors by an immunomagnetic technique. The young reticulocytes isolated had a larger mean corpuscular volume (MCV) but lower mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) than reticulocytes in peripheral blood. The cells were incubated in vitro for 5 days with hemolysis less than 5%. Maturation of reticulocytes was studied with 4 different systems: new methylene blue-stained smears and flow cytometry after auramine O, oxazine 750, or thiazole orange staining. The maturation process from reticulocytes to erythrocytes had a half-life of 30 hours estimated by visual counting. Reticulocytes analyzed with flow cytometers had half-lives of 20, 28, and 29 hours with Coulter Epics XL, Sysmex R-1000, and Bayer H*3 devices, respectively. Total maturation time was 4 to 5 days. During maturation, the reticulocyte MCV gradually decreased with a concomitant increase in reticulocyte MCHC. The reticulocytes synthesized hemoglobin, and the MCH of the cells increased approximately 7% during the incubation period. Taking into account loss of hemoglobin-containing vesicles during reticulocyte maturation, we estimated that more than 20% of hemoglobin in erythrocytes was synthesized in reticulocytes after release from bone marrow.

Iron uptake for hemoglobin synthesis was apparently increased in the reticulocytes of severely iron deficient anemic women, but was actually decreased significantly when expressed in terms of number of reticulocytes. The decreased synthesis could be restored to more than normal by supplementing with ferric iron or transferrin iron (normal plasma), but not with ferrous iron, which rather further reduced the already impaired hemoglobin synthesis.

Succinylacetone (SA) is an inhibitor of heme synthesis that acts on the enzyme delta-aminolevulinic acid dehydratase. When reticulocytes are incubated with 59Fe-transferrin (59Fe-Tf) in the presence of SA, there is an accumulation of 59Fe in the mitochondrion and in a cytosolic non-heme intermediate that has been described as a putative Fe transporter (Adams et al, Biochim Biophys Acta 1012:243, 1989). Considering these observations, the present study was designed to examine the intermediates of Fe metabolism in control and SA-treated reticulocytes. This investigation showed that in the cytosol of control cells, most 59Fe was incorporated into hemoglobin (Hb) with a minor amount entering ferritin. In addition, a previously unrecognized cytosolic intermediate was identified (band X) that was absent when heme synthesis was inhibited with SA. Upon reincubation of SA-treated reticulocytes with protoporphyrin IX, band X initially increased in intensity and then decreased later in the incubation. In contrast, when 59Fe-labeled control cells were reincubated in the presence of SA and unlabeled diferric Tf, there was a marked decrease in the intensity of band X. These experiments suggest that component X may be an intermediate involved in the transfer of heme in the cytosol. Alternatively, these data could also be interpreted as indicating that band X may be a short-lived hemoprotein. We have confirmed the presence of an 59Fe-containing molecule in the cytosol of SA-treated reticulocytes (band Y) that is not present in control cells. However, when cells were incubated with 59Fe-Tf plus SA and then chased in the presence of SA and unlabeled diferric Tf, there was no decrease in this cytosolic pool of Fe, suggesting that it was not a intermediate supplying Fe for either ferritin or heme synthesis. Finally, there is little low molecular weight (Mr) Fe in reticulocytes, and our studies suggest that the low-Mr Fe present does not behave as an intermediate. Moreover, after inhibition of heme synthesis with SA, 59Fe in the low-Mr compartment was markedly decreased, suggesting that this component may be heme or a low-Mr heme-containing molecule. Considering the apparent lack of a cytosolic Fe transporter in rabbit reticulocytes, an alternative model of intracellular Fe transport is proposed that does not implicate a potentially toxic intermediate pool of low-Mr Fe complexes.

1. A quantitative increase of organelles in early reticulocytes has been observed compared to that found in late erythroblasts of the peripheral rabbit embryo blood. 2. The increase is due to the formation of hemosomes, organelles taken as sites for final hemoglobin (Hb) biosynthesis or where the assembly of heme and globin polypeptides could occur. 3. These organelles derive indirectly from mitochondria whose internal membrane grows concomitantly to its differentiation, originating lamellated bodies that modify successively to prehemosomal vesicles, prohemosomes and hemosomes. 4. The occurrence of membrane synthesis for the formation of lamellated bodies could explain the increase of organelles per cell and, thereby, the enhancement of the Hb biosynthesis rate in peripheral embryo blood in relation to this biosynthesis rate in the liver, as had been biochemically ascertained by other authors.

