Biomedical subjects

E Puvion

Publications and source records attributed to E Puvion.

At least 19 recordsLinked to original sources

PubMed2001

Role of promyelocytic leukemia (PML) sumolation in nuclear body formation, 11S proteasome recruitment, and As2O3-induced PML or PML/retinoic acid receptor alpha degradation.

Promyelocytic leukemia (PML) is the organizer of nuclear matrix domains, PML nuclear bodies (NBs), with a proposed role in apoptosis control. In acute promyelocytic leukemia, PML/retinoic acid receptor (RAR) alpha expression disrupts NBs, but therapies such as retinoic acid or arsenic trioxide (As2O3) restore them. PML is conjugated by the ubiquitin-related peptide SUMO-1, a process enhanced by As2O3 and proposed to target PML to the nuclear matrix. We demonstrate that As2O3 triggers the proteasome-dependent degradation of PML and PML/RARalpha and that this process requires a specific sumolation site in PML, K160. PML sumolation is dispensable for its As2O3-induced matrix targeting and formation of primary nuclear aggregates, but is required for the formation of secondary shell-like NBs. Interestingly, only these mature NBs harbor 11S proteasome components, which are further recruited upon As2O3 exposure. Proteasome recruitment by sumolated PML only likely accounts for the failure of PML-K160R to be degraded. Therefore, studying the basis of As2O3-induced PML/RARalpha degradation we show that PML sumolation directly or indirectly promotes its catabolism, suggesting that mature NBs could be sites of intranuclear proteolysis and opening new insights into NB alterations found in viral infections or transformation.

Adenosine Triphosphatases↗

PubMed2000

DNA replication progresses on the periphery of nuclear aggregates formed by the BCL6 transcription factor.

The BCL6 proto-oncogene, frequently alterated in non-Hodgkin lymphoma, encodes a POZ/zinc finger protein that localizes into discrete nuclear subdomains. Upon prolonged BCL6 overexpression in cells bearing an inducible BCL6 allele (UTA-L cells), these subdomains apparently coincide with sites of DNA synthesis. Here, we explore the relationship between BCL6 and replication by both electron and confocal laser scanning microscopy. First, by electron microscope analyses, we found that endogenous BCL6 is associated with replication foci. Moreover, we show that a relatively low expression level of BCL6 reached after a brief induction in UTA-L cells is sufficient to observe its targeting to mid, late, and at least certain early replication foci visualized by a pulse-labeling with bromodeoxyuridine (BrdU). In addition, when UTA-L cells are simultaneously induced for BCL6 expression and exposed to BrdU for a few hours just after the release from a block in mitosis, a nuclear diffuse BCL6 staining indicates cells in G(1), while cells in S show a more punctate nuclear BCL6 distribution associated with replication foci. Finally, ultrastructural analyses in UTA-L cells exposed to BrdU for various times reveal that replication progresses just around, but not within, BCL6 subdomains. Thus, nascent DNA is localized near, but not colocalized with, BCL6 subdomains, suggesting that they play an architectural role influencing positioning and/or assembly of replication foci. Together with its previously function as transcription repressor recruiting a histone deacetylase complex, BCL6 may therefore contribute to link nuclear organization, replication, and chromatin-mediated regulation.

Cell Line↗

PubMed1999

Deletion of the fiber gene induces the storage of hexon and penton base proteins in PML/Sp100-containing inclusions during adenovirus infection.

The present study has documented changes in the in situ distribution of viral DNA and capsid proteins in 293 cells infected with fiber gene-deleted adenoviruses. It shows that infection results in the intense production of progeny viruses which appear morphologically intact although they are devoid of fiber-coding sequence in their genome and hence of fiber protein in their capsid. The data confirm, therefore, that fiber protein is not essential for the assembly of progeny viruses. The main contribution of our observations concerns specific intranuclear structures induced by infection with either wild-type or fiber gene-deleted viruses. These clear amorphous inclusions contain two cellular proteins, PML and Sp100, which in non-infected cells co-localize to a special type of nuclear bodies. PML and Sp100 nuclear bodies appear to directly modulate or to be altered in a wide variety of situations including viral infections, cell death and transformation. In cells infected with fiber gene-deleted viruses, the clear amorphous inclusions now accumulate non-used hexon and penton base proteins, whereas the absence of fiber protein prevents the assembly of capsid proteins in crystallin arrays. Taken together, the data suggest that the clear amorphous inclusions may correspond to storage sites of structural and regulatory proteins. Consequently, these virus-induced structures may promote the productive cycle of adenoviruses by regulating the amount of over-produced viral proteins and the shutoff of the host cell metabolism.

