The amyloid deposition in the form of extracellular fibrillar aggregates of amyloid-β (Aβ) peptide is a critical pathological event in Alzheimer’s disease. Here, we report a systematic investigation of the effects of environmental factors on the kinetics of Aβ fibrillization in vitro. The effects of Aβ42 peptide concentration, temperature, pH, added solvents and the ratio of Aβ40 and Aβ42 on the peptide fibrillization under agitated conditions was studied. The analysis show that the rate of fibril growth by monomer addition is not limited by diffusion but by rearrangement in the monomer structure, which is enhanced by low concentrations of fluorinated alcohols and characterized by the activation energy of 12 kcal/mol. Fibrillization rate decreases at pH values below 7.0 where simultaneous protonation of His 13 and 14 inhibits fibril formation. The lag period for Aβ42 was only twofold shorter and the fibril growth rate twofold faster than those of Aβ40. Lag period was shortened and the fibrillization rate was increased only at 90% content of Aβ42.

The progression of Alzheimer’s disease is causatively linked to the accumulation of amyloid-β aggregates in the brain, however, it is not clear how the amyloid aggregates initiate the death of neuronal cells. The in vitro toxic effects of amyloid peptides are most commonly examined using the human neuroblastoma derived SH-SY5Y cell line and here we show that differentiated neuron-like SH-SY5Y cells are more sensitive to amyloid peptides than non-differentiated cells, because the latter lack long neurites. Exogenous soluble amyloid-β 1–42 covered cell bodies and whole neurites in differentiated cells with dense fibrils, causing neurite beading and fragmentation, whereas preformed amyloid-β 1–42 fibrils had no toxic effects. Importantly, spontaneously fibrillizing amyloid-β 1–42 peptide exhibited substantially higher cellular toxicity than amyloid-β 1–40, which did not form fibrils under the experimental conditions. These results support the hypothesis that peptide toxicity is related to the active fibrillization process in the incubation mixture.

Many peptides and proteins can form fibrillar aggregates in vitro, but only a limited number of them are forming pathological amyloid structures in vivo. We studied the fibrillization of four peptides--Alzheimer's amyloid-β (Aβ) 1-40 and 1-42, amylin and insulin. In all cases, intensive mechanical agitation of the solution initiated fast fibrillization. However, when the mixing was stopped during the fibril growth phase, the fibrillization of amylin and insulin was practically stopped, and the rate for Aβ40 substantially decreased, whereas the fibrillization of Aβ42 peptide continued to proceed with almost the same rate as in the agitated conditions. The reason for the different sensitivity of the in vitro fibrillization of these peptides towards agitation in the fibril growth phase remains elusive.

Clinical use of CuO nanoparticles (NPs) as antibacterials can be hampered by their toxicity to human cells. We hypothesized that certain surface functionalizations of CuO NPs may render NPs toxic to bacteria, but still be relatively harmless to human cells. To control this hypothesis, the toxicity of differently functionalized CuO NPs to bacteria Escherichia coli vs human cells (THP-1 macrophages and HACAT keratinocytes) was compared using similar conditions and end points. CuO NPs functionalized with polyethylene glycol (CuO-PEG), carboxyl (CuO-COOH, anionic), ammonium (CuO-NH 4 + , cationic) and unfunctionalized CuO NPs and CuSO 4 (controls) were tested. In general, the toxicity of Cu compounds decreased in the following order: CuO-NH 4 + > unfunctionalized CuO > CuSO 4 > CuO-COOH > CuO-PEG. Positively charged unfunctionalized CuO and especially CuO-NH 4 + proved most toxic (24-h EC 50 = 21.7-47 mg/l) and had comparable toxicity to bacterial and mammalian cells. The multivariate analysis revealed that toxicity of these NPs was mostly attributed to their positive zeta potential, small hydrodynamic size, high Cu dissolution, and induction of reactive oxygen species (ROS) and TNF-α. In contrast, CuO-COOH and CuO-PEG NPs had lower toxicity to human cells compared to bacteria despite efficient uptake of these NPs by human cells. In addition, these NPs did not induce TNF-α and ROS. Thus, by varying the NP functionalization and Cu form (soluble salt vs NPs), it was possible to "target" the toxicity of Cu compounds, whereas carboxylation and PEGylation rendered CuO NPs that were more toxic to bacteria than to human cells envisaging their use in medical antibacterial products.

Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.