Browsing by Author "Marques-da-Silva, Dorinda"
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- Early disruption of the actin cytoskeleton in cultured cerebellar granule neurons exposed to 3-morpholinosydnonimine-oxidative stress is linked to alterations of the cytosolic calcium concentrationPublication . Tiago, Teresa; Marques-da-Silva, Dorinda; Samhan-Arias, A. K.; Aureliano, M.; Gutiérrez-Merino, CarlosCytoskeleton damage is a frequent feature in neuronal cell death and one of the early events in oxidantinduced cell injury. This work addresses whether actin cytoskeleton reorganization is an early event of SIN-1-induced extracellular nitrosative/oxidative stress in cultured cerebellar granule neurons (CGN). The actin polymerization state, i.e. the relative levels of G-/F-actin, was quantitatively assessed by the ratio of the fluorescence intensities of microscopy images obtained from CGN double-labelled with Alexa594- DNase-I (for actin monomers) and Bodipy-FL-phallacidin (for actin filaments). Exposure ofCGNto a flux of peroxynitrite as low as 0.5–1 M/min during 30 min (achieved with 0.1mMSIN-1) was found to promote alterations of the actin cytoskeleton dynamics as it increases the G-actin/F-actin ratio. Because L-type voltage-operated Ca2+ channels (L-VOCC) are primary targets in CGN exposed to SIN-1, the possible role of Ca2+ dynamics on the perturbation of the actin cytoskeleton was also assessed from the cytosolic Ca2+ concentration response to the L-VOCC’s agonist FPL-64176 and to the L-VOCC’s blocker nifedipine. The results showed that SIN-1 induced changes in the actin polymerization state correlated with its ability to decrease Ca2+ influx through L-VOCC. Combined analysis of cytosolic Ca2+ concentration and G-actin/Factin ratio alterations by SIN-1, cytochalasin D, latrunculin B and jasplakinolide support that disruption of the actin cytoskeleton is linked to cytosolic calcium concentration changes.
- Identification of new targets of S-nitrosylation in neural stem cells by thiol redox proteomicsPublication . Santos, Ana Isabel; Lourenco, Ana S.; Simão, Sónia; Marques-da-Silva, Dorinda; Santos, Daniela F.; Carvalho, Ana Paula Onofre de; Pereira, Ana Catarina; Izquierdo-Álvarez, Alicia; Ramos, Elena; Morato, Esperanza; Marina, Anabel; Martínez-Ruiz, Antonio; Araújo, InêsNitric oxide (NO) is well established as a regulator of neurogenesis. NO increases the proliferation of neural stem cells (NSC), and is essential for hippocampal injury-induced neurogenesis following an excitotoxic lesion. One of the mechanisms underlying non-classical NO cell signaling is protein S-nitrosylation. This post-translational modification consists in the formation of a nitrosothiol group (R-SNO) in cysteine residues, which can promote formation of other oxidative modifications in those cysteine residues. S-nitrosylation can regulate many physiological processes, including neuronal plasticity and neurogenesis. In this work, we aimed to identify S-nitrosylation targets of NO that could participate in neurogenesis. In NSC, we identified a group of proteins oxidatively modified using complementary techniques of thiol redox proteomics. S-nitrosylation of some of these proteins was confirmed and validated in a seizure mouse model of hippocampal injury and in cultured hippocampal stem cells. The identified S-nitrosylated proteins are involved in the ERK/MAPK pathway and may be important targets of NO to enhance the proliferation of NSC.
- Polyoxovanadate inhibition of Escherichia coli growth shows a reverse correlation with Ca2+-ATPase inhibitionPublication . Marques-da-Silva, Dorinda; Fraqueza, Gil; Lagoa, Ricardo; Vannathan, Anjana Anandan; Mal, Sib Sankar; Aureliano, ManuelRecently, a global analysis of the structure-activity-relationship of a series of polyoxometalates (POMs) revealed that the most active POMs were ascribed to be polyoxovanadates (POVs), especially decavanadate (V-10), which was very active against certain bacteria (Bijelic et al., Chem. Commun., 2018). The present study explores this observation and compares the effects of three POVs namely MnV11, MnV13 and V-10 against Escherichia coli growth. It was observed that MnV11 presents the lowest growth inhibition (GI(50)) value for Escherichia coli followed by the MnV13 compound, being about 2 times lower than that of V-10 respectively, the values obtained were 0.21, 0.27 and 0.58 mM. All three compounds were more effective than vanadate alone (GI(50) = 1.1 mM) and also than decaniobate, Nb-10 (GI(50) > 10 mM), an isostructural POM of V-10. However, the POVs exhibiting the highest antibacterial activity (MnV11) were shown to have the lowest Ca2+-ATPase inhibitor capacity (IC50 = 58 mM) whereas decavanadate, which was also very active against this membranar ATPase (IC50 = 15 mM), was less active against bacterial growth, suggesting that POV inhibition of ion pumps might not be associated with the inhibition of Escherichia coli growth.