Adenoviridae↗

PubMed1998

Vav binding to heterogeneous nuclear ribonucleoprotein (hnRNP) C. Evidence for Vav-hnRNP interactions in an RNA-dependent manner.

The vav proto-oncogene is exclusively expressed in hematopoietic cells and encodes a 95-kDa protein that contains multiple structural domains. Vav is involved in the expansion of T and B cells, in antigen-mediated proliferative responses, and in the induction of intrathymic T cell maturation. It becomes rapidly and transiently tyrosine-phosphorylated upon triggering of a large number of surface receptors and catalyzes GDP/GTP exchange on Rac-1. We now provide evidence for the specific interaction of Vav with heterogeneous nuclear ribonucleoprotein (hnRNP) C. Vav and hnRNP C interact both in vivo and in vitro mediated through the carboxyl Src homology 3 domain of Vav and the proline-rich motif located in the nuclear retention sequence of hnRNP C. More importantly, Vav-hnRNP C complexes are present in living hematopoietic cells and both proteins localize in the nuclei, mainly on perichromatic fibrils but also on clusters of interchromatin granules. The Vav-hnRNP C interaction is regulated by poly(U) RNA, although a basal association is still detected in the absence of RNA. Furthermore, RNA homopolymers differentially alter the binding affinity of Vav to hnRNP C and hnRNP K. We propose that Vav-hnRNP interactions may be established in an RNA-dependent manner.

Cell Line↗

PubMed1998

Pilot ultrastructural evaluation of human preauricular skin before and after high-energy pulsed carbon dioxide laser treatment.

BACKGROUND: Carbon dioxide laser resurfacing has recently come into favor for the treatment of photodamaged skin. While the clinical and histologic effects of high-energy short-pulse carbon dioxide lasers on human skin have been investigated, the ultrastructural effects of these lasers have not been documented. Our objective was to study the ultrastructural effects of a high-energy pulsed carbon dioxide laser on photodamaged human skin. OBSERVATIONS: Before laser surgery, the ultrastructural changes characteristic of photodamaged skin were evident. Immediately after treatment, there was extensive coagulation necrosis of the epidermis and papillary dermis. Thirty days after treatment, there was no evidence of intercellular or intracellular edema, and ordered differentiation of the epidermal keratinocytes, with a loss of keratinocyte dysplasia, was seen. Increased numbers of desmosomes and tonofibrils were noted. New deposition of collagen was present in the papillary dermis. The ultrastructural findings seen at 90 days after treatment were similar to those seen at 30 days, apart from increased organization of collagen fibers in the papillary dermis. CONCLUSIONS: Treatment with the high-energy pulsed carbon dioxide laser appears to reverse the epidermal and dermal changes of photoaging on an ultrastructural level. These changes appear morphologically to be consistent with previously described clinical and histologic changes following laser resurfacing.

Aged↗

PubMed1998

Fluorescent and radiolabeling of polysaccharides: binding and internalization experiments on vascular cells.

Glycosaminoglycans (GAGs) such as heparan sulfates are complex carbohydrate polymers. These structural components of the extracellular matrix are essential for the adhesion, migration, and regulation of cellular growth. To understand the physiological role of GAGs and GAG analogues, a practical approach consists of labeling and detecting them in cell extracts, or analyzing binding domains and their distributions into the cells. We propose a convenient and reliable method for preparing and labeling amino-enriched, polysaccharides with the fluorescent derivative 5-[(4,6-dichlorotriazine-2-yl)amino]-fluorescein (DTAF). Radioiodination is then performed on the DTAF moiety. This method was applied to polysaccharides known to inhibit vascular smooth-muscle cell (SMC) proliferation such as functionalized dextrans derived from poly(alpha 1-6 glucose) and fucan, poly(L-fucose 4-sulfate) extracted from brown seaweed. Using autoradiography and confocal microscopy, we observed the fixation and internalization of labeled antiproliferative products in SMCs from rat aorta. These probes can be useful for the understanding of polysaccharide-cell interactions. In addition, the method presented here can be applied to various synthetic or natural biomedical materials.

