Identification of coiled body-like structures in meristematic cells of Pisum sativum cotyledonary buds.

This study focused on two types of nuclear bodies visible in plant cells that were previously thought to be similar to the coiled bodies (CBs) of animal cells: the nucleolus-associated body (NAB) and dense body (DB). We show that both NABs and DBs share common features with animal CBs: they consist of ribonucleoproteins, are silver-stainable, and lack DNA. Immunoelectron microscopy shows that only the NABs are rich in snRNAs and fibrillarin, two markers characteristic of animal CBs. This suggests that NABs rather than DBs are the plant counterparts of the CBs of animal cells. These structures appear most frequently in cells blocked in G0-1, their frequency gradually declining with resumption of the cell cycle and nucleolar activity. During this reactivation period, NABs are released from the nucleolus to the nucleoplasm, suggesting that they may act as nuclear transport or sorting structures.

AgNOR proteins from morphologically intact isolated nucleoli.

AgNOR staining has been proposed as a useful tool for the diagnosis and prognosis of cancer. The AgNOR proteins, however, have not yet been clearly identified and characterized, possibly due to the partial character of the results obtained when studying the proteins extracted from altered nucleoli isolated by "standard" methods. In the present study, we analysed, on western blots, the AgNOR staining profiles obtained with protein extracts from Ehrlich tumor cell nucleoli isolated by a recent procedure that preserves the nucleolar ultrastructure. In addition to the well-known C23 and B23 protein bands, we readily detected an extra band at approximately 125 Kda. By immunoblotting, we showed that this polypeptide may be related to the nucleolar phosphoprotein pp135 evidenced in rat-cell nucleoli. By immunoelectron microscopy, we detected this protein in the dense fibrillar component and fibrillar center of the nucleoli as well as the coiled bodies. The distribution coincides with the cytochemical AgNOR staining pattern obtained at the ultrastructural level.

Ultrastructural nucleolar alterations induced by an ametantrone--poly r(A-U) complex.

The current study has documented changes in the ultrastructure as well as in the intranucleolar distribution of rDNA and rRNA in RT4 (human transitional cell bladder carcinoma) cell nucleoli following a 3-h exposure to toxic doses of 50 microM ametantrone (AMT), 200 microM poly (adenylate-uridylate) (poly r(A-U) or an AMT/poly r(A-U) combination with an AMT/polyribonucleotide ratio of 1:4 and a poly r(A-U) concentration of 200 microM. While the main nucleolar components (fibrillar center (F), dense fibrillar component (D), granular component (G) and interstices (I) can be discerned following all treatments, the nucleoli exhibit: compaction, segregation, a decrease in the number of F, an increase in the size of remaining F, margination of intranucleolar chromatin and retention of intranucleolar pre-rRNA and rRNA. The relative abilities of the test agents to induce nucleolar compaction are AMT/poly r(A-U) > poly r(A-U) > AMT > sham-treated, while the abilities of the test agents to induce the remaining nucleolar changes are AMT/poly r(A-U) > or = AMT > poly r(A-U) > sham-treated cells. Poly r(A-U) and the induced interferon induce nucleolar compaction, while AMT produces nucleolar segregation. These results are consistent with a model in which the poly r(A-U) and/or the AMT inhibit DNA transcription and rRNA processing as well as the release of nascent preribosomes from the nucleolus.

Ultrastructural distribution of DNA within plant meristematic cell nucleoli during activation and the subsequent inactivation by a cold stress.

We have investigated the precise location of DNA within the meristematic cell nucleolus of Zea mays root cells and Pisum sativum cotyledonary buds, in the course of their activation and induced inactivation following a subsequent treatment at low temperature. For this purpose, we combined the acetylation method, providing an excellent distinction between the various nucleolar components, with the in situ terminal deoxynucleotidyl transferase-immunogold technique, a highly sensitive method for detecting DNA at the ultrastructural level. In addition to the presence of DNA in the condensed chromatin associated with the nucleolus, we demonstrated that a significant label was detected in the nucleolus of quiescent cells in both plant models. Evident labels were also found in the dense fibrillar component of actived nucleoli. Whereas in inactivated nucleoli no significant label was observed within the dense fibrillar component, an intense label was seen over the large heterogeneous fibrillar centres only during inactivation. The granular component was never significantly labelled. These results appear to indicate that the DNA present in the dense fibrillar component of activated nucleoli withdraws from this structure during its inactivation and becomes incorporated in the large fibrillar centres. These observations suggest that in plant cells inactivation of rRNA genes is clearly accompanied by changes in the conformation of ribosomal chromatin.

Ultrastructural nucleolar alterations induced by an ametantrone/polyr(A-U) complex.

