The effect of the Bacillus thuringiensis exotoxin on the fine nucleolar morphology and ultrastructure

Depending on the dose administred to the experimental mice, the Bacillus thuringiensis exotoxin produces striking changes in the nucleolar morphology of hepatocytes such as the formation of ring-shaped nucleoli, micronucleoli and the segregation of nucleolar components. Such changes are apparently related to the decrease and inhibition of nucleolar biosynthetic activities in the production of the nucleolar RNA. In addition, the Bacillus thuringiensis exotoxin causes the formation of nucleolar peripheral dense plaques and, at higher concentrations, the segregation of two distinctly separated granular areas in the nucleolus. Both these light and dense granular areas showed positive staining with Bernhard's EDTA procedure for the preferential demonstration of RNA-containing structures. In some segregated nucleoli the granular components of light granular areas seemed to leave the nucleolus. The presence of discontinuous filamentous shell around micronucleoli produced by the high dose of exotoxin suggests the nucleolar origin of nuclear granular bodies which are surrounded by similar but continuous filamentous shell characteristic of these structures.     

Effect of the lysosomotropic preparation suramin on the ultrastructural and functional characteristics of rat liver cells in acute toxic hepatitis

A study was made of the influence of suramin and of a combination of suramin and CCl4 on the ultrastructure of hepatocytes, structural and functional state of lysosomes and functional activity of the rat's liver. The latter was characterized by alanine aminotransferase level in blood. Increasing amounts of autophagosomes as well as lysosomal fusion disturbances were registered 24 and 48 hours after a single suramin administration. The combined suramin and CCl4 administration resulted even in a higher damage of hepatocytes. A decrease in the intralysosomal rate of proteolysis in hepatocytes, was also shown in addition to the impairment of liver functional activity. Some ultrastructural features of compensation of the insufficiency of the hepatocyte vacuolar apparatus were noted during suramin and CCl4 administration. Changes in the nucleolar structure were noticed in hepatocytes administered both suramin and CCl4. It is suggested that these changes may involve a compensatory increase in nucleologenesis due to the decrease in the activity of the protein-synthesizing apparatus in hepatocytes.     

Spectrally Resolved Morphometry of the Nucleus in Hepatocytes Stained by Four Histological Methods

A novel concept of spectrally resolved morphometry for histological specimens was developed using light microscopy combined with spectrally resolved imaging. The spectroscopic characteristics of rat hepatocytes stained by Haematoxylin and Eosin, Romanowsky–Giemsa, periodic acid–Schiff and Masson's trichrome were assessed. Light intensity in the range 450–850 nm was recorded from 10000 pixels of nuclear domains of each stained cell and represented as light transmittance spectra and optical density. In order to identify spectral shifts caused by stain– macromolecule interactions, we compared the spectra of individual stain components with those of DNA and bovine serum albumin. Chromatin and interchromatin areas were classified spectrally using a chosen spectral library followed by morphometric calculations of nuclear domains for each staining method. The spectral fingerprints of Masson's trichrome stain distinguished the nucleolus from the rest of the nuclear c hromatin, enabling the demarcation and calculation of the nucleolar area. Spectrally resolved imaging of human hepatocytes stained by Masson's trichrome stain revealed marked differences between the nucleolar area in normal human hepatocytes compared with hepatocellular carcinoma. Masson's trichrome stain also distinguished the nucleolar area in human breast carcinoma cells and keratinocytes.     

不同生殖期鳜肝脏超微结构变化的观察

应用透射电镜对生殖季节与非生殖季节鳜肝脏超微结构的变化进行了观察。鳜肝细胞含有单个卵圆形的核,核仁清楚;细胞质内含有粗面内质网、线粒体、糖原颗粒和脂滴等细胞器和内含物。胆小管由相邻的数个肝细胞质膜凹陷围成,而肝血窦则由内皮细胞的胞质构成。还发现了贮脂细胞、枯否氏细胞和成纤维细胞。胆小管腔和窦周隙内浸润许多由肝细胞发出的微绒毛结构。鳜肝细胞的超微结构在产卵前后呈现明显变化：产卵前的肝细胞内富含线粒体、糖原颗粒和脂滴,粗面内质网发达;而产卵后的肝细胞内核仁发生迁移,部分细胞核囊泡化,糖原颗粒和脂滴排空,少数肝细胞具双核结构。非生殖期多数肝细胞核含有双核仁结构,胞质内溶酶体数量增多。     

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures

Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.     