Plumbous ion a potent inhibitor of hemoglobin synthesis by reticulocytes in vivo, is not as effective in inhibiting in vitro globin synthesizing systems prepared from these cells. The reticulocyte is considerably more sensitive to Pb2+ than are leukemic leukocytes, HeLa cells or bacteria. In fact, protein synthesis in leukemic leukocytes is actually stimulated by Pb2+. The synthesis of non-heme protein as well as hemoglobin is inhibited by Pb2+ in reticulocytes and the synthesis of alpha chains is inhibited to a greater degree tcids into reticulocytes. All of these observations are consistent with the biosynthesis of heme rather than any of the steps in protein biosynthesis being the locus for inhibition of hemoglobin synthesis by low levels of plumbous ion. Also, there is marked biological variations in the susceptibilities of reticulocytes from different rabbits to Pb2+.

A morphological study of hemoglobin biosynthesis activity in rabbit-embryo liver and peripheral blood was comparatively developed. Orthochromatic erythroblasts and reticulocytes of the same maturing degree were analysed through thin sections, as to their organellar constitution and behaviour regarding iron incorporation. It was found that peripheral blood erythroid cells contain mainly hemosomes, organelles taken as sites of final hemoglobin molecule biosynthesis. The ratio between the mean number of hemosomes in blood erythroid cell sections and that in liver erythroid cell sections reaches a little more than 6:1. Inversely, late liver erythroid cells are predominantly constituted by mitochondria that directly or indirectly participate in hemosome formation. The ratio between the mean number of mitochondria in blood erythroid cell sections and that in liver erythroid cell sections reaches 1:11. Besides this, the iron incorporation activity is higher in peripheral blood erythroid cells than in liver erythroblasts and reticulocytes. It is apparent that erythroid cells in the liver accumulate heme, given the ratio of the means of iron incorporation activity per cell to hemosome per cell (3.67), while blood erythroid cells, with a 1.08 ratio, synthesize heme, which is immediately afterwards integrated into the globin chains. The sudden increase in the formation of hemosomes when liver orthochromatic erythroblasts and reticulocytes enter the peripheral blood, reflecting an enhancement of hemoglobin synthesis, agrees with biochemical findings of other authors.

The effect of cobalt on haemoglobin synthesis was studied in reticulocytes obtained from marmosets, new world monkeys. By means of different inhibitors the conditions necessary for either cobalt-induced stimulation or inhibition were determined. Cobalt-induced inhibition of globin synthesis was not reversible by the addition of either delta-amino levulinic acid or haem. Globia synthesis, however, in iron-deficient reticulocytes was stimulated by the addition of cobalt. Cobalt did not apparently require complexing with protoporphyrin for activity, since the effect was observed whether the haem-biosynthetic pathway was inhibited or not. The data have been interpreted to suggest cobalt operates by affecting a rate controlling step in the synthesis of globin.

A pancreatic deoxyribonuclease preparation, shown to be free of significant ribonuclease activity, inhibits hemoglobin synthesis in a rabbit reticulocytelysate. Preincubation with DNAase (15 min at 25 degrees C) is required to obtain a marked inhibitory effect (nearly 70%). It has been shown that DNAase does not significantly interfere with the elongation or termination steps of translation, but it seems to prevent reinitiation of globin polypeptide chains allowing polysome run-off. In particular, the formation of the 40S/met-tRNAmetf complex is greatly reduced in the DNAase-treated lysate. At the moment the mechanism by which DNAase inhibits initiation is not clear.