In the present study we examined the ultrastructural modifications as well as the precise distribution of DNA and RNA in RT4 cell nucleoli following a 3-h exposure to nontoxic or toxic doses of ametantrone (AMT), poly(adenylate-uridylate) (polyr(A-U), or an AMT/polyr(A-U) combination. While distribution of nucleic acids within the various nucleolar components is not modified following all treatments, the nucleoli exhibit several ultrastructural changes: redistribution of the nucleolar components, decrease in the number of fibrillar centers, and increase in the size of the fibrillar centers. The relative frequencies of the test agents to induce the apparition of nucleoli of compact type are AMT/polyr(A-U) > AMT approximately polyr(A-U) > sham-treated, while the abilities of the test agents to induce the nucleolar segregation are AMT/polyr(A-U) approximately AMT > polyr(A-U) > sham-treated cells. These ultrastructural changes are characteristics of drugs that intercalate into DNA and inhibit rDNA transcription as well as rRNA processing and release of nascent preribosomes from the nucleolus.

Reversibility of thiamine deficiency-induced partial necrosis and mitochondrial uncoupling by addition of thiamine to neuroblastoma cell suspensions.

Culture of neuroblastoma cells in the presence of low thiamine concentration (16 nM) and of the transport inhibitor amprolium leads to the appearance of signs of necrosis: the chromatin condenses, the oxygen consumption decreases and is uncoupled, the mitochondrial cristae are disorganized, the thiamine diphosphate-dependent dehydrogenase activities are impaired. When 10 microM thiamine are added to these cells, the basal respiration increases, the coupled respiration is restored and mitochondrial morphology is recovered within 1 h. Addition of succinate, which is oxidized via a thiamine diphosphate-independent dehydrogenase, to digitonin-permeabilized cells immediately restores a coupled respiration. Our results suggest that the slowing of the citric acid cycle is the cause of the biochemical lesion induced by severe thiamine deficiency and that part of the mitochondria remain functional.

Polyamine and intestinal properties in adult rats.

We questioned whether polyamines coming from the diet or produced by intestinal microflora or by intracellular metabolism influence intestinal functions. Therefore, we compared pathogen-free rats and germ-free rats receiving a diet with low polyamine content and either treated or not treated with difluoromethylornithine (DFMO) and/or methylglyoxal bis (guanylhydrazone) (MGBG). Wet weight, protein content, DNA content, sucrase (EC 3.2.1.48), maltase (EC 3.2.1.20) and lactase (EC 3.2.1.23) specific activities, amounts of putrescine, spermidine and spermine were measured in the mucosa of the proximal and distal intestine. Body weight was also determined. Rats without microflora had a higher specific activity of maltase and higher amounts of spermidine and spermine but lower lactase specific activity than pathogen-free animals; the low-polyamine diet given to germ-free rats had little effect on the functional variables measured (decrease of maltase and lactase specific activities) and did not modify the amounts of polyamines. DFMO and/or MGBG administered to germ-free rats receiving a low-polyamine diet induced modifications of most of the variables studied. Body weight and wet weight of proximal and distal intestine decreased, disaccharidase specific activities decreased, and amounts of polyamines changed according to the inhibitor used. Thus, our results showed that the deprivation of polyamine supply from microflora or from the diet failed, under our experimental conditions, to affect the intestinal properties analysed but exogenous and endogenous polyamine restriction altered general properties of the organism as well as intestinal functions.

Isolation of nucleoli from ELT cells: a quick new method that preserves morphological integrity and high transcriptional activity.

We have developed a quick new method for isolating nucleoli which, unlike the methods in current use, preserves the nucleolar ultrastructure. Until now, the isolation process has generally been assumed to empty one of the three major compartments of the nucleolus, the fibrillar center, of its content. We have used the AgNOR staining and in vitro transcription assay to test the degree of structural and functional preservation of the isolated nucleoli. Our results demonstrate the value of our procedure as a reliable tool for biochemical and ultrastructural studies on the nucleolus. Moreover, these proprieties prompt us to investigate the rRNA synthesis, using a nonisotopic approach, within morphologically intact isolated nucleoli. Thus, we show that newly synthesized rRNA transcripts are located not only in the dense fibrillar component, but also indubitably in the fibrillar center.

Localization of nucleic acids in hepatocyte nucleoli of rats upon D-galactosamine-induced block of transcription.

The precise localization of DNA and RNA within rat hepatocyte nucleoli during the process of D-galactosamine-induced nucleolar segregation has been studied by using sensitive methods for their detection: osmium-ammine staining and terminal deoxynucleotidyl transferase reaction for DNA, and immunoelectron microscopy with anti-RNA antibodies, RNase-gold, and autoradiography with tritiated orotic acid for RNA. The blocking of transcription was followed by the disappearance of intranucleolar condensed chromatin. Agglomerates of thin extended DNA filaments were found to change their location to the nucleolar periphery and to coalesce with each other. At the last stage of nucleolar segregation they were concentrated at the pole of the nucleolar fibrillar remnant while the rest of the nucleolus did not contain any DNA. No DNA was found in the dense fibrillar component of both intact and treated hepatocyte nucleoli. During the process of nucleolar segregation the bulk of the nucleolar RNA was found within the so-called spherical bodies. This RNA appeared to be synthesized shortly before or even after drug administration. The results obtained are in agreement with the hypothesis that the fibrillar centers are the site of nucleolar transcription. They also show that uncompleted molecules of pre-rRNA whose synthesis has been blocked are segregated from the rest of nucleolar RNA species into the spherical bodies.