Amino Acids↗

PubMed1997

The ultrastructural localization of 60-kDa Ro protein and human cytoplasmic RNAs: association with novel electron-dense bodies.

The 60-kDa Ro ribonucleoprotein is an important target of humoral autoimmune responses. However, the ultrastructural locations of the 60-kDa Ro protein and its associated small cytoplasmic RNAs (Y RNAs) have not been previously determined, and the functions of the Ro protein and RNAs are not known. In this study, the cellular locations of the 60-kDa Ro protein and the Ro Y1 and Y4 RNAs are determined by immunoelectron microscopy and in situ hybridization electron microscopy, respectively. Both Ro protein and Y RNAs are concentrated in discrete areas of the nucleoplasm, nucleolus, and cytoplasm of cultured cells and human skin sections. The 60-kDa Ro protein and Y RNAs are also present diffusely in the cytoplasm, where they occur in ribosome-rich regions, and in the nucleus. The presence of Ro ribonucleoprotein components in nucleoli and in ribosome-rich cytoplasmic areas suggests a potential for the involvement of Y RNAs and/or 60-kDa Ro protein in ribosome synthesis, assembly, or transport. Double labeling experiments show that Ro protein and Y RNAs colocalize in the nucleoplasm, nucleolus, and cytoplasm. In addition, aggregates of Y RNA occur unassociated with 60-kDa Ro protein, and aggregates of 60-kDa Ro protein occur unassociated with Y RNA. Aggregates of both Ro protein and Y RNAs label previously unreported nuclear and cytoplasmic electron-dense bodies. We propose that these distinctive Ro-associated electron-dense bodies may represent structure(s) important for cellular transport and/or Ro function.

Cell Line↗

PubMed1997

Early stages of pre-rRNA formation within the nucleolar ultrastructure of mouse cells studied by in situ hybridization with a 5'ETS leader probe.

The first cleavage in the processing of the rRNA primary transcript in mammals occurs within the 5'-terminal region of the 5' external transcribed spacer (5'ETS), which makes the upstream portion of this spacer a selective marker of nascent transcripts. Moreover, short treatments with low doses of actinomycin D (AMD), which selectively suppress pre-rRNA synthesis and allow processing of preformed pre-rRNAs, result in the production of prematurely terminated transcripts essentially spanning the 5'ETS leader region. To gain further insight into the intranucleolar localization of early stages of preribosome formation we analyzed the distribution of this specific pre-rRNA segment by in situ hybridization at the ultrastructural level in AMD-treated or in control 3T3 mouse cells. In control cells, 5'ETS leader rRNA was detected at the border of the fibrillar centers and over the dense fibrillar component, in agreement with previous data suggesting that rRNA gene transcription takes place at the border of the fibrillar centers before a rapid transfer of the nascent trancript to the dense fibrillar component. Observation of cells subjected to a short treatment with low doses of AMD fully supports this conclusion, with the prematurely terminated 5'ETS leader-containing transcripts detected at the border of enlarged fibrillar centers. With prolonged periods of AMD treatment even the partial transcription of rRNA genes is blocked and fibrillar centers of typically segregated nucleoli show no positive signals with the 5'ETS leader probe. We also analyzed in parallel the intranucleolar distribution of U3 small nucleolar RNA, which is involved in 5'ETS processing, by hybridization with biotinylated antisense oligonucleotides. Distribution of U3 roughly paralleled that of 5'ETS leader rRNA in untreated cells. However, U3 RNA persisted in the dense fibrillar component of segregated nucleoli whatever the conditions of drug treatment, i.e., even after a thorough chase of the rRNA precursors from this nucleolar compartment.

3T3 Cells↗

PubMed1997

Human and mouse MOK2 proteins are associated with nuclear ribonucleoprotein components and bind specifically to RNA and DNA through their zinc finger domains.

