Corrosion Behavior of Coated Low Carbon Steel in a Simulated PEMFC Environment.

Here, potential metallic bipolar plate (BP) materials were manufactured by laser coating NiCr-based alloys with different Ti additions on low carbon steel substrates. The titanium content within the coating varied between 1.5 and 12.5 wt%. Our present study focussed on electrochemically testing the laser cladded samples in a milder solution. The electrolyte used for all of the electrochemical tests consisted of a 0.1 M Na2SO4 solution (acidulated with H2SO4 at pH = 5) with the addition of 0.1 ppm F-. The corrosion resistance properties of the laser-cladded samples was evaluated using an electrochemical protocol, which consisted of the open circuit potential (OCP), electrochemical impedance spectroscopy (EIS) measurements, and potentiodynamic polarization, followed by potentiostatic polarization under simulated proton exchange membrane fuel cell (PEMFC) anodic and cathodic environments for 6 h each. After the samples were subjected to potentiostatic polarization, the EIS measurements and potentiodynamic polarization were repeated. The microstructure and chemical composition of the laser cladded samples were investigated by scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX) analysis.

Electrochemical Evaluation of Protective Coatings with Ti Additions on Mild Steel Substrate with Potential Application for PEM Fuel Cells.

In this work, the corrosion behavior of NiCr(Ti) protective coatings deposited on mild steel substrates through laser cladding technology is studied as an alternative new material for metallic bipolar plates used in PEMFC applications. For electrochemical testing, a solution consisting of 0.5 M H2SO4 + 2 ppm F- at room temperature is used as an electrolyte. The fluoride ions are added to simulate the conditions in the PEM fuel cell due to degradation of the proton exchange membrane and fluoride release. A saturated calomel electrode (SCE) is used as a reference electrode and a platinum mesh as the counter electrode. Scanning electron microscopy (SEM) and optical microscopy (OM) are used for studying the morphology of the protective coatings and the effect of Ti addition. The electrochemical evaluation consisted of measuring the open circuit potential (OCP), followed by electrochemical impedance spectroscopy measurements (EIS) and potentiodynamic polarization. It is found that the coatings with 5% Ti, 7% Ti and 10% Ti addition comply with the conditions of the US DOE regarding corrosion performance to be used as materials for the manufacture of the bipolar plates.

Influence of the Laser Cladding Parameters on the Morphology, Wear and Corrosion Resistance of WC-Co/NiCrBSi Composite Coatings.

To enhance the sliding wear and corrosion behavior of steels with low carbon content, cermet composite coatings are usually deposited on their surface by various deposition processes. Laser cladding, compared to other deposition techniques such as electroplating, arc welding, and thermal spraying, has numerous advantages to produce such protective coatings. The paper presents the optimization of laser cladding deposition speed versus energy density in order to obtain WC-Co/NiCrBSi coatings with Ni-Al addition free of defects and reduced porosity deposited on low carbon steel substrate. The microstructure and chemical composition were investigated by SEM combined with EDX analysis while XRD was performed in order to examinate the phases within the coatings. In order to investigate the cladding speed influence on the coatings, hardness measurements, POD (pin on disk) wear tests and corrosion tests in 3.5% NaCl solution were carried out. The results showed that an optimal cladding speed has a crucial impact on the microstructure, composition, and hardness. It was found out that optimizing the cladding deposition speed proved to be effective in enhancing the sliding wear resistance and corrosion behavior by controlling the iron content within the coatings.

The anti-apoptotic activity associated with phosphatidylinositol transfer protein alpha activates the MAPK and Akt/PKB pathway.

The conditioned medium (CM) from mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein alpha (PI-TPalpha; SPIalpha cells) demonstrates an increased anti-apoptotic activity compared with CM from wild type NIH3T3 (wtNIH3T3) cells. As previously shown, the anti-apoptotic activity acts by activating a G protein-coupled receptor, most probably a cannabinoid 1 (CB1)-like receptor as the activity was blocked by both pertussis toxin and rimonabant [M. Schenning, C.M. van Tiel, D. Van Manen, J.C. Stam, B.M. Gadella, K.W. Wirtz and G.T. Snoek, Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor, J. Lipid Res. 45 (2004) 1555-1564]. The CB1 receptor appears to be expressed in mouse fibroblast cells, at levels in the order SPIalpha>wtNIH3T3>SPIbeta cells (i.e. wild type cells overexpressing PI-TPbeta). Upon incubation of SPIbeta cells with the PI-TPalpha-dependent anti-apoptotic factors, both the ERK/MAP kinase and the Akt/PKB pathway are activated in a CB1 receptor dependent manner as shown by Western blotting. In addition, activation of ERK2 was also shown by EYFP-ERK2 translocation to the nucleus, as visualized by confocal laser scanning microscopy. The subsequent activation of the anti-apoptotic transcription factor NF-kappaB is in line with the increased resistance towards UV-induced apoptosis. On the other hand, receptor activation by CM from SPIalpha cells was not linked to phospholipase C activation as the YFP-labelled C2-domain of protein kinase C was not translocated to the plasma membrane of SPIbeta cells as visualized by confocal laser scanning microscopy.