Chicken erythroblasts transformed by a temperature-sensitive erythroblastosis virus (tsAEV) can terminally differentiate at the non-permissive temperature (42 degrees C). Morphological changes, as well as production of hemoglobin were shown to occur during maturation. The above system, as well as fractions of normal bone marrow cells were used to study the expression of cell surface antigens on erythroid cells. Four distinct reactivities were found with monoclonal antibodies (MAbs) and polyvalent sera: The MAb 4.5A5 was found to react with erythroblasts only. The MAb 4-M-12-26 reacted with tsAEV erythroblasts shifted to the non-permissive temperature (42 degrees C). The MAb 4.6C1 reacted weakly with erythroblasts and strongly with reticulocytes. The anti-Ery sera reacted with reticulocytes and erythrocytes. In addition the reactivity of MAb 4-M-12-26 was found only on virus-producing erythroblasts, though this MAb was found to react with myeloid cells which produce a helper virus of the same class.

Avian vitellogenin has been studied as an iron carrier for hemoglobin synthesis by reticulocytes. The Fe-vitellogenin uptake by the immature red cells is progressive with time, following an unspecific iron uptake process. The iron uptake from Fe-vitellogenin was in proportion to the immature red cells present and the radioactive iron was found in the hemoglobin synthesized by these cells. These results open up the possibility of assigning a secondary role to the Fe-vitellogenin in the avian erythropoiesis, added to the classical iron transport function for egg production.

2,3-Bisphosphoglycerate inhibited protein synthesis in reticulocyte lysates with 50% inhibition at 2 mM. Glycerate 2,3-P2 increased the Mg2+ optimum for protein synthesis by chelation of Mg2+, but Mg2+ addition did not completely reverse the inhibition, suggesting an additional site of action. eIF-2 has been used to examine the activity of casein kinase II in reticulocyte lysates in response to glycerate 2,3-P2. When glycerate 2,3-P2 was increased to 4mM, phosphorylation of eIF-2 beta was increasingly inhibited. Thus inhibition of phosphorylation of translational components by casein kinase II can be correlated with inhibition of globin synthesis at physiological concentrations of glycerate 2,3-P2.

Rabbit reticulocytes contain two RNA isoaccepting species for histidine as resolved by various chromatographic methods, while rabbit liver contains only one. These isoacceptors cannot be distinguished on the basis of coding properties, consistent with the "Wobble Hypothesis" (Smith, D.W.E., Meltzer, V.N., and McNamara, A.L. (1974) Biochim. Biophys. Acta 349, 366-375). Their function in hemoglobin synthesis in reticulocyte lysates has been investigated. Each of the tRNA isoacceptors of reticulocytes and the tRNA species of liver can incorporate histidine into positions in hemoglobin encoded by both of the histidine code words, CAC and CAU, and it is likely that each can incorporate histidine into all of the histidine-containing positions of hemoglobin. Even in experiments in which the two histidine tRNA species of reticulocytes are placed together in a lysate and are therefore in competition with each other, each incorporates histidine into all of the histidine-containing positions. There is no evidence that any residues are incorporated preferentially by either of the tRNA species. The two species are attached to reticulocyte ribosomes in the same proportion as they occur in the reticulocyte, also suggesting that neither of them is used preferentially in hemoglobin synthesis. The first of the two reticulocyte histidine isoacceptors and the histidine tRNA of rabbit liver contain Q base.

The synthesis of alpha and non-alpha chains of human hemoglobin (Hb) was studied in reticulocytes and in BFUe-derived cell colonies from patients with alpha chain or beta chain deficiencies. The subjects included normal adults (alpha alpha/alpha alpha) with or without a beta chain variant (Hb S, Hb Leslie) or an alpha chain variant (Hb G-Georgia); alpha-thalassemia-2 heterozygotes (alpha 0 alpha/alpha alpha) with an alpha chain variant (G-Georgia or G-Philadelphia); an alpha-thalassemia-1 heterozygote (alpha 0 alpha 0/alpha alpha); alpha-thalassemia-2 homozygotes (alpha 0 alpha 0/alpha 0 alpha) with a beta chain variant (Hb S), an alpha chain variant (G-Philadelphia), a Hb S homozygosity with Hb G-Philadelphia, or a Hb G-Philadelphia homozygosity; and three black beta +-thalassemia homozygotes. Data from reticulocyte in vitro synthesis analysis showed the expected deficiencies. However, similar analyses of the Hb synthesized in cell colonies (even from the black beta-thalassemia homozygotes) gave (nearly) balanced sigma alpha/sigma non-alpha ratios. It is speculated that this balanced synthesis is due to a most effective proteolysis in the immature erythroblast which rapidly removes free alpha or beta chains. The levels of Hb F and Hb A2 were considerably increased in these proerythroblasts; a two- to threefold increase in the synthesis of Hb A2 was observed over that seen in the reticulocytes.

