Biomedical subjects

M Thiry

Publications and source records attributed to M Thiry.

At least 37 records · Page 2Linked to original sources

PubMed1997

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.

Antineoplastic Agents↗

PubMed1997

The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids.

We used a 32P-labeled pCMV-CAT plasmid DNA to estimate the DNA uptake efficiency and unlabeled pCMV-CAT plasmid DNA to quantify the CAT activity after transfection of COS cells using each of the three following cationic compounds: [1] vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine, and previously described as diC14-amidine [1]), [2] lipofectin (a 1:1 mixture of N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and dioleylphosphatidylethanolamine (DOPE)), and [3] DMRIE-C (a 1:1 mixture of N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide (DMRIE) and cholesterol). Surprisingly, a high CAT activity was observed with vectamidine although the DNA uptake efficiency was lower as compared to lipofectin and DMRIE-C. Transmission electron microscopy (TEM) revealed endocytosis as the major pathway of DNA-cationic lipid complex entry into COS cells for the three cationic lipids. However, the endosomal membrane in contact with complexes containing vectamidine or DMRIE-C often exhibited a disrupted morphology. This disruption of endosomes was much less frequently observed with the DNA-lipofectin complexes. This comparison of the three compounds demonstrate that efficient transfection mediated by cationic lipids is not only correlated to their percentage of uptake but also to their ability to destabilize and escape from endosomes.

Amidines↗

PubMed1997

Reassembly of functional nucleoli following in situ unraveling by low-ionic-strength treatment of cultured mammalian cells.

In order to determine the most persistent components of the nucleolus that might serve as "core" nucleolar elements, we studied the reactivity of nucleoli in living mammalian cells subjected to hypotonic buffer saline followed by the incubation of the cells in an isotonic medium. To document as precisely as possible the fine structural changes which occurred, the cells were examined by video-enhanced optical microscopy, fluorescence confocal laser scanning microscopy, and electron microscopy combined with cytochemistry. Light microscopic autoradiography was used to demonstrate the transcriptional characteristics of the reassembled nucleoli. It was shown that all the major compartments of the intact nucleolus could be substantially affected by reduction of the osmolarity of the environmental media. The dynamic events of the nucleolar unraveling in low-salt buffers occurred in the following order: dispersion of the nucleolar pars granulosa, disassociation of the fibrillar complexes into discrete fibrillar centers (FCs) and the dense fibrillar component (DFC), and the almost complete unraveling of the DFC and FCs. At the terminal stages of nucleolar dispersion, the nuclear interior was mainly composed of a loose filamentous meshwork, and none of the typically discerned nucleolar constituents was recognized. Nevertheless, when hypotonically treated cells were returned to isotonic conditions, the nucleolar bodies rapidly began to reassemble. Within 1-2 h of cell incubation under isotonicity, the nucleoli not only became clearly visible, but also reconstituted to their initial size, shape, and position within the nucleus. The ultrastructure and functional activity of the reassembled nucleoli were also found to be fully comparable to those of the untreated controls. These data indicate that the architectural composition of the interphase nucleolus is strictly controlled by the cell. As far as could be determined, none of the usual substructures of the intact nucleolus that could be substituted by complete reassembly of the nucleolar bodies in normotonic conditions, including FCs and the DFC, remained clearly preserved in the terminal stage of nucleolar unraveling. We concluded that the integrity of the nucleolus was mainly preserved by the nuclear or nucleolar matrix system rather than by any other nucleolar structural domains.

Animals↗

PubMed1997

"Exolysosomes," enzyme-containing vesicles in the ecdysial space of molting crabs.

Free vesicle-like bodies (VLBs) present in the ecdysial space of cuticle regions undergoing degradation during preecdysis of the Atlantic shore crab Carcinus maenas have been interpreted either as infectious organisms or as secretion structures associated with degradation of the old cuticle. Ultrastructural, cytochemical, and immunocytological investigations were performed to test these hypotheses and to see whether VLBs are peculiar to this crab species. Similar VLBs were systematically found in two other preecdysial crabs, Cancer pagurus and Macropipus puber. In Car, maenas, they originate during early premolt inside Golgi buddings and are often gathered into large vacuoles in epidermal cells. The histochemical azo-dye technique and a cerium-based cytochemical method revealed acid phosphatase activity in both the ecdysial space and the VLBs, while Feulgen's method and immunocytological labeling always failed to reveal any DNA or RNA in either the ecdysial space or the VLBs. We conclude that VLBs are not infectious organisms but "extracellular" cuticle-degrading organelles of lysosomal origin and propose to coin them "exolysosomes."

Acid Phosphatase↗

PubMed1997

Experimental induction of prenucleolar bodies (PNBs) in interphase cells: interphase PNBs show similar characteristics as those typically observed at telophase of mitosis in untreated cells.

