UPEI-100, a conjugate of lipoic acid and apocynin, mediates neuroprotection in a rat model of ischemia/reperfusion

Previous work in our laboratory has provided evidence that preadministration of apocynin and lipoic acid at subthreshold levels for neuroprotection enhanced the neuroprotective capacity when injected in combination. Therefore, the present investigation was designed to determine whether a co-drug consisting of lipoic acid and apocynin functional groups bound by a covalent bond, named UPEI-100, is capable of similar efficacy using a rodent model of stroke. Male rats were anesthetized with Inactin (100 mg/kg iv), and the middle cerebral artery was occluded for 6 h or allowed to reperfuse for 5.5 h following a 30-min occlusion (ischemia/reperfusion, I/R). Preadministration of UPEI-100 dose-dependently decreased infarct volume in the I/R model (P < 0.05), but not in the middle cerebral artery occlusion model of stroke. Using the optimal dose, we then injected UPEI-100 during the stroke or at several time points during reperfusion, and significant neuroprotection was observed when UPEI-100 was administered up to 90 min following the start of reperfusion (P < 0.05). A time course for this neuroprotective effect showed that UPEI-100 resulted in a decrease in infarct volume following 2 h of reperfusion compared with vehicle. The time course of this neuroprotective effect was also used to study several mediators along the antioxidant pathway and showed that UPEI-100 increased the level of mitochondrial superoxide dismutase and oxidized glutathione and decreased a marker of lipid peroxidation due to oxidative stress (HNE-His adduct formation). Taken together, the data suggest that UPEI-100 may utilize similar pathways to those observed for the two parent compounds; however, it may also act through a different mechanism of action.     

Hydrogen peroxide signalling

Recent biochemical and genetic studies confirm that hydrogen peroxide is a signalling molecule in plants that mediates responses to abiotic and biotic stresses. Signalling roles for hydrogen peroxide during abscisic-acid-mediated stomatal closure, auxin-regulated root gravitropism and tolerance of oxygen deprivation are now evident. The synthesis and action of hydrogen peroxide appear to be linked to those of nitric oxide. Downstream signalling events that are modulated by hydrogen peroxide include calcium mobilisation, protein phosphorylation and gene expression. Calcium and Rop signalling contribute to the maintenance of hydrogen peroxide homeostasis.     

Improved Detection of Common Variants Associated with Schizophrenia and Bipolar Disorder Using Pleiotropy-Informed Conditional False Discovery Rate

Several lines of evidence suggest that genome-wide association studies (GWAS) have the potential to explain more of the “missing heritability” of common complex phenotypes. However, reliable methods to identify a larger proportion of single nucleotide polymorphisms (SNPs) that impact disease risk are currently lacking. Here, we use a genetic pleiotropy-informed conditional false discovery rate (FDR) method on GWAS summary statistics data to identify new loci associated with schizophrenia (SCZ) and bipolar disorders (BD), two highly heritable disorders with significant missing heritability. Epidemiological and clinical evidence suggest similar disease characteristics and overlapping genes between SCZ and BD. Here, we computed conditional Q–Q curves of data from the Psychiatric Genome Consortium (SCZ; n = 9,379 cases and n = 7,736 controls; BD: n = 6,990 cases and n = 4,820 controls) to show enrichment of SNPs associated with SCZ as a function of association with BD and vice versa with a corresponding reduction in FDR. Applying the conditional FDR method, we identified 58 loci associated with SCZ and 35 loci associated with BD below the conditional FDR level of 0.05. Of these, 14 loci were associated with both SCZ and BD (conjunction FDR). Together, these findings show the feasibility of genetic pleiotropy-informed methods to improve gene discovery in SCZ and BD and indicate overlapping genetic mechanisms between these two disorders.     

