A further study on nucleolar silver stained granules in some mononuclear-lymphoid bone marrow cells

Nucleolar silver stained granules (SSGs) representing nucleolus organizer regions (NORs) were investigated in human as well as rabbit bone marrows after visualization with standardized silver reaction for non-histone nucleolar argyrophilic proteins. The results indicated that few mononuclear lymphoid blast-like cells in investigated bone marrows are characterized by large irregularly shaped nucleoli which contain a larger number of SSGs than myeloblasts or proerythroblasts as well as immature or stimulated lymphocytes. Since according to previous studies the number of nucleolar SSGs decreased in the course of the erythroid, granulocytic and lymphocytic differentiation and maturation, a possibility exists that the described mononuclear lymphoid blast-like cells are even less differentiated and immature than committed stem cells for mentioned cell lines.     

Nonhistone nuclear antigens reactive with autoantibodies. Immunofluorescence studies on distribution in synchronized cells

Sera from patients with certain autoimmune diseases tht contained autoantibodies to nonhistone nuclear antigens were used as reagents in an indirect immunofluorescent study. The distribution of these nuclear antigens was determined in synchronized human B lymphoid cells. Autoantibodies to Sm antigen, nuclear ribonucleoprotein complex and SS- B antigen were used. Although all three nonhistone antigens appeared to show speckled nuclear straining patterns in the Go phase, different patterns of staining were present at other periods of the cell cycle. The SS-B antigen showed a distinctly nucleolar localization during the G1/early S phase. These studies demonstrate that autoantibodies occurring in certain human diseases can be useful reagents for the immunohistological localization of nuclear macromolecules and for tracing their pathways during different phases of cell growth and differentiation.     

Studies on satellite nucleoli in human blastic cells of acute leukaemias.

Blastic cells of acute lymphoid, acute myeloid and acute myelomonocytic leukaemias were studied by means of indirect immunofluorencence to provide more information on the presence of satellite nucleoli in blood cells. According to results, satellite nucleoli were found in a small but constant number of blastic cells disregarding their type and type of acute leukaemia. Satellite nucleoli exhibited a positive immunoreaction for fibrillarin and protein B23 which are characteristic for main nucleolar components. These findings suggest that satellite nucleoli contain fibrillar centers as well as dense fibrillar and granular components or at least proteins characteristic for these nucleolar components. Similarly as in normal and pathological cells of completely different origin, in blastic cells of acute leukaemias the number of satellite nucleoli per cells ranged between 1 and 2.     

Nucleolar immunofluorescence in human hematological malignancies.

Rabbit antibodies to the nuclear Tris extract of HeLa cells which have been shown by the indirect immunofluorescence technique to localize in nucleoli of a variety of human malignant tumors but not in a number of nontumor tissues also produced bright fluorescence in nucleoli of tumor cells in several hematological malignancies. The tumors studied included Hodgkins malignant lymphoma, non-Hodgkins malignant lymphoma, acute myeloid and acute myelomonocytic leukemia, chronic lymphatic and chronic myeloid leukemia. In contrast, none of the corresponding normal cell lines in the bone marrow exhibited bright nucleolar fluorescence. In addition, neither the cells of patients with acute infectious mononucleosis nor lymphoid hyperplasia exhibited bright nucleolar fluorescence. These studies suggest that antibodies to HeLa cell nucleolar antigens may be useful in immunodiagnosis of human malignancies.     

Characteristics of the silver-stained nucleoli in blast elements of various diameters from patients with acute leukemia

Using silver staining, a study was made of the activity of ribosomal cistrons (RC) in proliferating and non-proliferating subpopulations of bone marrow and peripheral blood blasts, taken from 33 patients with acute non-lymphoblastic leukemia and from 17 patients with acute lymphoblastic leukemia. All the blasts including non-proliferating ones were revealed to synthesize ribosomal RNA (rRNA). The RC activity of large blasts was higher than that of small ones. A significant difference (P less than 0.01) in RC activity between lymphoid and non-lymphoid blasts was found. The relation between nucleolar organizer activity of blasts and their proliferating potentials is discussed, in addition to a possibility for practical use of the above mentioned data.     