Changes in the ultrastructure of rat hepatocyte nucleoli during bilateral adrenalectomy and after administration of cortisol

Nucleolar ultrastructure of the rat hepatocytes after bilateral adrenalectomy and cortisol stimulation has been studied by the electron-microscopic method Traits of nucleolar inactivation (a decrease of granular component enlargement of fibrillar centres, condensation of peri- and intranucleolar chromatin, etc.) are observed in hepatocyte nucleoli 5 days after adrenalectomy. Cortisol stimulation of hepatocytes of the adrenalectomized rats leads to nucleolar activation (4h, 5h, 8h after the hormone injection). Adrenalectomy with following cortisol injection is a useful model to study inactivation and activation of ribosomal genes in the target cells.     

Nucleolar lesions induced by ribonuclease in living cultured cells

The nucleolar lesions provoked by the action of ribonuclease (RNase) on living chick embryo fibroblasts were studied by means of microcinematographic analysis and at the ultrastructural level using oxidized diaminobenzidine as a differentially contrasting stain for nucleic acids. This study has shown that the induction of nucleolar dispersion by RNase was only the beginning of a series of discrete steps. The following sequences are described: dispersion of the nucleolus into fragments, their reassembly, and the emission of spherules which appear of chromatin origin. At that step nucleoli are typically segregated. The alteration of the nucleolar associated chromatin seemed to be primordial in these processes. Moreover, the large mass of heterochromatin intimately associated with the nucleolus and which has been considered to be a part of the nucleolar organizer region apparently plays a chief part in the reassembly of the nucleolar fragments into a segregated nucleolus. Ribonuclease is compared to other drugs known to act on nucleolar DNA.     

Nucleolar lesions induced by ribonuclease in living cultured cells

The nucleolar lesions provoked by the action of ribonuclease (RNase) on living chick embryo fibroblasts were studied by means of microcinematographic analysis and at the ultrastructural level using oxidized diaminobenzidine as a differentially contrasting stain for nucleic acids. This study has shown that the induction of nucleolar dispersion by RNase was only the beginning of a series of discrete steps. The following sequences are described: dispersion of the nucleolus into fragments, their reassembly, and the emission of spherules which appear of chromatin origin. At that step nucleoli are typically segregated. The alteration of the nucleolar associated chromatin seemed to be primordial in these processes. Moreover, the large mass of heterochromatin intimately associated with the nucleolus and which has been considered to be a part of the nucleolar organizer region apparently plays a chief part in the reassembly of the nucleolar fragments into a segregated nucleolus. Ribonuclease is compared to other drugs known to act on nucleolar DNA.     

Further observations on ultrastructural changes in hepatocytes of mice infected with Schistosoma mansoni

The effect of hepatic granulomas initiated by eggs of Schistosoma mansoni on the ultrastructure of hepatocytes of murine hosts was studied. Specimens of infected livers were collected at half week intervals, starting at week 7 postinfection and terminating at week 9 postinfection. Only the hepatocytes adjacent to granulomas showed any alteration in structure. The most obvious change was the proliferation of smooth-surfaced endoplasmic reticulum, especially striking in samples collected 8 1/2 and 9 weeks postinfection. The hypothesis was presented that the material secreted by the egg of the organism might be responsible for what appeared to be a morphologic detoxification response by the hepatocytes. Other alterations evident in the hepatocytes were an increase in the lysosomal population, mitochondrial changes and a slight hypertrophy of the Golgi complexes. Previous related studies by other investigators were explored and the findings compared with the results of this study.     

Studies on satellite nucleoli in rat hepatocytes and Novikoff hepatoma cells

Summary The present study was undertaken to provide information on the presence and frequency of satellite nucleoli in cells with increased nucleolar biosynthetic activity. The number of hepatocytes containing satellite nucleoli was analyzed in rat liver, regenerating liver 18 h after partial hepatectomy and in Novikoff hepatoma ascites cells. In comparison with hepatocytes of normal liver, the number of both stimulated hepatocytes and malignant hepatoma cells containing satellite nucleoli was significantly reduced. The results also indicated that whereas most satellite nucleoli contain protein C23, a smaller percentage contain protein B23.     

Alteration of microtubule physiology in hepatocytes by insulin

Incubation of primary cultures of rat hepatocytes with insulin caused an alteration in several parameters of microtubule physiology. There was a transient decrease in total cellular tubulin levels and, at the same time, an apparent stabilization of microtubule polymers which was also transient. The level of microtubule polymer, however, was not altered, which indicated that an insulin-inducible decrease in the level of nonpolymerized tubulin was responsible for the decrease in total cellular tubulin. These changes were followed by an increase in tubulin mRNA levels and, shortly thereafter, by an increase in tubulin protein synthesis. This study demonstrates that microtubule physiology in hepatocytes is modulated by insulin and, further, suggests a model by which insulin-induced changes in microtubule physiology may play a role in regulating other insulin responses.     