The human and murine MOK2 ortholog genes that are preferentially expressed in brain and testis tissues encode two different Krüppel-like zinc finger proteins. In this paper, we show that the MOK2 proteins are mainly associated with nuclear ribonucleoprotein components, including the nucleoli and extranucleolar structures, and exhibit specific RNA homopolymer binding activities. Moreover, we have identified an identical 18-bp specific DNA binding sequence for both MOK2 proteins using a pool of random sequence oligonucleotides. The DNA binding domain is localized in the seven adjacent zinc finger motifs, which show 94% identity between human and murine proteins. Taken together, these results establish that the MOK2 proteins are able to recognize both DNA and RNA through their zinc fingers. This dual affinity and the subnuclear localization suggest that MOK2 may play roles in transcription, as well as in the posttranscriptional regulation processes of specific genes.

Animals↗

PubMed1997

Identification of transcription factories in nuclei of HeLa cells transiently expressing the Us11 gene of herpes simplex virus type 1.

Nuclear distribution and migration of herpes simplex virus type 1 Us11 transcripts were studied in transient expression at the ultrastructural level and compared to that of RNA polymerase II protein. Transcription was monitored by autoradiography following a short pulse with tritiated uridine. Us11 transcripts accumulated mainly over the foci of intermingled RNP fibrils as demonstrated by the presence of silver grains localizing incorporated radioactive uridine superimposed to these structures in which the presence of Us11 RNA and poly(A) tails was previously demonstrated. Silver grains were also scattered over the remaining nucleoplasm but not in the clusters of interchromatin granules, and over the dense fibrillar component of the nucleolus as in control, nontransfected HeLa cells. Pulse-chase experiments revealed the transient presence of migrating RNA in the clusters of interchromatin granules. RNA polymerase II was revealed by immunogold labeling following the use of two monoclonal antibodies: mAb H5, which recognizes the hyperphosphorylated form of the carboxy-terminal domain (CTD) of the molecule, and mAb 7C2, which recognizes both its hyperphosphorylated and unphosphorylated forms. The two mAbs bind to the newly formed Us11 transcription factories and the clusters of interchromatin granules of transfected cells. In control cells, however, clusters of interchromatin granules were labeled with mAb H5 but not with mAB 7C2. Taken together, our data demonstrate the involvement of the clusters of interchromatin granules in the intranuclear migration of Us11 RNA in transient expression. They also suggest the occurrence of changes in the accessibility of the RNA polymerase II CTD upon expression of the Us11 gene after transfection by exposing some epitopes, otherwise masked in nontransfected cells.

Cell Nucleus↗

PubMed1996

Ultrastructure of the nucleus in relation to transcription and splicing: roles of perichromatin fibrils and interchromatin granules.

In the present article we summarize our results concerning the in situ ultrastructural organization of the nuclear steps of mRNA production using several cellular models including normal noninfected cells, herpes simplex virus type 1 and adenovirus type 5 infected cells, and transiently transfected cells. We confirm that perichromatin fibrils are the in situ morphological expression of nascent transcripts and the main support of splicing. Special emphasis is devoted to the clusters of interchromatin granules which seem to be involved in several central functions including accumulations of snRNP components as well as sorting and/or regulation of export of different RNA species.

Animals↗

PubMed1996

Non-isotopic electron microscope in situ hybridization for studying the functional sub-compartmentalization of the cell nucleus.

Post-embedding electron microscope in situ hybridization using gold particles as label permits the clear identification of the cellular structures which contain the nucleic acid molecules under study. It has yielded information on the distribution of defined nucleic acid sequences of different origins-cellular or viral, DNA or RNA, single- or double-stranded molecules-which has revolutionized the study of the nucleus. Application of this powerful technique in combination with other refined techniques to studies on transcription and replication of cellular and viral genes has augmented our knowledge of the functional organization of the cell nucleus. One can now ask mechanistically meaningful questions concerning the successive steps of gene replication and expression not only under normal conditions of cell growth, but also when the cellular metabolism is altered by a drug treatment or a viral infection. This chapter aims (a) to present the established methods of post-embedding electron microscope in situ hybridization for localizing, precisely and specifically, a nucleic acid target in its normal environment and (b) to present some contributions of this technique to investigations of the functional compartmentalization of the cell nucleus and to elucidate the cell-virus relationships in infected cells.

Animals↗

PubMed1996

Intracellular distribution of ampicillin in murine macrophages infected with Salmonella typhimurium and treated with (3H)ampicillin-loaded nanoparticles.

