Percorrer por autor "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.
- The growth curve method to rapidly derive the antibacterial potential of polyoxovanadatesPublication . Marques-da-Silva, Dorinda; Mal, Sib Sankar; Aureliano, Manuel; Lagoa, RicardoIn previous studies (Marques-da-Silva et al., 2019), we measured the minimum inhibitory concentrations (MICs) of three polyoxovanadates, namely V10, MnV11, and MnV13, against Escherichia coli. MICs were obtained following the standard method, which requires a 16–20 h culture and might neglect the effects of the compounds’ metabolism during incubation. In this work, we studied the action of those compounds against Enterococcus faecalis by monitoring the bacterial growth kinetics, and we observed that the inhibition was evident from the beginning of the exponential phase. Notably, data collected from just a 7 h culture was enough to identify the compounds with stronger antibacterial activity according to standard MICs.
- 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.