Kiyokazu Morioka

Autophagy in embryonic erythroid cells: its role in maturation

Yolk sac-derived embryonic erythroid cells differentiate synchronously in the peripheral blood of Syrian hamster. The stage of differentiation on day 10 of gestation is equivalent to polychromatophilic erythroblast stage and that on day 13 is equivalent to the reticulocyte stage in adult animals. The cytoplasm of embryonic erythroid cells became scant and devoid of most organelles on day 12 of gestation. In addition, there were very few non-erythroid cells in circulation before day 13. Thus the embryonic erythroid cells serve a pure and synchronous system to study the mechanisms of terminal differentiation. The number of mitochondria in the embryonic erythroid cells decreased to about 10% of the initial number during the period between day 10 and day 12 of gestation. In contrast, the frequency of autophagy of mitochondria increased 4.6-fold in the same period. The cytochrome c content of the cell decreased as the mitochondria became extinct. However, release of cytochrome c into the cytoplasm was not detectable through day 10-13 of gestation, suggesting that the mitochondria were digested within a closed compartment. Decomposed mitochondria and ferritin particles were detected in lysosomes by electron microscopy on and after day 12 of gestation, which also suggested digestion in a closed compartment. Mitochondrial ATP synthase subunit c, which is known to be a protease-refractory protein, was retained in the cells even after the disappearance of mitochondria, indicating that most of the mitochondria were not extruded from the cells. The digestion of mitochondria in autolysosomes may allow the cells to escape from rapid apoptotic cell death through concomitant removal of mitochondrial death-promoting factors such as cytochrome c.

Temporal Characteristics of the Differentiation of Embryonic Erythroid Cells in Fetal Peripheral Blood of the Syrian Hamster

The morphological changes in erythroid cells and their nuclei in the circulation of fetuses of the Syrian hamster were investigated by use of an image‐processing system. The analysis included monitoring of nuclear condensation, nuclear periphralization (access of the nucleus to the cell membrane), enucleation, density of cells, and changes in cell size from day 9 of gestation to day 5 after birth. The yolk‐sac‐derived erythroid cells made rapid progress in nuclear condensation on day 11, while this process proceeded at a much lower rate after day 12 of gestation. The peripheralization of nuclei started on day 10 and reached a maximum on day 11. The frequency of enucleated cells was below 2% on day 11, while it increased to 30% on day 12. Extruded nuclei, most of which were accompanied by a small quantity of cytoplasm, appeared in the circulation on day 12. The most frequently observed diameter of enucleated erythrocytes, which was 10–10.5 μm on day 12, fell gradually to 8–9 μm on day 14. By contrast, the shift from fetal liver erythrocytes to adult erythrocytes occurred in a discontinuous manner. Adult‐type erythrocytes were detected after birth with diameters of 5.5–6 μm. Our data allows us to present the schedule of morphological changes during embryonic erythropoiesis and show that the developmental behavior of “primitive” yolk‐sac‐derived erythroid cells is more closely correlated with that of the “definitive” fetal liver cells than has been considered to be the case to date.

Serum factors stimulating DNA synthesis in the isolated nuclear system from rat liver.

Abstract 3H-TTP incorporation into DNA by the isolated rat liver nuclei was stimulated by the rat serum in proportion to its concentration. Dialysis and gel-filtration of the serum indicated the presence of two factors: one is low-molecular and another is high-molecular. The high-molecular factor is thermolabile while the low-molecular one is thermostable. The latter is resistant to pronase-treatment and can not be adsorbed on charcoal. The sera from normal and partially hepatectomized rats showed similar stimulatory effect.

Acceptors of poly(ADP-ribosylation) in differentiation inducer-treated and untreated Friend erythroleukemia cells

Acceptors of poly(ADP-ribosylation) were identified and compared between inducer-treated and untreated Friend erythroleukemia cells. When permeabilized Friend cells were pulse labeled with 0.6 microM [32P]NAD for 1 min and labeled proteins analyzed by SDS-polyacrylamide gel electrophoresis, nucleosome core histones were found to be the primary acceptors, with an additional minor radioactive peak at a position corresponding to Mr = 170 000. Friend cells induced to differentiate by DMSO treatment showed a similar distribution of radioactivity, but with a 60% reduction in the overall level of poly(ADP-ribosylation) under identical labeling conditions. When isolated nuclei were pulse labeled with 0.6 microM [32P]NAD, radioactive peaks were not restricted mainly at the positions of core histones but widely dispersed in the area from 10 to 50 kDa with another peak at 170 kDa. Increase of NAD concentration resulted in the overall shift of peaks to higher molecular weight positions. When pulse-labeled nuclei or permeable cells were chased with 1 mM NAD, radioactive peaks migrated to positions of very high molecular weight (greater than Mr = 180 000). Remarkable suppression of poly(ADP-ribose) synthesis was observed when DMSO, hexamethylene bisacetamide, butyric acid, or hemin were used as the inducers.