Recently, it was shown that a short exposure of living mammalian cells to low ionic strength buffers (hypotonic shock) caused partial or almost complete unraveling of interphase nucleoli. However, when the cells were released from the hypotonic shock and transferred to normal isotonic medium, functionally active and structurally integral nucleoli were reassembled at their initial positions within interphase nuclei. Here, we show further that this process is accompanied by the appearance of numerous discrete extranucleolar bodies, which have striking similarities to the prenucleolar bodies (PNBs) observed in untreated cells at telophase of mitosis. (1) Like PNBs at mitosis, hypotonically induced interphase PNBs are composed of RNA-positive granules and fibrils, contain the major nucleolar protein B23 and silver-binding proteins, but lack DNA and RNA polymerase I transcription factor UBF. (2) As for mitotic PNBs, disappearance of the interphase PNB counterparts coincides with the increase in size of reconstructed nucleoli. (3) Addition of actinomycin D does not prevent assembly of interphase PNBs, but does arrest their coalescence with the chromosomal nucleolus-organizing regions and blocks the complete reformation of nucleoli. It is concluded that the assembly of PNBs generally observed at telophase of mitosis can be induced experimentally in nuclei of interphase mammalian cells in vivo. At interphase, this process is probably initiated by changes in the intracellular ionic environment.

Animals↗

PubMed1997

Electron tomography of metaphase nucleolar organizer regions: evidence for a twisted-loop organization.

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60-80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

Nucleic acid detection by in situ molecular immunogold labeling procedures.

We have recently combined immunogold labeling procedures with molecular biology methods to pinpoint the precise locations of nucleic acids in biological material at the ultrastructural level. These new immunocytological approaches involve the incorporation of labeled nucleotides in the nucleic acids present at the surface of ultrathin sections prior to immunogold labeling. The antibodies used recognize a nucleoside analogue (bromodeoxyuridine) or a hapten (biotin) employed to label nucleotides. Examples of high-resolution detection include DNA or RNA present in different substructures of cell nuclei, and in particular, in adenovirus-induced intranuclear regions of HeLa cells. In addition to being highly sensitive and specific, these new methods offer the possibility of studying the spatial distribution of nucleic acids in very well preserved, readily recognizable structures.

Cell Nucleus↗

PubMed1995

Nucleic acid compartmentalization within the cell nucleus by in situ transferase-immunogold techniques.

In the present review, we report on recent results obtained by in situ transferase-immunogold techniques as to the ultrastructural distribution of DNA and RNA within the cell nucleus. Special emphasis is placed on the various nucleolar components and the various enigmatic structures of the extranucleolar region: interchromatin granules, coiled bodies, and simple nuclear bodies. These data are discussed in the light of our current understanding of the functional organization of the cell nucleus.

Cell Compartmentation↗

PubMed1995

Extracellular matrix proteins and basement membrane: their identification in bovine ovaries and significance for the attachment of cultured preantral follicles.

Described in the present paper is the immunolocalization of the extracellular matrix proteins (e.g., fibronectin, collagen Types I and III) in the bovine ovary, with special attention to preantral follicles. In addition, we have shown, histochemically and ultrastructurally, that mechanically isolated bovine preantral follicles are surrounded by an intact basement membrane. After 24 h of culture in serum-free medium, only 20.4% of these follicles attached to a plastic substrate. We showed that covering the plastic with extracellular matrix proteins (i.e., fibronectin, collagen Type I and matrigel) significantly increased the percentage of attached follicles to 76.0, 65.2 and 80.4%, respectively, while laminin had no effect (18.6%). When preantral follicles were embedded within three-dimensional collagen gels, no loss of follicles was observed. Restoring surface interactions between preantral follicles and the extracellular matrix in vitro, either in a two- or a three-dimensional system, might be important for maintaining follicular viability and growth in the future.

Journal Article↗

PubMed1995

Robert Feulgen Prize Lecture 1995. New approaches to in situ detection of nucleic acids.

The present paper reviews recent results obtained by different molecular biology-based, immunocytological approaches to the localization and identification of nucleic acids in sections of biological material. Examples of sensitive, high-resolution detection methods for RNA, DNA or specialized DNA regions are presented. Special emphasis is placed on the potential values and limitations of these new methods.

Animals↗

PubMed1995

Effect of high osmolarity acclimation on tolerance to hyperosmotic shocks in L929 cultured cells.

Application of abrupt, large hyperosmotic shocks induces in L929 cultured cells changes similar to those previously described in other cell types, notably a hypercondensation of the nuclear chromatin. This paper shows that; 1) this phenomenon is concomitant with a complete disappearance of deoxyribonucleic acid, as visualized by immunogold labelling, from the nucleoplasmic spaces; 2) acclimation to high osmolarities (600 mOsm) by addition to the culture medium of NaCl, sorbitol or proline protects the cells from these effects, which appear to be largely attenuated-acclimated cells also survive much better to the osmotic shock than do control cells and; 3) the best protection seems to be provided by sorbitol and NaCl. Proline acclimation is less effective. These effects are discussed in terms of increased tolerance to NaCl load induced at the level of different macromolecules by so-called 'compensatory' organic compounds.

Animals↗

PubMed1995

Differential distribution of single-stranded DNA, double-stranded DNA, and RNA in adenovirus-induced intranuclear regions of HeLa cells.

