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.

Ribonucleoprotein components in liver cell nuclei as visualized by cryoultramicrotomy.

The interphase nucleus of the normal rat hepatocyte has been studied in ultrathin frozen sections after glutaraldehyde fixation and the modification of various staining procedures known to be specific for DNA structures (Moyne's thallium stain, Gautier's osmium-ammine) or preferential for RNP carriers and basic proteins (regressive stains based on the use of EDTA or citrate, negatively charged colloidal iron). The results are comparable to those obtained after classical dehydration and embedding. Particular attention has been paid to the nucleolus and extranucleolar RNP components, such as perichromatin fibrils and granules, as well as interchromatin granules. A striking observation was the uneven size and the strongly increased number of perichromatin granules, and the appearance of a contiguous interchromatin net, containing nucleoproteins. Cryoultramicrotomy without embedding appears to be very useful for the exploration of the nucleus in thick sections which remain sufficiently transparent even with the usual accelerating voltages.

Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix.

Biochemical fractionation was combined with high resolution electron microscopic autoradiography to study the localization in rat liver nuclear matrix of attached DNA fragments, in vivo replicated DNA, and in vitro synthesized DNA. In particular, we determined the distribution of these DNA components with the peripheral nuclear lamina versus more internally localized structural elements of isolated nuclear matrix. Autoradiography demonstrated that the bulk of in vivo newly replicated DNA associated with the nuclear matrix (71%) was found within internal matrix regions. A similar interior localization was observed in isolated nuclei and in situ in whole liver tissue. Likewise, isolated nuclear lamina contained only a small amount (12%) of the total matrix-bound, newly replicated DNA. The structural localization of matrix-bound DNA fragments was examined following long-term in vivo labeling of the DNA. The radioactive DNA fragments were found predominantly within interior regions of the matrix structure (77%), and isolated nuclear lamina contained less than 15% of the total nuclear matrix-associated DNA. Most of the endogenous DNA template sites for the replicative enzyme DNA polymerase alpha (approximately 70%) were also sequestered within interior regions of the matrix. In contrast, a majority of the endogenous DNA template sites for DNA polymerase beta (a presumptive repair enzyme) were closely associated with the peripheral nuclear lamina. A similar spatial distribution for both polymerase activities was measured in isolated nuclei before matrix fractionation. Furthermore, isolated nuclear lamina contained only a small proportion of total matrix-bound DNA polymerase alpha endogenous and exogenous template activities (3-12%), but a considerable amount of the corresponding beta polymerase activities (47-52%). Our results support the hypothesis that DNA loops are both anchored and replicated at nuclear matrix-bound sites that are predominantly but not exclusively associated with interior components of the matrix structure. Our results also suggest that the sites of nuclear DNA polymerase beta-driven DNA synthesis are uniquely sequestered within the characteristic peripheral heterochromatin shell and associated nuclear envelope structure, where they may potentially participate in DNA repair and/or replicative functions.

Rapid effect of heat shock on two heterogeneous nuclear ribonucleoprotein-associated antigens in HeLa cells.

During severe heat shock, which known to interrupt both splicing of RNA transcripts and nucleocytoplasmic transport, it is to be expected that the substructure of heterogeneous nuclear ribonucleoproteins (hnRNP) is altered in some way. Recently, we have shown that such a stress actually induces rapid alterations at the level of individual proteins (Lutz, Y., M. Jacob, and J.-P. Fuchs. 1988 Exp. Cell Res. 175:109-124). Here we report further investigations on two related 72.5-74-kD hnRNP proteins whose behavior is also rapidly modified by a heat shock at 45 degrees C, whereas no effect is observed at 42 degrees C. Using a monoclonal antibody, we show that in situ the antigens are available only when the cells are heat shocked at 45 degrees C. Subcellular fractionation shows that in normal cells the antigens are associated with the bulk of hnRNP (50-200S). During heat shock, whereas the overall characteristics of the bulk of preexisting hnRNP are unchanged, these antigens rapidly switch to a subpopulation of hnRNP with larger average size (50 to less than 300S) and increased stability. Structural analysis of the associated hnRNP in normal and stressed cells shows that in both cases the antigens are associated with the nuclear matrix subcomplex of hnRNP, which in situ is part of the internal nuclear matrix. Such hnRNP antigens, which are rapidly redistributed during a heat shock at the upper temperature range of the stress response, might well be involved in splicing and/or transport control.