To the nucleolar density and size in apoptotic human leukemic myeloblasts produced in vitro by Trichostatin A

The present study was designed to provide more information on nucleoli in apoptotic cells,which were represented in the present study by cultured leukemic myeloblasts (Kasumi-1 cells). The apoptotic process in these cells was produced by trichostatin A (TSA) that is a histone deacetylase inhibitor with strong cytostatic effects. The selected TSA concentration added to cultures facilitated to study apoptotic and not-apoptotic cells in one and the same specimen. The nucleolar diameter and density were determined using computer assisted measurement and densitometry in specimens stained for RNA. In comparison with not-apoptotic cells, in apoptotic cells, nucleolar mean diameter did not change significantly and nucleolar RNA density was also not apparently different. On the other hand, the cytoplasmic RNA density in apoptotic cells was markedly reduced. Thus it seemed to be possible that the transcribed RNA remained "frozen"within the nucleolus but its transport to the cytoplasm decreased or stopped. However, the possibility of the RNA degradation in the cytoplasm of apoptotic cells based on the present study cannot be eliminated. At this occasion it should be added that AgNORs reflecting nucleolar biosynthetic and cell proliferation activity in apoptotic cells decreased in number or disappeared. The presented results also indicated that large nucleoli intensely stained for RNA need not be necessarily related to the high nucleolar biosynthetic or cell proliferation activity and may be also present in apoptotic cells responding to the cytostatic treatment.     

Nucleolar RNA synthesis in heterokaryons derived from senescent and pre-senescent human fibroblasts

Normal human fibroblasts, serially passaged in vitro, demonstrated decreasing synthesis of ribosomal RNA (rRNA). Senescent and pre-senescent WI38 cells were fused with one another in order to study age-related factors affecting the production of nucleolar RNA. Autoradiograms revealed that the young nucleus of a dichronic heterokaryon (2 nuclei of different ages) had an impaired ability to produce nucleolar RNA, while the young nucleus of a monochrome heterokaryon (2 nuclei of the same age) was not affected. Old nuclei of dichronic heterokaryons, and old monochronic heterokaryons displayed the same nucleolar RNA synthesis rate as did their single, unfused counterparts.     

A comparative study of the main components of the nucleolus in different populations of rat trophoblast cells during differentiation. II. The nucleoli of the trophospongium cells, the glycogen cells and the secondary giant cells

A comparative study was performed of the arrangement of different nucleolar components during differentiation of trophoblast cell populations in the junctional zone of placenta (glycogen cells and trophospongium) and in the secondary giant cells. Each cell type is characterized by specific interrelation of nucleolar components. Some glycogen cells show signs of segregation of nucleolar components: strands of nucleolar components with fibrillar centers (FCs) are displaced to the periphery of the nucleolus and contact with the perinucleolar chromatin. Large reticular nucleoli in trophospongium cells contain many FCs which are gathered into several "chains" by strands of dense fibrillar component. Such a "chain" has also been found in nucleoli of secondary giant cells, with greater number of FCs in each "chain". Relationship between the arrangement of nucleolar components and the level of cell differentiation is discussed.     

New evidence for nucleolar dominance in hybrids of Drosophila arizonae and Drosophila mulleri

Drosophila mulleri (MU) and D. arizonae (AR) are cryptic species of the mulleri complex, mulleri subgroup, repleta group. Earlier cytogenetic studies revealed that these species have different regulatory mechanisms of nucleolar organizing activity. In these species, nucleolar organizing regions are found in both the X chromosome and the microchromosome. In the salivary glands of hybrids between MU females and AR males, there is an interspecific dominance of the regulatory system of the D. arizonae nucleolar organizer involving, in males, amplification and activation of the nucleolar organizer from the microchromosome. The authors who reported these findings obtained hybrids only in that cross-direction. More recently, hybrids in the opposite direction, i.e., between MU males and AR females, have been obtained. The purpose of the present study was to evaluate, in these hybrids, the association of the nucleoli with the chromosomes inherited from parental species in order to cytogenetically confirm the dominance patterns previously described. Our results support the proposed dominance of the AR nucleolar organizer activity over that of MU, regardless of cross-direction.     