Abundant Genetic Overlap between Blood Lipids and Immune-Mediated Diseases Indicates Shared Molecular Genetic Mechanisms

Epidemiological studies suggest a relationship between blood lipids and immune-mediated diseases, but the nature of these associations is not well understood. We used genome-wide association studies (GWAS) to investigate shared single nucleotide polymorphisms (SNPs) between blood lipids and immune-mediated diseases. We analyzed data from GWAS (n~200,000 individuals), applying new False Discovery Rate (FDR) methods, to investigate genetic overlap between blood lipid levels [triglycerides (TG), low density lipoproteins (LDL), high density lipoproteins (HDL)] and a selection of archetypal immune-mediated diseases (Crohn’s disease, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, celiac disease, psoriasis and sarcoidosis). We found significant polygenic pleiotropy between the blood lipids and all the investigated immune-mediated diseases. We discovered several shared risk loci between the immune-mediated diseases and TG (n = 88), LDL (n = 87) and HDL (n = 52). Three-way analyses differentiated the pattern of pleiotropy among the immune-mediated diseases. The new pleiotropic loci increased the number of functional gene network nodes representing blood lipid loci by 40%. Pathway analyses implicated several novel shared mechanisms for immune pathogenesis and lipid biology, including glycosphingolipid synthesis (e.g. FUT2) and intestinal host-microbe interactions (e.g. ATG16L1). We demonstrate a shared genetic basis for blood lipids and immune-mediated diseases independent of environmental factors. Our findings provide novel mechanistic insights into dyslipidemia and immune-mediated diseases and may have implications for therapeutic trials involving lipid-lowering and anti-inflammatory agents.     

Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis

Ethylene is a plant hormone that regulates many aspects of growth and development. Despite the well-known association between ethylene and stress signalling, its effects on stomatal movements are largely unexplored. Here, genetic and physiological data are provided that position ethylene into the Arabidopsis guard cell signalling network, and demonstrate a functional link between ethylene and hydrogen peroxide (H(2)O(2)). In wild-type leaves, ethylene induces stomatal closure that is dependent on H(2)O(2) production in guard cells, generated by the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase AtrbohF. Ethylene-induced closure is inhibited by the ethylene antagonists 1-MCP and silver. The ethylene receptor mutants etr1-1 and etr1-3 are insensitive to ethylene in terms of stomatal closure and H(2)O(2) production. Stomata of the ethylene signalling ein2-1 and arr2 mutants do not close in response to either ethylene or H(2)O(2) but do generate H(2)O(2) following ethylene challenge. Thus, the data indicate that ethylene and H(2)O(2) signalling in guard cells are mediated by ETR1 via EIN2 and ARR2-dependent pathway(s), and identify AtrbohF as a key mediator of stomatal responses to ethylene.     

Production of reactive oxygen species and reactive nitrogen species by angiosperm stigmas and pollen: potential signalling crosstalk?

Angiosperm stigmas exhibit high levels of peroxidase activity when receptive to pollen. To explore possible function(s) of this peroxidase activity we investigated amounts of reactive oxygen species (ROS), particularly hydrogen peroxide, in stigmas and pollen. Because nitric oxide (NO) was recently implicated in pollen tube growth, we also investigated amounts of NO in pollen and stigmas. Reactive oxygen species accumulation was assessed with confocal microscopy and light microscopy using ROS probes DCFH2-DA and TMB, respectively. NO was assayed using the NO probe DAF-2DA and confocal microscopy. Stigmas from various different angiosperms were found to accumulate ROS, predominantly H2O2, constitutively. In Senecio squalidus and Arabidopsis thaliana high amounts of ROS/H2O2 were localized to stigmatic papillae. ROS/H2O2 amounts appeared reduced in stigmatic papillae to which pollen grains had adhered. S. squalidus and A. thaliana pollen produced relatively high amounts of NO compared with stigmas; treating stigmas with NO resulted in reduced amounts of stigmatic ROS/H2O2. Constitutive accumulation of ROS/H2O2 appears to be a feature of angiosperm stigmas. This novel finding is discussed in terms of a possible role for stigmatic ROS/H2O2 and pollen-derived NO in pollen-stigma interactions and defence.     

