Characterization of the promoter controlling Mona/Gads expression in the megakaryocytic lineage

The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a 'mirror' inverted duplication of the 'right' end of chromosome 1a (approximately 25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over approximately 105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb 'switch' region) was found. By contrast, XC155 contains two large (approximately 13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp 'satellite' DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after >150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp 'satellite' DNA bound by telomeric repeats.     

Protein kinase C in erythroid and megakaryocytic differentiation: possible role in lineage determination

Erythroid differentiation of normal human hematopoietic progenitor cells was drastically inhibited by phorbol ester, 12-myristate 13-acetate (PMA), an agent known to activate the class of serine-threonine kinases, protein kinase C (PKC). This inhibition was accompanied by augmented megakaryocytic differentiation as demonstrated by expression of megakaryocyte-specific mRNAs and proteins. These effects of PMA were reversed by two specific antagonists of PKC. Analysis of single colonies transferred from cultures not containing PMA to PMA-containing cultures indicated that, in this system, PMA exerts megakaryocytic differentiating activity directly on cells which may have already initiated a progression toward the erythroid pathway of differentiation. These results suggest that modulation of PKC activity plays a role in erythroid and megakaryocytic differentiation, and may constitute an important selective signal between these pathways during normal blood cell development.     

Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages

Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.     

Satellite association frequency and number of nucleoli depend on cell cycle duration and NOR-activity

Summary In human lymphocyte cultures the frequencies of satellite associations in first, second, and third mitoses were investigated using the BUDR-method. A marked decrease of the association frequency with increasing numbers of cell cycles was found. The number of nucleoli seen in interphase is correlated with the satellite association frequency in the respective metaphase. Satellite association is positively correlated to Ag-staining intensity of the NORs. Individual differences in satellite associations are due to differences in NOR activity and in lymphocyte activation. BUDR diminishes somewhat the Ag-staining intensity of the NORs but has no effect on satellite association frequencies. The main reason for the decrease of satellite association frequency in second and third lymphocyte mitoses is presumably a certain dislocation of the original chromosome position during mitosis and a decreased possibility of association during the short interphases. The high association frequency in first mitosis resembles the chromosome position in the long interphase of G₀-lymphocytes.     

Ring shaped nucleoli in liver cells of rats after treatment with Actinomycin D

Summary The incidence of ring shaped nucleoli was studied by means of light and electron microscopical methods in liver cells of rats injected intravenously with Actinomycin D in a dose of 150 g per kg body weight.Ring shaped nucleoli were most frequent 20 minutes after administration of Actinomycin D. The number of fragmented nucleoli was increased 20 minutes after administration of the drug and did not decrease even after 72 hours.Fibrillar ring like inclusions within nuclei and more perichromatin granules at the periphery of nucleoli were observed after treatment with Actinomycin D.

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Zusammenfassung Die Wirkung von Aktinomycin D (150 g/kg) auf die Leberzellen der Ratte wurde licht- und elektronenenmikroskopisch untersucht. 20 min nach intravenöser Aktinomycin-D-Gabe wurden ringörmige Nukleolen beobachtet. Die Zahl der fragmentierten Nukleolen war 20 min nach der Applikation gesteigert. Ihre relative Zahl änderte sich nach Aktinomycingabe nicht. Nach Behandlung der Tiere mit Aktinomycin D kann man in den Kernen der Leberzellen fibrilläre ringförmige Einschlüsse und eine größere Menge von perichromatin-granules an der Peripherie der Nukleolen beobachten.

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The authors are indebted to M. Mikoláová, V. Povolná, I. erníková and Dr. M. Titlbach for their help, to Dr. J. Chaloupka for providing Actinomycin D and to Professor Busch and Professor Wolf for many helpful suggestions.     

Dense distribution of nuclear alkaline protease to nucleoli with increase in rapidly growing cells in rats

Neutral and alkaline proteases (chymotrypsin-like serine proteases) are tightly bound to chromatin in nuclei of various tissues of rats, and rapidly growing cells are abundant in the latter enzyme. Activities per mg DNA of these enzymes were measured with fractions of euchromatin, heterochromatin, nucleoli and extranucleoli from normal and regenerating livers, and Rhodamine sarcoma. With normal liver, both enzymes, respectively, were almost evenly distributed among the fractions, except that alkaline protease was significantly higher in nucleoli. The nucleolar alkaline protease increased by 30-60% with regenerating liver and by higher than 100% with the tumor.     

