Simultaneous high resolution localization of Ag-NOR proteins and nucleoproteins in interphasic and mitotic nuclei

Summary Silver stainable proteins of the Nucleolar Organizer Regions (Ag-NOR proteins) of human breast cancer tissues have been localized at the electron microscopical level with a new method which combines a simple and reproducible one step Ag-NOR staining method combined with an acetylation procedure. This new method allows the fine identification of nucleolar components, particularly those which are stained by silver.In order to determine the cytochemical nature of the components associated with Ag-NOR proteins, the EDTA regressive preferential staining procedure for ribonucleoproteins has been applied to sections. By this means the precise localization of the Ag-NOR proteins was studied simultaneously with that of ribonucleoprotein within interphasic nucleoli and mitotic chromosomes.In interphasic nucleoli, stainable Ag-NOR proteins were localized in fibrillar centres and part of the dense fibrillar component. No silver deposits were seen on perichromatin or interchromatin fibrils and granules.In metaphasic nuclei, Ag-NOR proteins were only found on roundish fibrillar ribonucleoprotein structures, which could correspond to secondary constrictions. No silver deposits were seen on the well defined ribonucleoprotein sheet surrounding the chromosomes.In telophasic nuclei, Ag-NOR proteins were seen on the central part of roundish ribonucleoprotein fibrillar structures integrated in decondensing chromosomes. These structures have been interpreted as the nucleolar organizer regions around which rRNA synthesis resumes.In interphasic and mitotic nuclei, Ag-NOR proteins were never found within condensed chromatin but always in association with ribonucleoprotein components.The new method proposed here appears to be a useful tool for the simultaneous study of the localization of ribonucleoprotein and Ag-NOR proteins during the cell cycle.     

Palmitate increases Nur77 expression by modulating ZBP89 and Sp1 binding to the Nur77 proximal promoter in pancreatic β-cells

Nur77 is a stress sensor in pancreatic β-cells, which negatively regulates glucose-stimulated insulin secretion. We recently showed that a lipotoxic shock caused by exposure of β-cells to the saturated fatty acid palmitate strongly increases Nur77 expression. Here, using dual luciferase reporter assays and Nur77 promoter deletion constructs, we identified a regulatory cassette between −1534 and −1512 bp upstream from the translational start site mediating Nur77 promoter activation in response to palmitate exposure. Chromatin immunoprecipitation, transient transfection and siRNA-mediated knockdown assays revealed that palmitate induced Nur77 promoter activation involves Sp1 recruitment and ZBP89 release from the gene promoter.     

Three-dimensional organization of pKi-67: a comparative fluorescence and electron tomography study using FluoroNanogold.

The monoclonal antibody (MAb) Ki-67 is routinely used in clinical studies to estimate the growth fraction of tumors. However, the role of pKi-67, the protein detected by the Ki-67 MAb, remains elusive, although some biochemical data strongly suggest that it might organize chromatin. To better understand the functional organization of pKi-67, we studied its three-dimensional distribution in interphase cells by confocal microscopy and electron tomography. FluoroNanogold, a single probe combining a dense marker with a fluorescent dye, was used to investigate pKi-67 organization at the optical and ultrastructural levels. Observation by confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli. Double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy studies confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250-300 nm in diameter, which are themselves composed of 30-50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin.     

Textural analysis of nuclear mitotic apparatus antigen (NuMA) spatial distribution in interphase nuclei from human drug-resistant CEM lymphoblasts.

In tumour cell lines, the resistance of cancer cells to a variety of structurally unrelated chemotherapeutic drugs is termed multidrug-resistance or MDR. We reported previously [6] that MDR leukemic cells displayed nuclear texture changes, as assessed by image cytometry. The nature of these changes remained uncertain but they could be associated with alterations of the nuclear matrix which could serve an important role in DNA organization and chromatin structure. Therefore, we have compared the textural features observed in G0/G1 nuclei from human leukemic CEM cells and their MDR variant CEM-VLB, after staining of either DNA by Feulgen method or nuclear matrix by immunodetection of NuMA antigen on DNase treated samples. Chromatin or NuMA distributions within the nucleus were evaluated by image cytometry. Changes in textural parameters indicate that modifications of NuMA distribution observed in MDR cells are parallel to those observed at the whole chromatin level (i.e., a more decondensed and coarse texture with increase of Energy and Long-run sections and decrease of Contrast and Short-run sections). Moreover, Optical Densities measurements indicate that MDR cells seem to contain less NuMA, a datum confirmed by immunoblotting of nuclear proteins. In conclusion, chromatin changes observed by image cytometry in drug-resistant human leukemic CEM cells appear associated with modifications of the nuclear matrix structure.     

Production of human monoclonal antibodies against various antigens of erythrocyte stages of Plasmodium falciparum

Peripheral blood lymphocytes from 20 individuals living in a malaria endemic area (Burkina Faso) were transformed with Epstein-Barr virus. No antigen specific selection, nor stimulation of B-cells were performed prior to transformation. 20 cell lines were established, 14 secreted polyclonal antibodies directed against erythrocytic stages of Plasmodium falciparum. 4 lines were cloned and the supernatant analysed and characterised against Plasmodium falciparum antigens.