IGF-1 protects against dexamethasone-induced cell death in insulin secreting INS-1 cells independent of AKT/PKB phosphorylation.

Appropriate insulin secretion depends on beta-cell mass that is determined by the balance between cell proliferation and death. IGF-1 stimulates proliferation and protects against apoptosis. In contrast, glucocorticoids promote cell death. In this study we examined molecular interactions of the glucocorticoid dexamethasone (dexa) with IGF-1 signalling pathways in insulin secreting INS-1 cells. IGF-1 (50 ng/ml) increased the growth rate and stimulated BrdU incorporation, while dexa (100 nmol/l) inhibited cell growth, BrdU incorporation and induced apoptosis. Dexa-induced cell death was partially antagonized by IGF-1. This protection was further increased by LY294002 (10 micromol/l), an inhibitor of PI3 kinase. In contrast, MAP kinase inhibitor PD98059 (10 micromol/l) significantly reduced the protective effect of IGF-1. The analysis of signalling pathways by Western blotting revealed that dexa increased IRS-2 protein abundance while the expression of PI3K, PKB and ERK remained unchanged. Despite increased IRS-2 protein,IRS-2 tyrosine phosphorylation stimulated by IGF-1 was inhibited by dexa. Dexa treatment reduced basal PKB phosphorylation. However, IGF-1-mediated stimulation of PKB phosphorylation was not affected by dexa, but ERK phosphorylation was reduced. LY294002 restored IGF-1-induced ERK phosphorylation. These data suggest that dexa induces apoptosis in INS-1 cells by inhibiting phosphorylation of IRS-2, PKB and ERK. IGF-1 counteracts dexa-mediated apoptosis in the presence of reduced PKB but increased ERK phosphorylation.

SGK1-dependent upregulation of connective tissue growth factor by angiotensin II.

Angiotensin II has previously been shown to trigger fibrosis, an effect involving connective tissue growth factor (CTGF). The signaling pathways linking angiotensin II to CTGF formation are, however, incompletely understood. A gene highly expressed in fibrosing tissue is the serum- and glucocorticoid-inducible kinase SGK1. The present study explored whether SGK1 is transcriptionally regulated by angiotensin II and participates in the angiotensin II-dependent regulation of CTGF expression. To this end, experiments have been performed in human kidney fibroblasts and mouse lung fibroblasts from gene-targeted mice lacking SGK1 (sgk1-/-) and their wild-type littermates (sgk1+/+). In human renal fibroblasts, SGK1 and CTGF protein expression were enhanced by angiotensin II (10 nM) within 4 h. In sgk1+/+ mouse fibroblasts, SGK1 transcript levels were significantly increased after 4 h of angiotensin II treatment. Angiotensin II stimulated both transcript and protein abundance of CTGF in fibroblasts from sgk1+/+ mice, effects significantly blunted in fibroblasts of sgk1-/- mice. In conclusion, angiotensin II stimulates the expression of SGK1, which is in turn required for the stimulating effect of angiotensin II on the expression of CTGF. Thus, SGK1 presumably contributes to the profibrotic effect of angiotensin II.

Dexamethasone increases Na+/K+ ATPase activity in insulin secreting cells through SGK1.

Glucocorticoids blunt insulin release, an effect partially due to activation of Kv channels. Similar to those channels Na+/K+ ATPase activity repolarizes the plasma membrane. The present study explored whether glucocorticoids increase the Na+/K+ ATPase activity in pancreatic beta-cells. The glucocorticoid dexamethasone (100 nmol/l for 1 day) significantly increased Na+/K+ ATPase alpha1/beta1-subunit transcript levels and ouabain-sensitive outward current reflecting Na+/K+ ATPase activity in INS-1 cells, effects blunted by glucocorticoid-receptor-blocker RU487 (1 micromol/l). Dexamethasone (100 nmol/l) increased K+ current in beta-cells from wild type mice but not from knockout mice lacking functional serum and glucocorticoid inducible kinase SGK1. Thus, glucocorticoids indeed up-regulate Na+/K+ ATPase activity, an effect requiring SGK1.

Aberrant receptor-mediated endocytosis of Schistosoma mansoni glycoproteins on host lipoproteins.

