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.     

Control of nucleolar growth during interphase in higher plant meristem cells

Summary The evolution of nuclear and nucleolar sizes throughout interphase have been studied in synchronous caffeine-labeled binucleate cells of onion root meristems by using silver impregnation and stereological methods over semithin sections. Nucleus and nucleolus grow independently, since nucleolus enlarges at its fastest rate in G 1, while nucleus grows mostly in two periods: onset of replication and G 2. Nucleolar size in the cycle seems to be a genecontrolled function, hardly affected by protein synthesis inhibition. Hence, there is a biphasic response to cycloheximide (CHM) in the fast growing nucleoli of both early and late G 1 with an initial stimulation later counterbalanced by a depressed rate, so that nucleolar size in S was similar to control shortly afterwards the start of the CHM treatment. The initial enlargement under CHM was due to an increase of all nucleolar structural components, i.e., fibrillar, granular, vacuolar, and lacunar regions. No cycloheximide effect whatsoever was detected in S and G 2 nucleoli.Abbreviations CHM cycloheximide - F fibrillar component - G granular component - L lacunae - V vacuoles - VN nuclear volume - VNu nucleolar volume - VvNu volume density of the nucleoli     

蚕豆核仁残体的电镜研究

本文报道了有丝分裂过程中蚕豆(Vicia faba)根端分生组织细胞内一种由核仁物质组成的特殊结构,我们将其称作核仁残体。经常规染色后,可在前期末正在分散的核仁物质中看到由直径15—20nm 的颗粒和纤维组成的核仁残体;前中期时,核仁残体附着在染色体上,其电子密度低于染色体。Bernhard 染色结果表明,核仁残体的主要成分是核糖核蛋白(RNP)颗粒和纤维,一些核仁残体中存在着与染色质染色反应相似的被漂白区。有的核仁残体附着在中、后期染色体上,有的游离存在于细胞质中。本文讨论了核仁残体的成分及其本质等问题。     

正常造血细胞的核仁组成区计数定量研究

本文应用银染技术对正常造血细胞的核仁组成区进行了计数定量研究。结果显示,在粒系和红系中,随细胞的成熟,细胞中簇状ＡｇＮＯＲ的数量逐渐减少．而点状ＡｇＮＯＲ数量逐渐增多,无分裂能力的成熟细胞仅有少数点状ＡｇＮＯＲ。淋巴细胞中为一完全集结的银染颗粒,而巨核细胞内为各自分离的点状银染颗粒。本结果为正常造血细胞的核仁组成区提供了基础数值。     

The nucleolar structure and the activity of NopA100, a nucleolin-like protein, during the cell cycle in proliferating plant cells

For the purpose of gaining knowledge of the relationships between cell proliferation and ribosome biogenesis, as two fundamental mutually interconnected cellular processes, studies were performed on cell populations synchronized in their cell-cycle progression by treatment with hydroxyurea, followed by sampling at different times after its removal. A structural rearrangement of the nucleolus was observed throughout the interphase, along with changes in the relative amounts of different nucleolar subcomponents. A structural model of nucleolar organization was associated with each interphase period. Throughout interphase, the nucleolin-like protein, NopA100, was immunodetected in the dense fibrillar component of the nucleolus, preferentially near fibrillar centers and its levels were shown to increase from G1 to G2. A western blotting analysis of soluble nuclear protein extracts with anti-NopA100 antibody resulted in the intense labeling of a 100-kDa band, but also of a series of proteins related to it, suggesting that NopA100 undergoes a physiological process of proteolytic maturation, similar to that described for mammalian nucleolin, but not reported in other biological model systems. Physiological proteolysis of NopA100, related to cell-cycle progression, was confirmed after the nuclei extracted from synchronized cells were treated with the protease inhibitor, leupeptin, which resulted in an increase of the 100-kDa band at the expenses of the decrease of some other bands, according to the cell-cycle stages. We therefore conclude that there is a relationship between the increase in nucleolar activity, cell-cycle progression, nucleolar structure, the activity of NopA100, and the proteolysis of this nucleolin-like protein.     

Proliferation, apoptosis and thymic involution.