Nucleolar variation in response to nutritional condition in juvenile pejerrey Odontesthes bonariensis (Valenciennes)

The variations of the nucleolar characteristics size and number were studied in the gill and liver of Odontesthes bonariensis under different feeding regimes in order to test the hypothesis that functional genomes at the nucleolus, which determine variation of size (heterogeneity) in juvenile fishes, are modulated by environmental factors such as food availability. The programme that controls genetic changes of the nucleolar characteristics of the diploid cells of O. bonariensis was different from that described in the diploid cells of cyprinid fishes. No variations of the mean nucleolar number per nucleus were observed in liver or gill cells in relation to the feeding regime. Conversely, significant reduction of the mean nucleolar volume per nucleus ( V _MN) and change in the shape of the frequency distribution of the total nucleolar volume per cell was evident in both cell types in response to starvation or food restriction. Tissue specificity was observed in relation to the absolute values of the nucleolar characteristics; however, the relative behaviour was comparable in digestive and non-digestive tissues. The time-course reduction of V _MN in gill and liver cells of restrictively fed fish followed a negative exponential. A good correlation between V _MN and the condition index of the fish ( K ) was observed after long-term food restriction; however, a faster response of V _MN was observed after short-term fasting events. Results supported the stated hypothesis and showed nucleolar activity as a direct, rapid and sensitive indicator of the nutritional status of the fish.     

Targeting the nucleolus as a therapeutic strategy in human disease

The nucleolus is the site of ribosome biogenesis, one of the most resource-intensive processes in eukaryotic cells. Accordingly, nucleolar morphology and activity are highly responsive to growth signaling and nucleolar insults which are collectively included in the actively evolving concept of nucleolar stress. Importantly, nucleolar alterations are a prominent feature of multiple human pathologies, including cancer and neurodegeneration, as well as being associated with aging. The past decades have seen numerous attempts to isolate compounds targeting different facets of nucleolar activity. We provide an overview of therapeutic opportunities for targeting nucleoli in different pathologies and currently available therapies.     

Placement of genes for ribosomal RNA within the nucleolar organizing body of Zea mays

A maize strain that carried a reciprocal translocation between chromosome 6 and a B chromosome (TB6a) was used in this study. The break in chromosome 6 transected the nucleolar organizing body at approximately the cytological midpoint, and the break in the B was one third the distance from its distal end. As a result both chromosomes 6-B and B-6 contained a portion of the nucleolar organizing body. Because of nondisjunction of chromosome B-6 at the second microspore division after meiosis, crosses between plants carrying six to eight of these chromosomes and homozygous for chromosome 6-B, produced progeny that had between one and about nine chromosomes B-6. Thus a quantitative series of nucleolar organizing body fragments was produced. Molecular hybridization experiments with ribosomal-RNA and DNAs extracted from these plants revealed 1) that genes coding for rRNA were located in the nucleolar organizer fragments on either side of the original translocation breakpoint and 2) that with each additional nucleolar organizer fragment provided by the chromosomes B-6, there is a proportional increase in ribosomal-DNA content. The most important conclusion to be derived from these studies is that the vast majority, if not all, of the ribosomal-RNA genes are unambiguously located within the nucleolar organizing body [with possibly a small percentage of them in the adjacent achromatic gap (Givens and Phillips, 1973, abstract)]. This placement is consistent with that of Givens and Phillips who used a quite different cytogenetic approach. The preciseness of previous determinations in Drosophila, and Xenopus allowed their placement only to the region of the nucleolar organizer. This study showed no evidence for a disproportionate replication of rDNA as a function of different amounts of nucleolar organizing material.Abbreviations rDNA ribosomal-DNA - rRNA ribosomal-RNA - NO nucleolar organizer     

Attachment of DNA to nucleolar and nuclear skeletal structures as visualized by Kleinschmidt molecular spreading

Co-isolated residual nuclear shells and residual nucleoli from membrane-depleted rat liver nuclei were spread according to Kleinschmidt's method. Comparison of the spread residual structures isolated from nuclear shells and spread pore complex-lamina isolated from nuclear envelopes showed that these residual structures are morphologically identical. Furthermore, our nuclear shell isolation procedure allowed visualization of DNA strands bound to a granular component of the lamina. The fragmentation of nuclear shells allowed us to obtain well-spread nucleolar remnants, in which we observed DNA strands anchored on a residual nucleolar network attached to the lamina. The different molecular features revealed by the spreading of residual nucleolar structures suggest that both non-transcribing nucleolar DNA and active ribosomal genes are linked to the nucleolar network. Although the exact nature of this network remains to be defined, the results of the present study strongly suggest that the DNA molecules of the chromosomes bearing ribosomal genes have many sites of attachment to a non-chromatin nucleolar network which can be referred to as a nucleolar skeletal complex.     