An unusual form of beta thalassemia is described in two children of unrelated families. Its main features are a severe anaemia with a low reticulocyte count associated with an erythroblastic hyperplasia; these characteristics indicate a completely ineffective erythropoiesis. The results of the study of haemoglobin synthesis performed on the bone marrow in vitro showed a greater imbalance in chain synthesis than that typically found in Cooley's anaemia. Ultrastructural studies revealed, in erythroblasts, all the features observed in Cooley's anaemia, although these features were more widely encountered. In addition, two peculiar findings were noted: (1) many inclusion bodies, which were partially or totally surrounded by smooth membranes, and which may indicate an autophagic phenomenon; and (2) a spongy appearance of the chromatin in rare erythroblasts of one of the two patients which resembled that found in congenital dyserythropoietic anaemia type I. These findings suggest that the great imbalance in the synthesis of the haemoglobin chain is responsible for the presence of an increased number of inclusion bodies and results in the death of nearly all the late erythroblasts. Furthermore, the present results point out the need for further study of the synthesis of the globin chains in atypical congenital dyserythropoietic anaemias.

The polyadenylic acid-containing messenger ribonucleic acid from rabbit reticulocyte polyribosomes, isolated by a rapid and very gentle procedure (Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084), sediments in a sucrose gradient in three sharp peaks, at 9 S, 17 to 18 S, and 28 S. The alpha and beta globin messenger activity follows the absorbance profile in the sucrose gradients and has its major peak at 17 to 18 S. The larger messengers are more active than 9 S messenger by approximately 2-fold per mass unit of ribonucleic acid or by at least 8-fold per molecule. The major 17 to 18 S form of globin messenger was examined further and was shown to be a 1:1 complex of 9 S messenger and 18 S ribosomal ribonucleic acid. The effect of 18 S ribosomal ribonucleic acid on translation of purified 9 S globin messenger was analyzed in a messenger-dependent protein-synthesizing system (Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084). In the absence of exogenous ribosomal ribonucleic acid, 9 S messenger is inefficiently translated; a large excess of messenger is required to saturate the system; and globin is synthesized mainly on di- and monoribosomes. Exogenous liver or reticulocyte 18 S ribosomal ribonucleic acid potentiates 9 S messenger translation and renders it at least 10 times more efficient. The potentiation reaction can also be accomplished by increasing the concentration of ribosomes in the assay system. However, transfer or messenger ribonucleic acids cannot carry out this reaction. It is proposed that 9 S globin messenger ribonucleic acid is an inactive molecule which is normally potentiated by specific reversible base pairing with an accessible region of ribosomal ribonucleic acid contained in a 40 S ribosomal subunit. The potentiated messenger interacts with initiation factors and with other ribosomal subunits to synthesize protein. Potentiation is the first specific function in protein synthesis demonstrated for the ribosomal ribonucleic acid portion of ribosomes.

