Tyrosine nitration pattern during <i>Trypanosoma cruzi</i> trypomastigotes adhesion to extracellular matrix.

<p>Trypomastigotes (1x10⁹) were incubated with ECM (1.5 mg) in phenol red free-MEM, supplemented with 2% FBS, at 37°C and 5% CO₂, for different incubation times. Proteins (50 μg) were resolved in 6–16% gradient SDS-PAGE. The immunoblotting was performed using the antibodies: rabbit anti-TyrNO₂ 1:2,000, mouse anti-α-tubulin 1:5,000 as a load control and the secondary HRP conjugated anti-rabbit or anti-mouse 1:8,000 antibodies. The figure is representative of three independent experiments.</p

Presence of S-nitrosylated proteins in <i>Trypanosoma cruzi</i> trypomastigotes during adhesion to the extracellular matrix.

<p>Trypomastigotes (1x10⁹) were incubated with ECM (1.5 mg) in phenol red free-MEM, supplemented with 2% FBS, for 2 h at 37°C and 5% CO₂. Parasites were submitted to the immunofluorescence protocol. S-nitrosylated proteins are stained in red, alpha-tubulin in green, and nucleus and kinetoplast in blue (DAPI). The white bar represents 3.2 μm.</p

The effect of nitric oxide availability on <i>Trypanosoma cruzi</i> protein S-nitrosylation.

<p>Trypomastigotes (1x10⁹) were incubated with ECM (1.5 mg) in phenol red free-MEM, supplemented with 2% FBS, for 2 h at 37°C and 5% CO₂, in presence or absence of 100 μM cPTIO or CysNO. (A) Total protein S-NO was quantified in the parasite lysate using the Saville-Griess method. Asterisks represent a p<0.01 according to one-way ANOVA. (B) Parasite cell viability (1x10⁷/well in a 96 well plate) was carried out by following the reduction of WST-1 reagent (Roche) as described by the manufacturer. Differences are not statistically significant. Values are the mean of three independent experiments.</p

Presence of tyrosine nitrated proteins in <i>Trypanosoma cruzi</i> trypomastigotes during adhesion to the extracellular matrix.

<p>Trypomastigotes (1x10⁹) were incubated with ECM (1.5 mg) in phenol red free-MEM, supplemented with 2% FBS, for 2 h at 37°C and 5% CO₂. Parasites were submitted to the immunofluorescence protocol. Tyrosine nitrated proteins are stained in red, alpha-tubulin is stained in green, nucleus and kinetoplast are stained in blue (DAPI). The white bar represents 3.2 μm. The image is representative of two experiments.</p

Nitric oxide synthase activity during <i>Trypanosoma cruzi</i> trypomastigotes adhesion to extracellular matrix.

<p>Trypomastigotes (1x10⁹) were incubated with ECM (1.5 mg) in phenol red free-MEM, supplemented with 2% FBS, for 2 h at 37°C and 5% CO_2. The inhibitor L-NAME (10 mM) was added after the extract preparation. (A) •NO concentration in the medium was measured employing a modified fluorescent reaction of DAF-2 (Measure-iT High-Sensitivity Nitrite Assay Kit). (B) NOS activity was measured in <i>T</i>. <i>cruzi</i> extracts by the conversion of [³H]arginine to [³H]citrulline in <i>T</i>. <i>cruzi</i> extracts. The immunoblots were probed with anti-tubulin antibody to guarantee that the protein loading in each track was similar. The levels of intracellular L-Arginine (C) and L-citrulline (D) were obtained by the capillary electrophoresis method. Points marked with an asterisk represent a p<0.001 (one-way ANOVA) and with a dot represent p<0.001 in unpaired t-Student test. Results in (A) and (B) are the mean of three independent experiments and (C) and (D) are the mean of five independent experiments.</p

Validation of <i>T</i>. <i>cruzi</i> cysteine nitrosylated proteins.

<p>(A). S-Nitrosylated proteins from ECM-incubated and ECM-non-incubated parasites were isolated by biotin switch and streptavidin pull down methodology and submitted to immunoblotting using anti-mucin II antibodies. Control of protein input and negative control (the biotin switch methodology in the absence of ascorbate) are shown. <i>T</i>. <i>cruzi</i> control (Ty) and treated with ECM for 2 hours (Ty + ECM). (B) Quantification of the results presented in (A). The asterisk represents a p<0.001 according to <i>t-Student</i> test.</p

S-nitrosylation pattern during <i>Trypanosoma cruzi</i> trypomastigotes adhesion to extracellular matrix.

<p>Trypomastigotes (1x10⁹) were incubated with ECM (1.5 mg) in phenol red free-MEM, supplemented with 2% FBS, at 37°C and 5% CO₂, for different incubation times. Proteins (50 μg) were resolved in 6–16% gradient SDS-PAGE. The immunoblotting was performed using the antibodies: rabbit anti-SNO 1:2,000, and the secondary HRP conjugated anti-rabbit 1:8,000. The Ponceau’s staining was used as a load control. Arrows indicate protein bands in which the signal intensity is decreased on the group incubated with the extracellular matrix in relation to the equivalent group incubated with growth media alone. The figure is representative of three experiments.</p

Validation of <i>T</i>. <i>cruzi</i> tyrosine nitrated proteins.

<p>Proteins of interest were immunoprecipitated (IP) with specific antibodies (anti histone H2A, anti-histone H4, anti-enolase, anti-alpha tubulin, anti-beta tubulin or anti-paraflagellar rod proteins antibodies) and submitted to immunoblotting employing anti-nitro-tyrosine antibodies. A 150 kDa band of the blotting (A, B and D) or anti-tubulin antibodies (C) are shown as controls of the protein loading. <i>T</i>. <i>cruzi</i> control (Ty) and treated with ECM for 2 hours (Ty + ECM).</p

cGMP responses during <i>Trypanosoma cruzi</i> trypomastigotes adhesion to extracellular matrix.

<p>Trypomastigotes (1x10⁹) were incubated with ECM (1.5 mg) in phenol red free-MEM, supplemented with 2% FBS, for 2 h at 37°C and 5% CO₂. cGMP levels were measured in <i>T</i>. <i>cruzi</i> extracts by competitive ELISA. The asterisk represents a p<0.001 according to <i>t-Student</i> test. Values are the mean of four independent experiments.</p