Evidence for the existence of a nucleolar skeleton attached to the pore complex-lamina in human fibroblasts

This work deals with the types of nuclear skeletal structures obtained from human fibroblast nuclei isolated by different procedures. It is confirmed that, in somatic vertebrate cells, the pore complex-lamina is always observed, whereas the presence of internal nucleolar and extranucleolar residual structures depends upon the method of nuclear isolation used. Furthermore, the results reported here argue for the existence of a nucleolar skeleton different from the nucleolar matrix often observed in different cell types by other investigators. The conditions of nuclear isolation which allow us to visualize this nucleolar skeleton without any other internal residual structures are described. The attachment of the nucleolar skeleton to the lamina suggested by the present data is considered in relation to the in situ position of nucleoli near the nuclear envelope.     

Molecular cloning and functional expression of nucleolar phospholipid hydroperoxide glutathione peroxidase in mammalian cells

We cloned a full-length cDNA for phospholipid hydroperoxide glutathione peroxidase (PHGPx) including exon Ib from rat and mouse testis. The nuclear signal sequence of the N terminal of rat nuclear PHGPx possessed a different sequence from that previously reported for rat sperm nuclei GPx (SnGPx). Expression of this PHGPx-YFP (yellow fluorescent protein) fusion protein including a novel nuclear signal sequence was exclusively localized in nucleolus; although YFPs fused with only a novel nuclear signal sequence were distributed in the whole nucleus, indicating that preferential translocation of nucleolar PHGPx into nucleoli was required for the nuclear signal sequence and internal sequence of PHGPx. Low level expression of nucleolar PHGPx was detected in several tissues, but the expression of nucleolar PHGPx was extensively high in testis. Immunohistochemical analysis with anti-nucleolar PHGPx indicated that expression of nucleolar PHGPx was observed in the nucleoli in the spermatogonia, spermatocyte, and spermatid. Overexpression of 34kDa nucleolar PHGPx in RBL2H3 cells significantly suppressed cell death induced by actinomycin D and doxorubicin that induced damage in the nucleolus. These results indicated that nucleolar PHGPx plays an important role in prevention of nucleolus from damage in mammalian cells.     

Differentiation of Hashimoto's thyroiditis from thyroid neoplasms in fine needle aspirates

In order to refine the cytodiagnostic criteria for distinguishing Hashimoto's thyroiditis from thyroid neoplasms, aspirates from six cases of Hashimoto's thyroiditis, five Hürthle cell neoplasms and one papillary carcinoma associated with Hashimoto's thyroiditis were reevaluated. Distinguishing characteristics were cell arrangements, nuclear chromatin pattern and nucleolar appearance. Hashimoto's thyroiditis was characterized by flat sheets and clusters of epithelial cells with oncocytic changes or occasionally by cohesive tissue fragments with cells well oriented one to the other. Thyroid neoplasms were characterized by loosely cohesive, syncytial-type tissue fragments with crowded overlapping cells poorly oriented one to the other and/or numerous isolated single cells. The nuclear chromatin of Askanazy cells in Hashimoto's thyroiditis was bland and even while that of neoplastic cells was finely granular, coarsely granular or irregularly clumped. Macronucleoli were present in Hürthle cell tumors but not in the Askanazy cells of Hashimoto's thyroiditis. Epithelial cellularity, lymphoid cellularity, cellular polymorphism and nuclear pleomorphism were not useful criteria for making the differential diagnosis between the two conditions. An admixture of epithelial cells and lymphoid cells indicated Hashimoto's thyroiditis but was not helpful in ruling out an associated neoplasm.     

Double immunolocalization of major nucleolar proteins, fibrillarin and B23, in dividing mammalian cultured cells.

Using specific autoimmune sera to the nucleolar protein fibrillarin and monoclonal antibodies to B23/nucleophosmin, we localized early and late nucleolar rRNA-processing factors in cycling human HeLa and pig PK cells. It was shown that, at the electron microscopic level, fibrillarin was located over the nucleolar fibrillar compartment, but was absent in the fibrillar centres. During mitosis, fibrillarin was located within the same domains as B23, namely, the cytoplasm, the perichromosomal layer, prenucleolar bodies, and the nucleolar cytoplasmic derivatives, but the kinetics of the two proteins during mitosis was essentially different. Thus, fibrillarin dissociated from the nucleolar remnant at prophase of mitosis or following actinomycin D treatments after B23, but was found to be more prominent within the perichromosomal layer at metaphase, and earlier migrated to the reassembled nucleoli at telophase. In contrast to B23, fibrillarin was found to be resistant to the treatment with 2 M NaCl.