L-Azetidine-2-carboxylic acid is the naturally occurring lower homologue of L-proline. Reticulocytes from anemic rabbits incubated with DL-[14-C]azetidine-2-carboxylic acid synthesized radiolabeled hemoglobin, which when isolated from cell lysates co-chromatographed with unlabeled hemoglobin on Sephadex G-100 columns. Amino acid analysis of hemoglobin from reticulocytes incubated with DL-[14-C]-azetidine-2-carboxylic acid suggested that the homologue was incorporated into hemoglobin intact and unaltered. Alternatively, another amino acid analogue, 1-aminocyclopentane-[1-14-C]carboxylic acid, which is purported to be a valine antagonist, was not incorporated into hemoglobin under these conditions. Incubation of reticulocytes with 1, 5, and 10 mM L-azetidine-2-carboxylic acid reduced L-[U-14-C]proline (0.10 mM) incorporation into hemoglobin by 25, 58, and 72%, respectively. Conversely, 1.45 and 145 muM L-proline reduced radiolabeled azetidine-2-carboxylic acid (0.8 mM) in corporation into hemoglobin by 45 and 92%, respectively. Incorporation of L-[U-14-C]leucine and L-[U-14-C]lysine (0.1 mM each) into hemoglobin was unaffected at these concentrations of L-azetidine-2-carboxylic acid. These results suggest that L-azetidine-2-carboxylic acid is incorporated into hemoglobin without reducing the rate of globin synthesis in rabbit reticulocytes in vitro. The alpha and beta chains of hemoglobin into which [14-C]azetidine-2-carboxylic acid had been incorporated in rabbit reticulocytes in vitro were resolved electrophoretically on sodium dodecyl sulfate-polyacrylamide gels. The ratio of total radioactivity in the alpha and beta chains separately extracted from gels was in good agreement with the known 7:4 ratio of prolyl residues in the respective chains. Autoradiograms of two-dimensional tryptic peptide maps of rabbit globin into which either [14-C]azetidine-2-carboxylic acid or [14-C]proline had been incorporated showed nearly identical patterns of radioactivity. These results suggest that azetidine-2-carboxylic acid substitutes specifically for prolyl residues during in vitro hemoglobin synthesis in rabbit reticulocytes.

Actinomycin D inhibits hemoglobin synthesis in rabbit reticulocyte lysates by affecting initiation of globin polypeptide chains. To obtain information on the distribution of actinomycin-sensitive structures among the components of the translation machinery, a reticulocyte lysate is incubated with [3H]actinomycin. The tritium label is found associated with ribosomes actively engaged in protein synthesis and with a 15 S cytoplasmic particle. During cell-free incubation or when initiation is inhibited by aurintricarboxylic acid, the labeling of the 15 S particles increases, suggesting that they are relased from ribosomes into the cytoplasm and play a role in the initiation process. The 15 S cytoplasmic particles contain a DNAase-sensitive component, probably complexed with ribonucleic acids and proteins. A possible role of these DNA-containing particles in the initiation step of translation is discussed.

The major iron-bearing cytosol components of human reticulocytes identified after incubation with 59 Fe-125I-transferrin have been studied further. Component C previously found to behave consistently as an intermediate in the iron transport pathway to haem is shown to consist entirely of ferritin. After a short pulse of labelled transferrin incubation, chase experiments showed a fall of ferritin label with time and a corresponding increase in haemoglobin-iron incorporation. There was no loss of ferritin to the culture medium. Restriction of iron uptake by reticulocytes using both p-hydroxymercuribenzoate inhibition of uptake and incubation with progressively lower saturations of iron-transferrin gave linearly related incorporation of 59Fe into ferritin and haemoglobin at all levels of iron uptake, thus negating the concept of ferritin as an 'overspill' form of reticulocyte iron. The results suggest that cytosol ferritin is an obligatory intermediate in reticulocyte iron transport.

In this work it is demonstrated the NH3 potentially available from the oxidation of amino acids by the reticulocyte is utilized for the new-synthesis of serine via transamination reactions with hydroxypyruvate and phosphohydroxypyruvate. These compounds are derived from glucose, which furnishes in this manner the carbon skeleton of serine. It was shown that serine is mainly degraded via glycine, while glycine is in the main formed from serine. Furthermore it was found that serine synthesis is localized in the cytosol, while the reversible transformation of serine to glycine takes place in the mitochondria. A transfer of methylenetetrahydrofolate occurs in both directions across the mitochondrial membrane. A concentrative uptake of serine by the mitochondria was found, while glycine is transported slowly in both directions. On the basis of the data an overall balance is given of the quantitative relations between protein breakdown and hemoglobin synthesis as well as for the new-formation and utilization of serine and glycine. The new-formation of serine may amount to about one tenth of the glucose utilized by the reticulocyte and furnishes about one-half of the serine and glycine moieties required for the synthesis of heme and globin. The remainder is provided by the energy-dependent protein breakdown.

