Cementum protein 1 transfection does not lead to ultrastructural changes in nucleolar organization of human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Transfection of cementum protein 1 (CEMP1) into human gingival fibroblasts (HGFs) notably increases cell metabolism and results in overexpression of molecules related to biomineralization at transcriptional and protein levels. Therefore, HGF-CEMP1 cells are considered as putative cementoblasts. This represents a significant advance in periodontal research because cementum neoformation is a key event in periodontal regeneration. In addition, it is well known that important changes in cell metabolism and protein expression are related to nucleolar structure and the function of this organelle, which is implicated in ribosome biogenesis. The aim of this study was to determine the effect of transfecting CEMP1 gene in human HGF on the ultrastructure of the nucleolus. MATERIAL AND METHODS: Cells were processed using the conventional technique for transmission electron microscopy, fixed with glutaraldehyde, postfixed with osmium tetraoxide, and embedded in epoxy resin. Semi-thin sections were stained with Toluidine blue and observed by light microscopy. Thin sections were stained with uranyl acetate and lead citrate. For ribonucleoprotein detection, the staining method based on the regressive effect of EDTA was used. In addition, the osmium ammine technique was used for specific staining of DNA. RESULTS: The results obtained in this study suggest that transfection of CEMP1 into HGFs does not produce changes in the general nucleolar ultrastructure because the different components of the organelle are present as fibrillary centers, and dense fibrillar and granular components compared with the control. CONCLUSION: The transfection of CEMP1 into HGFs allows these cells to perform cementoblast-like functions without alteration of the ultrastructure of the nucleolus, evaluated by the presence of the different compartments of this organelle involved in ribosomal biogenesis.

Further ultrastructural characterization of the intranuclear ring-shaped bodies of the plant Lacandonia schismatica.

Ring-shaped bodies are found in the nucleus of Lacandonia schismatica, a rare plant with the sexual organs inverted. They are 0.5-microm-diameter structures that present an electron-dense external ring surrounding a central core. Ultrastructural studies indicate that these bodies contain RNA. The external ring is labeled with antibodies against small nuclear ribonucleoproteins, suggesting that they may be involved in pre-mRNA metabolism. In the present work we further characterized these intranuclear ring-shaped structures by serial-sectioning analysis. Moreover, we tested the presence of additional molecular elements related to pre-mRNA metabolism, such as SR proteins and poly(A)(+) RNA, using immunoelectron microscopy and ultrastructural in situ hybridization. Our results show that these nuclear bodies are spherical. They contain SR proteins involved in splicing and postsplicing events and little to no poly(A)(+) RNA. We also found similar nuclear bodies in other plant and animal species. Therefore, ring-shaped bodies in L. schismatica are spherical, highly compartmentalized nuclear structures that may be involved in pre-mRNA metabolism.

New evidence that Lacandonia granules are ultrastructurally related to perichromatin and Balbiani ring granules.

Lacandonia granules are abundant non-typical extranucleolar ribonucleoprotein particles found in the nucleus of Lacandonia schismatica, a rare plant showing spatial inversion of sex organs. In the present study, changes in the number of Lacandonia granules during flower development, and the presence of SR proteins and poly(A)+ RNA in the nuclei of L. schismatica were analyzed by electron microscopy, immunoelectron microscopy and ultrastructural in situ hybridization. Our results show an important reduction in the number of Lacandonia granules in the nuclei of cells of opened (post-anthesis) in relation to unopened (pre-anthesis) flowers, where granules are very abundant. The SR family of splicing factors and poly(A)+ RNA are present in both perichromatin fibers and Lacandonia granules. The developmental behavior, the presence of SR proteins, recently involved in post-splicing events, poly(A)+ RNA and the reported absence of snRNPs splicing factors in Lacandonia granules, suggest that these particles are involved in postranscriptional events as storage and/or transport of mRNAs. A similar situation is present in other nuclear RNP as perichromatin granules present in mammals and Balbiani ring granules of salivary glands of Chironomus. Based on similarities in morphological, developmental behavior, immunocytochemistry and in situ hybridization results, we conclude that Lacandonia, perichromatin and Balbiani ring granules may be also functionally similar structures.