Pietro Chistolini

Distribution of GD3 in DPPC Monolayers: A Thermodynamic and Atomic Force Microscopy Combined Study

Gangliosides are the main component of lipid rafts. These microdomains, floating in the outer leaflet of cellular membrane, play a key role in fundamental cellular functions. Little is still known about ganglioside and phospholipid interaction. We studied mixtures of dipalmitoylphosphatidylcholine and GD3 (molar fraction of 0.2, 0.4, 0.6, 0.8) using complementary techniques: 1), thermodynamic properties of the Langmuir-Blodgett films were assessed at the air-water interface (surface tension, surface potential); and 2), three-dimensional morphology of deposited films on mica substrates were imaged by atomic force microscopy. Mixture thermodynamics were consistent with data in the literature. In particular, excess free energy was negative at each molar fraction, thus ruling out GD3 segregation. Atomic force microscopy showed that the height of liquid-condensed domains in deposited films varied with GD3 molar fraction, as compatible with a lipid aggregation model proposed by Maggio. No distinct GD3-rich domain was observed inside the films, suggesting that GD3 molecules gradually mix with dipalmitoylphosphatidylcholine molecules, confirming DeltaG data. Morphological analysis revealed that the shape of liquid-condensed domains is strongly influenced by the amount of GD3, and an interesting stripe-formation phenomenon was observed. These data were combined with the thermodynamic results and interpreted in the light of McConnells model.

Native metastable prefibrillar oligomers are the most neurotoxic species among amyloid aggregates

Many proteins belonging to the amyloid family share the tendency to misfold and aggregate following common steps, and display similar neurotoxicity. In the aggregation pathway different kinds of species are formed, including several types of oligomers and eventually mature fibers. It is now suggested that the pathogenic aggregates are not the mature fibrils, but the intermediate, soluble oligomers. Many kinds of aggregates have been described to exist in a metastable state and in equilibrium with monomers. Up to now it is not clear whether a specific structure is at the basis of the neurotoxicity. Here we characterized, starting from the early aggregation stages, the oligomer populations formed by an amyloid protein, salmon calcitonin (sCT), chosen due to its very slow aggregation rate. To prepare different oligomer populations and characterize them by means of photoinduced cross-linking SDS-PAGE, Energy Filtered-Transmission Electron Microscopy (EF-TEM) and Circular Dichroism (CD) spectroscopy, we used Size Exclusion Chromatography (SEC), a technique that does not influence the aggregation process leaving the protein in the native state. Taking advantage of sCT low aggregation rate, we characterized the neurotoxic potential of the SEC-separated, non-crosslinked fractions in cultured primary hippocampal neurons, analyzing intracellular Ca(2+) influx and apoptotic trend. We provide evidence that native, globular, metastable, prefibrillar oligomers (dimers, trimers and tetramers) were the toxic species and that low concentrations of these aggregates in the population was sufficient to render the sample neurotoxic. Monomers and other kind of aggregates, such as annular or linear protofibers and mature fibers, were totally biologically inactive.

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir–Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.

Immunogold localisation of P-glycoprotein in supported lipid bilayers by transmission electron microscopy and atomic force microscopy

In this study, purified P-glycoprotein molecules, a membrane drug pump responsible for the multidrug resistance phenomenon, were incorporated in model membranes deposited onto solid supports, according to the method described by Puu and Gustafson (1997). The insertion of proteins into planar supported model membranes is of interest, as the films are fundamental in biosensor applications and for the investigation of how proteins conform and aggregate in a lipid environment. In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different hydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir–Blodgett lipid monolayers on mica. After the labelling of P-glycoprotein with two well-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) were observed by transmission electron microscopy (TEM) and samples (b) by atomic force microscopy (AFM).The comparative analysis performed by TEM and AFM allowed us to demonstrate the successful insertion of P-glycoprotein in the model membranes and their stability under different environmental conditions (vacuum, air and water). P-glycoprotein appeared to maintain, after purification and insertion in lipid bilayers, a good part of its conformational features as shown by the P-glycoprotein segments bearing the specific monoclonal antibody epitopes.