Thiamine deficiency--induced partial necrosis and mitochondrial uncoupling in neuroblastoma cells are rapidly reversed by addition of thiamine.

Culture of neuroblastoma cells in a medium of low-thiamine concentration (6 nM) and in the presence of the transport inhibitor amprolium leads to the appearance of overt signs of necrosis; i.e., the chromatin condenses in dark patches, the oxygen consumption decreases, mitochondria are uncoupled, and their cristae are disorganized. Glutamate formed from glutamine is no longer oxidized and accumulates, suggesting that the thiamine diphosphate-dependent alpha-ketoglutarate dehydrogenase activity is impaired. When thiamine (10 microM) is added to the cells, the O2 consumption increases, respiratory control is restored, and normal cell and mitochondrial morphology is recovered within 1 h. Succinate, which is oxidized via the thiamine diphosphate-independent succinate dehydrogenase, is also able to restore a normal O2 consumption (with respiratory control) in digitonin-permeabilized thiamine-deficient cells. Our results therefore suggest that the slowing of the citric acid cycle is the main cause of the biochemical lesion induced by thiamine deficiency as observed in Wernicke's encephalopathy.

Thiamine deficiency in cultured neuroblastoma cells: effect on mitochondrial function and peripheral benzodiazepine receptors.

When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16-fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 +/- 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high-affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low-affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine-deficient neuroblastoma cells.

Structure, function and assembly of the nucleolus.

Most events of ribosome biogenesis--such as transcription of the ribosomal RNA (rRNA) genes, processing of their primary transcripts into mature rRNAs and assembly with ribosomal and nonribosomal proteins to form the preribosomes--are confined to a special nuclear compartment, the nucleolus. Immunogold labelling and in situ hybridization at the ultrastructural level are providing novel insights into structure-function relationships of the nucleolus, and in vitro systems are beginning to shed light on the molecular mechanisms involved in the reforming of nucleoli after mitosis.

Ultrastructural distribution of DNA within the ring-shaped nucleolus of human resting T lymphocytes.

The precise distribution of DNA within the resting human T lymphocyte nucleolus has been investigated, at the ultrastructural level, by cytochemical and molecular immunocytochemical techniques. The nucleolus is partially enveloped by a layer of condensed chromatin which, at several places, penetrates into the nucleolar body until in close contact with the fibrillar centers. Morphometric analysis reveals that 32% of the fibrillar center surface is essentially occupied by condensed chromatin. Using the in situ terminal deoxynucleotidyl transferase-immunogold procedure for detecting DNA, we further show that evident label is exclusively found over the condensed chromatin and over the fibrillar centers, whereas no significant label is detected over the dense fibrillar component of the nucleolus.

Ultrastructural distribution of DNA within the nucleolus of various animal cell lines or tissues revealed by terminal deoxynucleotidyl transferase.

We have used the highly sensitive in situ terminal deoxynucleotidyl transferase method, applied to ultrathin sections, to investigate the location of DNA within nucleoli of various animal cells. In all the nucleoli studied, intense labelling is revealed over the peri- and intranucleolar condensed chromatin. Gold particles are also consistently found over the fibrillar centres, especially at their periphery, namely in the border area between the fibrillar centres and the dense fibrillar component, whereas the dense fibrillar component itself seems to be free of label in nucleoli in which these two compartments can be distinguished. We conclude that, in transcriptionally active nucleoli of this type, DNA is a characteristic constituent of the fibrillar centres, distinguishing them functionally from the dense fibrillar component. Some nucleoli exhibit neither fibrillar centres nor a dense fibrillar component, but have a single, albeit heterogeneous accumulation of fibrillar material; gold particles are consistently seen over some parts of this fibrillar compartment. This suggests that certain parts of the fibrillar material are functionally similar to the fibrillar centres of those nucleoli that possess them.

Location of DNA within the nucleolus of rat oocytes during the early stages of follicular growth.

We have investigated the DNA distribution within the rat oocyte nucleolus during the early stages of follicular growth by means of the in situ terminal deoxynucleotidyl transferase method. In the fibrillogranular nucleolus, label is visualized on small clumps of peri- and intranucleolar chromatin. Such labeled clumps are frequently observed inside the interstices surrounding the fibrillar centers. Label is also consistently found in the fibrillar centers whereas the dense fibrillar component and the granular component are devoid of gold particles. These results contradict earlier data but conform with other recent immunocytochemical observations, obtained in nucleoli of a variety of somatic cell types, concerning the correlation between structure and function in the nucleolus.