The thymus reaches its maximal size at the age of 1 month in ICR mice and thereafter, the thymic cortex undergoes an exponential decline. This study was designed to compare the proliferation and apoptosis of thymocytes in different parts of the thymus of ICR female mice at the beginning and after the rapid phase of decline of the thymic cortical cellularity. The pattern of proliferation and apoptosis of the thymus was studied in situ in 1-month-old ICR female mice (10 mice) compared to mice at 7 months of age (10 mice). Staining for argyrophylic nucleolar organizer region by histochemistry was used to determine the proportion of type 2 thymocytes, which are considered as cells at S phase of the cell cycle. The mean number of type 2 cells in four random samples of 50 cells in each part of the thymus was defined as the proliferation index of this part of the thymus. In situ detection of apoptosis of thymocytes was carried out using the Apoptag kit, which can detect a single cell apoptosis. The mean number of apoptotic cells in five randomly selected fields of each part of the thymus was defined as the apoptotic index of this part of the thymus. The proliferation index of the peripheral cortex of the 1-month-old mice was 3.6 times higher than the proliferation index of the deep cortex and 5.8 times higher than the proliferation index of the medulla (P < 0.0001). The proliferation index of the peripheral cortex of the 7-month-old mice was reduced by 45% compared to the 1-month-old mice (P < 0.005). The apoptotic index of the corticomedullary junction of the 1-month-old mice was six times higher than the apoptotic index of the cortex and 18 times higher than the apoptotic index of the medulla. The apoptotic index of the thymic cortex was elevated by 66% in the 7-month-old mice compared to the 1-month-old mice (P < 0.0001). We conclude that there is a reduction of the proliferation index and an elevation of the apoptotic index of the thymic cortex in adult mice compared to young mice. These changes might account for the reduction of thymic cortical cellularity during thymic involution.     

An EM Study on the Nucleolar Residual in Meristematic Cells of Vicia faba

A special structure which originates from the nucleolus and persists during mitosis is observed in meristematic cells of Vicia faba, and is called nucleolar residual in this paper. The result of conventional staining showed that the nucleolar residual (NR) was a medium electron-dense structure that could be distinguished from chromosomes. At the end of prophase, the NR was observed in the dispersing nucleolar material, which consisted principally of granules and fibrils about 15 to 20 nm in diameter. In prometaphase, the NR, composed mainly of the similar granules and fibrils, was found attached to chromosomes. Results of Bernhard's technique for RNP (ribonucleoprotein) preferential staining indicated that the main chemical component of the NR was RNP, and sometimes bleached regions showing the same reaction as chromatin in Bernhard's staining appeared in the structure. In metaphase and anaphase some of the NRs were attached to chromosomes while others existed randomly in the cytoplasm. The NR is supposed, according to its cytochemical features and its behavi0ur during the nucleolar disintegration, to be a structure composed mainly of nucleolar matrix material or nucleolar skeleton.     

Localisation of nucleolus-organising regions in interphase cells

Summary A technique based on the use of silver solutions, which selectively stains the nucleolus-organising regions (NORs) in chromosomes, was applied to interphase Ehrlich tumour cells. The results indicate that nucleolar fibrillar centres correspond to the NORs.     

Nucleoli in large (giant) bi- and multinucleate cells after apoptosis-inducing photodynamic treatment

The present experimental study was undertaken to provide information on nucleolar changes accompanying the apoptotic process in large or giant binucleate and multinucleate cells (LBMNCs). Such cells were present in a small but constant percentage in cultures of HL-60 cells. The apoptotic process was induced by photodynamic treatment (PDT) by means of 5-aminolaevulinic acid (ALA) as the precursor of the photosensitizer protoporphyrin IX and irradiation with broad spectrum blue light (BL). Nucleolar changes in LBMNCs were characterized by marked reduction or disappearance of silver stained particles representing AgNORs in nucleoli including the large ones. In addition, PDT also significantly reduced the number of nucleoli regardless of their size. These changes apparently reflected the decrease or cessation of nucleolar biosynthetic activities and resembled those which were previously observed in naturally maturing bone marrow megakaryocytes (Janoutová et al., 2001).     

一例硬皮病人自发抗核仁抗体的免疫荧光及其与银染的比较研究

从硬皮病人血清中筛选出一例含有自发抗核仁抗体的血清,利用这个血清对核仁抗原的性质及其在细胞中随分裂周期不同产生的分布变化做了初步研究,并把结果与核仁嗜银蛋白做了比较。间接免疫荧光染色及细胞化学分析表明,这种核仁抗原的性质是蛋白质,其分布与嗜银蛋白相似,在间期,抗原呈颗粒状簇集在核仁中,而在分裂中期,抗原颗粒与染色体NORs部位接合,但有证据指出,这种抗原蛋白与核仁嗜银蛋白有所不同,同时还发现,经长时间秋水仙素处理诱导产生微核化的多核细胞中尽管微核的数目远多于细胞中NORs的数目,免疫荧光染色和银染都显示出每个微核中类核仁小体的存在。这说明(1)类核仁小体也是由核仁物质构成;(2)某些类核仁小体的产生可能与NORs无关。对这个现象的意义进行了讨论。     

Some measurements on amphibian oocyte nucleoli

Summary The dry mass of nucleoli from yolky oocytes of T. cristatus, as determined by interference microscopy is about 150 g. Less than 10% of this is attributable to RNA that is removable by ribonuclease from formalin-fixed nucleoli. There is a linear relationship between nucleolar dry mass and size. There are basic proteins in oocyte nucleoli and these are bound to the nucleolar RNA. The concentrations of cytochemically detectable RNA and basic protein are higher in the nucleolar core than in the cortex. The ratio of RNA to basic protein is higher in the core than in the cortex, as determined by microdensitometry of nucleoli stained with gallocyanine and fast green. The significance of these observations in relation to the role of the nucleolus in ribosome biosynthesis is discussed.This study was supported in part by Science Research Council Grant No. BSR/4468. We wish to thank Dr. Colin Muir of the Department of Zoology, University of St. Andrews for his helpful advice on interferometry.     

