Constitutive overexpression of Cu/Zn superoxide dismutase exacerbates kainic acid-induced apoptosis of transgenic-Cu/Zn superoxide dismutase neurons.

Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene resides on chromosome 21 and is overexpressed in patients with Down syndrome. Cultured neurons of transgenic Cu/Zn SOD (Tg-Cu/Zn SOD) mice with elevated activity of Cu/Zn SOD were used to determine whether constitutive overexpression of Cu/Zn SOD creates an indigenous oxidative stress that predisposes the Tg-Cu/Zn SOD neurons to added insults. Neurons from three independently derived Tg-Cu/Zn SOD strains showed higher susceptibility than nontransgenic neurons to kainic acid (KA)-mediated excitotoxicity, reflected by an earlier onset and enhanced apoptotic cell death. This higher susceptibility of transgenic neurons to KA-mediated apoptosis was associated with a chronic prooxidant state that was manifested by reduced levels of cellular glutathione and altered [Ca2+]i homeostasis. The data are compatible with the thesis that overexpression of Cu/Zn SOD creates chronic oxidative stress in the transgenic neurons, which exacerbates their susceptibility to additional insults such as KA-mediated excitotoxicity.

Behavioral stress modifies hippocampal plasticity through N-methyl-D-aspartate receptor activation.

Behavioral stress has detrimental effects on subsequent cognitive performance in many species, including humans. For example, humans exposed to stressful situations typically exhibit marked deficits in various learning and memory tasks. However, the underlying neural mechanisms by which stress exerts its effects on learning and memory are unknown. We now report that in adult male rats, stress (i.e., restraint plus tailshock) impairs long-term potentiation (LTP) but enhances long-term depression (LTD) in the CA1 area of the hippocampus, a structure implicated in learning and memory processes. These effects on LTP and LTD are prevented when the animals were given CGP39551 (the carboxyethylester of CGP 37849; DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, before experiencing stress. In contrast, the anxiolytic drug diazepam did not block the stress effects on hippocampal plasticity. Thus, the effects of stress on subsequent LTP and LTD appear to be mediated through the activation of the NMDA subtype of glutamate receptors. Such modifications in hippocampal plasticity may contribute to learning and memory impairments associated with stress.

Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements.

The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis.

In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase.

Rab8 is a small GTP-binding protein that plays a role in vesicular transport from the trans-Golgi network to the basolateral plasma membrane in polarized epithelial cells (MDCK), and to the dendritic surface in hippocampal neurons. As is the case for most other rab proteins, the precise molecular interactions by which rab8 carries out its function remain to be elucidated. Here we report the identification and the complete cDNA-derived amino acid sequence of a murine rab8-interacting protein (rab8ip) that specifically interacts with rab8 in a GTP-dependent manner. Rab8ip displays 93% identity with the GC kinase, a serine/threonine protein kinase recently identified in human lymphoid tissue that is activated in the stress response. Like the GC kinase, rab8ip has protein kinase activity manifested by autophosphorylation and phosphorylation of the classical serine/threonine protein kinase substrates, myelin basic protein and casein. When coexpressed in transfected 293T cells, rab8 and the rab8ip/GC kinase formed a complex that could be recovered by immunoprecipitation with antibodies to rab8. Cell fractionation and immunofluorescence analyses indicate that in MDCK cells endogenous rab8ip is present both in the cytosol and as a peripheral membrane protein concentrated in the Golgi region and basolateral plasma membrane domains, sites where rab8 itself is also located. In light of recent evidence that rab proteins may act by promoting the stabilization of SNARE complexes, the specific GTP-dependent association of rab8 with the rab8ip/GC kinase raises the possibility that rab-regulated protein phosphorylation is important for vesicle targeting or fusion. Moreover, the rab8ip/GC kinase may serve to modulate secretion in response to stress stimuli.

A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana.

We describe here the cloning and characterization of a cDNA encoding a protein kinase that has high sequence homology to members of the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK or MEKK) family; this cDNA is named cATMEKKI (Arabidopsis thaliana MAP kinase or ERK kinase kinase 1). The catalytic domain of the putative ATMEKK1 protein shows approximately 40% identity with the amino acid sequences of the catalytic domains of MAPKKKs (such as Byr2 from Schizosaccharomyces pombe, Ste11 from Saccharomyces cerevisiae, Bck1 from S. cerevisiae, MEKK from mouse, and NPK1 from tobacco). In yeast cells that overexpress ATMEKK1, the protein kinase replaces Ste11 in responding to mating pheromone. In this study, the expression of three protein kinases was examined by Northern blot analyses: ATMEKK1 (structurally related to MAPKKK), ATMPK3 (structurally related to MAPK), and ATPK19 (structurally related to ribosomal S6 kinase). The mRNA levels of these three protein kinases increased markedly and simultaneously in response to touch, cold, and salinity stress. These results suggest that MAP kinase cascades, which are thought to respond to a variety of extracellular signals, are regulated not only at the posttranslational level but also at the transcriptional level in plants and that MAP kinase cascades in plants may function in transducing signals in the presence of environmental stress.

