Ultrastructural localization of antibody in differentiating plasma cells.

Antibody was localized by electron microscopy within differentiating and mature plasma cells of the spleens of hyperimmunized rabbits. Horseradish peroxidase was used as antigen. Intracellular antibody to peroxidase was revealed in glutaraldehyde-fixed tissue by coupling it with its antigen and then revealing the sites of peroxidase activity cytochemically. Antibody first appears in the perinuclear space of hemocytoblasts where it persists through differentiation into immature plasma cells, but it disappears from this site in mature plasma cells. Concomitant with the development of the ergastoplasm, antibody accumulates in many but not all of its cisternae. Antibody is present in the lamellar portion of the Golgi apparatus in all phases of plasmacytic differentiation. Mature plasma cells exhibit two types of antibody distribution, a concentration into large spherical intracisternal granules or an overflowing into all parts of the cytoplasm.

Mechanisms of transcription in nucleoli of amphibian oocytes as visualized by high-resolution autoradiography.

In oocytes of Pleurodeles waltlii, the method of Miller and Beatty has been combined with a method of high-resolution autoradiography especially suitable for the study of isolated molecules. In vitro labeling of RNA by tritiated precursors was carried out with increasing incubation times (1, 4, 15, 24, 48, and 72 h). Silver grains were present over ribonucleoprotein fibrils in amounts sufficient for quantitative analysis of nucleolar DNA transcription. Statistical analysis of the data revealed that: (a) The units of any one nucleolus exhibited a large degree of heterogeneity in their number of grains. (b) There was a parallelism between the increasing grain number and the ribonucleoprotein-fibril lengthening as observed along the transcription unit.

Procedure for isolating micronuclei from rat kangaroo cultured cells containing individualized chromosomes.

Micronuclei are small interphase nuclei containing part of the genome; the DNA content of the smallest micronuclei is equivalent to one chromosome. For analysis by biochemical method and by cytofluorometry of interphase micronuclei containing a single chromosome, several isolation and purification procedures were tested and checked by fluorescent microscopy using the DNA dye Hoechst 33 342 and electron microscopy. Micronucleation of rat kangaroo epithelial cells was induced by colchicine treatment for three days. Micronuclei were isolated in a low ionic strength buffer containing collagenase, with concomitant mechanical shocks. Eighty % of the micronuclei were released after 3 to 7 min, with minimum nuclear breakage. Subsequent filtration through several polycarbonate filters 12, 8 and 5 micron in diameter enabled purification of the smallest micronuclei without aggregates or debris. Micronuclear morphology was well preserved, as shown by electron microscope observations. Therefore, we established the optimal conditions allowing gentle mass isolation of individual micronuclei of cultured PtK1 cells, compatible with flow cytometry analysis.

Structural aspects of intranuclear matrix disintegration upon RNase digestion of HeLa cell nuclei.

The organization of the intranuclear elements observed in histone-depleted (2 M NaCl-extracted) HeLa cell nuclei was investigated by means of electron microscopy and two-dimensional gel electrophoresis. This work was mainly aimed at verifying whether or not an intranuclear skeleton or matrix existed, which could explain the stable attachment of RNA to the residual nuclear structure after high-salt extraction, and its three-dimensional organization. We compared the ultrastructure and the polypeptide composition of RNA-containing and RNA-depleted (RNase-treated) nuclear residues, and we visualized intermediate stages of RNase action on the intranuclear material. We showed that this material was made of two types (fibrillar and granular) of salt-resistant RNP components equally sensitive to RNase when the enzyme was used prior to high-salt extraction. At least in our material and under our experimental conditions, no intranuclear matrix could be distinguished from the residual RNP material. Our results further suggest that formation of such a matrix is a path-dependent phenomenon.

Silver staining of the nucleolar organizer regions (NORs) on Lowicryl and cryo-ultrathin sections.

Nucleolar organizer region (NOR) silver staining was applied to sections of fixed material. A positive reaction on cryo-ultrathin sections was found as well as on semithin and ultrathin Lowicryl sections. Repeatable staining that was easy to control was obtained by a one-step procedure after aldehyde-Carnoy fixation. Fixation of the material by formaldehyde and glutaraldehyde alone in cacodylate buffer also maintained reaction selectivity when ammonium chloride was used after fixation. Enzymatic digestion by pronase, RNase A, DNase I, or micrococcal nuclease was applied to ultrathin Lowicryl sections. Pronase digestion removed the silver-stained proteins, whereas digestion by the nucleases did not. A routine procedure is proposed for easy NOR silver staining of sections that preserves a good tissue ultrastructure and is also compatible with cytochemical and immunological investigations.

Localization of a gene: the nucleolar organizer.

