Different concentrations of Mg++ ions affect nuclear matrix protein distribution during thermal stabilization of isolated nuclei.

The nuclear matrix, a proteinaceous network believed to be a scaffolding structure determining higher-order organization of chromatin, is usually prepared from intact nuclei by a series of extraction steps. In most cell types investigated the nuclear matrix does not spontaneously resist these treatments but must be stabilized before the application of extracting agents. Incubation of isolated nuclei at 37C or 42C in buffers containing Mg++ has been widely employed as stabilizing agent. We have previously demonstrated that heat treatment induces changes in the distribution of three nuclear scaffold proteins in nuclei prepared in the absence of Mg++ ions. We studied whether different concentrations of Mg++ (2.0-5 mM) affect the spatial distribution of nuclear matrix proteins in nuclei isolated from K562 erythroleukemia cells and stabilized by heat at either 37C or 42C. Five proteins were studied, two of which were RNA metabolism-related proteins (a 105-kD component of splicing complexes and an RNP component), one a 126-kD constituent of a class of nuclear bodies, and two were components of the inner matrix network. The localization of proteins was determined by immunofluorescent staining and confocal scanning laser microscope. Mg++ induced significant changes of antigen distribution even at the lowest concentration employed, and these modifications were enhanced in parallel with increase in the concentration of the divalent cation. The different sensitivity to heat stabilization and Mg++ of these nuclear proteins might reflect a different degree of association with the nuclear scaffold and can be closely related to their functional or structural role.

Postmortem diagnosis of anaphylaxis in presence of decompositional changes.

Eosinophil and activated mast cell identification in the spleen combined with mast cell tryptase determination in postmortem serum may diagnose fatal anaphylaxis with a high degree of certainty. Mast cell tryptase measurement and significance in corpses with decompositional changes remains however an issue of controversy. Analogously, immunohistochemistry in corpses with decompositional changes may be influenced by several mechanisms, including protein alteration, antigen diffusion and unspecific antibody binding to disrupted protein structures. The authors present an autopsy case involving a 55-year-old woman who unintentionally received clarithromycin. Due to difficult in administrative procedures, the postmortem examination was performed 96 h after death. Mast cell tryptase was measured in postmortem serum from femoral, aortic and right heart blood. The obtained results were consistent with mast cell activation. Histochemistry (Pagoda Red) and immunohistochemistry (anti-tryptase antibodies) allowed splenic eosinophils and mast cells to be detected. Based on the results of all postmortem investigations, the hypothesis of anaphylaxis following accidental clarithromycin administration was formulated.