Padé-Laplace method for analysis of fluorescence intensity decay.

This novel approach to the analysis of multiexponential functions is based on the combined use of the Laplace transform and Padé approximants (Yeramian, E., and P. Claverie. 1987. Nature (Lond.). 326:169-174). It is similar in principle to the well-known Isenberg method of moments (Isenberg, I. 1983. Biophys. J. 43:141-148) traditionally applied to the analysis of fluorescence decay. The advantage of the Padé-Laplace method lies in its ability to detect the number of components in a multiexponential function as well as their parameters. In this paper we modified the original method so that it can be applied to the analysis of multifrequency phase/modulation measurements of fluorescence decay. The method was tested first on simulated data. It afforded recovery up to four distinct lifetime components (and their fractional contributions). In the case of simulated data corresponding to continuous lifetime distributions (nonexponential decay), the results of the analysis by the Padé-Laplace method indicated the absence of discrete exponential components. The method was also applied to real phase/modulation data gathered on known fluorophores and their mixtures and on tryptophan fluorescence in phospholipase A2. The lifetime and fraction recoveries were consistent with those obtained from standard methods involving nonlinear least-square fitting

Cross-talk between calpain and caspase-3/-7 in cisplatin-induced apoptosis of melanoma cells: a major role of calpain inhibition in cell death protection and p53 status

4noreservedThe contribution of different proteolytic systems, in particular calpains and effector caspases, in apoptotic cell death is still controversial. In this paper, we show that during cisplatin-induced apoptosis of human metastatic melanoma cells, calpain activation, as measured in intact cells by two different fluorescent substrates, is an early event, taking place well before caspase-3/-7 activation, and progressively increasing during 48 h of treatment. Such activation appears to be independent from any intracellular calcium imbalance; in fact, an increase of cytosolic calcium along with emptying of the reticular stores occur only at very late stages, uniquely in frankly apoptotic, detached cells. Calpain activation proves to be an early and crucial event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples co-treated with the calpain inhibitors, MDL 28170, calpeptin and PD 150606, where a variable but significant reduction of both caspase-3/-7 activity and cell detachment is observed. Consistently, such a protective effect can be at least partially due to the impairment of cisplatin-induced p53 activation, occurring early in committed, preapoptotic cells. Furthermore, in late apoptotic cells, calpain activity is also responsible for the formation of a novel p53 proteolytic fragment (approximate to 26 kDa), whose function is so far to be elucidated.mixedDel Bello, Barbara; Moretti, Daniele; Gamberucci, Alessandra; Maellaro, EmiliaDel Bello, Barbara; Moretti, Daniele; Gamberucci, Alessandra; Maellaro, Emili