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.

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.

Effect of quinacrine on nuclear structure and RNA synthesis in cultured rat hepatocytes.

The effects of quinacrine, an antimetabolite which intercalates into DNA, on the ultrastructure of interphase nuclei and on RNA turnover were studied in primary cultures of rat hepatocytes. Procedures included ultrastructural cytochemical staining for ribonucleoprotein and DNA, autoradiography, and measurement of labeled uridine uptake and incorporation. Addition to the culture medium of a nontoxic dose (10 microM for 30 min) reduces the net accumulation of labeled uridine in RNA. This involves first heterogeneous RNA and then ribosomal RNA since their structural precursors, interchromatin fibrils and nucleolar fibrils, respectively, diminish in that order. Intranucleolar chromatin retracts, and perinucleolar chromatin becomes unusually condensed. A toxic dose (50 microM for 30 min) produces greater inhibition of tritiated uridine incorporation in RNA. This precedes and is not due to a drop in uridine uptake into the cells. Toxic doses produce unusually large clusters of interchromatin granules which are embedded in an unusual dense material which stains positively for ribonucleoprotein. Three regions of the chromatin are altered. (a) Perinuclear condensed chromatin retracts from the nuclear envelope, remaining attached by short DNA-containing bridges. (b) The normally dispersed nucleoplasmic chromatin condenses into a stainable network which retracts centrifugally. (c) Perinucleolar chromatin becomes a network of small highly condensed masses or bands interconnected by fibrils which are either decondensed or stretched. These alterations in chromatin structure probably form the basis of quinacrine-impaired nuclear metabolism.

Nuclear localization of the Epstein-Barr virus/C3d receptor (CR2) in the human Burkitt B lymphoma cell, Raji.

Epstein-Barr virus/C3d receptor (CR2) is a glycoprotein of mol. wt 140,000 expressed on the surface of Raji cells. We previously isolated phosphorylated CR2 from purified Raji cell nuclei. We have analyzed the nuclear localization of CR2 by electron microscope immunochemistry of thin sections of Raji cells and we have compared the binding properties of CR2 expressed on purified plasma membranes or nuclei. Anti-CR2 mAb immunogold labeling of thin sections of Raji cells identified CR2 at the nuclear surface and also within the nucleus. Nuclear envelope associated CR2 was localized mainly at nuclear pores. Within the nucleus, CR2 was associated with ribonucleoprotein (RNP) interchromatin fibrils. This labeling was preserved in nuclear matrix preparations. CR2 expressed on the surfaces of purified nuclei or on the cell surface interacted with soluble and particle-bound C3bi/C3d. Monoclonal anti-CR2 antibodies, which recognized extracellular domains of CR2, reacted differently with CR2 depending on its subcellular localization. The presence of CR2 in nuclei may be due to translocation of the cell surface CR2 and/or the presence of two distinct intracellular pathways for mature CR2.

Ultrastructural localization of viral DNA in thin sections of herpes simplex virus type 1 infected cells by in situ hybridization.

