Immunospecificity of antisera against nonhistone nuclear proteins of human lymphoid cells grown in culture

Rabbit antibodies were obtained to nonhistone protein--DNA complexes (dehistonized chromatin) prepared from two human lymphoblastoid cell lines: the Conception line from an American Burkitt lymphoma and NC-37 from a nonmalignant source. Both antisera showed a high degree of specificity for nuclear proteins of their respective cell lines. This specificity was evident in the reactivity of both whole chromatin and dehistonized chromatin using a quantitative micro-complement fixation assay. The results presented here suggest that DNA present in the antigen is necessary for maintaining the structure of the antigenic site.     

delta-Aminolevulinate synthase isozymes in the liver and erythroid cells of chicken

Antibodies raised against the purified chicken liver delta-aminolevulinate synthase showed a partial cross-reactivity with the chicken erythroid delta-aminolevulinate synthase. delta-Aminolevulinate synthase synthesized in vitro using polysomes from erythroid cells showed a subunit molecular weight of 55,000, whereas the enzyme synthesized in vitro using liver polysomes had a subunit molecular weight of 73,000. delta-Aminolevulinate synthase isolated from mitochondria of erythroid cells showed a molecular weight of 53,000, while the enzyme in liver mitochondria had a value of 65,000. These observations imply that the erythroid delta-aminolevulinate synthase differs from the hepatic enzyme.     

Immunofluorescent detection of erythrocyte sialoglycoprotein antigens on murine erythroid cells

A sialoglycoprotein fraction isolated from murine (DBA/2) erythrocytic ghosts (see companion article, Sarris and Palade, 1982, J. Cell. Biol. 93:583-590) was used to raise antibodies in rabbits. By immune-IgG (serum)-[125I] protein A overlays, the antibodies were found to react positively with the four sialoglycoprotein monomers (gp-2.1, gp-2.2, gp- 3.1, and gp-3.2) of the original fraction, with the sialoglycoproteins detected in erythrocytic ghosts (gp-2.1 and gp-3.1), with a diffuse component (probably a macroglycolipid) trailing around gp-3.1 in SDS polyacrylamide gel electrophoretograms of solubilized ghosts, and with a minor sialoglycoprotein hidden under this trail. IgG's isolated from immune and nonimmune rabbit sera were conjugated to tetramethylrhodamine isothiocyanate and used to survey, by fluorescence microscopy, the distribution of the cognate antigens on the three different erythroid lines known to succeed each other during the life span of the mouse. In the peripheral blood of the adult, the antibodies recognized only mature erythrocytes; they did not crossreact with either platelets, monocytes, or different types of granulocytes. In the spleen of adult anemic mice, the antibodies reacted weakly with proerythroblasts and strongly with all types of erythroblasts. In enucleating erythroblasts, antigens were preferentially segregated on the cell membrane of the nascent reticulocyte. In the 10-day-old embryo, antigens were already present on the primitive nucleated erythrocytes (produced by the blood islets of the yolk sack), and in the 14-d fetus they were found on all hepatic erythroblasts and derived non-nucleated erythrocytes. A positive immunoreaction was also obtained on Friend erythroleukemic cells, before or after induction by dimethyl sulfoxide. Nonimmune serum, or nonimmune IgGs gave negative reactions in all cases. The antibodies were species-specific: they did not crossreact with either human or rat erythrocytes.     

Comparison of chicken erythroid cell nuclear isolation methods using morphological,immunochemical and biochemical criteria

Chicken erythroid nuclei were prepared using four published methods. Our findings indicate that nuclei prepared by nitrogen cavitation are less likely to be contaminated with plasma membrane fragments than those made by procedures involving cell disruption by hypotonic lysis. However, globin gene sequences were much less sensitive to DNase I digestion in nuclei prepared by nitrogen cavitation. This suggests that the conformation of chromatin was altered by the cavitation procedure. Analysis of the proteins solubilized during limited DNase I digestion of nuclei prepared by both hypotonic lysis and cavitation revealed no appreciable differences in HMG proteins but a notable difference in the RNP-associated proteins and core histones.Abbreviations HMG high mobility group nonhistone chromosomal protein - RNP ribonucleoprotein - SSC 14 mM sodium citrate buffered saline pH 7.0 - PMSF phenylmethanesulfonyl fluoride - EDTA ethylenediaminetetraacetic acid - DTT dithiothreitol - PBS 10 mM sodium phosphate buffered saline pH 7.2 - NP-40 Nonidet P-40 (octylphenoxypolyethoxyethanol) - SS-DNA single-stranded DNA - RSB reticulocyte standard buffer, 0.01 M NaCl, 0.003 M MgCl₂, 0.01 M Tris-HCI, pH 7.4.     

