Auxin-induced reactive oxygen species production requires the activation of phosphatidylinositol 3-kinase

We recently reported that production of reactive oxygen species (ROS) is essential for auxin-induced gravitropic signaling. Here, we investigated the role of phosphatidylinositol 3-kinase and its product, PtdIns(3)P, in auxin-mediated ROS production and the root gravitropic response. Pretreatment with LY294002, an inhibitor of PtdIns 3-kinase activity, blocked auxin-mediated ROS generation, and reduced the sensitivity of root tissue to gravistimulation. The amount of PtdIns(3)P increased in response to auxin, and this effect was abolished by pretreatment with LY294002. In addition, sequestration of PtdIns(3)P by transient expression of the endosome binding domain in protoplasts abrogated IAA-induced ROS accumulation. These results indicate that activation of PtdIns 3-kinase and its product PtdIns(3)P are required for auxin-induced production of ROS and root gravitropism.     

Uronema marinum: identification and biochemical characterization of phosphatidylcholine-hydrolyzing phospholipase C

Phosphatidylcholine (PC)-specific phospholipase D (PC-PLD) and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) activities have been detected in Uronema marinum. Partial purification of PC-PLC revealed that two distinct forms of PC-PLC (named as mPC-PLC and cPC-PLC) were existed in membrane and cytosol fractions. The two PC-PLC enzymes showed the preferential hydrolyzing activity for PC with specific activity of 50.4 for mPC-PLC and 28.3 pmol/min/mg for cPC-PLC, but did not hydrolyze phosphatidylinositol or phosphatidylethanolamine. However, the biochemical characteristics and physiological roles of both enzymes were somewhat different. mPC-PLC had a pH optimum in the acidic region at around, pH 6.0, and required approximately 0.4 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC had a pH optimum in the neutral region at around, pH 7.0, and required 1.6 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC, but not mPC-PLC, showed a dose-dependent inhibitory effect on the luminal-enhanced chemiluminescence (CL) responses and the viability of zymosan-stimulated phagocytes of olive flounder, indicating that cPC-PLC may contribute to the parasite evasion against the host immune response. Our results suggest that U. marinum contains PC-PLD as well as two enzymatically distinct PC-PLC enzymes, and that mPC-PLC may play a role in the intercellular multiplication of U. marinum and cPC-PLC acts as a virulence factor, serving to actively disrupt the host defense mechanisms.     

Oxygen and hydrogen peroxide enhance light-induced carotenoid synthesis in Neurospora crassa

Previously, we found that intracellular reactive oxygen species (ROS) affect photomorphogenesis in Neurospora crassa. In this study, we investigated the physiological roles of ROS in the response to light and found that the exposure of mycelia to air was important for the light-induced carotenogenesis. Mycelia treated with a high concentration of O(2) gas and H(2)O(2) to release ROS showed an enhancement of light-induced carotenoid accumulation and the expression of gene related to light-inducible carotenogenesis. These results suggested that stimuli caused by the exposure of the mycelia to air containing O(2) gas triggered the light-induced carotenoid synthesis.     

Selective activation of adrenergic beta1 receptors induces heme oxygenase 1 production in RAW264.7 cells

We hypothesized that catecholamines through beta-adrenoceptor might modulate macrophage function. We showed that isoproterenol concentration-dependently induced HO-1 production through beta(1)-but not beta(2)-adrenoceptor. Production was increased by forskolin and inhibited by pretreatment with the PKA inhibitor, H-89. Furthermore, induction of HO-1 by isoproterenol effectively protected RAW264.7 cells from effects of glucose oxidase treatment, which was abrogated either by HO-1 inhibitor, ZnPP IX and beta-adenoceptor antagonist, propranolol. Thus, stimulation of HO-1 production through beta(1)-adenoceptors, and via the PKA pathways by isoproterenol, can enable RAW264.7 cells to resist oxidant stress, suggesting that catecholamine hormones may be necessary, at least, to maximize defending role of macrophages.     

Co-exposure to benzo[a]pyrene and UVA induces phosphorylation of histone H2AX

Phosphorylation of histone H2AX (termed gamma-H2AX) was recently identified as an early event after induction of DNA double strand breaks (DSBs). We have previously shown that co-exposure to benzo[a]pyrene (BaP), a wide-spread environmental carcinogen, and ultraviolet A (UVA), a major component of solar UV radiation, induced DSBs in mammalian cells. In the present study, we examined whether co-exposure to BaP and UVA generates gamma-H2AX in CHO-K1 cells. Single treatment with BaP (10(-9)-10(-7)M) or UVA ( approximately 2.4 J/cm(2)) did not result in gamma-H2AX, however, co-exposure drastically induced foci of gamma-H2AX in a dose-dependent manner. gamma-H2AX could be detected even at very low concentration of BaP (10(-9)M) plus UVA (0.6J/cm(2)), which did not change cell survival rates. NaN(3) effectively inhibited the formation of gamma-H2AX induced by co-exposure, indicating the contribution of singlet oxygen. This is the first evidence that co-exposure to BaP and UVA induced DSBs, involving gamma-H2AX.     