Venous blood was obtained from five sickle cell trait donors with relatively high hemoglobin S concentrations (40% of total hemoglobin) and five donors with unusually low hemoglobin S concentrations (25 to 30%). A fraction of cells with 15 to 20% reticulocytes was isolated from the blood and incubated with [3H]leucine in a medium supporting protein synthesis for various times from 1.25 to 60 min. Previous studies showed an imbalance in globin chain synthesis in reticulocytes of "low hemoglobin S" donors which suggested the presence of an alpha-thalassemia gene; reticulocytes of "high hemoglobin S" donors had balanced globin chain synthesis (DeSimone, J., Kleve, L., Longley, M.A., and Shaeffer, J. (1974) Biochem. Biophys. Res. Commun. 59, 564-569). In the present study the soluble phase of the 3H-labeled reticulocytes was examined by electrophoresis on strips of cellulose acetate. The tetramer hemoglobins A and S were separated from each other and from a small pool of free, newly synthesized alpha and beta chains. Kinetics of labeling studies showed that the free alpha and beta chains were intermediates in tetramer hemoglobin assembly. The distribution of radioactivity between the alpha and beta chains of each of the electrophoretically isolated components were determined by separation of their globin chains on CM-cellulose columns. After 5 min of 3H-labeling of the reticulocytes from donors with 40% hemoglobin S the ratio of newly synthesized alpha chains to beta chains in the tetramer hemoglobins A and S ranged from 0.37 to 0.58. This ratio increased with longer labeling times. Almost all of the radioactivity of the free chain intermediates was in the alpha chain. These results confirmed the presence of a significant pool of newly synthesized alpha chains and a normal pattern of hemoglobin assembly in which initially unlabeled alpha chains combined with labeled beta chains when the cells were exposed to [3H]leucine. Conversely, in the reticulocytes of donors with 25 to 30% hemoglobin S the ratio of newly synthesized alpha chains to beta chains in the completed hemoglobins A and S ranged from 0.96 to 1.37 and remained unchanged throughout the 3H-labelling period. The radioactivity of the free alpha chain pool was substantially less that the total radioactivity of the betaA and betaS chain pools. These results confirmed the existence of a decreased pool size of soluble alpha chain intermediates and a pattern of hemoglobin assembly consistent with the presence of the alpha-thalassemia gene.

1. Peripheral blood reiculocytes of normal and bled rabbits and of rabbits with phenylhydrazine-induced anemia, were morphologically analysed, through silver sections, for a relative evaluation of hemoglobin (Hb) biosynthesis activity. 2. Reticulocytes of maturation degrees within the range of 35-60 polysomes/microns2, were compared as to their mean numbers of hemosomes (sites of heme integration into the globin chains), and mitochondria (indirect precursors for hemosome formation). 3. The results on the mean numbers of hemosomes per reticulocyte section, correlated to several physiological data under those three conditions, suggested a close relationship between Hb biosynthesis activity and hemosome frequency. 4. In bled rabbits, reticulocytes showing a low mean number of hemosomes (means hB/section = 0.32), as compared to reticulocytes of normal rabbits (means hN/section = 0.70) and to reticulocytes of rabbits with hemolytic anemia (means hH/section = 2.10), gave rise to a new erythrocyte population characterized by a low Hb content. 5. Hb concentration differences were verified by confronting hematological data before bleeding with those obtained after the regression of anemia.

Exposure of reticulocytes to 25 mM spermidine and spermine stimulates the incorporation of 14C leucine into hemoglobin and globin polypeptide chains. These two polyamines stimulate beta chain synthesis to a greater extent than they stimulate alpha chain synthesis, thus resulting in a decreased synthetic alpha/beta globin ratio. Because of the low permeability of the red cell membrane to these polycations, less than 1.0% of extracellular spermidine and spermine accumulate within the erythrocyte, thus these effects occur at intracellular polyamine levels that are physiological. Putrescine progressively decreases hemoglobin synthesis at extracellular concentrations greater than 10 mM without affecting the synthetic alpha/beta ratio. These findings suggest a role for polyamines in the fine tuning of the alpha/beta globin ratio at the translational level.