BACKGROUND: Bilharzia is one of the major parasitic infections affecting the public health and socioeconomic circumstances in (sub) tropical areas. Its causative agents are schistosomes. Since these worms remain in their host for decades, they have developed mechanisms to evade or resist the immune system. Like several other parasites, their surface membranes are coated with a protective layer of glycoproteins that are anchored by a lipid modification. METHODS AND FINDINGS: We studied the release of glycosyl-phosphatidylinositol (GPI)-anchored proteins of S. mansoni and found them in the circulation associated with host lipoprotein particles. Host cells endocytosed schistosomal GPI-anchored proteins via their lipoprotein receptor pathway, resulting in disturbed lysosome morphology. In patients suffering from chronic schistosomiasis, antibodies attacked the parasite GPI-anchored glycoproteins that were associated with the patients' own lipoprotein particles. These immunocomplexes were endocytosed by cells carrying an immunoglobulin-Fc receptor, leading to clearance of lipoproteins by the immune system. As a consequence, neutral lipids accumulated in neutrophils of infected hamsters and in human neutrophils incubated with patient serum, and this accumulation was associated with apoptosis and reduced neutrophil viability. Also, Trypanosoma brucei, the parasite that causes sleeping sickness, released its major GPI-anchored glycoprotein VSG221 on lipoprotein particles, demonstrating that this process is generalizable to other pathogens/parasites. CONCLUSIONS: Transfer of parasite antigens to host cells via host lipoproteins disrupts lipid homeostasis in immune cells, promotes neutrophil apoptosis, may result in aberrant antigen presentation in host cells, and thus cause an inefficient immune response against the pathogen.

Serum- and glucocorticoid-inducible kinase 1 mediates salt sensitivity of glucose tolerance.

Excess salt intake decreases peripheral glucose uptake, thus impairing glucose tolerance. Stimulation of cellular glucose uptake involves phosphatidylinositide-3-kinase (PI-3K)-dependent activation of protein kinase B/Akt. A further kinase downstream of PI-3K is serum- and glucocorticoid-inducible kinase (SGK)1, which is upregulated by mineralocorticoids and, thus, downregulated by salt intake. To explore the role of SGK1 in salt-dependent glucose uptake, SGK1 knockout mice (sgk1(-/-)) and their wild-type littermates (sgk1(+/+)) were allowed free access to either tap water (control) or 1% saline (high salt). According to Western blotting, high salt decreased and deoxycorticosterone acetate (DOCA; 35 mg/kg body wt) increased SGK1 protein abundance in skeletal muscle and fat tissue of sgk1(+/+) mice. Intraperitoneal injection of glucose (3 g/kg body wt) into sgk1(+/+) mice transiently increased plasma glucose concentration approaching significantly higher values ([glucose]p,max) in high salt (281 +/- 39 mg/dl) than in control (164 +/- 23 mg/dl) animals. DOCA did not significantly modify [glucose]p,max in control sgk1(+/+) mice but significantly decreased [glucose]p,max in high-salt sgk1(+/+) mice, an effect reversed by spironolactone (50 mg/kg body wt). [Glucose]p,max was in sgk1(-/-) mice insensitive to high salt and significantly higher than in control sgk1(+/+) mice. Uptake of 2-deoxy-d-[1,2-(3)H]glucose into skeletal muscle and fat tissue was significantly smaller in sgk1(-/-) mice than in sgk1(+/+) mice and decreased by high salt in sgk1(+/+) mice. Transfection of HEK-293 cells with active (S422D)SGK1, but not inactive (K127N)SGK, stimulated phloretin-sensitive glucose uptake. In conclusion, high salt decreases SGK1-dependent cellular glucose uptake. SGK1 thus participates in the link between salt intake and glucose tolerance.

Dexamethasone induces cell death in insulin-secreting cells, an effect reversed by exendin-4.

Glucocorticoid excess induces hyperglycemia, which may result in diabetes. The present experiments explored whether glucocorticoids trigger apoptosis in insulin-secreting cells. Treatment of mouse beta-cells or INS-1 cells with the glucocorticoid dexamethasone (0.1 micromol/l) over 4 days in cell culture increased the number of fractionated nuclei from 2 to 7 and 14%, respectively, an effect that was reversed by the glucocorticoid receptor antagonist RU486 (1 micromol/l). In INS-1 cells, dexamethasone increased the number of transferase-mediated dUTP nick-end labeling-staining positive cells, caspase-3 activity, and poly-(ADP-) ribose polymerase protein cleavage; decreased Bcl-2 transcript and protein abundance; dephosphorylated the proapoptotic protein of the Bcl-2 family (BAD) at serine155; and depolarized mitochondria. Dexamethasone increased PP-2B (calcineurin) activity, an effect abrogated by FK506. FK506 (0.1 micromol/l) and another calcineurin inhibitor, deltamethrin (1 micromol/l), attenuated dexamethasone-induced cell death. The stable glucagon-like peptide 1 analog, exendin-4 (10 nmol/l), inhibited dexamethasone-induced apoptosis in mouse beta-cells and INS-1 cells. The protective effect of exendin-4 was mimicked by forskolin (10 micromol/l) but not mimicked by guanine nucleotide exchange factor with the specific agonist 8CPT-Me-cAMP (50 micromol/l). Exendin-4 did not protect against cell death in the presence of cAMP-dependent protein kinase (PKA) inhibition by H89 (10 micromol/l) or KT5720 (5 micromol/l). In conclusion, glucocorticoid-induced apoptosis in insulin-secreting cells is accompanied by a downregulation of Bcl-2, activation of calcineurin with subsequent dephosphorylation of BAD, and mitochondrial depolarization. Exendin-4 protects against glucocorticoid-induced apoptosis, an effect mimicked by forskolin and reversed by PKA inhibitors.