Short- and long-term correlations in repetitive movements.

Time is ubiquitous in most of human daily activities. Over several decades, research has focused on the capacity to produce regularly timed movements (such as walking or chewing) or to synchronize a motor response with a sensory event (such as in dancing or ensemble music production). However, our knowledge of the temporal mechanisms underlying such processes has not been completely elucidated. In psychological research, the finger-tapping task has been successfully employed to investigate these mechanisms. In 1886, Stevens developed the synchronization– continuation (SC) paradigm: first, participants tap with their finger synchronously to a sequence of metronomic clicks, then the metronome is switched off and participants have to continue tapping at the same rate. This paradigm allows to get insight into the ability both to synchronize actions to external stimuli and produce precisely timed, self-paced movements. Though finger tapping is a rather simple task, as a matter of fact we do not tap that regularly or accurately. The resulting variability is supposed to be the manifestation of control processes underlying motor performance. In his seminal work, Stevens (1886) reported that inter-tap interval (ITI) variability can be observed at different time scales: short-term fluctuations, defined as a ‘‘constant zigzag’’, and long-term drifts, described as ‘‘larger and more primary waves’’. Performance variability has been mainly characterized through descriptive statistical analysis assessing mean and variance in ITI series. These static measures are based on the assumption that the single series elements are independent; as a consequence they cannot provide exhausting information on the temporal structure in the data. It has been shown that the data from serial interval production are anti-correlated (Wing and Kristofferson 1973); it means that an interval longer than the mean is frequently followed by an interval shorter than the mean and vice versa. Long-term correlations (LTCs) are frequently observed as slow fluctuations in self-paced finger tapping when the stabilizing effect of an external reference is absent (Madison 2004; Gilden et al. 1995). The occurrence of LTCs implies that an interval longer (shorter) than the mean is followed by an even longer (shorter) interval, so that when a fluctuation starts it tends to persist over time. LTCs have been generally addressed as drift and, as such, they have been regarded more as noise to be removed rather than a feature of the timing process. Only recently this issue has aroused a vivid interest and has been revisited under a different perspective. In 1995, Gilden et al. showed that lifetime of slow fluctuations in finger tapping and other cognitive tasks followed a power law, evidencing the presence of the so-called ‘‘1/f noise’’. This is a kind of correlated noise observed in the most diverse fields, such as physics, geology, physiology and economics. It derives its name from the shape that the series power spectrum shows in a double logarithmic plot. Since then an animated debate has started on the nature of these LTCs and it is still far from being concluded (Thornton and Gilden 2005; Wagenmakers et al. 2004). In cognitive psychology, the subjective rhythmization (SR) or grouping is a well-known perceptual phenomenon that occurs when one listens to a succession of isochronous I. Ruspantini (&) P. Chistolini Technologies and Health Department, Istituto Superiore di Sanitá, Rome, Italy e-mail: Irene.ruspantini@iss.it

Organ transplantation in Italy: Analysis of donors and recipients

The Istituto Superiore di Sanità (The Italian National Institute of Health) has been collecting information regarding organ donation and transplantation in Italy. Herein we describe organ procurement and transplant activity in Italy in the years 1992–1997 and show some of the characteristics regarding both donors and transplanted patients who received kidneys, hearts, livers and lungs. Although transplantation in Italy has been very difficult because of shortage of organ donors, national rates have been improving year after year. The present situation should ameliorate further to enable Italy to reach the same level of other advanced European countries.

Discrete Simulations of Cadaver Kidney Allocation Schemes

The thesis of this study is that the use of discrete simulations is the most appropriate instrument to support the definition of any organs allocation procedure. We studied the influence of recipient pool size on the probability of obtaining a good match grade and the exchange rate between regional and/or interregional organizations for two different hypothesis of national allocation schemes. We also showed the potential of simulation methods at assessing the most appropriate adjustments for the optimization of any allocation scheme.