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In this year there is the 40th anniversary of the first publication of plant nitric oxide (NO) emission by Lowell Klepper. In the decades since then numerous milestone discoveries have revealed that NO is a multifunctional molecule in plant cells regulating both plant development and stress responses. Apropos of the anniversary, these authors aim to review and discuss the developments of past concepts in plant NO research related to NO metabolism, NO signaling, NO's action in plant growth and in stress responses and NO's interactions with other reactive compounds. Despite the long-lasting research efforts and the accumulating experimental evidences numerous questions are still needed to be answered, thus future challenges and research directions have also been drawn up.

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Hydrogen sulfide (H2S) is an important gasotransmitter in both animals and plants. Many physiological events, including responses to stress, have been suggested to involve H2S, at least in part. On the other hand, numerous responses have been reported following treatment with H2S, including changes in the levels of antioxidants and the activities of transcription factors. Therefore, it is important to understand and unravel the events that are taking place downstream of H2S in signaling pathways. H2S is known to interact with other reactive signaling molecules such as reactive oxygen species (ROS) and nitric oxide (NO). One of the mechanisms by which ROS and NO have effects in a cell is the modification of thiol groups on proteins, by oxidation or S-nitrosylation, respectively. Recently, it has been reported that H2S can also modify thiols. Here we report a method for the determination of thiol modifications on proteins following the treatment with biological samples with H2S donors. Here, the nematode Caenorhabditis elegans is used as a model system but this method can be used for samples from other animals or plants.

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Hydrogen sulfide (H2S) has been postulated to be the third gasotransmitter, and along with other reactive compounds such as reactive oxygen species (ROS) and nitric oxide (NO) it is thought to be a key signalling molecule. Enzymes which generate H2S, and remove it, have been characterised in both plants and animals and although it is inherently toxic to cells - inhibiting cytochrome oxidase for example - H2S is now being thought of as part of signal transduction pathways. But is it working as a signal in the sense usually seen for small signalling molecules, that is, produced when needed, perceived and leading to dedicated responses in cells? A look through the literature shows that H2S is involved in many stress responses, and in animals is implicated in the onset of many diseases, in both cases where ROS and NO are often involved. It is suggested here that H2S is not acting as a true signal, but through its interaction with NO and ROS metabolism is modulating such activity, keeping it in check unless strictly needed, and that H2S is acting as a referee to ensure NO and ROS metabolism is working properly.

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It has been hypothesized that radiation-induced oxidative stress is the mechanism for a wide range of negative impacts on biota living in radioactively contaminated areas around Chernobyl. The present study tests this hypothesis mechanistically, for the first time, by modelling the impacts of radiolysis products within the cell resulting from radiations (low linear energy transfer ß and γ), and dose rates appropriate to current contamination types and densities in the Chernobyl exclusion zone and at Fukushima. At 417 µGy h(-1) (illustrative of the most contaminated areas at Chernobyl), generation of radiolysis products did not significantly impact cellular concentrations of reactive oxygen species, or cellular redox potential. This study does not support the hypothesis that direct oxidizing stress is a mechanism for damage to organisms exposed to chronic radiation at dose rates typical of contaminated environments.

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Abscisic acid (ABA) and nitric oxide (NO) are both extremely important signalling molecules employed by plants to control many aspects of physiology. ABA has been extensively studied in the mechanisms which control stomatal movement as well as in seed dormancy and germination and plant development. The addition of either ABA or NO to plant cells is known to instigate the actions of many signal transduction components. Both may have an influence on the phosphorylation of proteins in cells mediated by effects on protein kinases and phosphatases, as well as recruiting a wide range of other signal transduction molecules to mediate the final effects. Both ABA and NO may also lead to the regulation of gene expression. However, it is becoming more apparent that NO may be acting downstream of ABA, with such action being mediated by reactive oxygen species such as hydrogen peroxide in some cases. However not all ABA responses require the action of NO. Here, examples of where ABA and NO have been put together into the same signal transduction pathways are discussed.