Nucleolus differentiates around the nucleolus organizer regions of the chromosomes or NORs. In the interphasic nucleoli the fibrillar centers are now considered as the NORs. The purpose of this editorial is to review the experimental data which allowed such identification. Our current concepts regarding this point result from three lines of evidence: 1) specific localization during nucleologenesis, 2) in situ hybridization with labeled ribosomal RNA or DNA, and 3) specific staining with silver.

Attachment of DNA to nucleolar and nuclear skeletal structures as visualized by Kleinschmidt molecular spreading.

Co-isolated residual nuclear shells and residual nucleoli from membrane-depleted rat liver nuclei were spread according to Kleinschmidt's method. Comparison of the spread residual structures isolated from nuclear shells and spread pore complex-lamina isolated from nuclear envelopes showed that these residual structures are morphologically identical. Furthermore, our nuclear shell isolation procedure allowed visualization of DNA strands bound to a granular component of the lamina. The fragmentation of nuclear shells allowed us to obtain well-spread nucleolar remnants, in which we observed DNA strands anchored on a residual nucleolar network attached to the lamina. The different molecular features revealed by the spreading of residual nucleolar structures suggest that both non-transcribing nucleolar DNA and active ribosomal genes are linked to the nucleolar network. Although the exact nature of this network remains to be defined, the results of the present study strongly suggest that the DNA molecules of the chromosomes bearing ribosomal genes have many sites of attachment to a non-chromatin nucleolar network which can be referred to as a nucleolar skeletal complex.

Localization by autoradiography of viral proteins in the parenchymal cells of the liver during frog virus 3 induced hepatitis of mice.

Frog virus 3 inoculated into mice induces an acute degenerative hepatitis. This hepatitis is of toxic origin since the virus is unable to multiply at 37 degrees C. The Kupffer cells, which are the target cells for FV3, reveal the presence of viral particles, viral DNA and proteins. Although the hepatocytes present early and drastic nuclear lesions, viral particles were never observed in these cells. Viral proteins however but not DNA, could be found inside parenchymal cells.

Anchorage of the nucleolus in the pore complex-lamina by a DNA-bearing structure masked in situ in rat liver nuclei.

We have developed a method by which nuclear shells containing nucleoli can be isolated from membrane-depleted rat liver nuclei. This method involves the removal of the internal chromatin. This chromatin is expelled from the nuclear shell using combinations of low and high ionic strength buffers. The expelled internal part is subsequently digested with DNase I or micrococcal nuclease. Examination by electron microscopy of the nuclear and the nucleolar structures at various steps of the isolation procedure shows that the nucleoli are anchored in the peripheral lamina by a pedicle that is continuous with an intranucleolar network. This network is masked in situ by nucleolar granules. The pedicle and the network which support the nucleolar DNA are composed mainly of non-histone proteins insoluble in 2M NaCl.

[Application of immunoelectron microscopy to the detection of viruses in a water medium]. / Application de l'Immuno-Microscopie-Electronique à la détection de virus en milieu hydrique.

Adenoviruses were immersed in demineralyzed and deionized water for 5 days. The water was subsequently analyzed by Immuno-Electron-Microscopy for detection of viral particles. An attempt of quantation was also made on control, untreated particles. Viruses dispersed in water can be detected by the technique employed but quantitation is limited by the heterogeneous dispersion of the particles on the grids.

Electron microscope fluoro-autoradiography: improvement of efficiency.

The fluorographic process for enhancement of the autoradiographic detection of beta-ray emitted by tritium has been successfully adapted to electron microscope autoradiography. An original scintillator plastic film helped to increase the detection yield of electrons up to 80% without alteration of the morphological aspect of the autoradiograms, and without increasing background fog or causing apparent loss of resolution.

Characterization of lamina-bound chromatin in the nuclear shell isolated from HeLa cells.

The structure and the polypeptide composition of the nuclear shell isolated from interphase HeLa cells have been investigated and compared to those of the intranuclear material. The isolated nuclear shell contains chromatin superstructures (28-32 nm thick fibres) made of tightly packed nucleosomes that resist low ionic strength conditions and that are associated with the three nuclear lamins. Chromatin in the nuclear shell exhibits very simple chemical composition. Especially, non-histone proteins are lacking. The results presented here rule out the possibility that the nuclear shell results from contamination of lamina by intranuclear elements. They suggest that the lamins are directly involved in the specific properties and in the organization of chromatin in the nuclear shell.

Nuclear RNA-associated proteins and their relationship to the nuclear matrix and related structures in HeLa cells.

