Superoxide generation by Nox1 in guinea pig gastric mucosal cells involves a component with p67(phox)-ability.

Nox1, a homologue of gp91(phox) subunit of the phagocyte NADPH oxidase, is responsible for spontaneous superoxide (O(2)(-)) generation in guinea pig gastric mucosal cells (GMC), but involvement of regulatory components (p67(phox), p47(phox), and Rac) which are essential in phagocytes remains unknown. Here, we aimed to figure out how Nox1 of GMC achieves an active oxidase status. GMC in primary culture show low O(2)(-) generation but acquire a 9-fold higher activity when cultured with Helicobacter pylori lipopolysaccharide (LPS), in correlation with a far increased Nox1 expression. Investigation into the O(2)(-)-generating ability of LPS-induced Nox1 in cell-free reconstitution assays showed that: 1) Nox1 is unable to generate O(2)(-) per se; 2) the combination of Nox1 with GMC cytosol is insufficient for a significant O(2)(-) generation; 3) the combination with neutrophil cytosol enables Nox1 to act like gp91(phox), i.e., to produce O(2)(-) appreciably in response to myristate stimulation; 4) Nox1 prefers NADPH to NADH under the in vitro assay with neutrophil cytosol plus myristate (K(m)=10.4 microM); 5) substitution of neutrophil cytosol by a set of recombinant cytosolic components (rp67(phox), rp47(phox), Rac2) is, however, ineffective and still requires GMC cytosol. Thus, Nox1 probably requires an additional cytosolic factor(s). In contrast, GMC cytosol enables cytochrome b(558) to generate plenty of O(2)(-), on condition that rp47(phox) is added. This result suggests that GMC cytosol contains a component with p67(phox)-ability, and also Rac, but lacks p47(phox). These data indicate that GMC Nox1 requires at least a p67(phox) counterpart and Rac to acquire NADPH oxidase activity.

Stimulation of human bronchial epithelial cells by IgE-dependent histamine-releasing factor.

An IgE-dependent histamine-releasing factor (HRF p23; also known as translationally controlled tumor protein or p23) stimulates the release of histamine, IL-4, and IL-13 from a subpopulation of highly allergic donor basophils. It has also been shown to act as a chemoattractant for eosinophils. To elucidate novel functions of HRF p23 in airway inflammation, we examined the effects of human recombinant HRF p23 (hrHRF) on bronchial epithelium and found that hrHRF stimulated the secretions of IL-8 and granulocyte/macrophage colony-stimulating factor by both primary cultures of human bronchial epithelial cells and BEAS-2B cells. In response to hrHRF, these cells induced IL-8 mRNA expression within 4 h. H2O2, but not IL-1 beta or tumor necrosis factor-alpha, stimulated secretion of HRF p23 by BEAS-2B cells, suggesting that oxidative stress may trigger the release of HRF p23 from bronchial epithelial cells. Bronchoalveolar lavage (BAL) from healthy volunteers contained only trivial or undetectable amounts of HRF p23. Significantly higher amounts of HRF p23 were recovered from BAL fluid taken from asthmatic patients, and the amounts of HRF p23 were further elevated in patients with idiopathic eosinophilic pneumonia. Our results demonstrate for the first time that HRF p23 can stimulate nonimmune epithelium. HRF p23 derived from bronchial epithelial cells may regulate complex cytokine networks in eosinophil-dependent inflammation of the human airway.

Effects of a soy protein diet on exercise-induced muscle protein catabolism in rats.

OBJECTIVE: We examined effects of dietary soy protein isolate on muscle calpain activity and myosin heavy chain (MHC) degradation in rats performing an acute running exercise. METHODS: In rats fed a 20% casein diet, the treadmill running exercise, fixed at 80 kg/m, transiently increased calpain activity in gastrocnemius muscles in parallel with the release of creatine kinase into plasma. The fixed running also caused an accumulation of immunoreactive degradation fragments of MHC in the muscle. Feeding a 20% soy protein isolate diet as opposed to the control casein diet to rats significantly suppressed the running-induced activation of mu- and m-calpains, fragmentation of MHC, and release of creatine kinase into plasma (P < 0.05). RESULTS: Rats fed the soy protein isolate diet had significantly higher calpastatin activity in gastrocnemius muscle than did rats fed the casein diet (P < 0.05), indicating that this increase inhibits the exercise-induced autoactivation of calpain. Activities of proteasome, cathepsin B + L, and antioxidant enzymes and the levels of glutathione and thiobarbituric acid-reactive substances in the muscle did not differ between the diet groups at the end of the exercise. CONCLUSIONS: Our results suggest that diets containing soy protein prevent exercise-induced protein degradation in skeletal muscle, possibly through inhibiting the calpain-mediated proteolysis.

Dioxin stimulates synthesis and secretion of IgE-dependent histamine-releasing factor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is the most toxic man-made member of the class of environmental pollutants represented by polychlorinated dibenzo-p-dioxins and dibenzofurans. TCDD produces a wide variety of toxic effects. However, the downstream genes targeted by TCDD and their relation to the diversity of dioxin toxicity symptoms are poorly understood. To identify the target genes of TCDD, we used a cDNA representational difference analysis (RDA) to compare the mRNA patterns of mouse embryonic stem (ES) cells that had and had not been exposed to TCDD. Here we show that TCDD stimulated the expression of IgE-dependent histamine-releasing factor (HRF) mRNA via an aryl hydrocarbon receptor (AhR)-dependent pathway. TCDD also induced the synthesis and secretion of HRF. To our knowledge, this is the first example of HRF being a direct transcriptional target of a toxic agent. HRF has previously been shown to induce histamine release in a dose-dependent manner, at least in vitro. Thus, our data suggest that "endocrine-disrupting" agents may have the potential to influence allergic disorders in the human body.