Search

The regulator VicR of the two-component regulatory system VicRK is essential in several Gram-positive bacteria. However, the authors were able to generate an unconditional vicR insertional mutant of group A Streptococcus. This mutant grew well in rich media but not in non-immune human blood and serum, had attenuated virulence, and was unstable in mice. Complementation of the mutant with vicR expressed in trans restored its phenotype to wild-type. A vicK deletion mutant had a phenotype similar to that of the vicR mutant. Phagocytosis and killing of the vicR mutant were normal, suggesting that VicRK does not regulate processes involved in evasion of host defence. Microarray analysis showed that vicR inactivation down-regulated the transcription of 13 genes, including putative cell wall hydrolase gene pcsB and spy 1058-1060, which encode a putative phosphotransferase system enzyme II for carbohydrate transport, and upregulated the expression of five genes, including spy0183 and spy0184, which encode an osmoprotectant transporter OpuA. Consistent with microarray analysis, the vicR mutant took up more of the osmoprotectants betaine and proline and was sensitive to osmotic stress, indicating that vicR inactivation induced osmotic stress and increased susceptibility to osmotic pressure. Additionally, a spy1060 deletion mutant also displayed attenuated virulence. These results suggest that VicRK regulates processes involved in cell wall metabolism, nutrient uptake, and osmotic protection.

Activation of [D.sub.1]-like receptors ([D.sub.1] and/or [D.sub.5]) induces antioxidant responses; however, the mechanism(s) involved in their antioxidant actions are not known. We hypothesized that stimulation of the [D.sub.5] receptor inhibits NADPH oxidase activity, and thus the production of reactive oxygen species (ROS). We investigated this issue in [D.sub.5] receptor-deficient ([D.sub.5]-/-) and wild-type ([D.sub.5]+/+) mice. NADPH oxidase protein expression (gp[91.sup.phox], p[47.sup.phox], and Nox 4) and activity in kidney and brain, as well as plasma thiobarbituric acid-reactive substances (TBARS) were higher in [D.sub.5]-/- than in [D.sub.5]+/+ mice. Furthermore, apocynin, an NADPH oxidase inhibitor, normalized blood pressure, renal NADPH oxidase activity, and plasma TBARS in [D.sub.5]-/- mice. In HEK-293 cells that heterologously expressed human [D.sub.5] receptor, its agonist fenoldopam decreased NADPH oxidase activity, expression of one of its subunits (gp[91.sup.phox]), and ROS production. The inhibitory effect of the [D.sub.5] receptor activation on NADPH oxidase activity was independent of cAMP/PKA but was partially dependent on phospholipase D2. The ability of [D.sub.5] receptor stimulation to decrease ROS production may explain, in part, the antihypertensive action of [D.sub.5] receptor activation. hypertension

Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, [p22.sup.phox] and [gp91.sup.phox] as well as the [Ca.sup.2+] concentration-responsive [gp.syup.91phox] homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for [p22.sup.phox] was not detectable, both [gp91.sup.phox] and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to [p22.sup.phox] or [gp91.sup.phox] did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death. superoxide anion; diphenylene iodonium; [p22.sup.phox]; [gp91.sup.phox]; adenosine 3',5'-cyclic monophosphate response element; caspases

Activation of the phagocyte NADPH oxidase complex requires assembly of the cytosolic factors [p47.sup.PHOX], [p67.sup.PHOX], [p40.sup.PHOX], and Rac with the membrane-bound cytochrome [b.sub.558]. We recently established a direct interaction between [p67.sup.PHOX] and cytochrome [b.sub.558]. In the present study, we show that removal of the C-terminal domain of [p67.sup.PHOX] increased its binding to cytochrome [b.sub.558]. Whereas phosphorylated [p40.sup.PHOX] alone did not bind to cytochrome [b.sub.558], phosphorylated [p47.sup.PHOX] did, and, moreover, it allowed the binding of [p40.sup.PHOX] to the cytochrome. Furthermore, both increased the binding of [p67.sup.PHOX] to the cytochrome. Phosphorylated [p47.sup.PHOX] thus appears to increase the binding of [p67.sup.PHOX] to cytochrome [b.sub.558] by serving as an adapter, bringing [p67.sup.PHOX] into proximity with cytochrome [b.sub.558], whereas phosphorylated [p40.sup.PHOX] may increase the binding by inducing a conformational change that allows [p67.sup.PHOX] to interact fully with cytochrome [b.sub.558].

Evidence is rapidly accumulating that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates in both endothelium and vascular smooth muscle. We therefore explored the possibility of such an oxidase regulating growth of airway smooth muscle (AWSM). Proliferation of human AWSM cells in culture was inhibited by the antioxidants catalase and N-acetylcysteine, and by the flavoprotein inhibitor diphenylene iodonium (DPI). Membranes prepared from human AWSM cells generated superoxide anion ([O.sup.-.sub.2]) measured by superoxide dismutase-inhibitable lucigenin chemiluminescence, with a distinct preference for NADPH instead of reduced nicotinamide adenine dinucleotide as substrate. Chemiluminescence was also inhibited by DPI, suggesting the presence of a flavoprotein containing oxidase generating ([O.sup.-.sub.2]) as a signaling molecule for cell growth. Examination of human AWSM cells by reverse transcriptase-polymerase chain reaction consistently demonstrated transcripts with sequences identical to those reported for [p22.sup.phox]. Transfection with p22phox antisense oligonucleotides reduced human AWSM proliferation. Inhibition of NADPH oxidase activity with DPI prevented serum-induced activation of nuclear factor-[kappa]B (NF-[kappa]B), and overexpression of a superrepressor form of the NF-[kappa]B inhibitor I[kappa]B[alpha] significantly reduced human AWSM growth. These findings suggest that an NADPH oxidase containing [p22.sup.phox] regulates growth-factor responsive human AWSM proliferation, and that the oxidase signals in part through activation of the prototypical redox-regulated transcription factor NF-[kappa]B. superoxide anion; diphenylene iodonium; [p22.sup.phox]