Digitonin-permeabilization of astrocytes in culture monitored by trypan blue exclusion and loss of S100B by ELISA

Tramontina, Francine; Karl, Juliana Damm; Gottfried, Carmem; Mendez, Andreas Sebastian Loureiro; Gonçalves, Daniela; Portela, Luis Valmor; Gonçalves, Carlos‐Alberto

doi:10.1016/s1385-299x(00)00041-6

Cited by 40 publications

(30 citation statements)

References 15 publications

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“…The cell pellets were washed twice with phosphate buffered saline (300 μL, pH 7.5) and centrifuged (2,000 g, 5 min, 4°C). Each washed cell pellet was lysed in 40 μL of digitonin solution (1.5 mM in 1% DMSO in deionised water, prepared according to Tramontina et al, (Tramontina et al 2000) and centrifuged (14000 rpm, 5 min, 4°C). The lysed cells in digitonin solution were incubated in a water bath (37°C, 10 min), followed by gentle shaking for 10 min at room temperature and re-centrifuged (14, 000 rpm, 5 min, 4°C).…”

Section: Reduced Glutathionementioning

confidence: 99%

The selective cytotoxicity of the alkenyl glucosinolate hydrolysis products and their presence in Brassica vegetables.

et al. 2015

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Cruciferous vegetable consumption correlates with reduced risk of cancer. This chemopreventative activity may involve glucosinolates and their hydrolysis products.Glucosinolate-derived isothiocyanates have been studied for their toxicity and chemopreventative properties, but other hydrolysis products (epithionitriles, nitriles) have not been thoroughly examined. We report that these hydrolysis products differ in their cytotoxicity to human cells, with toxicity most strongly associated with isothiocyanates rather than epithionitriles and nitriles. We explored mechanisms of this differential cytotoxicity by examining the role of oxidative metabolism, oxidative stress, mitochondrial permeability, reduced glutathione levels, cell cycle arrest and apoptosis.2-Propenylisothiocyanate and 3-butenylisothiocyanate both inhibited cytochome P450 1A (CYP1A) enzyme activity in CYP expressing MCL-5 cells at high cytotoxic doses.Incubation of MCL-5 cells with non-cytotoxic doses of 2-propenylisothiocyanate for 24 h resulted in a dose-dependent inhibition of ethoxyresorufin O-deethylase, yet failed to affect CYP1A1 mRNA expression indicating interference with enzyme activity rather than inhibition of transcription. Increased reactive oxygen species (ROS) production was observed only for 2-propenylisothiocyanate treatment. 2-Propenylisothiocyanate treatment lowered reduced glutathione levels whereas no changes were noted with 3,4-epithiobutylnitrile. Cell cycle analysis showed that 2-propenylisothiocyanate induced a G2/M block whereas other hydrolysis products showed only marginal effects. We found that 2-propenylisothiocyanate and 3-butenylisothiocyanate induced cell death predominantly via necrosis whereas, 3,4-epithiobutylnitrile promoted both necrosis and apoptosis. Thus the activity of glucosinolate hydrolysis products includes cytotoxicity that is compound-class specific and may contribute to their putative chemoprotection properties.

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Section: Reduced Glutathionementioning

confidence: 99%

The selective cytotoxicity of the alkenyl glucosinolate hydrolysis products and their presence in Brassica vegetables.

et al. 2015

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“…Endogenous extracellular S100B was measuerd by ELISA as previously described (Tramontina et al, 2000). Briefly, 50 µL of sample plus 50 µL of barbital buffer (pH 8.6) were applied to microtiter plates previously coated with monoclonal anti-S100B in carbonate buffer and blocked with 1% bovine serum albumin.…”

Section: Elisa For S100bmentioning

confidence: 99%

Early Activation of Extracellular Signal-Regulated Kinase Signaling Pathway in the Hippocampus is Required for Short-Term Memory Formation of a Fear-Motivated Learning

Tramontina

Souza

et al. 2006

Cell Mol Neurobiol

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1.S100B is a calcium-binding protein expressed and secreted by astrocytes, which has been implicated in glial-neuronal communication. Extracellular S100B appears to protect hippocampal neurons against toxic concentrations of glutamate. Here we investigated a possible autocrine role of S100B in glutamate uptake activity. 2. Astrocyte cultures were prepared of hippocampi from neonate Wistar rats. [(3)H] Glutamate uptake was measured after addition of S100B protein, antibody anti-S100B or TRTK-12, a peptide that blocks S100B activity mediated by the C-terminal region. 3.Antibody anti-S100B addition decreased glutamate uptake measured 30 min after medium replacement, without affecting cell integrity or viability. Moreover, low levels of S100B (less than 0.1 ng/mL) stimulated glutamate uptake measured immediately after medium replacement. 4. This finding reinforces the importance of astrocytes in the glutamatergic transmission, particularly the role of S100B neuroprotection against excitotoxic damage.

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“…When the cell membrane loses its integrity, large molecules become permeable, indicating a possible sign of cell death. [40][41][42][43] To evaluate the cytotoxicity of polymeric NPs, the MTT assay was used, which evaluates mitochondrial integrity and is widely used to check the cell activity. Figure 7E and F shows the cytotoxic activity of polymeric NPs in K562 cells.…”

mentioning

confidence: 99%

Encapsulation of <em>Piper cabralanum </em>(Piperaceae) nonpolar extract in poly(methyl methacrylate) by miniemulsion and evaluation of increase in the effectiveness of antileukemic activity in K562 cells

Mendes¹,

Filgueiras²,

Siqueira³

et al. 2017

IJN

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This study aimed to synthesize and characterize nanoparticles (NPs) of poly(methyl methacrylate) (PMMA) and evaluate their ability to incorporate plant extracts with antitumor activity and low dissolution in aqueous media. The extract used was n -hexane partition of the methanol extract of Piper cabralanum (PCA-HEX). PMMA NPs were obtained using the mini-emulsion method, which was able to encapsulate almost 100% of PCA-HEX. The synthesized polymeric particles presented with a size of 200 nm and a negative charge. Cytotoxicity tests by MTT and trypan blue assays showed that NPs without PCA-HEX did not kill leukemic cells (K562 cells). NPs containing PCA-HEX were able to enhance cell death when compared to pure extract. The results showed that PMMA NPs could be useful as a drug delivery system as they can enhance the antitumor activity of the PCA-HEX extract by more than 20-fold. PMMA NPs containing plant extracts with antitumor activities may be an alternative to control the evolution of diseases such as leukemia.

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Digitonin-permeabilization of astrocytes in culture monitored by trypan blue exclusion and loss of S100B by ELISA

Cited by 40 publications

References 15 publications

The selective cytotoxicity of the alkenyl glucosinolate hydrolysis products and their presence in Brassica vegetables.

The selective cytotoxicity of the alkenyl glucosinolate hydrolysis products and their presence in Brassica vegetables.

Early Activation of Extracellular Signal-Regulated Kinase Signaling Pathway in the Hippocampus is Required for Short-Term Memory Formation of a Fear-Motivated Learning

Encapsulation of <em>Piper cabralanum </em>(Piperaceae) nonpolar extract in poly(methyl methacrylate) by miniemulsion and evaluation of increase in the effectiveness of antileukemic activity in K562 cells

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