Data acquisition and analysis of the time-resolved fluorescence anisotropy is typically a time consuming process preventing usage of this experimental method for monitoring of time-dependent phenomena. We describe a method for pseudo real-time monitoring of the limiting fluorescence anisotropy r(infinity) allowing to track changes of the membrane order occurring on the time scale of minutes. Principle and performance of the method is demonstrated in the time domain with the time-correlated single photon counting detection. DMPC liposomes stained with 1,6-diphenyl-1,3,5-hexatriene (DPH) have been used to test influence of the diffusion membrane potential on the membrane order during the temperature-induced phase transition in DMPC membranes. It has been found that the transmembrane field of the order of -70 mV increases the phase transition temperature by about 1.5 degrees C-2 degrees C. It is proposed that the full advantage of the method can be utilized with a gated detection, which besides a faster data acquisition brings additional advantage of excitation light suppression. The method can be also used for imaging.

• MeSH
• dimyristoylfosfatidylcholin chemie   MeSH
• liposomy MeSH
• membrány umělé * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dimyristoylfosfatidylcholin MeSH
• liposomy MeSH
• membrány umělé * MeSH

• MeSH
• kvasinky * MeSH
• Publikační typ
• kongresy MeSH
• souhrny MeSH

The rate and extent of uptake of the fluorescent probe diS-C3(3) reporting on membrane potential in S. cerevisiae is affected by the strain under study, cell-growth phase, starvation and by the concentration of glucose both in the growth medium and in the monitored cell suspension under non-growth conditions. Killer toxin K1 brings about changes in membrane potential. In all types of cells tested, viz. in glucose-supplied stationary or exponential cells of the killer-sensitive strain S6/1 or a conventional strain RXII, or in glucose-free exponential cells of both strains, both active and heat-inactivated toxin slow down the potential-dependent uptake of diS-C3(3) into the cells. This may reflect "clogging" of pores in the cell wall that hinders, but does not prevent, probe passage to the plasma membrane and its equilibration. The clogging effect of heat-inactivated toxin is stronger than that exerted by active toxin. In susceptible cells, i.e. in exponential-phase glucose-supplied cells of the sensitive strain S6/1, this phase of probe uptake retardation is followed by an irreversible red shift in probe fluorescence maximum lambda max indicating damage to membrane integrity and cell permeabilization. A similar fast red shift in lambda max signifying lethal cell damage was found in heat-killed or nystatin-treated cells.

• MeSH
• fluorescenční barviva metabolismus   MeSH
• fungální proteiny farmakologie   MeSH
• karbocyaniny metabolismus   MeSH
• killer faktory kvasinek MeSH
• membránové potenciály účinky léků   MeSH
• mykotoxiny farmakologie   MeSH
• nystatin farmakologie   MeSH
• Saccharomyces cerevisiae fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3,3'-dipropylthiacarbocyanine MeSH Prohlížeč
• fluorescenční barviva MeSH
• fungální proteiny MeSH
• K1 killer toxin MeSH Prohlížeč
• karbocyaniny MeSH
• killer faktory kvasinek MeSH
• mykotoxiny MeSH
• nystatin MeSH

• MeSH
• fluorescenční barviva * MeSH
• fluorescenční spektrometrie metody   MeSH
• karbocyaniny * MeSH
• membránové potenciály * MeSH
• Saccharomyces cerevisiae fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3,3'-dipropylthiacarbocyanine MeSH Prohlížeč
• fluorescenční barviva * MeSH
• karbocyaniny * MeSH