No abstract

During phagocytosis, gp91phox , the catalytic subunit of the phagocyte NADPH oxidase, becomes activated to produce superoxide, a precursor of microbicidal oxidants. Currently increasing evidence suggests that nonphagocytic cells contain similar superoxide-producing oxidases, which are proposed to play crucial roles in various events such as cell proliferation and oxygen sensing for erythropoiesis. Here we describe the cloning of human cDNA that encodes a novel NAD(P)H oxidase, designated NOX4. The NOX4 protein of 578 amino acids exhibits 39% identity to gp91 phox with special conservation in membrane-spanning regions and binding sites for heme, FAD, and NAD(P)H, indicative of its function as a superoxide-producing NAD(P)H oxidase. The membrane fraction of kidney-derived human embryonic kidney (HEK) 293 cells, expressing NOX4, exhibits NADHand NADPH-dependent superoxide-producing activities, both of which are inhibited by diphenylene iodonium, an agent known to block oxygen sensing, and decreased in cells expressing antisense NOX4 mRNA. The human NOX4 gene, comprising 18 exons, is located on chromosome 11q14.2-q21, and its expression is almost exclusively restricted to adult and fetal kidneys. In human renal cortex, high amounts of the NOX4 protein are present in distal tubular cells, which reside near erythropoietin-producing cells. In addition, overexpression of NOX4 in cultured cells leads to increased superoxide production and decreased rate of growth. The present findings thus suggest that the novel NAD(P)H oxidase NOX4 may serve as an oxygen sensor and/or a regulator of cell growth in kidney.

The superoxide-producing phagocyte NADPH oxidase can be activated by arachidonic acid (AA) or by phosphorylation of p47 phox under cell-free conditions. The molecular mechanism underlying the activation, however, has remained largely unknown. Here we demonstrate that AA, at high concentrations (50 -100 M), induces direct interaction between the oxidase factors p47 phox and p22 phox in parallel with superoxide production. The interaction, being required for the oxidase activation, is mediated via the Src homology 3 (SH3) domains of p47 phox (p47-(SH3) 2 ), which are intramolecularly masked in a resting state. We also show that AA disrupts complexation of p47-(SH3) 2 with its intramolecular target fragment (amino acids 286 -340) without affecting association of p47-(SH3) 2 with p22 phox , indicating that the disruption plays a crucial role in the induced interaction with p22 phox . Phosphorylation of p47 phox by protein kinase C partially replaces the effects of AA; treatment of the SH3 target fragment with PKC in vitro results in a completely impaired interaction with p47-(SH3) 2 , and the same treatment of the full-length p47 phox leads to both interaction with p22 phox and oxidase activation without AA, but to a lesser extent. Furthermore, phosphorylated p47 phox effectively binds to p22 phox and activates the oxidase in the presence of AA at low concentrations (1-5 M), where an unphosphorylated protein only slightly supports superoxide production. Thus AA, at high concentrations, fully induces the interaction of p47 phox with p22 phox by itself, whereas, at low concentrations, AA synergizes with phosphorylation of p47 phox to facilitate the interaction, thereby activating the NADPH oxidase.

No abstract

We examined the effect of alemtuzumab and basiliximab induction therapy on patient survival and freedom from bronchiolitis obliterans syndrome (BOS) in double lung transplantation. The United Network for Organ Sharing database was reviewed for adult double lung transplant recipients from 2006 to 2013. The primary outcome was risk-adjusted all-cause mortality. Secondary outcomes included time to BOS. There were 6117 patients were identified, of whom 738 received alemtuzumab, 2804 received basiliximab, and 2575 received no induction. Alemtuzumab recipients had higher lung allocation scores compared with basiliximab and no-induction recipients (41.4 versus 37.9 versus 40.7, p < 0.001) and were more likely to require mechanical ventilation before to transplantation (21.7% versus 6.5% versus 6.2%, p < 0.001). Median survival was longer for alemtuzumab and basiliximab recipients compared with patients who received no induction (2321 versus 2352 versus 1967 days, p = 0.001). Alemtuzumab (hazard ratio 0.80, 95% confidence interval 0.67-0.95, p = 0.009) and basiliximab induction (0.88, 0.80-0.98, p = 0.015) were independently associated with survival on multivariate analysis. At 5 years, alemtuzumab recipients had a lower incidence of BOS (22.7% versus 55.4 versus 55.9%), and its use was independently associated with lower risk of developing BOS on multivariate analysis. While both induction therapies were associated with improved survival, patients who received alemtuzumab had greater median freedom from BOS.