Chaperonin of Group I: Oligomeric Spectrum and Biochemical and Biological Implications.

Chaperonins play various physiological roles and can also be pathogenic. Elucidation of their structure, e.g., oligomeric status and post-translational modifications (PTM), is necessary to understand their functions and mechanisms of action in health and disease. Group I chaperonins form tetradecamers with two stacked heptameric rings. The tetradecamer is considered the typical functional complex for folding of client polypeptides. However, other forms such as the monomer and oligomers with smaller number of subunits than the classical tetradecamer, also occur in cells. The properties and functions of the monomer and oligomers, and their roles in chaperonin-associated diseases are still incompletely understood. Chaperonin I in eukaryotes occurs in various locations, not just the mitochondrion, which is its canonical place of residence and function. Eukaryotic Chaperonin I, namely Hsp60 (designated HSP60 or HSPD1 in humans) has, indeed, been found in the cytosol; the plasma-cell membrane; on the outer surface of cells; in the intercellular space; in biological liquids such as lymph, blood, and cerebrospinal fluid; and in secretions, for instance saliva and urine. Hsp60 has also been found in cell-derived vesicles such as exosomes. The functions of Hsp60 in all these non-canonical locales are still poorly characterized and one of the questions not yet answered is in what form, i.e., monomer or oligomer, is the chaperonin present in these non-canonical locations. In view of the steady increase in interest on chaperonopathies over the last several years, we have studied human HSP60 to determine its role in various diseases, its locations in cells and tissues and migrations in the body, and its post-translational modifications that might have an impact on its location and function. We also carried out experiments to characterize the oligomeric status of extramitochondrial of HSP60 in solution. Here, we provide an overview of our results, focusing on the oligomeric equilibrium and stability of the various forms of HSP60 in comparison with GroEL. We also discuss post-translational modifications associated with anti-cancer drugs to indicate the potential of Hsp60 in Medicine, as a biomarker and etiopathogenic factor.

Insulin-activated Akt rescues Aß oxidative stress-induced cell death by orchestrating molecular trafficking.

Increasing evidence indicates that Alzheimer's disease, one of the most diffused aging pathologies, and diabetes may be related. Here, we demonstrate that insulin signalling protects LAN5 cells by amyloid-ß42 (Aß)-induced toxicity. Aß affects both activation of insulin receptors and the levels of phospho-Akt, a critical signalling molecule in this pathway. In contrast, oxidative stress induced by Aß can be antagonized by active Akt that, in turn, inhibits Foxo3a, a pro-apoptotic transcription factor activated by reactive oxygen species generation. Insulin cascade protects against mitochondrial damage caused by Aß treatment, restoring the mitochondrial membrane potential. Moreover, we show that the recovery of the organelle integrity recruits active Akt translocation to the mitochondrion. Here, it plays a role both by maintaining unimpaired the permeability transition pore through increase in HK-II levels and by blocking apoptosis through phosphorylation of Bad, coming from cytoplasm after Aß stimulus. Together, these results indicate that the Akt survival signal antagonizes the Aß cell death process by balancing the presence and modifications of common molecules in specific cellular environments.

Employment of cationic solid-lipid nanoparticles as RNA carriers.

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. In this article, the suitability of cationically modified solid-lipid nanoparticles (SLN) as a nonviral vector for gene delivery was investigated, in order to obtain stable materials able to condense RNA. Cationic SLN were produced by microemulsion using Compritol ATO 888 as matrix lipid, Pluronic F68 as tenside, and dimethyldioctadecylammonium bromide (DDAB) as cationic lipid. The resulting particles were approximately 100 nm in size and showed a highly positive surface charge (+41 mV) in water. Size and shape were further characterized by scanning electron microscopy (SEM) measurements. Moreover, we utilized the sea urchin as a model system to test their applicability on a living organism. To evaluate cationic SLN ability to complex the in vitro transcribed Paracentrotus lividus bep3 RNA, we utilized both light scattering and gel mobility experiments, and protection by nuclease degradation was also investigated. By microinjection experiment, we demonstrated that the nanoparticles do not inference with the viability of the P. lividus embryo and the complex nanoparticles-bep3 permits movement of the RNA during its localization in the egg, suggesting that it could be a suitable system for gene delivery. Taken together, all these results indicate that the cationic SNL are a good RNA carrier for gene transfer system and the sea urchin a simple and versatile candidate to test biological properties of nanotechnology devices.

Ordering of agarose near the macroscopic gelation point.

Gel formation and spatial structure is an important area of study in polymer physics and in macromolecular and cellular biophysics. Agarose has a sufficiently complex gelation mechanism to make it an interesting prototype for many other gelling systems, including those involved in amyloid fibrillogenesis. Static (over a scattering vector range of 0.1-30 microm(-1)) and dynamic light scattering and rheology methods were used to follow the gelation kinetics of agarose at 0.5% in water or in the presence of 25 mM NaCl and quenched to temperatures of 20-43 degrees C. Light scattering results on gelling samples are fully described by a fractal aggregate model with four physically meaningful parameters. In all cases aggregates, with fractal dimensions at or near 3, form more rapidly and are smaller in characteristic size at lower quench temperatures. A region three to four times larger than the aggregate becomes depleted of agarose as the gelation proceeds. Below about 30 degrees C the aggregation process freezes spatial ordering rapidly, resulting in fragile macroscopic gels as determined by rheology. Salt effects are seen to be minimal and not important in the fundamental aggregation mechanism.