Irreversible AE1 tyrosine phosphorylation leads to membrane vesiculation in G6PD deficient red cells

Background. While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs. Methodology/Principal Findings. Our results show that treatment of G6PD deficient red cells with diamide (0.25 mM) or divicine (0.5 mM) causes: (1) an increase in the oxidation and tyrosine phosphorylation of AE1; (2) progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3) parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation. Conclusions/Significance. Collectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products

Low low-density lipoprotein (LDL), cholesterol and triglycerides plasma levels are associated with reduced risk of arterial occlusive events in chronic myeloid leukemia patients treated with ponatinib in the real-life. A Campus CML study

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Irreversible AE1 Tyrosine Phosphorylation Leads to Membrane Vesiculation in G6PD Deficient Red Cells

Background: While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs. Methodology/Principal Findings: Our results show that treatment of G6PD deficient red cells with diamide (0.25 mM) or divicine (0.5 mM) causes: (1) an increase in the oxidation and tyrosine phosphorylation of AE1; (2) progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3) parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation. Conclusions/Significance: Collectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products

No red blood cell damage and no hemolysis in G6PD-deficient subjects after ingestion of low vicine/convicine <em>Vicia faba</em> seeds

International audienc

Novel monoclonal antibody-based <i>Helicobacter pylori</i> stool antigen test

Background. A number of noninvasive tests have been developed to establish the presence of Helicobacter pylori infection. Although polyclonal antibody-based stool antigen testing has a good sensitivity and specificity, it is less accurate than urea breath testing. Recently, a monoclonal antibody-based stool antigen test demonstrated an excellent performance in diagnosing H. pylori infection in adults and in pediatric populations. Aim. To evaluate the diagnostic accuracy of a novel stool test based on monoclonal antibodies to detect H. pylori antigens in frozen human stool in the pretreatment setting. Patients and Methods. Stool specimens were prospectively collected from 78 patients undergoing gastroscopy and stored at −20°C until tested. Helicobacter pylori infection was evaluated by histology, rapid urease testing and urea breath tests (13C-UBT). Positivity of the three tests was considered the gold standard for H. pylori active infection. Patients with no positive test were considered negative. The gold standard was compare to the results of the monoclonal antibody stool antigen test. Frozen stool specimens were tested using a novel monoclonal-antibody-based enzyme immunoassay (HePy-Stool, Biolife-Italiana, Milan, Italy) . Results. The sensitivity and specificity of the monoclonal stool antigen test were 97%[95% confidence interval, (CI) 86–100] and 94% (95% CI: 81–99), respectively. Negative and positive predictive values were 97% (95% CI: 85–99), and 95% (95% CI: 83–99), respectively. The diagnostic accuracy was 96% (95% CI: 88–99). The likelihood ratio for a positive test was 17 and for a negative test was 0. Conclusions. Although the 13C-UBT is the most accurate among the available noninvasive tests, our results show that an H. pylori stool test using monoclonal antibody might be an excellent alternative

Detection of Chlamydiae pneumoniae but not Helicobacter pylori DNA in atherosclerosis plaques

Chronic infections have been associated with cardiovascular disease. We used bacterial culture, polymerase chain reaction (PCR), and immunohistochemical staining with anti-vacA and anticagA antibodies to search for Helicobacter pylori and Chlamydiae pneumoniae in atherosclerotic plaques obtained at endarterectomy. Serum IgG antibodies to H. pylori and C. pneumoniae were also determined. Thirty-two patients were enrolled. Anti-H. pylori and anti-C. pneumoniae IgG were present in 72% and 81%, respectively. Culture and PCR for H. pylori of vessel walls and plaques were negative. Atherosclerotic plaque and normal vessel sections from H. pylori-negative and- positive patients showed reactivity with anti-vacA and anti-cagA antibodies. C. pneumoniae DNA was amplified in three atherosclerotic lesions. These findings suggest that the association between H. pylori infection and atherosclerosis does not result from continuing direct effects of H. pylori antigens in the vessel walls. Antigens within vessel atherosclerotic plaques cross-react with H. pylori virulence factors and could act as cofactors in determining instability for the atherosclerotic plaques

Detection of <i>Chlamydiae pneumoniae</i> but not <i>Helicobacter pylori</i> DNA in atherosclerosis plaques

