Valorization of Apple Peels through the Study of the Effects on the Amyloid Aggregation Process of κ-Casein.

Waste valorization represents one of the main social challenges when promoting a circular economy and environmental sustainability. Here, we evaluated the effect of the polyphenols extracted from apple peels, normally disposed of as waste, on the amyloid aggregation process of κ-casein from bovine milk, a well-used amyloidogenic model system. The effect of the apple peel extract on protein aggregation was examined using a thioflavin T fluorescence assay, Congo red binding assay, circular dichroism, light scattering, and atomic force microscopy. We found that the phenolic extract from the peel of apples of the cultivar "Fuji", cultivated in Sicily (Caltavuturo, Italy), inhibited κ-casein fibril formation in a dose-dependent way. In particular, we found that the extract significantly reduced the protein aggregation rate and inhibited the secondary structure reorganization that accompanies κ-casein amyloid formation. Protein-aggregated species resulting from the incubation of κ-casein in the presence of polyphenols under amyloid aggregation conditions were reduced in number and different in morphology.

Different chloride electrochemical gradients across the plasma membrane in subcellular compartments of rat cerebellum granules.

The effects of GABA on intracellular Ca2+ have been studied in neonatal rat cerebellum granule cells (CGC) in culture by Oregon Green and two-photon excitation fluorescence microscopy. This technique allowed the study of [Ca2+]i both in cell bodies and neurites. Working with a perfusion chloride concentration corresponding to the average extracellular level, we found that GABA induced an increase in [Ca2+]i in the cell bodies in many of the cells studied with a maximum at day 4 in vitro. This effect disappeared after day 6. However, no increase in [Ca2+]i was ever found in neurites at standard [Cl-]e. On the other hand, an increase of [Ca2+]i was found also in neurites when [Cl-]e was close to zero. The [Ca2+]i increases were blocked by both bicuculline methiodide and nimodipine. The results indicate the presence of an outward directed electrochemical gradient for chloride in the cell bodies which results in depolarization by GABA via GABA(A) receptor activation. Calcium ion influx ensues due to activation of voltage-gated calcium channels (VGCC). This phenomenon may mediate the well-known trophic effect of GABA on these cells at this developmental stage, via an action of [Ca2+]i on the transcriptional activity of the nucleus. No calcium accumulation takes place in neurites due to either no or a reverse (hyperpolarizing) electrochemical gradient for chloride ions. Such a circumstance in later developmental stages may be of importance for the phasic component of GABA-mediated inhibition.