Spatial and temporal dependence of interspark interactions in femtosecond-nanosecond dual-pulse laser-induced breakdown spectroscopy.

A femtosecond air spark has recently been combined with a nanosecond ablative pulse in order to map the spatial and temporal interactions of the two plasmas in femtosecond-nanosecond orthogonal preablation spark dual-pulse laser-induced breakdown spectroscopy (LIBS). Good spatial and temporal correlation was found for reduced atomic emission from atmospheric species (nitrogen and oxygen) and increased atomic emission from ablated species (copper and aluminum) in the femtosecond-nanosecond plasma, suggesting a potential role for atmospheric pressure or nitrogen/oxygen concentration reduction following air spark formation in generating atomic emission enhancements in dual-pulse LIBS.

Cytosolic phospholipase A2 Group IValpha but not secreted phospholipase A2 Group IIA, V, or X induces interleukin-8 and cyclooxygenase-2 gene and protein expression through peroxisome proliferator-activated receptors gamma 1 and 2 in human lung cells.

It has been reported that interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) expression is regulated by peroxisome proliferator-activated receptor (PPAR)-gamma synthetic ligands. We have shown previously that cytosolic phospholipase A2 (cPLA2) is able to activate gene expression through PPAR-gamma response elements (Pawliczak, R., Han, C., Huang, X. L., Demetris, A. J., Shelhamer, J. H., and Wu, T. (2002) J. Biol. Chem. 277, 33153-33163). In this study we investigated the influence of cPLA2 and secreted phospholipase A2 (sPLA2) Group IIA, Group V, and Group X on IL-8 and COX-2 expression in human lung epithelial cells (A549 cells). We also studied the results of cPLA2 activation by epidermal growth factor (EGF) and calcium ionophore (A23187) on IL-8 and COX-2 reporter gene activity, mRNA level, and protein synthesis. cPLA2 overexpression and activation increased both IL-8 and COX-2 reporter gene activity. Overexpression and activation of Group IIA, Group V, or Group X sPLA2s did not increase IL-8 and COX-2 reporter gene activity. Methyl arachidonyl fluorophosphate, a cPLA2 inhibitor, inhibited the effect of A23187 and of EGF on both IL-8 and COX-2 reporter gene activity, steady state levels of IL-8 and COX-2 mRNA, and IL-8 and COX-2 protein expression. Small inhibitory RNAs directed against PPAR-gamma1 and -gamma2 blunted the effect of A23187 and of EGF on IL-8 and COX-2 protein expression. Moreover small inhibitory RNAs directed against cPLA2 decreased the effect of A23187 and EGF on IL-8 and COX-2 protein expression. These results demonstrate that cPLA2 has an influence on IL-8 and COX 2 gene and protein expression at least in part through PPAR-gamma.

Oxidative stress induces arachidonate release from human lung cells through the epithelial growth factor receptor pathway.

Oxidative stress is thought to be a factor influencing many inflammatory responses, including arachidonic acid (AA) release. We have studied the effect of hydrogen peroxide on AA and prostaglandin E(2) release, cytosolic phospholipase (cPLA(2)) steady-state mRNA, cPLA(2) protein levels, cPLA(2) enzyme activity, and cPLA(2) phosphorylation in a human lung epithelial cell line: A549 cells. Hydrogen peroxide caused a dose-dependent increase of A23187-stimulated AA and prostaglandin E(2) release, with a maximum effect at 1 h. This effect is associated with a maximum specific cPLA(2) activity at 1 h, and with a significant increase in cPLA(2) Serine 505 phosphorylation. All these effects were abolished, in a dose-related manner, by the epithelial growth factor receptor kinase inhibitor, AG 1478. To further investigate the pathway leading to the increase cPLA(2) phosphorylation, we used cells transfected with a Ras dominant negative vector and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and p38 kinase inhibitors. Cells transfected with the Ras dominant negative vector exhibited diminished hydrogen peroxide-induced AA release and cPLA(2) phosphorylation as compared with cells transfected with the Ras expression vector. Both MEK and p38 kinase inhibitors inhibited the hydrogen peroxide effect on AA release and specific cPLA(2) activity. Finally, cells stably transfected with an antisense cPLA(2) vector exhibited diminished A23187-stimulated AA release in response to hydrogen peroxide as compared with cells stably transfected with empty expression vector. Collectively, these data show that hydrogen peroxide increases cPLA(2) activity through its phosphorylation utilizing an epithelial growth factor/Ras/extracellular signal-regulated kinase and p38 pathway.

Identification of ARTS-1 as a novel TNFR1-binding protein that promotes TNFR1 ectodomain shedding.

Proteolytic cleavage of TNF receptor 1 (TNFR1) generates soluble receptors that regulate TNF bioactivity. We hypothesized that the mechanism of TNFR1 shedding might involve interactions with regulatory ectoproteins. Using a yeast two-hybrid approach, we identified ARTS-1 (aminopeptidase regulator of TNFR1 shedding) as a type II integral membrane protein that binds to the TNFR1 extracellular domain. In vivo binding of membrane-associated ARTS-1 to TNFR1 was confirmed by coimmunoprecipitation experiments using human pulmonary epithelial and umbilical vein endothelial cells. A direct relationship exists between membrane-associated ARTS-1 protein levels and concordant changes in TNFR1 shedding. Cells overexpressing ARTS-1 demonstrated increased TNFR1 shedding and decreased membrane-associated TNFR1, while cells expressing antisense ARTS-1 mRNA demonstrated decreased membrane-associated ARTS-1, decreased TNFR1 shedding, and increased membrane-associated TNFR1. ARTS-1 neither bound to TNFR2 nor altered its shedding, suggesting specificity for TNFR1. Although a recombinant ARTS-1 protein demonstrated selective aminopeptidase activity toward nonpolar amino acids, multiple lines of negative evidence suggest that ARTS-1 does not possess TNFR1 sheddase activity. These data indicate that ARTS-1 is a multifunctional ectoprotein capable of binding to and promoting TNFR1 shedding. We propose that formation of a TNFR1-ARTS-1 molecular complex represents a novel mechanism by which TNFR1 shedding is regulated.