We investigated in great detail the fine spatial distribution of nucleic acids within adenovirus-infected HeLa cells by various immunogold labeling procedures. To detect DNA, we used the in situ terminal deoxynucleotidyl transferase-immunogold technique. In addition to the expected evident label over the condensed host chromatin and the structures containing viral double- and single-stranded DNA, label was consistently revealed over round fibrillar spots. By contrast, other virus-induced substructures, such as compact rings, crystalloids, clear amorphous inclusions, and electron-dense amorphous inclusions, displayed no significant label. Except for the viral single-stranded DNA accumulation sites, identical labeling pattern was obtained with the in situ nick-translation-immunogold method. We further labeled the sections with anti-RNA antibodies. Label was present not only over the cytoplasm and the intranuclear fibrillogranular network but also quite obviously over the compact rings and interchromatin granule clusters. None was seen over the other nuclear structures of infected cells, notably over the fibrillar spots. We suggest that these fibrillar spots might be involved in the formation of the viral, non-encapsidated, double-stranded DNA storage site.

Adenoviridae↗

PubMed1995

Behavior of interchromatin granules during the cell cycle.

We investigated at the ultrastructural level, by different cytochemical and immunocytological approaches, the behavior of interchromatin granules (IGs) during interphase and mitosis in two cell lines (HEp-2 and Ehrlich tumor cells). Identical results were found in all two cell types. In interphase cells, IGs group into irregular clusters of varying size. They are frequently associated with coiled bodies and homogeneous fibrillar bodies. Analysis of serial sections reveals that IG clusters occupy distinct regions within the nucleoplasm. During prophase, the aggregation of granules in these clusters gives rise to compact, spherical, granular structures. These disperse in the mitotic cytoplasm at the breakdown of the nuclear envelope. At early telophase, some of them come into close contact with the periphery of reforming nuclei. IG clusters reappear in the daughter nuclei only after the chromosomes have decondensed during late telophase. Concomitantly, the cytoplasmic granular structures disappear. During the cell cycle, IG are silver-stainable and EDTA-positive. They are also constantly labeled by the polyadenylate nucleotidyl transferase-immunogold technique for detecting RNA. These results support the view that IGs persist throughout the whole cell cycle.

Acetylation↗

PubMed1995

The interchromatin granules.

In the present review, we report on the data obtained on the most prominent elements observed in the interchromatin spaces: the interchromatin granules (IGs). Special emphasis is placed on the recent contributions of immunocytochemical and in situ hybridization methods towards elucidating the composition and the behavior of these nuclear substructures during the cell cycle. The possible roles of IGs in the cell nucleus are also discussed.

Animals↗

PubMed1995

Eighteen years of vaccination against viral haemorrhagic septicaemia in France.

Viral haemorrhagic septicaemia (VHS) has been considered for many years to be a major cause of loss in the French trout industry. The high prevalence of VHS in certain geographic areas made a control strategy based on control policy unfeasible. This provided the impetus for immunoprophylaxis development that resulted in 3 successive types of vaccines: inactivated, live attenuated and recombinant vaccines. When delivered by intraperitoneal injection, the 2 propiolactone-inactivated VHS virus was immunogenic and/or protective for trout all of sizes, but it was not suitable for the practical immunization of alevin, the trout life stage that is the most sensitive to VHS. A carp cell-passed, attenuated variant of the VHS virus was effective after both immersion or injection delivery and met the practical requirements of juvenile vaccination. However, this vaccine was discarded because it retained some virulence that discouraged the launching of its commercialization. Then came the era of genetically engineered vaccines. The recombinant glycoprotein of VHSV produced in Escherichia coli or in Saccharomyces cerevisiae failed to protect fish whatever the route of delivery. A recombinant baculovirus vaccine was found to be immunogenic and protective against VHS, but only when delivered by injection. Due to its cost and route of delivery, the latter vaccine was not licensed. Simultaneously, the sudden occurrence of another rhabdovirosis, infectious haematopoietic necrosis (IHN), in France, rendered vaccination against VHS questionable. Indeed, no cross-protection between these 2 rhabdoviroses exists. If vaccination is still believed to be an effective control method for VHS, it should be based in the future upon an autoreplicative vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Cytochemical and immunocytochemical study of coiled bodies in different cultured cell lines.

We analyzed by different cytochemical and immunocytochemical approaches the biochemical compositon of coiled bodies in three different cultured cell lines. Coiled bodies are stained by the AgNOR staining method and by the EDTA regressive staining method preferential for ribonucleoprotein (RNP). Using the in situ polyadenylate nucleotidyl transferase-immunogold technique or anti-RNA antibodies, we decisively demonstrated the presence of appreciable amounts of RNA in coiled bodies. Neither the in situ terminal deoxynucleotidyl transferase-immunogold technique nor anti-DNA antibodies revealed any DNA in coiled bodies. Coiled bodies thus appear as distinct regions of cell nuclei involved in some steps of RNA metabolism but not directly in RNA synthesis. Their relationships with the dense fibrillar component of the nucleolus and with interchromatin granule clusters are discussed.

Animals↗