Effect of medium change on poly(ADP-ribose) synthesis in friend erythroleukemic cells

Abstract In Friend leukemic cells cultured in the presence of 5 mM hexamethylene bisacetamide, a potent differentiation-inducer, poly(ADP-ribose) synthesis was reduced to about one-third of that in control cells. Replacing the original culture medium with fresh medium resulted in a decrease of poly(ADP-ribose) synthesis in confluent control cultures, while cells induced to differentiate were not affected by the medium change. This is not attributable to the difference of the level of poly(ADP-ribose) synthesis in different cell cycle stages, since DNA synthesis and cell growth in differentiating cells were maintained at the same level with those of control cells. In control cultures, a medium change during the log-phase effected a prolongation in the rise of poly(ADP-ribose) synthesis. When conditioned medium was substituted during log-phase growth, poly(ADP-ribose) synthesis was stimulated in control cells. This stimulating effect was not lost by dialysis but was lost by heat-treatment or trypsin-digestion. Results suggest that poly(ADP-ribose) synthesis is regulated by some factor(s) released into the culture medium.

Novel Inducers and Inhibitors of Differentiation of Friend Erythroleukemia Cells: Application of an Opal Glass Transmission Method to Study of Erythroid Differentiation

The potencies of poly(ADP‐ribosylation)‐inhibitors in inducing erythroid differentiation of Friend erythroleukemia cells were surveyed. Picolinamide and m‐aminobenzamide were newly found to be inducers, whereas compounds related caffeine did not induce differentiation. In other series of experiments some bile acids suspected of being tumor promoters were found to inhibit the differentiation like typical tumor promoters such as phorbol esters. These modifications of erythroid differentiation were detected by an opal glass transmission method. This method is simpler than any previously reported methods, and is sufficiently reliable to use in determining hemoglobin in living cells as a quantitative marker of erythroid differentiation.

A conversion factor for cytoplasmic DNA polymerase of rat liver.

Abstract DNA polymerase of 6–8S prepared from rat liver cytosol was found to be dissociated into active 3.3S DNA polymerase by column chromatography on phosphocellulose. This 3.3S enzyme was converted into a 5S form in the presence of a factor derived from cytoplasm in both high and low ionic strength media, with an accompanied change in the template specificity. The converting activity seems to be associated with a thermolabile protein of about 4–5S.

Localization of myosin and actin in the pelage and whisker hair follicles of rat.

The combined effects of myosin II and actin enable muscle and nonmuscle cells to generate forces required for muscle contraction, cell division, cell migration, cellular morphological changes, the maintenance of cellular tension and polarity, and so on. However, except for the case of muscle contraction, the details are poorly understood. We focus on nonmuscle myosin and actin in the formation and maintenance of hair and skin, which include highly active processes in mammalian life with respect to the cellular proliferation, differentiation, and movement. The localization of nonmuscle myosin II and actin in neonatal rat dorsal skin, mystacial pad, hair follicles, and vibrissal follicles was studied by immunohistochemical technique to provide the basis for the elucidation of the roles of these proteins. Specificities of the antibodies were verified by using samples from the relevant tissues and subjecting them to immunoblotting test prior to morphological analyses. The myosin and actin were abundant and colocalized in the spinous and granular layers but scarce in the basal layer of the dorsal and mystacial epidermis. In hair and vibrissal follicles, nonmuscle myosin and actin were colocalized in the outer root sheath and some hair matrix cells adjoining dermal papillae. In contrast, most areas of the inner root sheath and hair matrix appeared to comprise very small amounts of myosin and actin. Hair shaft may comprise significant myosin during the course of its keratinization. These results suggest that the actin-myosin system plays a part in cell movement, differentiation, protection and other key functions of skin and hair cells.