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Effects of hydrogen sulfide (H(2)S) on plant physiology have been previously studied, but such studies have relied on the use of NaSH as a method for supplying H(2)S to tissues. Now new compounds which give a less severe H(2)S shock and a more prolonged exposure to H(2)S have been developed. Here the effects of one such compound, GYY4137, has been investigated to determine its effects on stomatal closure in Arabidopsis thaliana. It was found that both NaSH and GYY4137 caused stomatal opening in the light and prevented stomatal closure in the dark. Nitric oxide (NO) has been well established as a mediator of stomatal movements and here it was found that both NaSH and GYY4137 reduced the accumulation of NO in guard cells, perhaps suggesting a mode of action for H(2)S in this system. GYY4137, and future related compounds, will be important tools to unravel the effects of plant exposure to H(2)S and to determine how H(2)S may fit into plant cell signalling pathways.

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Recent advice to the general public is to increase intake of fresh fruit and vegetables, a message based on the assumed benefits of the antioxidant content of plant substances. Although there have been numerous studies on the reactive oxide species scavenging of fresh food products, few studies have focused on whether or not compounds in the diet can modulate the levels of nitric oxide (NO). Nitric oxide is a key signalling molecule that controls vasodilation and blood pressure, along with a range of other physiological events. Here, it is shown that commonly used food substances, such as cabbage, broccoli, kidney bean and oranges, all have the capacity to scavenge NO from solution, and therefore can potentially alter the level of NO in humans, with ramifications for the physiological systems that NO regulates. Using spinach, at least one element of the NO scavenging ability was shown to be heat-unstable, although heat-treating of other leaf materials had little effect, showing that NO scavenging will still occur after cooking. It is proposed that the NO scavenging of dietary components needs to be investigated more thoroughly before the full effects of increasing antioxidants through increased intake of fresh fruit and vegetables can truly be understood.

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Cell signalling is an immensely important topic in biological and biomedical sciences, and one which has an ever-increasing literature. As more and more is known about it, and more components are discovered, it is getting harder and harder to visualise how it all might work to create an holistic mechanism in the cell. To achieve a better understanding of a complex issue such as this, it is often useful to use an analogy which is familiar to the researcher to encourage better understanding. In this essay it is suggested that music, and the instruments used to produce it, can be used as such an analogy. Various elements and issues in cell signalling are discussed and musical comparisons are made. Clearly, the true understanding of cell signalling will come from systems biology and mathematical modelling, but it is proposed that this analogy might prove useful. The phrasing used may be considered a little loose and flamboyant for a scientific topic of such importance, but it is hoped that the discussion will not only be interesting but might also be useful in fostering debate and facilitating teaching in this area of molecular biology.

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BACKGROUND: Over the past 40 years, short-term psychodynamic psychotherapies (STPP) for a broad range of psychological and somatic disorders have been developed and studied. Four published meta-analyses of STPP, using different methods and samples, have found conflicting results. OBJECTIVES: This review evaluated the efficacy of STPP relative to minimal treatment and non-treatment controls for adults with common mental disorders. SEARCH STRATEGY: We searched CCDANCTR-Studies and CCDANCTR-References on 25/4/2005, CENTRAL, MEDLINE, CINAHL, EMBASE, PsycINFO, DARE and Biological Abstracts were also searched. We contacted triallists and checked references from papers retrieved. SELECTION CRITERIA: All randomised controlled trials (RCT) of adults with common mental disorders, in which a brief psychodynamic therapy lasting less than 40 hours in total, and provided in individual format, were included. DATA COLLECTION AND ANALYSIS: Three reviewers working in pairs evaluated studies. Studies were selected only if pairs of reviewers agreed they met inclusion criteria. A third reviewer was consulted if two reviewers could not reach consensus. Data were collected and entered into Review Manager. Study quality was assessed and scored by pairs of raters. Publication bias was assessed using a funnel plot. Sensitivity analyses were also conducted. MAIN RESULTS: 23 studies of 1431 randomised patients with common mental disorders were included. These studies evaluated STPP for general, somatic, anxiety, and depressive symptom reduction, as well as social adjustment. Outcomes for most categories of disorder suggested significantly greater improvement in the treatment versus the control groups, which were generally maintained in medium and long term follow-up. However, only a small number of studies contributed data for each category of disorder, there was significant heterogeneity between studies, and results were not always maintained in sensitivity analyses. AUTHORS' CONCLUSIONS: STPP shows promise, with modest to moderate, often sustained gains for a variety of patients. However, given the limited data and heterogeneity between studies, these findings should be interpreted with caution. Furthermore, variability in treatment delivery and treatment quality may limit the reliability of estimates of effect for STPP. Larger studies of higher quality and with specific diagnoses are warranted.