Developmentally related responses of maize catalase genes to salicylic acid.

The response of the maize catalase genes (Cat1, Cat2, and Cat3) to salicylic acid (SA) was examined at two distinct developmental stages: embryogenesis and germination. A unique, germination-related differential response of each maize catalase gene to various doses of SA was observed. During late embryogenesis, total catalase activity in scutella increased dramatically with 1 mM SA treatment. The accumulation of Cat2 transcript and CAT-2 isozyme protein provided the major contribution to the observed increase in total catalase activity. This increase was paralleled by the enhanced growth of germinated embryos at that stage. In a CAT-2 null mutant line, a full compensation of total catalase activity by the CAT-1 isozyme was observed in the presence of SA. This suggests that catalase is important for maintenance of normal cellular processes under stress conditions. SA at 1 mM, which enhances growth of precociously germinated embryos, appeared to inhibit seed germination at 1 day after inhibition. Furthermore, Cat2 transcript accumulation was inhibited at this stage. SA is probably not a direct signal for the induction of the catalase genes. Other signals, possibly germination-related regulator(s), might be responsible for the induction of the catalase genes. The effect of SA on the activity of purified catalase protein was also examined.

Oleic acid modulates mRNA expression of liver X receptor (LXR) and its target genes ABCA1 and SREBP1c in human neutrophils

Purpose: Regulation of liver X receptors (LXRs) is essential for cholesterol homeostasis and inflammation. The present study was conducted to determine whether oleic acid (OA) could regulate mRNA expression of LXRα and LXRα-regulated genes and to assess the potential promotion of oxidative stress by OA in neutrophils. Methods: Human neutrophils were treated with OA at different doses and LXR target gene expression, oxidative stress production, lipid efflux and inflammation state were analyzed. Results: We describe that mRNA synthesis of both LXRα and ABCA1 (a reverse cholesterol transporter) was induced by OA in human neutrophils. This fatty acid enhanced the effects of LXR ligands on ABCA1 and LXR expression, but it decreased the mRNA levels of sterol regulatory element-binding protein 1c (a transcription factor that regulates the synthesis of triglycerides). Although OA elicited a slight oxidative stress in the short term (15–30 min) in neutrophils, it is unlikely that this is relevant for the modulation of transcription in our experimental conditions, which involve longer incubation time (i.e., 6 h). Of physiological importance is our finding that OA depresses intracellular lipid levels and that markers of inflammation, such as ERK1/2 and p38 mitogen-activated protein kinase phosphorylation, were decreased by OA treatment. In addition, 200 μM OA reduced the migration of human neutrophils, another marker of the inflammatory state. However, OA did not affect lipid peroxidation induced by pro-oxidant agents. Conclusions: This work presents for the first time evidence that human neutrophils are highly sensitive to OA and provides novel data in support of a protective role of this monounsaturated acid against the activation of neutrophils during inflammation.

Endophytic colonization of barley (Hordeum vulgare) roots by the nematophagous fungus Pochonia chlamydosporia reveals plant growth promotion and a general defense and stress transcriptomic response

Plant crop yields are negatively conditioned by a large set of biotic and abiotic factors. An alternative to mitigate these adverse effects is the use of fungal biological control agents and endophytes. The egg-parasitic fungus Pochonia chlamydosporia has been traditionally studied because of its potential as a biological control agent of plant-parasitic nematodes. This fungus can also act as an endophyte in monocot and dicot plants, and has been shown to promote plant growth in different agronomic crops. An Affymetrix 22K Barley GeneChip was used in this work to analyze the barley root transcriptomic response to P. chlamydosporia root colonization. Functional gene ontology (GO) and gene set enrichment analyses showed that genes involved in stress response were enriched in the barley transcriptome under endophytism. An 87.5 % of the probesets identified within the abiotic stress response group encoded heat shock proteins. Additionally, we found in our transcriptomic analysis an up-regulation of genes implicated in the biosynthesis of plant hormones, such as auxin, ethylene and jasmonic acid. Along with these, we detected induction of brassinosteroid insensitive 1-associated receptor kinase 1 (BR1) and other genes related to effector-triggered immunity (ETI) and pattern-triggered immunity (PTI). Our study supports at the molecular level the growth-promoting effect observed in plants endophytically colonized by P. chlamydosporia, which opens the door to further studies addressing the capacity of this fungus to mitigate the negative effects of biotic and abiotic factors on plant crops.

