Thioacetamide (TA) is converted into a hyperacetylating agent which causes hepatic necrosis, regeneration, cirrhosis and cancerous transformation. One of the most characteristic toxicities of TA in rat is observed with a 50 mg/kg per day which induces nucleolar enlargement different from that in regenerating liver. From TA-treated liver, the nucleoli were isolated and characterized for an altered nucleolar signal transduction system. Immunochemistry revealed that the poisoned nucleoli had increased levels of both nucleolus specific proteins (nucleophosmin and nucleolin) and various signal molecules (CK2, Erk1/2, p38, protein kinases A and C, and cyclin A). Using flow cytometry, the nucleoli were found to be in G2-arrested nuclei. Manifestation of the nucleolar enlargement could be readily observed using an ex vivo hepatocyte culture. There were two types of nucleolar enlargement. One was observed in normal hepatocytes with light density of enlarged nucleoli. The other was in TA-treated hepatocytes with dense and compact density of enlarged nucleoli, which contained a 3 to 5-fold higher nudeophosmin content than the control. In vitro induction of nucleolar enlargement with TA was possible. As soon as the hepatocytes anchored on a collagen coat, exogeneous TA (higher than 1 microg/mL) could induce dense and compact nucleoli. However, when an exogeneous drug was added after monolayer formation (1 day), no drug-induced nucleolar enlargement was observed.
Animal ; Cells, Cultured ; Flow Cytometry ; G2 Phase/drug effects ; Hepatocytes/ultrastructure ; Hepatocytes/drug effects* ; Male ; Nucleolus Organizer Region/physiology ; Nucleolus Organizer Region/drug effects* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects ; Thioacetamide/toxicity*

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.
Alpha-Amanitin/pharmacology ; Animals ; Antibodies, Monoclonal/analysis/metabolism ; Antibodies, Protozoan/analysis/metabolism ; Dactinomycin/pharmacology ; Fluorescent Antibody Technique, Direct ; Gene Expression/*physiology ; Green Fluorescent Proteins/genetics ; Hela Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleic Acid Synthesis Inhibitors/pharmacology ; Nucleolus Organizer Region/drug effects/*metabolism ; Pol1 Transcription Initiation Complex Proteins/metabolism ; Protein Sorting Signals/physiology ; Protozoan Proteins/*biosynthesis/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Toxoplasma/*physiology ; Transfection