Nitric oxide, stomatal closure, and abiotic stress

Various data indicate that nitric oxide (NO) is an endogenoussignal in plants that mediates responses to several stimuli.Experimental evidence in support of such signalling roles forNO has been obtained via the application of NO, usually in theform of NO donors, via the measurement of endogenous NO, andthrough the manipulation of endogenous NO content by chemicaland genetic means. Stomatal closure, initiated by abscisic acid(ABA), is effected through a complex symphony of intracellularsignalling in which NO appears to be one component. ExogenousNO induces stomatal closure, ABA triggers NO generation, removalof NO by scavengers inhibits stomatal closure in response toABA, and ABA-induced stomatal closure is reduced in mutantsthat are impaired in NO generation. The data indicate that ABA-inducedguard cell NO generation requires both nitric oxide synthase-likeactivity and, in Arabidopsis, the NIA1 isoform of nitrate reductase(NR). NO stimulates mitogen-activated protein kinase (MAPK)activity and cGMP production. Both these NO-stimulated eventsare required for ABA-induced stomatal closure. ABA also stimulatesthe generation of H₂O₂ in guard cells, and pharmacological andgenetic data demonstrate that NO accumulation in these cellsis dependent on such production. Recent data have extended thismodel to maize mesophyll cells where the induction of antioxidantdefences by water stress and ABA required the generation ofH₂O₂ and NO and the activation of a MAPK. Published data suggestthat drought and salinity induce NO generation which activatescellular processes that afford some protection against the oxidativestress associated with these conditions. Exogenous NO can alsoprotect cells against oxidative stress. Thus, the data suggestan emerging model of stress responses in which ABA has severalameliorative functions. These include the rapid induction ofstomatal closure to reduce transpirational water loss and theactivation of antioxidant defences to combat oxidative stress.These are two processes that both involve NO as a key signallingintermediate. Key words: Abscisic acid, antioxidants, guard cells, hydrogen peroxide, nitric oxide, oxidative stress, stomata, water stress Received 19 June 2007; Revised 21 September 2007 Accepted 5 November 2007     

Harpin induces activation of the Arabidopsis mitogen-activated protein kinases AtMPK4 and AtMPK6

Mitogen-activated protein kinases (MAPKs) are key enzymes that mediate adaptive responses to various abiotic and biotic stresses, including pathogen challenge. The proteinaceous bacterial elicitor harpin (secreted by Pseudomonas syringae pv syringae) activates two MAPKs in suspension cultures of Arabidopsis var. Landsberg erecta. In this study, we show that harpin and exogenous hydrogen peroxide (H(2)O(2)) activate myelin basic protein kinases in Arabidopsis leaves. Using anti-AtMPK4 and anti-AtMPK6 antibodies, we identify the harpin-activated MAPKs in both leaves and suspension cultures as AtMPK4 and AtMPK6, and show that H(2)O(2), generated by Arabidopsis cells in response to challenge with harpin, activates only AtMPK6. However, treatments with catalase, which removes H(2)O(2), or diphenylene iodonium, which inhibits superoxide and H(2)O(2) production, do not inhibit harpin-induced activation of AtMPK4 or AtMPK6. In addition, activation of AtMPK4 but not AtMPK6 is inhibited by the MAPK kinase inhibitor PD98059. Neither harpin nor H(2)O(2) has any effect on AtMPK4 or AtMPK6 gene expression. In addition, the expression of AtMEKK1, AtMEK1, or AtMKK2, previously shown to be potential functional partners of AtMPK4, were not affected by either harpin or H(2)O(2) treatments. These data suggest that harpin activates several signaling pathways, one leading to stimulation of the oxidative burst and others leading to the activation of AtMPK4 or AtMPK6.