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281 results on '"Bubacco L."'

1. Number and Brightness analysis of alpha-synuclein oligomerization and the associated mitochondrial morphology alterations in live cells.

Author

Plotegher, N, Bubacco, L, and Gratton, Enrico

Subjects
Fluorescence, Live imaging, Lysosomes, Oligomers, Parkinsons disease, Protein aggregation, Calibration, Cell Line, Tumor, Cell Survival, Humans, Lysosomes, Mitochondria, Protein Multimerization, alpha-Synuclein
Abstract

BACKGROUND: Alpha-synuclein oligomerization is associated to Parkinsons disease etiopathogenesis. The study of alpha-synuclein oligomerization properties in live cell and the definition of their effects on cellular viability are among fields expected to provide the knowledge required to unravel the mechanism(s) of toxicity that lead to the disease. METHODS: We used Number and Brightness method, which is a method based on fluorescence fluctuation analysis, to monitor alpha-synuclein tagged with EGFP aggregation in living SH-SY5Y cells. The presence of alpha-synuclein oligomers detected with this method was associated with intracellular structure conditions, evaluated by fluorescence confocal imaging. RESULTS: Cells overexpressing alpha-synuclein-EGFP present a heterogeneous ensemble of oligomers constituted by less than 10 monomers, when the protein approaches a threshold concentration value of about 90nM in the cell cytoplasm. We show that the oligomeric species are partially sequestered by lysosomes and that the mitochondria morphology is altered in cells presenting oligomers, suggesting that these mitochondria may be dysfunctional. CONCLUSIONS: We showed that alpha-synuclein overexpression in SH-SY5Y causes the formation of alpha-synuclein oligomeric species, whose presence is associated with mitochondrial fragmentation and autophagic-lysosomal pathway activation in live cells. GENERAL SIGNIFICANCE: The unique capability provided by the Number and Brightness analysis to study alpha-synuclein oligomer distribution and properties, and the study of their association to intracellular components in single live cells is important to forward our understanding of the molecular mechanisms of Parkinsons disease and it may be of general significance when applied to the study of other aggregating proteins in cellular models.

Published
2014

3. DOPAL initiates αSynuclein-mediated impaired proteostasis in neuronal projections leading to enhanced vulnerability in Parkinson’s disease

Author

Masato, A., primary, Plotegher, N., additional, Thor, A., additional, Adams, S., additional, Sandre, M., additional, Cogo, S., additional, De Lazzari, F., additional, Fontana, C. M., additional, Martinez, P. A., additional, Strong, R., additional, Bellucci, A., additional, Bisaglia, M., additional, Greggio, E., additional, Valle, L. Dalla, additional, Boassa, D., additional, and Bubacco, L., additional

Published
2021
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8. Synapsin III is a key component of α-synuclein fibrils in Lewy bodies of Parkinson’s disease and dementia with Lewy bodies

Author

Longhena, F., Faustini, G., Varanita, T., Zaltieri, M., Porrini, V., Tessari, I., Poliani, P. L., Missale, C., Borroni, B., Padovani, A., Bubacco, L., Pizzi, M., Spano, Pf., and Bellucci, A.

Subjects
parkinson's disease, Dementia with Lewy bodies, alpha synuclein, parkinson's disease, alpha synuclein ,synapsin III, Dementia with Lewy bodies, synapsin III
Published
2017

23. A protein-based oxygen biosensor for high-throughput monitoring of cell growth and cell viability

Author

Strianese, M., Zauner, G., Tepper, A.W.J.W., Bubacco, L., Breukink, E.J., Aartsma, J., Canters, G.W., Tabares, L.C., Biochemistry of membranes, and Dep Scheikunde

Abstract

Fluorescently labeled hemocyanin has been previously proposed as an oxygen sensor. In this study, we explored the efficacy of this biosensor for monitoring the biological oxygen consumption of bacteria and its use in testing bacterial cell growth and viability of Escherichia coli, Pseudomonas aeruginosa, Paracoccus denitrificans, and Staphylococcus simulans. Using a microwell plate, the time courses for the complete deoxygenation of samples with different initial concentrations of cells were obtained and the doubling times were extracted. The applicability of our fluorescence-based cell growth assay as an antibacterial drug screening method was also explored. The results provide a proof-of-principle for a simple, quantitative, and sensitive method for high-throughput monitoring of prokaryotic cell growth and antibiotic susceptibility screening.

Published
2009

25. Kinetic and structural analysis of the early oxidation products of dopamine - Analysis of the interactions with alpha-synuclein

Author

Bisaglia M., Mammi S., and Bubacco L.

Abstract

Oxidative stress appears to be directly involved in the pathogenesis of several neurodegenerative disorders, including Alzheimer and Parkinson diseases. Nigral dopaminergic neurons are particularly exposed to oxidative stress because a pathological accumulation of cytosolic dopamine gives rise to various toxic molecules, including free radicals and reactive quinones. These latter species can react with proteins preventing them from exerting their physiological functions. Among the possible targets of quinones, alpha-synuclein is of primary interest because of its direct involvement in dopamine metabolism. Contrary to the neurotoxic processes, neuromelanin synthesis seems to play a protective role by its ability to sequester a variety of potentially damaging substances. In this study, we carried out a kinetic and structural analysis of the early oxidation products of dopamine. Specifically, considering the potential high toxicity of aminochrome for both cells and mitochondria, we focused our attention on its rearrangement to 5,6-dihydroxyindole. After the spectroscopic characterization of the products derived from the oxidation of dopamine, the structural information obtained was used to analyze the reactivity of quinones toward alpha-synuclein. Our results suggest that indole-5,6-quinone, rather than dopamine-o-quinone or aminochrome, is the reactive species. We propose that the observed reactivity could represent a general reaction pathway whenever cysteinyl residues are absent in proteins or if they are sterically protected.

Published
2007

29. Crystal structure of human C53A DJ-1

Author

Cendron, L., primary, Girotto, S., additional, Bisaglia, M., additional, Tessari, I., additional, Mammi, S., additional, Zanotti, G., additional, and Bubacco, L., additional

Published
2014
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32. Tyrosinase-catalyzed oxidation of fluorophenols

Author

Battaini, G., Monzani, E., Casella, L., Lonardi, E., Tepper, A.W.J.W., Canters, G.W., and Bubacco, L.

Abstract

The activity of the type 3 copper enzyme tyrosinase toward 2-, 3-, and 4-fluorophenol was studied by kinetic methods and H-1 and F-19 NMR spectroscopy. Whereas 3- and 4-fluorophenol react with tyrosinase to give products that undergo a rapid polymerization process, 2-fluorophenol is not reactive and actually acts as a competitive inhibitor in the enzymatic oxidation of 3,4-dihydroxyphenylalanine (L-dopa). The tyrosinase-mediated polymerization of 3- and 4-fluorophenols has been studied in detail. It proceeds through a phenolic coupling pathway in which the common reactive fluoro-quinone, produced stereospecifically by tyrosinase, eliminates an inorganic fluorine ion. The enzymatic reaction studied as a function of substrate concentration shows a prominent lag that is completely depleted in the presence Of L-dopa. The kinetic parameters of the reactions can be correlated to the electronic and steric effects of the fluorine substituent position. Whereas the fluorine electron withdrawing effect appears to control the binding of the substrates (K-m for 3- and 4-fluorophenols and K-1 for 2-fluorophenol), the k(cat) parameters do not follow the expected trend, indicating that in the transition state some additional steric effect rules the reactivity.

Published
2002

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