Canavanine inhibits vimentin assembly but not its synthesis in chicken embryo erythroid cells

In chicken embryo erythroid cells, newly synthesized vimentin first enters a Triton X-100 (TX-100)-soluble pool and subsequently assembles posttranslationally into TX-100-insoluble vimentin filaments (Blikstad I., and E. Lazarides, J. Cell Biol., 96:1803-1808). Here we show that incubation of chicken embryo erythroid cells in a medium in which arginine has been substituted by its amino acid analogue, canavanine, results in the inhibition of the posttranslational assembly of vimentin into the TX-100-insoluble filaments. Immunoprecipitation and subsequent SDS gel electrophoresis showed that the synthesis of canavanine- vimentin is not inhibited and that it accumulates in the TX-100-soluble compartment. Pulse-chase experiments with [35S]methionine demonstrated that while arginine-vimentin can be rapidly chased from the soluble to the cytoskeletal fraction, canavanine-vimentin remains in the soluble fraction, where it turns over. The effect of canavanine on the assembly of vimentin did not prevent the assembly of arginine-vimentin, as cells labeled with [35S]methionine first in the presence of canavanine and then in the presence of arginine contained labeled canavanine-vimentin only in the soluble fraction, and arginine-vimentin in both the soluble and cytoskeletal fractions. These results suggest that arginine residues play an essential role in the assembly of vimentin in vivo.     

Autophagy of mitochondria in rat bone marrow erythroid cells Relation to nuclear extrusion

Summary Late erythroblasts and reticulocytes from bone marrow of male Wistar rats were studied by electron-microscopic stereology. Late erythroblasts with morphological signs of nuclear extrusion (EN+erythroblasts) and late erythroblasts without these signs (EN-erythroblasts) were analysed separately. The volumes of mitochondria, autophagosomes, autophagocytosed mitochondria, autophagocytosed cytoplasm and degraded material inside autophagosomes were calculated per unit volume of cytoplasm.The results demonstrate that (1) the volume density of mitochondria in the cytoplasm decreases by 34% during maturation from (EN-)- to (EN+)-erythroblasts (p< 0.001) and by 60% during differentiation from (EN+)-erythroblasts to reticulocytes (p<0.001), (2) a fivefold increase in the volume density of autophagosomes in the cytoplasm is noted during maturation from (EN-)- to (EN+)-erythroblasts (p<0.01), whereas the value of this parameter remains essentially unchanged during the subsequent differentiation to reticulocytes, (3) no mitochondria are found inside autophagosomes of (EN-)-erythroblasts, whereas mitochondria occupy 26% and 35%, respectively, of the autophagosomal volume in (EN+)-erythroblasts and in reticulocytes.Our results show that autophagocytosis of mitochondria starts at the moment of nuclear extrusion and continues in the bone marrow reticulocytes.     

Induction of host nuclear DNA synthesis in coccidia-infected chicken intestinal cells

When Eimeria maxima (gamonts) infects villus epithelial cells of the chicken duodenum there is extensive cellular enlargement with no alteration in nuclear size. Feulgen DNA microspectrophotometric measurements indicated that the infected host-cell nucleus contains the same amount of DNA as an uninfected cell nucleus. Evidence is presented to indicate that second generation schizonts of E. necatrix develop in crypt epithelial cells that are displaced/migrate into the lamina propria. The developing parasite causes cellular and nuclear hypertrophy in these cells as does E. tenella in cecal cells of the chicken. In these two cases nuclear enlargement is accompanied by induced rounds of DNA synthesis in the host-cell. Analyses indicated that the DNA content of enlarged nuclei does not fall into classes that correspond to a geometric series 2:4:6:8:16: etc. times the DNA content of a 2C equivalent, and that nuclear size and DNA content in infected cells are not significantly correlated. Autoradiographic studies on E. necatrix infected chicks administered ³H-thymidine show that DNA synthesis takes place in the nuclei of cells containing all developing stages but not mature schizonts, and that this synthesis is not a continuous process. The data suggest that intestinal cells that are capable of undergoing cell division and therefore additional rounds of DNA synthesis, can be induced by coccidial infection in the absence of concomitant cell division.     

Histone H5 and H1 cross-reacting material is restricted to erythroid cells in chicken

Monoclonal H5 antibodies and a polyclonal antiserum, raised against the globular domain of chicken H5 (GH5) but which cross-reacts with histone H1(0) from mouse liver, were used to search for H5 or H1 (0)-like proteins in chicken embryo and adult tissue sections by indirect immunofluorescence. Chicken cell lines in culture were examined for H5 protein and H5 mRNA. Histone H5 was detected only in erythroid cells in tissue sections of chicken embryos or adult livers. H5 protein and H5 mRNA were found only in erythroid cells in culture. No cross-reacting proteins were detected in any other tissue or cell line examined.     

