Distinct labelling of fusion events in rat lactotrophs by FM 1-43 and FM 4-64 is associated with conformational differences.

AIM: Conformational analysis of fluorescent styryl dyes FM 1-43 and FM 4-64 was undertaken to clarify if distinct activity-dependent labelling of single lactotrophs vesicles and plasma membrane by two dyes is associated with their structural differences. METHODS: The activity-dependent labelling of single vesicles and plasma membrane by FM 1-43 and FM 4-64 was studied using confocal microscopy. The fluorescence intensity of vesicles fused with the plasma membrane, and the plasma membrane alone was measured; the ratio of their respective peak amplitudes was calculated. The conformational analysis of FM 1-43 and FM 4-64 was further undertaken by employing the Monte Carlo approach to search the conformational space of these molecules. RESULTS: In FM 1-43 staining of vesicles and plasma membrane, the ratio of the fluorescence peak amplitudes (vesicle vs. plasma membrane) was 2.6 times higher in comparison with FM 4-64 staining. In FM 4-64 molecule the low-energy conformations are distributed in three conformational states (consisting of 3, 4 and 2 conformers respectively) in which the proportion of the molecules residing in a given state is 62%, 28% and 9% respectively. In FM 1-43 the conformation distribution is limited to just one conformational state with three approximately equally populated conformers what can be explained by greater intrinsic rigidity of the molecule. CONCLUSIONS: The observed structural characteristics of FM 1-43 molecules may account for a higher increase in quantum yield and/or binding affinity upon incorporation of the dye into the vesicle matrix and therefore stronger fluorescence emission in comparison with FM 4-64.

High-molecular-mass receptors for ammodytoxin in pig are tissue-specific isoforms of M-type phospholipase A(2) receptor.

Studying the molecular basis of presynaptic neurotoxicity of ammodytoxin C, a secretory phospholipase A(2) from the venom of Vipera a. ammodytes snake, we demonstrated the existence of two high-molecular-mass ammodytoxin C-binding proteins in porcine tissues, one in cerebral cortex and the other in liver. These proteins differ considerably in stability and Western blotting properties. However, as shown by immunological analysis and tandem mass spectrometry sequencing of several internal peptides derived from the purified receptors, both belong to secretory phospholipase A(2) receptors of the M type, which are Ca(2+)-dependent multilectins homologous to the macrophage mannose receptor. Based on Southern blot analysis of genomic DNA and deglycosylation of the receptors, the difference between the two proteins most likely stems from the different posttranscriptional and posttranslational modifications of a single gene product. Our findings raise the possibility that the M-type receptors for secretory phospholipases A(2) may display different physiological properties in different tissues.

Immune response in lymphocyte cultures stimulated by oral bacteria preparations.

Lymphocyte cultures were used as an in vitro experimental model to get a deeper insight into immune response to oral bacteria in periapical granulomas. Lymphocytes isolated from leucocyte concentrate were in lymphocyte cultures stimulated by antigen preparations of oral bacteria. Lymphocyte subsets that have developed in lymphocyte cultures after a week of stimulation were analysed by flow cytometry. A significant increase in expression of INF-gamma molecules in CD3+ cells stimulated by antigen preparations of oral streptococci was found, compared with negative control. On the other hand we observed a significant increase in expression of IL-4 in CD3+ cells stimulated by antigens of anaerobic bacteria, compared with negative control. Our results show that antigens of oral streptococci in in vitro lymphocyte cultures induce the differentiation of T helper cells into Th2 cells and that antigen preparations of anaerobic bacteria induce the differentiation of T helper cells into Th1 cells. Furthermore, an increased expression of HLA-DR molecules on CD8+ T cells stimulated by antigens of oral streptococci was found, compared with negative control.