The ultrastructure and the polypeptide composition of residual nuclear substructures including nuclear matrices, nuclear ghosts, and residual envelopes were investigated by means of electron microscopy and two-dimensional gel electrophoresis. Nuclear matrices were prepared by digesting isolated nuclei with DNase I alone, followed by high-salt extraction in 2 M NaCl. Nuclear ghosts were obtained by high-salt extraction of nuclei previously digested with DNase and RNase in MgCl2-containing buffers. To prepare residual envelopes, nuclei were first digested with RNase in the presence of EDTA, then digested with Mg2+ -activated DNase I, and extracted in 2 M NaCl. The results of this comparative study support the conclusion that the intranuclear matrix is made of two distinct RNA-containing elements. One of these elements appears on ultrathin sections as a thin fibrillar network. It disappears from RNase-digested nuclei, together with numerous basic proteins, whatever the conditions of digestion. Although this element is present in extranucleolar territories, it is a major component of residual nucleoli. The second element appears as coarse-beaded fibers absent from the nucleolar areas. Its preservation in residual nuclear substructures depends on the presence of Mg2+ ions during RNase digestion of nuclei. It is enriched in two minor basic proteins of relative mass 49 000 and 70 000. The involvement of this fibrogranular element in heterogeneous nuclear RNA attachment to the nuclear matrix is discussed.

Preservation of chromosome integrity during micronucleation induced by colchicine in PtK1 cells.

Colchicine induces the formation of small nuclei called micronuclei which contain limited parts of the genome. Some of them exhibit a DNA content equivalent to that of a single chromosome. Our purpose was to study the preservation of chromosome integrity during this micronucleation in PtK1 cells. Observation of karyotypes obtained after 3 days of cell cycle restoration revealed that micronucleation did not affect chromosome integrity or the presence of each chromosome pair in the surviving cells. In 'early restoration' cells, all the chromosomes included a centromere and were represented in the karyotype, but at variable rates. Furthermore, flow cytometry analysis of micronucleated cells, intermediate in DNA rate between control PtK1 cells in G1 and those in G2/M phases, led us to consider the possibility of selective replication of some chromosomes during micronucleation. Using antibodies against the kinetochore proteins, we derived the presence of one centromeric region (1-2 spots) in the smallest micronuclei. Therefore, these data (karyotypes, number of chromosomes, DNA content and kinetochore proteins) seem to indicate that micronucleation does not induce chromosome damages or translocations. Micronuclei are a convenient tool for investigation of the role of the different chromosomes in the organization of the interphase nuclei.

Sorting of micronuclei from PtK1 cells.

We report here a procedure allowing to select micronuclei corresponding to defined individualized chromosomes in conditions which preserve their synthetic activity. The mammalian PtK1 cells, which possess six chromosome pairs, were micronucleated by colchicine. DNA of the micronucleated cells was labeled by the Hoechst 33342 fluorochrome under vital conditions. The micronuclei were isolated by a gentle procedure and their fluorescence was analysed by flow cytometry. The flow-cytometry parameters were determined for the analysis of non-fixed subdiploid fractions. We obtained five distinct peaks of fluorescence which have been sorted. The sorted micronuclei are different in each peak exhibiting different fluorescence intensity. Peak 3 contains the micronuclei with nucleoli and chromocenters that correspond to the X chromosome in this cell line.

Expression of integrated hepatitis B virus DNA in PLC/PRF/5, Hep 3B, and L6EC3 cell lines detected by in situ hybridisation.

The expression of the DNA of hepatitis B virus (HBV) was analysed in three cell lines (PLC/PRF/5, Hep 3B, L6EC3) which contain the HBV DNA integrated in their genome and release the viral surface antigen (HBsAg) in relation to cell growth. Using the in situ hybridisation technique and a cloned DNA probe specific for hepatitis B virus (PTKH9), the intracellular viral RNA localisation showed that for the three cell lines, HBV RNA are present in the different cell compartments according to the age of the culture. The nucleolar and nuclear localisation are visible in the early stages of the cell growth, whereas in the later stages viral RNA are found in the cytoplasm corresponding to the maximal production of the HBsAg. These observations suggest that the nucleolus is implicated in the expression of the integrated form of HBV genetic information, the regulation of which is linked to cell growth.

[Joint pediatric and psychiatric interviews of parents of leukemic children]. / Entretiens conjoints pédiatre-psychiatre avec des parents d'enfants leucémiques.

The authors (pediatricians and psychiatrists) report on a three-year-experience of joint interviews with parents of children with malignant diseases. These meetings, held at diagnosis and a few months afterwards, throw a light on the familial environment and help parents in expressing their feelings towards their traumatic situation.

Infection of cultivated CNS tissue with herpes virus, HSVII. A reappraisal.

Organized cultures of newborn rat and hamster cerebellum were infected with herpes virus type II, after 7 and 14 days "in vitro". 48 h after the infection, electron microscopic examination of the cultures showed that astrocytes contained numerous intranuclear and intracytoplasmic viral particles, while neurons remained apparently intact. The specificity of the infection for a given cell type is discussed.