Effects of vase solution pH and abscisic acid on the longevity of cut 'Baccara' roses

Abscisic acid (ABA) supplied in the vase solution can induce stomatal closure in the leaves of cut flowers, including roses (Rosa hybrida L.). This effect may be beneficial in reducing water deficit stress. Extracellular pH can affect active ABA concentrations in the apoplast of guard cells, with sap alkalisation enhancing the physiological activity of ABA. Accordingly, it was hypothesized that vase solution pH may affect ABA-mediated stomatal closure of cut roses. Two experiments were conducted to study the interaction of vase solution pH and ABA. In the first, cut 'Baccara' roses were held in vase solutions with +/- 10(-5) M ABA at pH 6, pH 7 and pH 8. In the second experiment, roses were held with +/- 10(-5) M ABA at pH 6 and pH 8 in the presence and absence of 1 mg l(-1) AgNO3 as a bactericide. Supply of ABA increased vase life and reduced vase solution usage of flowers held in low pH 6 solutions, indicating induction of stomatal closure. Conversely, ABA supplied at pH 8 was associated with reduced vase life. This negative result was associated with enhanced development of vase solution microbes at high pH, which overrode any potential pH-mediated ABA efficacy effects.

The relationship between antioxidant supplements and oxidative stress in renal transplant recipients: A review

Renal transplant recipients (RTRs) have elevated oxidative stress and a high incidence of cardiovascular morbidity and mortality. Although recent studies do not support the use of antioxidant supplements as a cardioprotectant in the general population, evidence suggests that RTRs may represent individuals that would benefit from this therapy. RTRs have elevated oxidative stress probably caused by the immunosuppressive therapy, and although only a small number of studies have examined the effects of antioxidant supplementation in these patients, most have reported beneficial findings. This review discusses these studies along with the rationale for the use of antioxidant supplements in RTRs and a call for more research to investigate this important topic.

Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis

The plant hormones abscisic acid (ABA), jasmonic acid (JA), and ethylene are involved in diverse plant processes, including the regulation of gene expression during adaptive responses to abiotic and biotic stresses. Previously, ABA has been implicated in enhancing disease susceptibility in various plant species, but currently very little is known about the molecular mechanisms underlying this phenomenon. In this study, we obtained evidence that a complex interplay between ABA and JA-ethylene signaling pathways regulate plant defense gene expression and disease resistance. First, we showed that exogenous ABA suppressed both basal and JA-ethylene-activated transcription from defense genes. By contrast, ABA deficiency as conditioned by the mutations in the ABA1 and ABA2 genes, which encode enzymes involved in ABA biosynthesis, resulted in upregulation of basal and induced transcription from JA-ethylene responsive defense genes. Second, we found that disruption of AtMYC2 (allelic to JASMONATE INSENSITIVE1 [JIN1]), encoding a basic helix-loop-helix Leu zipper transcription factor, which is a positive regulator of ABA signaling, results in elevated levels of basal and activated transcription from JA-ethylene responsive defense genes. Furthermore, the jin1/myc2 and aba2-1 mutants showed increased resistance to the necrotrophic fungal pathogen Fusarium oxysporum. Finally, using ethylene and ABA signaling mutants, we showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Collectively, our results indicate that the antagonistic interactions between multiple components of ABA and the JA-ethylene signaling pathways modulate defense and stress responsive gene expression in response to biotic and abiotic stresses.

Physiological responses to water stress in Eucalyptus cloeziana and E-argophloia seedlings

Effects of water stress duration and intensity on gas exchange and leaf water potential were investigated in 7-month-old seedlings of a humid coastal provenance (Gympie) and a dry inland (Hungry Hills) provenance of E. cloeziana F. Muell. and in a dry inland (Chinchilla) provenance of E. argophloia Blakely supplied with 100% (T-100), 70% (T-70), 50% (T-50) of their water requirements, or were watered only after they were wilted at dawn (T-0). Seedlings of E. argophloia had the highest midday net photosynthetic rate (A), stomata] conductance (g(s)), stomatal density and predawn leaf water potential (Psi(pd)) in all treatments. The E. cloeziana provenances did not differ in these attributes. The T-70 and T-50 treatments caused reductions in A of 30% in E. argophloia, and 55% in the E. cloeziana provenances. Under the T-0 treatment, E. argophloia maintained higher rates of gas exchange at all levels of water stress than E. cloeziana provenances. The estimates of Psi(pd) and midday water potential (Psi(md)) at which plants remained wilted overnight were respectively: -2.7 and -4.1 MPa for E. cloeziana (humid), -2.8 and -4.0 MPa for E. cloeziana (dry) and, -3.7 and -4.9 MPa for E. argophloia. Following stress relief, both A and g(s) recovered more quickly in E. argophloia and in the dry provenance of E. cloeziana than in the humid provenance. We conclude that E. argophloia is more drought tolerant and has a potential for cultivation in the humid and semi humid climates, whilst E. cloeziana has greater potential in the humid subtropical climates.