We have used in situ hybridization at the ultrastructural level to localize non-encapsidated and encapsidated herpes simplex virus type 1 (HSV-1) genomes in nuclei of infected rabbit fibroblasts. A biotinylated cloned subgenomic HSV DNA fragment was used as hybridization probe. The probe hybridized to the viral DNA accessible at the surface of Lowicryl sections was revealed by immunogold labeling. Non-encapsidated viral DNA was detected exclusively within the virus-induced central region of 4 h to 17 h infected nuclei. Localization of the probe either near the nuclear envelope or within marginated host chromatin was found only on HSV DNA which was packaged into viral nucleoids. The use in parallel of in situ hybridization with specific staining for DNA and autoradiography after tritiated thymidine incorporation, followed by either conventional fixation of the cells or the nucleoprotein loosening procedure, indicated that non-encapsidated viral DNA and marginated host chromatin formed two juxtaposed compartments without interpenetration even after experimentally produced mild dispersion of the nuclear components.

Structural organization of rat hepatocyte chromatin as visualized in thin frozen sections selectively stained for DNA.

We studied the structure of rat hepatocyte chromatin in situ using thin frozen sections selectively stained for DNA after aldehyde fixation. Our results indicate that intranucleolar chromatin is arranged into three different organization levels, confirming the observations on Epon-embedded chromatin. These are: completely extended DNA filaments, with a thickness of approximately 3 nm, clustered in loose, roundish agglomerates, very long fibers with a thickness ranging from 15 to 35 nm and compact chromatin clumps. Both the fibers and the chromatin clumps frequently appeared to be composed of nucleosome-like particles. In the extranucleolar chromatin, agglomerates of extended DNA filaments and long fibers were never visualized. In contrast to data from Epon-embedded chromatin, we noticed that in frozen sections neither the nucleolar nor the extranucleolar compact chromatin appear to be organized into discrete, 20 to 30 nm superordered fibers.

Replicating single-stranded adenovirus type 5 DNA molecules accumulate within well-delimited intranuclear areas of lytically infected HeLa cells.

The intranuclear compartments which contain adenovirus type 5 (Ad5) genomes during productive infection of HeLa cells were determined by high resolution when the rate of viral DNA replication is at its maximum. In situ hybridization was performed with and without denaturation of Ad5 DNA to reveal those portions of both double-stranded and single-stranded viral DNA and of single-stranded viral DNA alone, respectively, which were present at the surface of Lowicryl thin sections. Autoradiography after short pulse incorporation of tritiated thymidine revealed sites of active DNA replication whereas a pulse-chase experiment visualized the migration of the synthesized DNA. Immunocytochemistry determined distribution of the Ad5 72 kDa protein. Viral DNA was contained within multiple, well-delimited, compact fibrillar structures and their immediately surrounding, less well-defined, fibrillo-granular areas of the cell nucleoplasm. The compact fibrillar masses were primarily storage sites of single-stranded viral DNA and its accompanying 72 kDa protein, so they were named ssDNA accumulation sites. They contained intermittent viral replicative activity. Conversely, viral ssDNA and 72 kDa protein were rare in the surrounding fibrillo-granular areas in which Ad5 replication DNA is continuous, so we referred to them as peripheral replicative zones. Well-delimited subnuclear structures induced by viruses, therefore, represent accumulations of specific components of their replicating genomes.

Organization of the nucleolar RNP components in cultured rabbit fibroblasts following mild loosening of the nuclear content.

The organization of the ribonucleoprotein (RNP) components of the nucleolus was studied in ultrathin sections by a recent method of mild loosening of the nuclear content. This investigation was carried out in cultured rabbit fibroblasts in which the nucleoli contain clearly separated fibrillar and granular RNP components. In addition, the demarcation between these two particulate components was accentuated by infection with herpes simplex type 1 virus (HSV1), by recovery at 37 degrees C after a heat shock, and by actinomycin D treatment. Except in actinomycin D-treated cells, it was possible to identify two distinct components which are not visible in routinely prepared sections: (1) highly contrasted fibrillar clusters resulting from the loosening of the fibrillar component, (2) a granular network composed of nucleolar granules interconnected by a thin filament of about 5 nm in thickness. In actinomycin D-treated cells, only the granular zone was observed. High resolution autoradiography carried out both on standard fixed and on loosened cells following a 5 min labeling with tritiated uridine revealed that the fibrillar clusters correspond to sections of nucleolar transcription complexes. When the same labeling was followed by 3 h chase only the granular network was labeled indicating that it corresponds to the site of pre-ribosomal ribonucleic acid (pre-rRNA) maturation. Infection by HSV1 did not change this result. Preferential RNP staining and deoxyribonucleic acid (DNA) specific staining failed to reveal the thin filaments interconnecting the nucleolar granules. We suggest that the latter filaments serve as support for pre-rRNA processing and for storage of large pre-ribosomal subunits.

