Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p 72

In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis

Mitapivat, a pyruvate kinase activator, improves transfusion burden and reduces iron overload in M-thalassemic mice

Not available

Effects of hypoxia–reoxygenation stimuli on renal redox status and nuclear factor erythroid 2‐related factor 2 pathway in sickle cell SAD mice

International audienc

Pesticide toxicogenomics across scales: In vitro transcriptome predicts mechanisms and outcomes of exposure in vivo

In vitro Omics analysis (i.e. transcriptome) is suggested to predict in vivo toxicity and adverse effects in humans, although the causal link between high-throughput data and effects in vivo is not easily established. Indeed, the chemical-organism interaction can involve processes, such as adaptation, not established in cell cultures. Starting from this consideration we investigate the transcriptomic response of immortalized thyrocytes to ethylenthiourea and chlorpyrifos. In vitro data revealed specific and common genes/mechanisms of toxicity, controlling the proliferation/survival of the thyrocytes and unrelated hematopoietic cell lineages. These results were phenotypically confirmed in vivo by the reduction of circulating T4 hormone and the development of pancytopenia after long exposure. Our data imply that in vitro toxicogenomics is a powerful tool in predicting adverse effects in vivo, experimentally confirming the vision described as Tox21c (Toxicity Testing in the 21st century) although not fully recapitulating the biocomplexity of a living animal

Increased levels of ERFE-encoding FAM132B in patients with congenital dyserythropoietic anemia type II

Recessive mutations in SEC23B gene cause congenital dyserythropoietic anemia type II (CDAII),1 a rare hereditary disorder hallmarked by ineffective erythropoiesis, iron overload, and reduced expression of hepatic hormone hepcidin.2,3 Some erythroid regulators have been proposed as pathological suppressors of hepcidin expression, such as growth differentiation factor 15 (GDF15) in thalassemia, CDAI and II,4-6 even if alone it seems not necessary for physiological hepcidin suppression.7 The most recently described is the erythroblast-derived hormone erythroferrone (ERFE), a member of TNF-\u3b1 superfamily that specifically inhibits hepcidin production. ERFE-encoding FAM132B is an erythropoietin (EPO)-responsive gene in experimental models.8 However, the function of ERFE in humans remains to be investigated. This study provides the first analysis on ERFE expression in human model of dyserythropoietic anemia with ineffective erythropoiesis, such as CDAII. Our ex vivo and in vitro data indicate that ERFE over-expression in CDAII patients might be most likely related to both physiological and pathological mechanisms leading to hepcidin suppression in condition of dyserythropoiesis. Indeed, we clearly demonstrated that in two different genetic conditions sharing common clinical findings and similar pathogenesis, such as CDAII and BT-intermedia, FAM132B over-expression is related to the abnormal erythropoiesis. Nevertheless, the absence of a clear correlation between erythroferrone levels and CDAII iron balance suggest that ERFE cannot be the only erythroid regulator of hepcidin suppression, at least in CDAII patients

Two-Photon Microscopy Imaging of <em>thy1</em>GFP-M Transgenic Mice: A Novel Animal Model to Investigate Brain Dendritic Cell Subsets <em>In Vivo</em>

<div><p>Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for <em>in vivo</em> brain studies with two-photon fluorescence (TPF) microscopy. Mice of the <em>thy1</em>GFP-M line have been engineered for selective expression of green fluorescent protein (GFP) in neuronal populations. Here, we report that TPF microscopy reveals, at the brain surface of these mice, also motile non-neuronal GFP+ cells. We have analyzed the behavior of these cells <em>in vivo</em> and characterized in brain sections their immunophenotype.</p> <p>With TPF imaging, motile GFP+ cells were found in the meninges, subarachnoid space and upper cortical layers. The striking feature of these cells was their ability to move across the brain parenchyma, exhibiting evident shape changes during their scanning-like motion. In brain sections, GFP+ cells were immunonegative to antigens recognizing motile cells such as migratory neuroblasts, neuronal and glial precursors, mast cells, and fibroblasts. GFP+ non-neuronal cells exhibited instead the characteristic features and immunophenotype (CD11c and major histocompatibility complex molecule class II immunopositivity) of dendritic cells (DCs), and were immunonegative to the microglial marker Iba-1. GFP+ cells were also identified in lymph nodes and blood of <em>thy1</em>GFP-M mice, supporting their identity as DCs. Thus, TPF microscopy has here allowed the visualization for the first time of the motile behavior of brain DCs <em>in situ</em>. The results indicate that the <em>thy1</em>GFP-M mouse line provides a novel animal model for the study of subsets of these professional antigen-presenting cells in the brain. Information on brain DCs is still very limited and imaging in <em>thy1</em>GFP-M mice has a great potential for analyses of DC-neuron interaction in normal and pathological conditions.</p> </div

GFP-DCs in cervical lymph nodes of a <i>thy1</i>GFP-M mouse.

<p>(A) Confocal analysis of cryosectioned cervical lymph node shows the presence of numerous GFP-tagged cells (green). They surround the B cell follicles (dashed lines) and extend in the T cell zone (on the upper right of the figure), while they rarely occur in the subcapsular zone. CD3+ cells, visualized by immunohistochemistry, are here shown in red. (B) High magnification of CD3+ cells (red) and GFP+ cells (green) in the T cell zone. (C) Immunopositivity of GFP-tagged cells (green) to CD11c (red; white arrowheads). Note that the GFP is expressed in the cytoplasm of GFP+DCs.</p

Immunophenotypic analysis of the non-neuronal GFP-tagged cells in the brain of <i>thy</i>GFP-M mice.

<p>Confocal images of coronal brain sections showing non-neuronal GFP+ cells in several locations. (A–C) GFP+ cell into the lumen of a blood vessel (arrowhead) in layer II of the parietal cortex. Neuronal nuclei were stained with anti-NeuN, (B, C, red); the blood vessel is visualized in bright-field (insert A, C). The inset in A represents the area shown an high magnification in A–C. (D–F) GFP+ cell in the ependyma between hippocampus and thalamus. This cell is immunopositive to the dendritic cell marker anti-CD11c (E, red). Cell nuclei are stained with DAPI in F and represented in blue. (G–I) In the anterior hypothalamus a high number of small-sized GFP+ cells shows immunopositivity to the lymphocytes marker CD3+. Different morphologies of GFP+/CD3+ cells in the anterior hypothalamus. A small round (H, high magnification) GFP+/CD3+ cell and a GFP+/CD3+ cell showing an irregular shape (I, high magnification). Scale bars 10 µm.</p

<i>In vivo</i> observation of motile GFP-labeled cells in the cortex of <i>thy1</i>GFP-M mice.

<p>(A) GFP+ cell (white arrowhead) above the pial surface rapidly changing its morphology. Time-lapse sequence of maximum intensity projections of a set of optical sections acquired at 2 µm z-step. The right column shows the depth of the cell through a digital rotation of the corresponding images on the left. The white dotted lines indicate the dura and pia mater.The frame acquired at 45′ shows fluorescent cells (blue arrowheads) passing above the pia through the CSF. (B) GFP+ cell rolling inside a blood vessel on the pial surface. The blood vessel walls (shown in red) were labeled by the intravital dye SR101. Fluorescent cells showing motility at the pial surface (C) and deep in the brain parenchyma (D–E). E) GFP+ cells showing translation across the field of view. Scale bars 10 µm.</p