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Oxidative stress, resulting from an imbalance in the accumulation and removal of reactive oxygen species such as hydrogen peroxide (H(2)O(2)), is a challenge faced by all aerobic organisms. In plants, exposure to various abiotic and biotic stresses results in accumulation of H(2)O(2) and oxidative stress. Increasing evidence indicates that H(2)O(2) functions as a stress signal in plants, mediating adaptive responses to various stresses. To analyze cellular responses to H(2)O(2), we have undertaken a large-scale analysis of the Arabidopsis transcriptome during oxidative stress. Using cDNA microarray technology, we identified 175 non-redundant expressed sequence tags that are regulated by H(2)O(2). Of these, 113 are induced and 62 are repressed by H(2)O(2). A substantial proportion of these expressed sequence tags have predicted functions in cell rescue and defense processes. RNA-blot analyses of selected genes were used to verify the microarray data and extend them to demonstrate that other stresses such as wilting, UV irradiation, and elicitor challenge also induce the expression of many of these genes, both independently of, and, in some cases, via H(2)O(2).

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It has now become recognized that one of the key events in the induction of apoptosis, or programmed cell death, in both plants and animals is the release of cytochrome c from mitochondria. It is also known that oxidative stress imposed on cells can have a profound effect on the onset or progression of apoptosis. Here, we discuss how the redox status of cytochrome c, and thus its structure, can be altered by the presence of reactive oxygen species (ROS) and reduced glutathione (GSH). We suggest that cytochrome c will only induce programmed cell death if present in the cytoplasm in the oxidized state, and that the presence of high levels of cytoplasmic GSH maintain cytochrome c in an inactive (reduced) state, thus behaving as a fail-safe mechanism if cytochrome c is released by mitochondria when programmed cell death is not the required outcome. If the redox status of the cell is disturbed however, perhaps in the presence of hydrogen peroxide, GSH concentrations will drop, the cellular E(h) will rise, and cytochrome c will tend towards the oxidized state, allowing programmed cell death to proceed. Therefore, we propose that the redox state of cytoplasmic cytochrome c may be a key regulator of programmed cell death.

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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.

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Reactive oxygen species (ROS) were originally thought to only be released by phagocytic cells during their role in host defence. It is now clear that ROS have a cell signalling role in many biological systems, both in animals and in plants. ROS induce programmed cell death or necrosis, induce or suppress the expression of many genes, and activate cell signalling cascades, such as those involving mitogen-activated protein kinases.

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Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant-pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase-like activity in Arabidopsis cells, while the caspase-1 inhibitor, Ac-YVAD-CMK, blocked NO-induced cell death. A well-established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell-permeable cGMP analogue, 8Br-cGMP, although 8Br-cGMP alone did not induce cell death or potentiate NO-induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO-induced cell death in Arabidopsis. In-gel protein kinase assays showed that NO activates a potential mitogen-activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2-induced cell death, did not inhibit the effects of NO.