On the nucleoli of the dinoflagellate Prorocentrum

To date no nucleolus had been observed in Prorocentrum under the light microscope. The author failed to show the nucleoli of P. micans and P. cassubica with eosin in 70% alc or with methyl green-pyronin. But when these dinoflagellates were treated with an Ag-1 technique which had been improved for demonstrating NORs in unicellular organisms, nucleoli were stained dark brown or black, while all other parts showed no colour. When the materials were stained well, only the central part of the nucleolus was stained. Under the electron microscope, it was observed that all the silver grains were concentrated in the pars fibrosa of the nucleolus. P. cassubica had only one small oblate nucleolus attached to the nuclear envelope, with NOR usually in the shape of the letters O or C. P. micans had 1–7 nucleoli of various sizes and shapes with NORs in various complicated forms. The number of nucleoli bore a certain relationship to the living state of the dinoflagellate. One day after fresh medium was added, cells with 3 nucleoli were most common, and 28.5% of the individuals had 4–6 nucleoli. Cells having only one nucleolus accounted for 8.6%. 3 days after, cells with 2 nucleoli became dominant, and those with 4–6 decreased to 18.4%. After a month, cells with 1 nucleolus became most abundant, cells having 4 nucleoli decreased to 2.4%, and no cells had 5 or 6 nucleoli.     

Prorocentrum属涡鞭毛虫核仁的观察

迄今未能在光学显微镜下观察到Prerocentrum属的涡鞭毛虫有核仁。本文作者用伊红的酒精溶液和用甲基缘—派若宁法染色,也未能在Prerocentrum micans和Proro-centrum cassubica的细胞核中显示出核仁来。但是在用专门为显示单细胞生物的核仁组织者区（NOR）而改进了的Ag—1法进行染色时,这两种涡鞭毛虫的核仁都会被染作鲜明的深褐色或深黑色,而身体的所有其他部份,包括染色体,全都不着色。染色适当时可以看出,实际上只是核仁的中央部分被染上色。在电镜下可见,此时所有的银粒全部是集中在核仁的纤维区中。染色的结果表明,Prorocentrum cassbica只有一个扁园形的小核仁,后者是贴附在核膜上,其NOR通常是作O形或C形。与P.cassubica不同,P.micans的核仁的数量变化很大,可以有一个至七个;其核仁的大小与形状同样也变化很大;其NOR的形状也复杂多变。发现P.micans的核仁数量与个体的生活状况有一定的关联:向老的培养液中加入等量的新的培养液一天以后,具有三个核仁的个体是最多的（占三分之一）,具有4—6个核仁的个体占28.5％,只有一个核仁的个体只占8.6％;加入新培养液三天后,具两个核仁的个体变成是最多的（占38.8％）,具4—6个核仁的个体降为占18.4％;加入新培养液一个月以后,只有一个核仁的个体是最多的（占3     

RNA synthesis in the circular nucleoli of hepatocytes]

By analysis of serial sections it has been revealed that the so-called ring-like nucleoli of hepatocytes consist of a cavity with an amorphous contents surrounded by fibrillar and granular material. Such nucleoli are sometimes encountered in normal animals; the number of ring-like nucleoli increases considerably in chronic pathological process caused by repeated CCl4 injections. The capacity of RNA synthesis in the ring-like nucleoli was revealed by means of electron-microscopic autoradiography.     

Fine structure of nucleoli in micronucleated cells

The correlation between the number of nucleolus organizing regions (NOR) on metaphase chromosomes and the number of nucleoli was studied in normal and micronucleate cells. Many micronuclei, but not all, were able to form complete nucleoli with fibrillar and granular RNP components and fibrillar centers. Micronuclei which failed to form complete nucleoli often contained multiple electron-dense bodies of fibrillar material. These structures, which were much smaller than nucleoli, reacted with nucleolus-specific antibodies and the Ag-As method in the same way as complete nucleoli, but lacked fibrillar centers and granular RNP components. The data suggest that these nucleolus-like ‘blobs’ contain nucleolar material which, following mitosis, has been enclosed in micronuclei which do not contain nucleolus organizing chromosomes. No evidence was found for the activation of latent NORs not expressed in mononucleate cells.