蚕豆染色体周边RNP形成过程的电镜研究

本文运用Bernhard染色方法研究了蚕豆根端分生组织细胞中染色体周边RNP的超微结构以及这种周边RNP在有丝分裂前期到中期的形成过程。我们观察到,在前期核仁解体过程中,来自核仁的RNP物质结合于染色体表面,形成染色体周边RNP。前期末时,大量核仁RNP颗粒向周围扩散并进一步结合于染色体表面,使染色体周边RNP有所增加。中期染色体的周边RNP明显多于前期,由直径15-20 nm的RNP颗粒构成。RNP物质在染色体周边的分布是不均匀的。姊妹染色单体之间往往有较多的RNP物质存在。本文观察结果表明染色体周边RNP来源于核仁RNP。     

Studies on nucleoli of maturing frog erythroblasts

Summary Frog erythroblasts were studied in summer animals with a very active as well as reduced erythropoiesis due to experimental hibernation, the latter being administered in order to get more information on the frequency of various nucleolar types in maturing cells. The results suggest that nucleoli with nucleolonemata are a transitional nucleolar type between compact and ringshaped nucleoli. Since micronucleoli represent final nucleolar maturation changes and compact nucleoli are present in most immature cells, the sequence of nucleolar changes based on the frequency of investigated nucleolar types is as follows: compact nucleolinucleoli with nucleolonemataringshaped nucleolimicronucleoli. The experimental hibernation produces a shift of nucleoli to less active and maturer nucleolar types in all stages of the erythroblastic maturation. In addition, the experimental hibernation produces the formation of ringshaped nucleoli in the first stages of the erythroblastic maturation which in summer animals usually contain compact nucleoli and/or nucleoli with distinct nucleolonemata.     

In vivo production of the nucleolar channel system in post menopausal endometrium

Summary Endometrial biopsies from seven postmenopausal women on hormone replacement therapy have been examined for the presence of the unique nuclear structure, the nucleolar channel system. Its identification in five of the patients has demonstrated that the nucleolar channel system can be produced by an appropriate oestrogen and progestagen treatment and is not otherwise dependent on ovulation.     

Nucleolar localization of parafibromin is mediated by three nucleolar localization signals

Parafibromin is a putative tumor suppressor encoded by HRPT2 and implicated in parathyroid tumorigenesis. We previously reported a functional bipartite nuclear localization signal (NLS) at residues 125-139. We now demonstrate that parafibromin exhibits nucleolar localization, mediated by three nucleolar localization signals (NoLS) at resides 76-92, 192-194 and 393-409. These NoLS represent clusters of basic amino acids arginine and lysine, similar to those found in other nucleolar proteins, as well as being characteristic of NLSs. While parafibromin's bipartite NLS is the primary determinant of nuclear localization, it does not mediate nucleolar localization. In contrast, the three identified NoLSs play only a minor role in nuclear localization, but are critical for the nucleolar localization of parafibromin.     

Nucleolar Stress Induces Ubiquitination-independent Proteasomal Degradation of PICT1 Protein

The nucleolar protein PICT1 regulates tumor suppressor p53 by tethering ribosomal protein L11 within the nucleolus to repress the binding of L11 to the E3 ligase MDM2. PICT1 depletion results in the release of L11 to the nucleoplasm to inhibit MDM2, leading to p53 activation. Here, we demonstrate that nucleolar stress induces proteasome-mediated degradation of PICT1 in a ubiquitin-independent manner. Treatment of H1299 cells with nucleolar stress inducers, such as actinomycin D, 5-fluorouridine, or doxorubicin, induced the degradation of PICT1 protein. The proteasome inhibitors MG132, lactacystin, and epoxomicin blocked PICT1 degradation, whereas the inhibition of E1 ubiquitin-activating enzyme by a specific inhibitor and genetic inactivation fail to repress PICT1 degradation. In addition, the 20 S proteasome was able to degrade purified PICT1 protein in vitro. We also found a PICT1 mutant showing nucleoplasmic localization did not undergo nucleolar stress-induced degradation, although the same mutant underwent in vitro degradation by the 20 S proteasome, suggesting that nucleolar localization is indispensable for the stress-induced PICT1 degradation. These results suggest that PICT1 employs atypical proteasome-mediated degradation machinery to sense nucleolar stress within the nucleolus.