GABA shunt deficiencies and accumulation of reactive oxygen intermediates: insight from Arabidopsis mutants

In plants, succinic semialdehyde dehydrogenase (SSADH)-deficiency results in the accumulation of reactive oxygen intermediates (ROI), necrotic lesions, dwarfism, and hypersensitivity to environmental stresses. We report that Arabidopsis ssadh knockout mutants contain five times the normal level of gamma-hydroxybutyrate (GHB), which in SSADH-deficient mammals accounts for phenotypic abnormalities. Moreover, the level of GHB in Arabidopsis is light dependent. Treatment with gamma-vinyl-gamma-aminobutyrate, a specific gamma-aminobutyrate (GABA)-transaminase inhibitor, prevents the accumulation of ROI and GHB in ssadh mutants, inhibits cell death, and improves growth. These results provide novel evidence for the relationship between the GABA shunt and ROI, which may, in part, explain the phenotype of SSADH-deficient plants and animals.     

Quaternary structure of LOV-domain containing polypeptide of Arabidopsis FKF1 protein

Flavin-binding, Kelch repeat, F-box (FKF1) protein is a photoreceptor to regulate flowering of Arabidopsis. The protein has a light, oxygen and voltage (LOV)-sensing domain binding a flavin mononucleotide. The photo-activation of the domain is an indispensable step to initiate the cellular signaling for flowering. In the present study, a LOV-containing polypeptide of FKF1 was prepared by an overexpression system, and the quaternary structure of it was studied by size exclusion chromatography and small-angle X-ray scattering. The apparent molecular weight from chromatography suggested a globular trimeric or an anisotropic-shaped dimeric association of the polypeptide in solution. The scattering experiment demonstrated a dimeric association of the polypeptides with an elongated molecular shape displaying the radius of gyration of 27 A and the maximum dimension of 94 A. The molecular shape simulated from scattering profiles suggests an antiparallel association of the LOV domains in the dimer. Though the absorption spectrum of blue-light irradiated polypeptide was stable in the photoactivated state for a long period, the scattering profiles showed very small changes between the dark and light conditions. Based on the homologies in the amino-acid sequences and the scattering profiles, these results are discussed in connection with the structures and function of LOV domains of phototropin.     

Effects of disialoganglioside GD3 on the mitochondrial membrane potential

GD3 is an intracellular mediator of apoptotic signaling. Although GD3 is known to directly act on mitochondria, the dynamic responses of individual mitochondria to GD3 remain to be elucidated. In the current study, the membrane potential of single mitochondria is observed in the presence of GD3 or its analogues. Here, we report that (1) GD3 specifically induces gradual depolarizations of the inner membrane by a mechanism that differs from the permeability transition, and (2) the GD3-induced depolarizations are suppressed by cyclosporin A. These results suggest that GD3 depolarizes mitochondria by a mechanism distinct from but relevant to the permeability transition.     

Valproic acid induces apoptosis dependent of Yca1p at concentrations that mildly affect the proliferation of yeast

Valproic acid (VPA) inhibited the growth of yeast in a dose-dependent manner with complete inhibition attained at 100 mM. When cells were exposed to 25 mM VPA, the wild-type died showing apoptotic markers, while yca1Delta deleted of YCA1 encoding yeast caspase 1 survived. On the other hand, when cells were exposed to 50 mM VPA, both the wild-type and yca1Delta died showing morphological features similar to those of the autophagic death of cdc28 which was also independent of YCA1. Thus, these results suggested that yeast cells die via YCA1-dependent apoptosis when their proliferative activity is mildly impaired.     

PrP(106-126) activates neuronal intracellular kinases and Egr1 synthesis through activation of NADPH-oxidase independently of PrPc

Prion diseases are characterised by severe neural lesions linked to the presence of an abnormal protease-resistant isoform of cellular prion protein (PrPc). The peptide PrP(106-126) is widely used as a model of neurotoxicity in prion diseases. Here, we examine in detail the intracellular signalling cascades induced by PrP(106-126) in cortical neurons and the participation of PrPc. We show that PrP(106-126) induces the activation of subsets of intracellular kinases (e.g., ERK1/2), early growth response 1 synthesis and induces caspase-3 activity, all of which are mediated by nicotinamide adenine dinucleotide phosphate hydrogen-oxidase activity and oxidative stress. However, cells lacking PrPc are similarly affected after peptide exposure, and this questions the involvement of PrPc in these effects.