Intranuclear distribution of U1 and U2 snRNAs visualized by high resolution in situ hybridization: revelation of a novel compartment containing U1 but not U2 snRNA in HeLa cells.

We have examined the intranuclear distribution of U1 and U2 small nuclear RNAs (snRNAs) in HeLa cells by electron microscope in situ hybridization using biotinylated DNA probes reacting at the surface of thin sections of Lowicryl-embedded cells. U1 and U2 snRNAs colocalized on perichromatin fibrils, clusters of interchromatin granules and coiled bodies. The perichromatin granules were just occasionally labeled. In addition, we identified a novel nuclear domain associated with the clusters of interchromatin granules which contains U1 but not U2 snRNA. This new compartment termed "interchromatin granule-associated zone" has a fibrillar texture, does not contain DNA and might be the equivalent of the A snurposomes described in germinal vesicles of amphibians (Wu et al., J. Cell Biol. 113, 465-483 (1991)). We propose that the interchromatin granule-associated zones might be sites of the final maturation of the U1-pre-snRNP particle before its transfer to interchromatin granules and its subsequent assembly in the spliceosome.

Cytochemical study of the localization and organization of parental herpes simplex virus type I DNA during initial infection of the cell.

Changes in the location and structural organization of parental herpes simplex virus type 1 (HSV-1) DNA during its migration from the extracellular space to the interior of the nucleus of the target cell were examined by in situ hybridization using an HSV-1 DNA probe, specific DNA staining, and autoradiography after infection of cells with tritium-labeled viruses. In situ hybridization was carried out on denatured DNA to reveal as much as possible of the HSV-1 sequence present at the surface of the sections, and also on non-denatured DNA which revealed the presence of single-stranded portions of parental DNA, both prior to and during its intracellular migration. The results from in situ hybridization and autoradiography demonstrated that a short interval of about 15 min separated the initial contact of the viruses with the cells from the entry of parental viral DNA into the nucleus. In transit, morphologically intact nucleoids were released into the cytoplasm, and swollen nucleoids which contained partially decondensed viral DNA became detectable in the juxtanuclear cytoplasm and the periphery of the nucleus among the cell chromatin fibers. Completely decondensed parental viral DNA fibers could not be distinguished structurally from cellular DNA, but their position could be revealed by the in situ hybridization label. The infective DNA became randomly distributed within all compartments of the nucleus except the matrix-associated clusters of interchromatin granules.

Appearance of host-specific nucleolar proteins in intranuclear "dense bodies" following herpes simplex infection.

Nucleolar modifications induced by herpes simplex virus type 1 (HSV1) infection were studied at the ultrastructural level with special attention to the fate of a family of proteins serologically related to the nucleolar 100 kDa protein. Immunocytochemical techniques revealed that antigenic sites related to these proteins were associated with nucleoli in a pattern similar to that observed with non-infected cells. In addition, the "dense bodies" induced by HSV infection were heavily decorated by antibodies to the 100 kDa protein. Neither DNA nor RNA was detectable in the latter by cytochemical techniques. Therefore, it appears that "dense bodies" are exclusively proteinaceous and contain at least one host protein implicated in ribosomal RNA synthesis. An accumulation of 100 kDa protein in extra-nucleolar structures might account for previously reported defects in ribosomal RNA expression during HSV infection.

Alterations of nucleolar ultrastructure and ribosome biogenesis by actinomycin D. Implications for U3 snRNP function.

