A relative morphological evaluation of hemoglobin biosynthesis in peripheral blood reticulocytes of normal and anemic rabbits.

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.

The mitochondrial function in hemosome formation and hemoglobin biosynthesis.

1. Rabbit-kidney epithelial cell cultures were induced to synthesize hemoglobin, by previously mixing cell suspensions with solutions containing reticulocyte free globin, hemoglobin and anemic rabbit blood plasma. As control, a solution without globin was used. 2. After a 24 hr culture growth period, hemoglobin was absent, as stated through electrophoresis, suggesting hemoglobin denaturation: mitochondria interacted with the incorporated material and particles resembling ferritin molecules were found within 48 hr. 3. Mitochondria modified remarkably giving rise to lamellated bodies which recomposed to form prohemosomes, presumably containing globin and newly synthesized heme: hemoglobin was still absent up to 72 hr. 4. After 96 hr hemosomes developed and hemoglobin, apparently constituted by reticulocyte globin, was detected.

The marginal band and its role in the ellipsoidal shape of Geochelone carbonaria erythrocytes.

Erythrocytes from a chelonian (Geochelone carbonaria) were submitted to physical and chemical treatments. Observations of their change in shape from ellipsoidal to spherical were made by means of light microscopy. A 100% change to the spherical shape was attained with erythrocytes under high temperature treatment (42 degrees C) for 45 min., and at room temperature (29 degrees C) plus colchicine, for 60 min. Erythrocytes kept at room temperature present changes in shape while those kept at low temperature (10 degrees C) did not exhibit significant changes in their original ellipsoidal shape, but displayed an intact marginal band (MB). By direct examination the diameter of microtubules was found to be nearly 300 A in the dissociated form and 0.70 mu when bundled. In cross section the value found for microtubule diameter was up to 210 A. These measures are compatible with those found by several other authors. Counting of microtubules by ultrathin sectioning indicated approximately 70 tubules per cell, a value that is higher than that found in other species. By means of electron microscopy the marginal band can be observed intact or dissociated into several dense rings formed by parallel microtubules running nearly or accompanying the periphery of the stroma. Temperature and colchicine are responsible for the depolymerization of the microtubules. In the stroma of spherical erythrocytes a dense peripheric fold can be observed but microtubules were never found. It is suggested therefore that the role of the marginal band is essential to maintain the ellipsoidal shape of chelonian erythrocytes.

Induction of hemosome formation and hemoglobin biosynthesis in HeLa cells.

Tissue cultures of HeLa cells in Hank's medium suspension, previously mixed with a solution containing reticulocyte polysomes and/or free globin, hemoglobin and blood plasma from bled rabbits, were homogenized, and an electrophoretic pattern of hemoglobin band was obtained from the supernatant; hemoglobin denatures under the tissue culture conditions. HeLa cells show typical hemosomes, identidical to those found in immature erythrocytes of embryos, and adult mammals with hemolytic anemias. When HeLa cell suspensions are mixed with solutions void of anemic blood plasma, or containing normal rabbit blood plasma, anomalous organelles are formed in the cells, and no hemoglobin biosynthesis occurs.

Comparative ultrastructure of late rabbit-embryo erythroid cells in liver and peripheral blood.

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.