Identification of proteins in activated human neutrophils susceptible to tyrosyl radical attack. A proteomic study using a tyrosylating fluorophore.

Tyrosyl radicals cross-linked to protein tyrosine residues (tyrosylated proteins) represent hallmarks of neutrophil-mediated injury at the inflammatory locus. Yet the proteins targeted by tyrosyl radicals in an intact cellular system remain to be elucidated. Here, we show that tyrosyl radicals generated by human neutrophils after activation by phorbol 12-myristate 13-acetate (PMA), interferon-gamma (IFN-gamma) or TNF-alpha could act in an autocrine manner by cross-linking to endogenous proteins. We have identified the tyrosylated proteins by using a membrane-impermeable tyrosine analogue, tyramine coupled to fluorescein (TyrFluo), in combination with proteomics techniques. Confocal microscopy images indicated that initially the tyrosylated proteins were localized in patches at the cell surface to become internalized subsequently. In the neutrophil membrane-associated proteome, lactoferrin was the prime target of tyrosylation. Out of three isoforms identified, an 80 kDa neutral isoform was tyrosylated more extensively than the 85 kD basic isoform, particularly after PMA activation. Although all three stimuli induced tyrosylation of the filamentous component vimentin, additional tyrosylated vimentin fragments were detected after IFN-gamma- and TNF-alpha-stimulation. Moreover, upon activation the bulk of vimentin behaved as a dimer (M(r) 120 kDa) being slightly tyrosylated, yet phosphorylated at Thr-425 possibly as a requirement for its externalization. Unexpectedly, bovine catalase added to end tyrosyl radicals formation was detected as a highly tyrosylated neutrophil-associated protein. A moderate stimulus-dependent tyrosylation of ATP synthase-beta, alpha-enolase, glyceraldehyde 3-phosphate dehydrogenase, cytokeratin-10, filamin-A, and annexin-I was also observed. When the membrane-permeable probe (acetylTyrFluo) was used, protein tyrosylation was not observed indicating that the intracellular proteins were well protected against oxidative attack. This study shows that human neutrophils can modulate their proteome via a tyrosine oxidation pathway induced by pro-inflammatory mediators.

Activated neutrophils oxidize extracellular proteins of endothelial cells in culture: effect of nitric oxide donors.

Protein oxidation of human umbilical-vein endothelial cells (HUVEC) in culture was examined under various conditions of oxidative stress. Extracellular protein (ECP) oxidation was assessed by determining dityrosine bond formation, which is indicated by the covalent coupling of the membrane-impermeable tyramine-fluorescein conjugate (TyrFluo) to HUVEC proteins. The acetylated membrane-permeable form of TyrFluo (acetylTyrFluo) was used for the determination of intracellular protein (ICP) oxidation. Oxidative stress was induced by exposing the HUVEC to PMA-activated human neutrophils, to a horseradish peroxidase/hydrogen peroxide (HRP/H(2)O(2)) system or to H(2)O(2) alone. Coupling of the probes was determined by confocal laser scanning microscopy and by Western blotting using anti-fluorescein antibody. Diethylamine nitric oxide (DEANO) was used to determine the effect of NO on the tyrosyl radical formation in proteins. The oxidative burst generated by activated neutrophils for 15 min, resulted in inducing dityrosine formation in ECP of HUVEC. Similar results were obtained with HRP/H(2)O(2), but H(2)O(2) alone did not have any effect on ECP. In the presence of DEANO (0.1 mM or higher), ECP oxidation was almost completely inhibited. This indicates that NO may protect endothelial cells against protein oxidation by activated neutrophils under pro-inflammatory conditions. Activated neutrophils did not oxidize ICP of HUVEC, which strongly suggests that the effect of the oxidative burst was restricted to the proteins exposed to the medium.