A method is described for the determination of the pH of intracellular water based on the distribution of [14C]benzoate (0.01 mM) between intra- and extra-cellular water. Benzoate at higher concentrations (2-10mM) enters the yeast cell in the undissociated form, and its neutralization within the cell can cause a shift of the pH of the intracellular water by more than 1 pH unit. Benzoate causes an accumulation of the two hexose monophosphates of yeast glucose fermentation and a decrease in intermediates beyond phosphofructokinase, suggesting inhibition at this stage. Benzoate also causes a concomitant fall in [ATP]. Phosphofructokinase is inhibited to a greater extent than hexokinase at acid pH. There is a relationship between intracellular pH, phosphofructokinase inhibition and CO2 production, suggesting that the antifungal action of benzoate is caused by an accumulation of benzoate at low external pH, which lowers the intracellular pH into the range where phosphofructokinase is sensitive. The subsequent inhibition of glycolysis causes a fall in [ATP] and thus restricts growth.

Oxidizing agents are powerful activators of factors responsible for the transcriptional activation of cytokine-encoding genes involved in tissue injury. In this study we show evidence that STAT3 is a transcription factor whose activity is modulated by H2O2 in human lymphocytes, in which endogenous catalase had previously been inhibited. H2O2-induced nuclear translocation of STAT3 to form sequence-specific DNA-bound complexes was evidenced by immunoblotting of nuclear fractions and electrophoretic mobility shift assays, and vanadate was found to strongly synergize with H2O2. Moreover, anti-STAT3 antibodies specifically precipitated a protein of 92 kDa that becomes phosphorylated on tyrosine upon lymphocyte treatment with H2O2. Phenylarsine oxide, a tyrosine phosphatase inhibitor, and genistein, a tyrosine kinase inhibitor, cooperated and cancelled, respectively, the H2O2-promoted STAT3 nuclear translocation. Evidence is also presented, using Fe2+/Cu2+ ions, that.OH generated from H2O2 through Fenton reactions could be a candidate oxygen reactive species to directly activate STAT3. Present data suggest that H2O2 and vanadate are likely to inhibit the activity of intracellular tyrosine phosphatase(s), leading to enhanced STAT3 tyrosine phosphorylation and hence its translocation to the nucleus. These results demonstrate that the DNA binding activity of STAT3 can be modulated by oxidizing agents and provide a framework to understand the effects of oxidative stress on the JAK-STAT signaling pathway.

Interleukin (IL)-1β is a pleiotropic cytokine implicated in a variety of activities, including damage of insulin-producing cells, brain injury, or neuromodulatory responses. Many of these effects are mediated by nitric oxide (NO) produced by the induction of NO synthase (iNOS) expression. We report here that IL-1β provokes a marked repression of genes, such as fragile X mental retardation 1 (FMR1) and hypoxanthine phosphoribosyltransferase (HPRT), having a CpG island in their promoter region. This effect can be fully prevented by iNOS inhibitors and is dependent on DNA methylation. NO donors also cause FMR1 and HPRT gene silencing. NO-induced methylation of FMR1 CpG island can be reverted by demethylating agents which, in turn, produce the recovery of gene expression. The effects of IL-1β and NO appear to be exerted through activation of DNA methyltransferase (DNA MeTase). Although exposure of the cells to NO does not increase DNA MeTase gene expression, the activity of the enzyme selectively increases when NO is applied directly on a nuclear protein extract. These findings reveal a previously unknown effect of IL-1β and NO on gene expression, and demonstrate a novel pathway for gene silencing based on activation of DNA MeTase by NO and acute modification of CpG island methylation.

Neutrophils are mobilized to the vascular wall during vessel inflammation. Published data are conflicting on phagocytic nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activation during the hypertensive state, and the capacity of angiotensin II (Ang II) to modulate the intracellular redox status has not been analyzed in neutrophils. We here describe that Ang II highly stimulates endogenous and extracellular O2- production in these cells, consistent with the translocation to the cell membrane of the cytosolic components of NADPH oxidase, p47phox, and p67phox. The Ang II–dependent O2- production was suppressed by specific inhibitors of AT1 receptors, of the p38MAPK and ERK1/2 pathways, and of flavin oxidases. Furthermore, Ang II induced a robust phosphorylation of p38MAPK, ERK1/2, and JNK1/2 (particularly JNK2), which was hindered by inhibitors of NADPH oxidase, tyrosine kinases, and ROS scavengers. Ang II increased cytosolic Ca2+ levels—released mainly from calcium stores—enhanced the synthesis de novo and activity of calcineurin, and stimulated the DNA-binding activity of the transcription factor NF-κB in cultured human neutrophils. Present data demonstrate for the first time a stimulatory role of Ang II in the activation of phagocytic cells, underscore the relevant role of ROS as mediators in this process, and uncover a variety of signaling pathways by which Ang II operates in human neutrophils.

15-Deoxy-delta(12,14)-prostaglandin J(2) (15dPGJ(2) has been recently proposed as a potent anti-inflammatory agent. However, the mechanisms by which 15dPGJ(2) mediates its therapeutic effects in vivo are unclear. We demonstrate that 15dPGJ(2) at micromolar (2.5-10 microm) concentrations induces the expression of heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, at both mRNA and protein levels in human lymphocytes. In contrast, troglitazone and ciglitazone, two thiazolidinediones that mimic several effects of 15dPGJ(2) through their binding to the peroxisome proliferator-activated receptor (PPAR)-gamma, did not affect HO-1 expression, and the positive effect of 15dPGJ(2) on this process was mimicked instead by other cyclopentenone prostaglandins (PG), such as PGD(2) (the precursor of 15dPGJ(2)) and PGA(1) and PGA(2) which do not interact with PPAR-gamma. Also, 15dPGJ(2) enhanced the intracellular production of reactive oxygen species (ROS) and increased xanthine oxidase activity in vitro. Inhibition of intracellular ROS production by N-acetylcysteine, TEMPO, Me(2)SO, 1,10-phenanthroline, or allopurinol resulted in a decreased 15dPGJ(2)-dependent HO-1 expression in the cells. Furthermore, buthionine sulfoximine, an inhibitor of reduced glutathione synthesis, or Fe(2+)/Cu(2+) ions enhanced the positive effect of 15dPGJ(2) on HO-1 expression. On the other hand, the inhibition of phosphatidylinositol 3-kinase or p38 mitogen-activated protein kinase, or the blockade of transcription factor NF-kappaB activation, hindered 15dPGJ(2)-elicited HO-1 expression. Collectively, the present data suggest that 15dPGJ(2) anti-inflammatory actions at pharmacological concentrations involve the induction of HO-1 gene expression through mechanisms independent of PPAR-gamma activation and dependent on ROS produced via the xanthine/xanthine oxidase system and/or through Fenton reactions. Both phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase signaling pathways also appear implicated in modulation of HO-1 expression by 15dPGJ(2).