Myosin, parvalbumin and myofibril expression in barbel (Barbus barbus L.) lateral white muscle during development.

Histo- and immunohistochemical techniques have recently been used to study the fibre type and myosin expression in fish muscle during development. In the present work, embryonic, larval and adult myosin isozymes (heavy and light chains) and parvalbumin isotypes were analyzed, from fertization to the adult stage, by polyacrylamide gel electrophoresis of barbel (Barbus barbus L.) trunk muscle extracts. The examined myosins display the sequential transitions from embryonic to larval and adult forms characteristic of higher vertebrates. They are characterized by specific heavy chains but their light chains differ only by the LC1/LC3 stoichiometry with LC3 exceeding LC1 after 10 days. Sarcoplasmic parvalbumins show considerable and unforeseen developmental transitions in their isotype distribution: the PA II isotype first appears after hatching and becomes the predominant form until the length reaches about 6 cm. One month after hatching, the amount of PA II then decreases and the synthesis of PA III and IV further increases to reach the typical adult pattern at a size of 18 cm. These observations show that the distribution of parvalbumin isotypes reflects the stage of development. It suggests a specific role for each isotype in relation to muscle activity. Microscopy illustrates the progressive development of somites, muscles cells, and myofibrils, which accelerates at hatching when movements increase.

Distinguishing the sites of pre-rRNA synthesis and accumulation in Ehrlich tumor cell nucleoli.

The precise location of transcribing rRNA genes within Ehrlich tumor cell nucleoli has been investigated using two approaches: high-resolution autoradiography of cells pulse-labelled with tritiated uridine, varying the exposure time, and in situ-in vitro transcription coupled with an immunogold labelling procedure. When autoradiographic preparations are exposed for a short time, silver grains are found associated almost exclusively with interphasic cell nucleoli. Labelling of extranucleolar areas requires longer exposure. Within the nucleolus, the first sites to be revealed are in the dense fibrillar component. Prolonging exposure increases labelling over the dense fibrillar component, with label becoming more and more apparent over the fibrillar centers. Under these conditions, however, labelling does not extend into the granular component, and no background is observed. Initiation of transcription on ultrathin cell sections occurs preferentially at the borders of condensed chromatin blocks and in their close vicinity. The condensed chromatin areas themselves remain unlabelled. Inside most nucleoli, gold-particle clusters are mainly detected in the fibrillar centers, especially at their periphery, whereas the dense fibrillar component and the granular component remain devoid of label. These results, together with previous observations made on the same cell type, clearly indicate that the fibrillar centers are the sites of rRNA gene transcription in Ehrlich tumor cell nucleoli, while the dense fibrillar component is the site of pre-rRNA accumulation.

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level.

Nucleoli are the morphological expression of the activity of a defined set of chromosomal segments bearing rRNA genes. The topological distribution and composition of the intranucleolar chromatin as well as the definition of nucleolar structures in which enzymes of the rDNA transcription machinery reside have been investigated in mammalian cells by various immunogold labelling approaches at the ultrastructural level. The precise intranucleolar location of rRNA genes has been further specified by electron microscopic in situ hybridization with a non-autoradiographic procedure. Our results indicate that the fibrillar centers are the sole nucleolar structures where rDNA, core histones, RNA polymerase I and DNA topoisomerase I are located together. Taking into account the potential value and limitations of immunoelectron microscopic techniques, we propose that transcription of the rRNA genes takes place within the confines of the fibrillar centers, probably close to the boundary regions to the surrounding dense fibrillar component.

Cytochemical distinction of various nucleolar components in insect cells.

The fine structure of the insect Sf9 cell nucleolus has been investigated by means of different cytochemical and immunocytochemical techniques at the electron microscope level. Apart from a few perinucleolar condensed chromatin clumps, the insect cell nucleolus comprises two compartments. The first of these consists of a roundish compact zone formed of fibrillar material. The other is composed of fibrillar and granular structures organized into a network separated by interstitial spaces. But, unlike mammalian cell nucleoli, any fibrillar center has been observed in the Sf9 cell nucleolus, even after actinomycin D treatment. We also show that the compact fibrillar zone of Sf9 cell nucleoli contains silver-stainable material and DNA. In actinomycin D-treated cells, a preferential contact of this compact fibrillar zone with condensed chromatin has been visualized. Finally, silver-stainable material has been found to persist throughout the whole mitosis. These results suggest that the compact fibrillar zone at the insect Sf9 cell nucleolus should, at least partly, correspond to the fibrillar center of mammalian cell nucleoli.