Cytochemistry of the microtrabecular network in compact nucleoli of hepatocytes treated with cycloheximide

Summary Compact nucleoli without the segregation of nucleolar components were produced in hepatocytes by the treatment of experimental rats with cycloheximide to facilitate a cytochemical study on the organization of nucleolar components in such nucleoli. The extraction of pepsin pretreated specimens with nucleases (deoxyribonuclease and ribonuclease) demonstrated that compact nucleoli are characterized by a relatively uniform distribution of RNP components which mask a microtrabecular intranucleolar network. This network apparently consists of proteins and contains fine DNA filaments.     

The Ag-NOR proteins and transcription and duplication of ribosomal genes in mammalian cell nucleoli

The relationship between the Ag-NOR (silver-stained Nucleolar Organizer Region) proteins and the functional-structural organization of the nucleolar ribosomal chromatin was studied in regenerating and cortisol-stimulated rat hepatocytes. Statistical analysis of Ag-NOR proteins, carried out with an automated image analyzer, indicated that in regenerating rat hepatocytes the quantity of Ag-NOR proteins mainly increased between the 4th and 12th h of regeneration, reaching a level twice that of resting hepatocytes. Also the synthesis of pre-ribosomal RNA (pre-rRNA) was stimulated after the 4th h of regeneration. Cycloheximide administered to rats at a dose of 0.025 mg/100 g body weight (bw) prevented any increase in Ag-NOR proteins but did not hinder the stimulation of pre-rRNA synthesis. In 8 h cortisol-stimulated hepatocytes no significant change in amount of Ag-NOR protein was observed whereas pre-rRNA synthesis was highly increased as in 12 h regenerating hepatocytes. These results indicated that in rat hepatocytes Ag-NOR proteins and stimulation of pre-rRNA synthesis are not related. The relationship between the Ag-NOR proteins and the distribution of the completely extended intranucleolar ribosomal chromatin was also studied in regenerating rat hepatocytes. At 12 h after partial hepatectomy an increased amount of completely extended ribosomal chromatin was observed, contemporaneously with an increased quantity of Ag-NOR proteins. These ribosomal chromatin changes preceded the beginning of DNA synthesis and were prevented by cycloheximide-induced inhibition of protein synthesis.     

Activation of nucleolar DNA and ribosome biosynthesis in hepatocytes under the effect of glucocorticoids and high density lipoproteins

The structural bases of cooperative effect of glucocorticoids and HDL brings about the activation of protein biosynthesis in hepatocytes. Using surviving rat liver it was shown that these two compounds together activate the gene expression which was indicated by increased 3H-uridine incorporation into the total RNA pool. The enhanced incorporation of 14C-leucine into proteins in these experiments confirms protein biosynthesis acceleration. With the use of liver perfusion technique it was morphologically demonstrated that the earliest changes in hepatocyte genome take place in nucleoli. The increase of nucleolar dimensions and granular component reflects the activation of ribosomal precursors synthesis. Considerable number of ribosomes in the hepatocyte perinuclear space indicates their active transport across the numerous nuclear membrane pores into the cytoplasm. In the first place and more prominently in hepatocytes the protein synthesis "for export" is stimulated, which was proved by the dynamics of ribosome accumulation on the membranes of endoplasmic reticulum according the perfusion duration. The kupffer cells play a significant role in HDL transcytosis and in the realization of their cooperative effect with glucocorticoids.     

Stressing on the nucleolus in cardiovascular disease

The nucleolus is a multifunctional organelle with multiple roles involving cell proliferation, growth, survival, ribosome biogenesis and stress response signaling. Alteration of nucleolar morphology and architecture signifies an early response to increased cellular stress. This review briefly summarizes nucleolar response to cardiac stress signals and details the role played by nucleolar proteins in cardiovascular pathophysiology. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.     

Detection of fibrillarin in nucleolar remnants and the nucleolar matrix

In order to gain further insights into the fundamental structure of the nucleolus, nucleolar remnants of Xenopus and chickens were examined for the presence of fibrillarin and nucleolus organizer region (NOR) silver staining. Nucleolar remnants of Xenopus nucleated red blood cells were found to contain easily detectable amounts of fibrillarin and NOR silver staining. Upon examination of various tissues, fibrillarin and NOR silver staining were detected in nucleoli of Xenopus liver hepatocytes and within nucleoli of oocytes and follicle cells from ovaries of mature female toads. By comparison, nucleolar remnants of adult chicken nucleated red blood cells contained only trace amounts of fibrillarin and NOR silver staining, whereas red blood cell nucleolar remnants of immature chicks had easily detectable amounts of fibrillarin and NOR silver staining. Nucleoli from hepatocytes of both adult and immature chickens demonstrated comparable levels of fibrillarin and NOR silver staining. Since fibrillarin was found in nucleolar remnant structures, we tested for (and detected) its presence in residual nucleoli of in situ nuclear matrix derived from HeLa cells. These findings are discussed in terms of the basic structural and functional organization of the nucleolus.