An adaptogenic role for omega-3 fatty acids in stress; a randomised placebo controlled double blind intervention study (pilot) [ISRCTN22569553]

Background There is evidence for an adaptive role of the omega -3 fatty acid, docosahexaenoic acid (DHA) during stress. Mechanisms of action may involve regulation of stress mediators, such as the catecholamines and proinflammatory cytokines. Prevention of stress-induced aggression and hostility were demonstrated in a series of clinical trials. This study investigates whether perceived stress is ameliorated by DHA in stressed university staff. Methods Subjects that scored ≥ 17 on the Perceived Stress Scale were randomised into a 6-week pilot intervention study. The diet reactive group was supplemented with 6 g of fish oil containing 1.5 g per day DHA, while the placebo group was supplemented with 6 g a day of olive oil. The groups were compared with each other and a wider cross sectional study population that did not receive either active or placebo intervention. Results There was a significant reduction in perceived stress in both the fish oil and the placebo group from baseline. There was also a significant between-group difference between the fish oil group and the no-treatment controls in the rate of stress reduction (p < 0.05). However, there was not a significant between-group difference between the fish oil and the placebo group, nor the placebo group and the control group. These results are discussed in the context of several methodological limitations. The significant stress reductions in both the fish oil and the placebo group are considered in view of statistical regression, an effect likely to have been exaggerated by the time course of the study, a large placebo effect and the possibility of an active effect from the placebo. Conclusion There were significant differences (p < 0.05) in the fish oil group compared with no-treatment controls. This effect was not demonstrated in the placebo group. As a pilot study, it was not sufficiently powered to find the difference between the fish oil group and the placebo group significant. Further work needs to be undertaken to conclusively demonstrate these data trends. However, the findings from this research support the literature in finding a protective or 'adaptogenic' role for omega-3 fatty acids in stress.

Hypothalamic paraventricular nucleus neurons regulate medullary catecholamine cell responses to restraint stress

Both physical and psychological stressors recruit catecholamine cells (CA) located in the ventrolateral medulla (VLM) and the nucleus of the solitary tract (NTS). In the case of physical stressors, this effect is initiated by signals that first access the central nervous system at or below the level of the medulla. For psychological stressors, however, CA cell recruitment depends on higher structures within the neuraxis. Indeed, we have recently provided evidence of a pivotal role for the medial amygdala (MeA) in this regard, although such a role must involve a relay, as MeA neurons do not project directly to the medulla. However, some of the MeA neurons that respond to psychological stress have been found to project to the hypothalamic paraventricular nucleus (PVN), a structure that provides significant input to the medulla. To determine whether the PVN might regulate medullary CA cell responses to psychological stress, animals were prepared with unilateral injections of the neurotoxin ibotenic acid into the PVN (Experiment 1), or with unilateral injections of the retrograde tracer wheat germ agglutinin-gold (WGA-Au) into the CA cell columns of the VLM or NTS (Experiment 2). Seven days later, animals were subjected to a psychological stressor (restraint; 15 minutes), and their brains were subsequently processed for Fos plus appropriate cytoplasmic markers (Experiment 1), or Fos plus WGA-Au (Experiment 2). PVN lesions significantly suppressed the stress-related induction of Fos in both VLM and NTS CA cells, whereas tracer deposits in the VLM or NTS retrogradely labeled substantial numbers of PVN cells that were also Fos-positive after stress. Considered in concert with previous results, these data suggest that the activation of medullary CA cells in response to psychological stress may involve a critical input from the PVN. (C) 2004 Wiley-Liss, Inc.

Evidence for a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as a-lipoic acid and alpha-tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with alpha-lipoic acid and alpha-tocopherol, alone or in combination, prior to incubation with H2O2 or staurosporine. alpha-lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H2O2 and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with a-lipoic acid and/or a-tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of BcI-2, Bax, HSP70 or pERK1/2 or pJNK. alpha-lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with alpha-lipoic acid and alpha-tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of alpha-lipoic acid as an antioxidant.