The intracellular distribution of (3H)ampicillin-loaded polyisohexylcyanoacrylate nanoparticles was studied in murine macrophages (peritoneal cells and the J774 cell line) infected by Salmonella typhimurium C5, using ultrastructural autoradiography. Ampicillin penetration and retention into the cells obviously increased by means of nanoparticles. After short-term (2-4 h) treatment with the nanoparticle formulation, numerous intracellular bacteria were seen to be in the process of destruction. The tritium labelling was located in the cell cytoplasm and inside vacuoles in which bacteria undergoing degradation were often present. After long-term (12 h) treatment, numerous spherical bodies (d: 100 nm to 500 nm) and larger forms (2 microns) were seen in the vacuoles. Radioactivity was mainly found to be localized on the spherical bodies, indicating marked damaging action of the ampicillin on the bacterial walls. The targeting of ampicillin therefore allowed its penetration into the macrophages and vacuoles infected with S. typhimurium.

Ampicillin↗

PubMed1996

The osmium ammine-SO2 staining method for studying the in situ configuration of viral genomes in ultrathin sections of DNA virus infected cells.

The Feulgen-like osmium ammine-SO2 method developed by Cogliati and Gautier (CR Acad Sci Ser D 1973, 276, 3041-3044) stains the DNA at the ultrastructural level. Compared to several other techniques for detecting DNA, this method remains the only one revealing the configuration of the DNA molecules within the cell whatever their compactness. In the present article we summarize the results we obtained with the osmium ammine method in the study of the fate of viral genomes along the infectious cycles in several DNA virus infected cells including adenovirus, herpes simplex virus, simian virus 40 and poxvirus. The results are discussed in relation to the replicative and transcribing activities of viral DNA.

Adenoviridae Infections↗

PubMed1995

Immunocytochemistry, autoradiography, in situ hybridization, selective stains: complementary tools for ultrastructural study of structure-function relationships in the nucleus. Applications to adenovirus-infected cells.

A significant amount of new information on structure-function relationships in nuclei of adenovirus-infected cells has accumulated during the last decade as a result of the combined use of several new cytochemical techniques. Localization of viral DNA on ultrathin sections of infected cells has been investigated at the ultrastructural level by using specific DNA staining and immunocytochemistry with monoclonal anti-DNA antibodies. Both techniques, however, concomitantly visualize cellular and viral DNA. The specific stain for DNA reveals the configuration of the DNA molecules in the different nuclear substructures, whatever their synthetic activities. The immunodetection of DNA reveals that specific antibodies strongly bind to DNA of condensed host chromatin and to both encapsidated and nonencapsidated inactive viral genomes. However, the observation of an abnormally low level of labeling over the substructures in which synthetic activities of viral genomes are known to be intense demonstrates a serious limitation of this technique for the detection of active DNA. Postembedding in situ hybridization is the most useful method for identifying with certainty the structures containing defined nucleic acid sequences. By using a biotinylated viral DNA probe, in situ hybridization provides specific identification of structures containing either viral DNA or viral RNA molecules. In addition, with appropriate pretreatment of the sections, it is possible to reveal either all the viral DNA--that is, both double- and single-stranded DNA molecules (dsDNA, ssDNA)--or more specific species such as only ssDNA or only dsDNA molecules. The replicative and transcriptional activities of viral genomes are determined by high-resolution autoradiography. Autoradiography after a short pulse incorporation of appropriate radioactive precursors by infected cells reveals the sites of cellular and viral DNA replication or transcription. A short pulse followed by chase periods of different durations reveals the progressive migration of the cellular and viral synthesized products. The in situ distribution of the viral 72 kDa DNA-binding protein, a highly phosphorylated protein which protects the viral ssDNA, is revealed either by immunocytochemistry with specific antibodies or by the bismuth staining method which stains all highly phosphorylated proteins, including both cellular and viral proteins. The combined results of all these cytochemical procedures reveal the composition and functions of some of the structures induced by adenovirus infection. They demonstrate that viral genomes engaged in replication lead to the formation of the replicative foci in which two compartments rapidly develop, one of which results from the aggregation of single strands of viral DNA and their accompanying 72 kDa protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviruses, Human↗

PubMed1995

Adenovirus infection induces rearrangements in the intranuclear distribution of the nuclear body-associated PML protein.

