Synergistic effects of micromolar concentrations of Zn2+ and Ca2+ on membrane fusion

Resonance Energy Transfer between N-(7-nitro-2,1,3 benzoxadiazol -4 yl) phosphatidyl ethanolamine and N-Lissamine-Rhodamine B sulfonyl) phosphatidyl ethanolamine embedded in two different populations of small unilamellar vesicles made of phosphatidyl serine has been used to study the fusion process induced by Zn2+ and Ca2+. Lipid intermixing demonstrating fusion of liposome membranes can already be observed at 125 and 250 mumol/l of Zn2+. After short time pre-incubations with micromolar concentrations of Zn2+ as low as 150 mumol/l, Ca2+ induces an instantaneous increase of vesicle fusion. The lipid intermixing induced by micromolar concentrations of Ca2+ (250-500 mumol/l) could be increased up to 4 times when pre-incubated with 150 or 200 mumol/l of Zn2+. The effect of 1 mM of Ca2+ alone on lipid intermixing can be mimicked by 150 mumol/l of Zn2+ followed by 500 mumol/l of Ca2+. Our data demonstrate that Zn2+ and Ca2+ act synergistically to affect cation-induced membrane fusion. We suggest that Zn2+ specifically alters the physical state of phospholipid membranes making them more prone to calcium-triggered fusion.     

Thrombocyte aggregation in plasma and whole blood during hyperventilation (hypocapnia) in cats

Platelet aggregation in platelet rich plasma (PRP) and whole blood was simultaneously studied in acute experiments on cats in hypocapnic conditions. ADP-induced aggregation increase was determined in PRP and whole blood. Contradictory results were obtained during platelet aggregation induced by collagen and arachidonic acid: increased aggregation in PRP and decreased aggregation in whole blood. The data obtained suggest that ADP is a risk factor for the onset of intravascular thrombosis.     

A new statistical method for identifying interconnections between neuronal network elements

A new method is proposed to analyse dependencies in point processes, which takes into account specific character of neuronal activity. Simulation modelling of neuronal network revealed that the estimated weight of connection depends monotonically on the value of the model synaptic strength. In contrast to the crosscorrelation, the method allows for nonlinear interconnections and does not require point processes to be stationary and samples to be large. Examples are presented of the method's application to neurophysiological data analysis.     

The immunohistochemical demonstration of thyroglobulin in human thyroid tumour xenografts using a monoclonal antibody

A monoclonal antibody (RBU/01) was raised against human thyroglobulin and its suitability for the immunohistochemical staining of thyroglobulin was determined on fixed, wax-embedded tissue, using the peroxidase anti-peroxidase (PAP) method. The antibody was then used to demonstrate the expression of human thyroglobulin in sections of a human follicular carcinoma of the thyroid which had been grown in immunodeficient mice. It is concluded that the immunohistochemical evaluation of the xenografts with the antibody provides useful information on this xenograft system as a potential model for thyroid carcinoma.     

Promotion and inhibition of vesicle fusion by polylysine

Polylysine induced rapid aggregation of large unilamellar vesicles composed of phosphatidylcholine-cardiolipin (1:1 molar ratio) but not their fusion. Application of the terbium-dipicolinic acid fusion assay showed that addition of polylysine at nanomolar concentrations enabled a significant lowering of the Ca2+ threshold concentration for vesicle fusion from 9 to 1 mM. Analysis of the kinetics of fusion with a mass-action kinetic model showed that polylysine enhanced significantly the rate of aggregation but affected only slightly the rate of fusion per se. Maximal enhancement of overall fusion rates occurred at a charge ratio (polylysine/cardiolipin) of about 0.5. At larger polylysine concentrations, e.g., at charge ratios greater than 3, polylysine inhibited vesicle fusion.     

A proton nuclear magnetic resonance study of gamma-glutamyl-amino acid cyclotransferase in human erythrocytes

Spin-echo NMR spectroscopy was shown to be a reliable technique for the monitoring of the in situ cleavage of gamma-Glu-Ala by gamma-glutamyl-amino acid cyclotransferase in whole erythrocytes and hemolysates. Of particular importance was the difference in chemical shifts between peptide resonances and those of the constituent amino acids. Using lysates of varying dilution, it was shown that the specific activity of the enzyme was not concentration-dependent, thus suggesting a lack of cytosolic low-molecular-weight-effectors or enzyme dissociation. Furthermore, the initial velocities of the reaction as a function of substrate concentration obeyed Michaelis-Menten kinetics with a Km = 2.0 +/- 0.3 mmol/l and Vmax = 137 +/- 7 mmol/h/l of cell water in 1H2O medium. Similar analysis in 2H2O medium revealed a solvent kinetic isotope effect of 1.9 +/- 0.4 at low substrate concentrations. The implications of this observation for the mechanism of the reaction are discussed. Cleavage of the peptide by a suspension of intact erythrocytes was at a rate 300 times less than the corresponding lysate flux, thus indicating the rate limitation by transport in the coupled system.     

Cell Cycle activation and ouabain-sensitive ion movements of 3T3 and C3H-10T½ fibroblasts

The introduction of either PGF_2α (10^?7 M) or TPA (10^?7 M) stimulated, ouabain-sensitive ⁸⁶Rb⁺ influx at 30 min in postconfluent 3T3-4 mouse fibroblast cultures by 117% and 124%, respectively. Both TPA and PGF_2α at these concentrations stimulated the incorporation of ³H-TdR into DNA. TPA had the greatest stimulatory effect, which was similar to that obtained with 10% fetal calf serum. In accord with the idea that modulation of membrane processes such as Na⁺/K⁺ pump activity in fibroblasts may reflect important events related to the initiation of DNA synthesis, it was observed that in both 3T3-4 and C3H-1 0T½ cells there were parallel increases in ³H-TdR incorporation and ouabain-sensitive ⁸⁶Rb⁺ influxes with 10^?7 M TPA, whereas PGF_2α stimulated a significant increase in ³H-TdR incorporation in 3T3-4 but not C3H-10T½ cells and only marginal increases in ouabain-sensitive ⁸⁶Rb⁺ influx in both. Therefore, although there appears to be a close correlation between Na⁺/K⁺ pump activation and subsequent S-phase entry following TPA stimulation, a similar correlation for PGF_2α cannot be confirmed.