Immunospecificity of nonhistone chromatin proteins tightly bound to DNA from chicken thrombocytes and erythrocytes

Erythroid cell-specific antisera capable of detecting tightly bound nonhistone chromatin protein-DNA complexes were obtained by injecting rabbits with dehistonized chicken reticulocyte chromatin. The antisera showed no crossreactivity with chromatin of thrombocytes which are regarded as cells genealogically closely related with erythrocytes. The lack of thrombocyte chromatin immunoactivity was not caused by conformational constrains. Tightly bound nonhistone protein-DNA complexes isolated from thrombocyte chromatin showed no immunological similarity with these of erythrocyte chromatin.     

Correlation of chromatin composition with metabolic changes in nuclei of primitive erythroid cells from chicken embryos

Changes in levels of biosynthesis of DNA, RNA, and histones were compared with relative proportions of each histone class during primitive erythropoiesis in embryonic chicks. We confirmed that erythrocyte-specific histone 5 (H5) was substantial in the earliest accessible, erythroblast-enriched stage and that it doubled in relative amount between polychromatic and orthochromatic stages to about 1 mol per 2 mol of each nucleosomal histone, still considerable less than in adult definitive erythrocytes. No other histones changed during primitive erythropoiesis, but the molar proportion of histone 1 (H1) always exceeded that of H5 in these cells, unlike definitive erythrocytes. The increase in content of H5 was accompanied by continued decline in synthesis of the other histones and DNA. The accumulation of H5 during development appears to occur in steps corresponding to the maturation of the primitive and definitive erythroid cell lines. Lysine-rich histones were more easily extracted from nuclei of the erythrosynthesis in whole cells and in isolated nuclei.     

Transferrin endocytosis and iron uptake by developing erythroid cells in the chicken (Gallus domesticus)

Summary The mechanism of iron uptake by avian erythroid cells was investigated using cells from 7 and 15-day chicken embryos, and chicken serum transferrin and conalbumin (ovotransferrin) labelled with¹²⁵I and⁵⁹Fe. Endocytosis of the protein was determined by incubation of the cells with Pronase at 4°C to distinguish internalized from surface-bound protein.Iron was taken up by the cells by receptor-mediated endocytosis of transferrin or conalbumin. The receptors had the same affinity for serum transferrin and conalbumin. Endocytosis of diferric transferrin and conalbumin and exocytosis of apo-protein occurred at the same rates, indicating that iron donation to the cells occurred during the process of intracellular cycling of the protein. The recycling time was approximately 4 min. The rate of endocytosis of diferric protein varied with incubation temperature and at each temperature the rate of endocytosis was sufficient to account for the iron accumulated by the cells. These results and experiments with a variety of inhibitors confirmed the role of endocytosis in iron uptake.The mean cell volumes, receptor numbers and iron uptake rates of 7-day embryo cells were approximately twice those of 15-day embryo cells but the protein recycling times were approximately the same. Hence, the level of transferrin receptors is probably the main determinant of the rate of iron uptake during development of chicken erythroid cells.Transferrins from a variety of mammalian species were unable to donate iron to the chicken cells, but toad (Bufo marinus) transferrin could do so at a slow rate. The mechanism of iron uptake by developing chicken erythroid cells appears to be similar to that described for mammalian cells, although receptor numbers and iron uptake rates are lower than those reported for mammalian cells at a similar stage of development.Abbreviations BSS Hanks balanced salt solution - PBS phosphate buffered saline - MCV mean corpuscular volume - CCCP carbonyl cyanide-M-chlorophenyl hydrazone     

Selective synthesis and modification of nuclear proteins during maturation of avian erythroid cells.

The synthesis of the nuclear proteins of duck erythroid cells at different stages of maturation has been investigated. Synthesis of histone fractions H1, H2a, H2b, H3, and H4 is restricted to the erythroblasts, while synthesis of H5 can be detected even at later stages of maturation after DNA synthesis has ceased. The synthesis of nonhistone nuclear proteins (NHNP), on the other hand, occurs in cells at all stages of maturation although their rates of synthesis decline as the cells mature. The same size classes of NHNP appear to be synthesized in erythroblasts and in early- and midpolychromatic erythrocytes. In late polychromatic erythrocytes the synthesis of a new group of NHNP of molecular weights ranging from 54,000 to 130,000 was observed. This group of proteins does not accumulate in the mature erythrocyte, indicating that their relative proportions are very small.Turnover of histone-bound phosphate was found to occur mainly at the erythroblast stage, except for histone H2a which was actively phosphorylated even at more advanced stages of maturation. Phosphorylation of most of the histones appears to be coupled to histone (and coordinate DNA) synthesis.Incorporation of radioactive acetate into histones occurs at all stages, but the rate of acetylation decreases four- to fivefold with maturation. Although the RNA synthetic activity of erythroid cells also decreases with age, experiments involving the use of RNA polymerase inhibitors suggest that the mechanisms that control RNA synthesis and histone acetylation are not tightly coupled.