We have studied at the ultrastructural level the localization of PML protein in response to adenovirus infection in HeLa cells. In nuclei of noninfected cells or at the early stage of infection, PML accumulated at the border of nuclear bodies as previously described [Koken et al. (1994) EMBO J. 13, 1073-1083]. Interestingly, we demonstrate herein that PML is also a component of the interchromatin granule-associated zones, recently described structures containing U1 snRNP [Visa et al. (1993) Eur. J. Cell Biol. 60, 308-321], suggesting that PML protein might be involved in some steps of splicing events. However, as the infection progressed these two cellular PML-containing structures disappeared but significant amounts of PML accumulated within two virus-induced structures, essentially the clear amorphous inclusions, but also the protein crystals. Relocalization of this protein to virus-induced structures may reflect an inactivation of PML functions.

Adenoviruses, Human↗

PubMed1995

Sequestration of PML and Sp100 proteins in an intranuclear viral structure during herpes simplex virus type 1 infection.

We investigated the intranuclear distribution of PML and Sp100 in HeLa cells at the ultrastructural level and examined their relocalization in response to herpes simplex virus type 1 (HSV-1) infection. In the absence of infection, we observed that both are components, not only of nuclear bodies, but also of interchromatin granule-associated zones, which suggests a potential role for PML and Sp100 in splicing events. Prolonged HSV-1 infection induced dramatic changes in nuclear organization which consisted of the morphological disappearance of some nuclear structures (nuclear bodies, interchromatin granule-associated zones, coiled bodies) and of the development of a centrally located electron-translucent viral region which pushed the cellular clusters of interchromatin granules to the nuclear border. Concomitantly, dense bodies, concentric arrays of reduplicated inner nuclear membrane, and translucent patches containing a few viral capsids occurred at the nuclear border. PML and Sp100 were exclusively detected over the finely granular material of the viral translucent patches which also contains small amounts of p80-coilin and U1 and U2 snRNAs. An antiserum raised against capsid proteins intensely labeled the viral translucent patches at the level of their finely granular material and enclosed viral capsids. Our data, therefore, suggest that these viral structures, in addition to being the site of accumulation of viral capsid proteins and, possibly, a capsidworks, are also a site of sequestration of cell factors including PML and Sp100. Viral capsid proteins could interfere with and inactivate PML and Sp100 and be implicated in the shutoff of host cell metabolism induced by HSV-1 infection.

Antibodies, Viral↗

PubMed1995

p80-coilin: a component of coiled bodies and interchromatin granule-associated zones.

We investigated at the electron microscope level the fate of the three intranuclear structures known to accumulate snRNPs, and which correspond to the punctuate immunofluorescent staining pattern (the coiled bodies, the clusters of interchromatin granules and the interchromatin granule-associated zones) after exposure to either a low salt medium which induces a loosening and partial spreading of nucleoprotein fibers or a high ionic strength salt medium and subsequent DNase I digestion, in order to obtain DNA-depleted nuclear matrices. The loosened clusters of interchromatin granules and the coiled bodies could no longer be distinguished from surrounding nucleoprotein fibers solely by their structure, but constituents of the clusters of interchromatin granules could be detected by in situ hybridization with both U1 and U2 DNA probes, and constituents of the coiled bodies were detectable mainly with the U2 DNA probe. The interchromatin granule-associated zones, the electron-opacity and compactness of which were preserved despite the loosening treatment, remained labeled with the U1 DNA probe only. In DNA-depleted nuclear matrices, the snRNA content of the coiled bodies, the clusters of interchromatin granules and their associated zones, which were all easily recognizable within the residual nuclear ribonucleoprotein network, was unmodified. The data indicate, therefore, that the loosening procedure as well as the high salt extraction procedure preserve the snRNA content of all three spliceosome component-accumulation sites and reveal that interchromatin granule-associated zones are elements of the nuclear matrix. The p80-coilin content coiled bodies was also preserved whatever the salt treatment used. An intriguing new finding is the detection of abundant p80-coilin within the interchromatin granule-associated zones, both before and after either low or high salt treatment of cells. Therefore, p80-coilin is an integral constituent of the interchromatin granule-associated zones.

Cell Nucleus↗