We have studied, at the electron microscope level, the reorganizations of nucleolar ultrastructure induced by actinomycin D (AMD) in different conditions of drug treatment associated with an inhibition of rRNA synthesis. We have analyzed in parallel the localizations of ribosomal genes, of their transcripts, of various pre-rRNA intermediates, as well as of U3 RNA and fibrillarin by in situ hybridization with nucleic acid probes and immunocytological detection on thin sections of human and mouse cells. Consistent with previous observations, dense fibrillar component (DFC) and granular component (GC) appear to contain distinct pre-rRNA species at different stages of their processing. DFC appears as a major site of U3 RNA accumulation, but a very substantial fraction of nucleolar U3 RNA is also found in GC, colocalizing with partially processed pre-rRNAs. Remarkably, the major nucleolar components retain their ultrastructural appearance when extensively depleted of their pre-rRNA moiety, and ribosomal genes are always detected over fibrillar center (FC), even after extended AMD treatments which result in the characteristic segregation of nucleolar components. Moreover, while for GC the U3 RNA and pre-rRNA contents evolve in parallel following the cessation of rRNA synthesis, a dramatic uncoupling is observed for DFC. The persistent presence of U3 RNA and fibrillarin after pre-rRNA depletion suggests that DFC could represent an anchorage site for U3 snRNPs, before their entering another cycle of pre-rRNA processing reactions.

Ultrastructural localization of nucleic acids through several cytochemical techniques on osmium-fixed tissues: comparative evaluation of the different labelings.

Several cytochemical techniques, such as sodium tungstate, acid hydrolysis phosphotungstic acid (HAPTA), ethylenediaminetetraacetic acid (EDTA), RNase-gold, and osmium-ammine, have been applied for the ultrastructural demonstration of nucleic acids on sections of tissues fixed in glutaraldehyde postfixed with osmium tetroxide and embedded in Epon. In order to obtain specific results, the sections had to be treated with sodium metaperiodate prior to performing the labeling protocol. The results for each method were identical to those obtained on nonosmicated tissues; the main difference being the enhancement in the ultrastructural preservation, which allowed for higher resolution. In addition to these techniques, and for comparative evaluations, DNA was also revealed by the DNase-gold approach on nonosmicated tissue sections. The consistency in the results, obtained over the nucleus with either EDTA or the RNase-gold complex for revealing RNA and those obtained with either osmium-ammine or DNase-gold for revealing DNA, supports the high specificity of the RNase-gold, DNase-gold, and osmium-ammine techniques. Furthermore, these results demonstrate the possibility of performing various cytochemical techniques on tissues processed for routine electron microscopy.

Colloidal alpha-stannic acid and negative iron colloid as differential electron stains for surface proteins.

Colloidal alpha-stannic acid and a negative iron colloid obtained from ferric hydroxide and potassium ferrocyanide, both negative sols being stable within a wide pH range, were refined as surface protein electron markers. Because of the relatively small size of its particles, colloidal alpha-stannic acid was used for staining all surface proteins. According to the pH at which the negative iron colloid was applied, it revealed either all surface proteins, or because of its large colloidal particles, stained basic proteins. This differential staining capability of the iron colloidal has been demonstrated previously on various control preparations (Puvion E, Blanquet PR: J Microsc 12:171, 1971). Controls on the affinity of the two colloids to surface amino groups were carried out on rat liver, mouse fibroblasts, HeLa and KB cells, Ehrlich and Zajdela ascites cells subjected to prior enzymatic and chemical treatments (incubation with neuraminidase or phospholipase C, esterification, acetylation or lipid extraction). At any pH below 9, the two sols stained proteins in the outer hydrophilic leaflet of esterified cells with relative selectivity, but the alpha-stannic acid showed them more accurately. The iron sol did reveal at high pH protein components of high isoionic point on the surfaces of rat hepatocytes and ascites cells which had only been treated with neuraminidase.

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)

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.