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NADPH oxidase is classically regarded as a key enzyme of neutrophils, where it is involved in the pathogenic production of reactive oxygen species. However, NADPH oxidase-like enzymes have recently been identified in non-neutrophil cells, supporting a separate role for NADPH-oxidase derived oxygen species in oxygen sensitive processes. This article reviews the current literature surrounding the potential role of NADPH oxidase in the oxygen sensing processes which underlie hypoxic pulmonary vasoconstriction, systemic vascular smooth muscle proliferation, carotid and airways chemoreceptor activation, erythropoietin gene expression, and oxytropic responses of plant cells.

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Hydrogen peroxide (H(2)O(2)) is generated in plants after exposure to a variety of biotic and abiotic stresses, and has been shown to induce a number of cellular responses. Previously, we showed that H(2)O(2) generated during plant-elicitor interactions acts as a signaling molecule to induce the expression of defense genes and initiate programmed cell death in Arabidopsis thaliana suspension cultures. Here, we report for the first time the identification by RNA differential display of four genes whose expression is induced by H(2)O(2). These include genes that have sequence homology to previously identified Arabidopsis genes encoding a late embryogenesis-abundant protein, a DNA-damage repair protein, and a serine/threonine kinase. Their putative roles in H(2)O(2)-induced defense responses are discussed.

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Proctolin caused dose-dependent (1-200 nM) contraction of the isolated hindgut of S. gregaria which was abolished by [alpha-methyl-L-tyrosine(2)]-proctolin (1 microM). In comparison, cycloproctolin (5 microM) reduced the proctolin maximum response by 41%. Hindgut homogenates contained [(3)H]proctolin binding sites with a K(d) value of 660 nM, a B(max) value of 23.8 pmol/mg protein and a Hill coefficient of 0.934. Cycloproctolin (IC(50,) 220 nM; K(i), 204 nM), unlabeled proctolin (IC(50) 680 nM) and [alpha-methyl-L-tryosine(2)]-proctolin (IC(50) 3.1 microM, K(i), 2.9 microM) but not SchistoFLRFamide (1 nM-10 microM) were capable of displacing bound [(3)H]proctolin.

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Elicitation of Arabidopsis thaliana (L.) Heynh. suspension cultures with the bacterial protein harpin (from Pseudomonas syringae pv. syringae) induced the activation of two kinases of 39 and 44 kDa, as demonstrated by in-gel kinase assays using myelin basic protein (MBP) as a substrate. Both these kinases appeared to be tyrosine-phosphorylated upon activation, as demonstrated by treatment with tyrosine phosphatase and immunoprecipitation using an anti-phosphotyrosine monoclonal antibody. An inhibitor of mammalian mitogen-activated protein kinase (MAPK) activation, PD98059, inhibited harpin-induced MBPK activation, but did not inhibit the activity of these kinases. PD98059 also inhibited harpin-induced programmed cell death and defence gene expression, suggesting the involvement of harpin-induced MAPKs in defence responses in Arabidopsis thaliana.

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Programmed cell death is increasingly viewed as a key component of the hypersensitive disease resistance response of plants. The generation of reactive oxygen species (ROS) such as H2O2 triggers a cell death programme in Arabidopsis suspension cultures following challenge with the bacterial elicitor harpin. Both harpin and exogenous H2O2 initiate a cell death pathway that requires gene expression, and also act as signalling molecules to induce the expression of plant defence genes encoding enzymes such as phenylalanine ammonia-lyase (PAL), glutathione S-transferase (GST) and anthranilate synthase (ASA1), an enzyme of phytoalexin biosynthesis in Arabidopsis. H2O2 induces the expression of PAL1 and GST but not that of ASA1. Harpin initiates two signalling pathways, one leading to increased ROS generation and expression of PAL1 and GST mRNA, and another leading to increased GST and ASA1 expression, independent of H2O2.

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