Ruolo delle specie reattive dell'ossigeno e di caveole/raft lipidici nella biosegnalazione in linee cellulari umane

Membrane lipid rafts are detergent-resistant microdomains containing glycosphingolipids, cholesterol and glycosylphosphatidylinositol-linked proteins; they seem to be actively involved in many cellular processes including signal transduction, apoptosis, cell adhesion and migration. Lipid rafts may represent important functional platforms where redox signals are produced and transmitted in response to various agonists or stimuli. In addition, a new concept is emerging that could be used to define the interactions or amplification of both redox signalling and lipid raft-associated signalling. This concept is characterized by redox-mediated feed forward amplification in lipid platforms. It is proposed that lipid rafts are formed in response to various stimuli; for instance, NAD(P)H oxidase (Nox) subunits are aggregated or recruited in these platforms, increasing Nox activity. Superoxide and hydrogen peroxide generation could induce various regulatory activities, such as the induction of glucose transport activity and proliferation in leukaemia cells. The aim of our study is to probe: i) the involvement of lipid rafts in the modulation of the glucose transporter Glut1 in human acute leukemia cells; ii) the involvement of plasma membrane caveolae/lipid rafts in VEGF-mediated redox signaling via Nox activation in human leukemic cells; iii) the role of p66shc, an adaptor protein, in VEGF signaling and ROS production in endothelial cells (ECs); iv) the role of Sindecan-2, a transmembrane heparan sulphate proteoglycan, in VEGF signaling and physiological response in ECs and v) the antioxidant and pro-apoptotic activities of simple dietary phenolic acids, i. e. caffeic, syringic and protocatechuic acids in leukemia cells, characterized by a very high ROS content. Our results suggest that the role played by NAD(P)H oxidase-derived ROS in the regulation of glucose uptake, proliferation and migration of leukaemia and endothelial cells could likely occur through the control of lipid raft-associated signalling.

Adattamento e acclimatazione a diverse temperature di lieviti psicrofili obbligati e facoltativi e di lieviti mesofili. Studio della produzione di acidi grassi polinsaturi omega-3 e omega-6 per via fermentativa.

Adaptation and acclimation to different temperatures of obligate psychrophilic, facultative psychrophilic and mesophilic yeasts. Production of ω-3 and ω-6 polyunsaturated fatty acids by fermentative way. Obligate psychrophilic, facultative psychrophilic and mesophilic yeasts were cultured in a carbon rich medium at different temperatures to investigate if growth parameters, lipid accumulation and fatty acid composition were adaptive and/or acclimatory responses. Acclimation of facultative psychrophiles and mesophiles to lower temperature negatively affected their specific growth rate. Obligate psychrophiles exhibited the highest biomass yield (YX/S), followed by facultative psychrophiles, then by mesophiles. The growth temperature did not influence the YX/S of facultative psychrophiles and mesophiles. Acclimation to lower temperature caused the increase in lipid yield (YL/X) in mesophilic yeasts, but did not affect YL/X in facultative psychrophiles. Similar YL/X were found in both facultative and obligated psychrophiles, suggesting that lipid accumulation is not a distinctive character of adaptation to permanently cold environments. The extent of unsaturation of fatty acids was one major adaptive feature of the yeasts which colonize permanently cold ecosystems. Remarkable amounts of α-linolenic acid were found in obligate psychrophiles at the expenses of linoleic acid, whereas it was generally scarce or absent in all the others strains. Increased unsaturation of fatty acids was also an acclimatory response of mesophiles and facultative psychrophiles to lower temperature. It’s well known that omega-3 polyunsaturated fatty acids (PUFAs) display a variety of beneficial effects on various organ systems and diseases, therefore a process for the microbial production of omega-3 PUFAs would be of great interest. This work sought also to investigate if one of the better psychrophilic yeast, Rhodotorula glacialis DBVPG 4785, stimulated by acclamatory responses, produced omega-3 PUFAs. In fact, the adaptation of psychrophilic yeasts to cold niches is related to the production of higher amounts of lipids and to increased unsaturation degree of fatty acids, presumably to maintain membrane fluidity and functionality at low temperatures. Bioreactor fermentations of Rhodotorula glacialis DBVPG 4785 were carried out at 25, 20, 15, 10, 5, 0, and -3°C in a complex medium with high C:N ratio for 15 days. High biomass production was attained at all the temperatures with a similar biomass/glucose yield (YXS), between 0.40 and 0.45, but the specific growth rate of the strain decreased as the temperature diminished. The coefficients YL/X have been measured between a minimum of 0.50 to a maximum of 0.67, but it was not possible to show a clear effect of temperature. Similarly, the coefficient YL/S ranges from a minimum of 0.22 to a maximum of 0.28: again, it does not appear to be any significant changes due to temperature. Among omega-3 PUFAs, only α-linolenic acid (ALA, 18:3n-3) was found at temperatures below to 0°C, while, it’s remarkable, that the worthy arachidonic acid (C20:4,n-6), stearidonic acid (C20:4,n-3) C22:0 and docosahexaenoic acid (C22:6n-3) were produced only at the late exponential phase and the stationary phase of batch fermentations at 0 and -3°C. The docosahexaenoic acid (DHA) is a beneficial omega-3 PUFA that is usually found in fatty fish and fish oils. The results herein reported improve the knowledge about the responses which enable psychrophilic yeasts to cope with cold and may support exploitation of these strains as a new resource for biotechnological applications.