Chronic infections have been associated with cardiovascular disease. We used bacterial culture, polymerase chain reaction (PCR), and immunohistochemical staining with anti-vacA and anticagA antibodies to search for Helicobacter pylori and Chlamydiae pneumoniae in atherosclerotic plaques obtained at endarterectomy. Serum IgG antibodies to H. pylori and C. pneumoniae were also determined. Thirty-two patients were enrolled. Anti-H. pylori and anti-C. pneumoniae IgG were present in 72% and 81%, respectively. Culture and PCR for H. pylori of vessel walls and plaques were negative. Atherosclerotic plaque and normal vessel sections from H. pylori-negative and- positive patients showed reactivity with anti-vacA and anti-cagA antibodies. C. pneumoniae DNA was amplified in three atherosclerotic lesions. These findings suggest that the association between H. pylor infection and atherosclerosis does not result from continuing direct effects of H. pylori antigens in the vessel walls. Antigens within vessel atherosclerotic plaques cross-react with H. pylori virulence factors and could act as cofactors in determining instability for the atherosclerotic plaques

Differential Proteomic Analysis of Hepatocellular Carcinoma

10nonenoneCORONA G; DE LORENZO E; ELIA C; SIMULA MP; AVELLINI C; BACCARANI U; LUPO F; TIRIBELLI C; COLOMBATTI A; TOFFOLI GCorona, G; DE LORENZO, E; Elia, C; Simula, Mp; Avellini, C; Baccarani, Umberto; Lupo, F; Tiribelli, C; Colombatti, Alfonso; Toffoli, G

Time course of AE1 tyrosine phosphorylation, oxidation and clustering.

<p>GSH levels measured in control (CTRL) and G6PD deficient (G⁻) RBCs upon diamide treatment, expressed as µM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015847#pone-0015847-g001" target="_blank">Fig. 1A</a>). Amounts of hemichromes (HMC) measured in isolated membranes of control and G6PD deficient RBCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015847#pone-0015847-g001" target="_blank">Fig. 1B</a>). HMCs were quantified by Vis spectrometry and expressed as nmoles/ml. Quantitative densitometry of AE1 phosphorylation levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015847#pone-0015847-g001" target="_blank">Fig. 1C</a>) and of the oxidatively cross-linked AE1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015847#pone-0015847-g001" target="_blank">Fig. 1D</a>) in control and G⁻ RBCs. Quantification of AE1 phosphorylation levels and of oxidized AE1 was performed with an IR fluorescence detection scanner (Odyssey, Licor, USA) of anti-phosphotyrosine (apTyr) and anti-AE1 (aBd3) western blots with Odyssey V3.0 software and expressed as fluorescence arbitrary units. The western blots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015847#pone-0015847-g001" target="_blank">figure 1C and 1D</a> show the areas used for AE1 tyrosine phosphorylation and oxidation quantifications. Membrane proteins were solubilized in presence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015847#pone-0015847-g001" target="_blank">Fig. 1C</a>) or absence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015847#pone-0015847-g001" target="_blank">Fig. 1D</a>) of the reducing agent (DTT). Percentages of clustered AE1 after gel filtration separation of the high molecular weight protein complexes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015847#pone-0015847-g001" target="_blank">Fig. 1E</a>). Membrane proteins were extracted from control and G6PD deficient RBCs by 1% triton-X100, the supernatant was applied to a 40×1 cm column filled with Sepharose CL-6B to isolate the high molecular weight membrane protein complexes. AE1 was quantified by eosine maleimide fluorescence detection. Clustered AE1 was quantified as percentage of total AE1 eluted from the column. The chromatogram in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015847#pone-0015847-g001" target="_blank">figure 1E</a> shows the two peaks corresponding to clustered and non-clustered AE1. The western blots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015847#pone-0015847-g001" target="_blank">figure 1E</a> show the presence of aggregated AE1 (aBd3), its tyrosine phosphorylation (apTyr) and Syk (aSyk) in the high molecular weight fraction. Membrane proteins were solubilized in absence (lanes 1 and 2) or in presence (lane 3) of the reducing agent (DTT). Control and G6PD deficient RBCs were treated with 0.25 mM diamide (Dia) in presence or absence of Syk inhibitors 10 µM (Syk I.) at different incubation times (0–600 minutes). Values are means of 3 experiments. All differences observed between control and G6PD deficient RBCs were significant (p<0.01). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015847#pone-0015847-g001" target="_blank">Figure 1C and 1E</a> (only in G6PD deficient and after 60 minutes) the changes caused by Syk inhibitors were significant (p<0.01).</p

Confocal microscopy images of control and G6PD deficient RBCs.

<p>Panels A D: hemichrome (HCM) autofluorescence; Panels B, E staining with anti-band 3 antibody (aBd3); Panels C, F staining with anti-phosphotyrosine antibody (apTyr). Panels A, B, C control RBCs (CTRL). Panels D, E, F G6PD deficient red cells (G⁻). Yellow arrows indicate clusters containing hemicromes and co-stained with anti AE1 and anti-phosphotyrosine antibodies. Bar: 20 µm.</p