The diatom-derived aldehyde decadienal affects life cycle transition in the ascidian Ciona intestinalis through nitric oxide/ERK signalling

Polyunsaturated aldehydes (PUAs) are fatty-acid-derived metabolites produced by some microalgae, including different diatom species. PUAs are mainly produced as a wound-activated defence mechanism against microalgal predators or released from senescent cells at the end of a bloom. PUAs, including 2,4-trans-decadienal (DD), induce deleterious effects on embryonic and larval development of several planktonic and benthic organisms. Here, we report on the effects of DD on larval development and metamorphosis of the ascidian Ciona intestinalis. Ciona larval development is regulated by the cross-talking of different molecular events, including nitric oxide (NO) production, ERK activation and caspase 3-dependent apoptosis. We report that treatment with DD at the competence larval stage results in a delay in metamorphosis. DD affects redox balance by reducing total glutathione and NO levels. By biochemical and quantitative gene expression analysis, we identify the NO-signalling network affected by DD, including the upregulation of ERK phosphatase mkp1 and consequent reduction of ERK phosphorylation, with final changes in the expression of downstream ERK target genes. Overall, these results give new insights into the molecular pathways induced in marine organisms after exposure to PUAs during larval development, demonstrating that this aldehyde affects key checkpoints of larval transition from the vegetative to the reproductive life stage. & 2015 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited

Propagators and Path Integrals

Path-integral expressions for one-particle propagators in scalar and fermionic field theories are derived, for arbitrary mass. This establishes a direct connection between field theory and specific classical point-particle models. The role of world-line reparametrization invariance of the classical action and the implementation of the corresponding BRST-symmetry in the quantum theory are discussed. The presence of classical world-line supersymmetry is shown to lead to an unwanted doubling of states for massive spin-1/2 particles. The origin of this phenomenon is traced to a `hidden' topological fermionic excitation. A different formulation of the pseudo-classical mechanics using a bosonic representation of \gam_5 is shown to remove these extra states at the expense of losing manifest supersymmetry.Comment: 35 pages, latex, no figures, additional references; to be published in Nucl. Phys.

Sviluppo larvale e metamorfosi nell’ascidia Ciona intestinalis: ruolo del monossido di azoto

Dottorato di Ricerca in Biologia animale, Ciclo XXV, a.a. 2011-2012Sviluppo larvale e metamorfosi nell’ascidia Ciona intestinalis: ruolo del monossido di azoto Lo sviluppo larvale e la metamorfosi dell’ascidia Ciona intestinalis rappresentano un processo molto complesso mediante il quale la larva natante si trasforma in un individuo giovane sessile attraverso una profonda ricostruzione del piano corporeo e la regressione della coda. A livello molecolare, avvengono alcuni processi quali l'attivazione della caspasi-3-simile e della MAPK (Chambon et al., 2002, 2007) e la produzione di monossido di azoto (NO) (Comes et al., 2007). In particolare, è stato dimostrato che l'espressione spaziale di ossido nitrico sintasi (NOS) e di NO è molto dinamica durante lo sviluppo di ciona. Il segnale si sposta rapidamente lungo tutta la larva coinvolgendo il sistema nervoso centrale e la coda nei suoi diversi compartimenti. Inoltre, l'NO regola la regressione della coda, agendo sulla apoptosi mediata da caspasi-3-simile. Il progetto di tesi é stato focalizzato ad uno studio approfondito del ruolo dell'NO durante lo sviluppo larvale e la metamorfosi di C. intestinalis, fornendo nuovi elementi sui bersagli molecolari dell'NO in relazione allo sviluppo ed alla risposta allo stress. Una serie di esperimenti, volti ad esaminare l'andamento e la velocità di metamorfosi in diverse condizioni dei livelli di NO, hanno rivelato che l'NO prodotto nelle larve di ciona contribuisce alla fosforilazione della MAP chinasi ERK, un evento chiave e necessario affinché la metamorfosi avvenga. In particolare, diminuendo i livelli endogeni di NO, trattando le larve alla schiusa con un inibitore della NOS o un agente che lega l'NO, si ottiene un rallentamento della metamorfosi ed una concomitante riduzione della fosforilazione di ERK. Al contrario, un aumento di NO mediante l'uso di un donatore di NO, determina una accelerazione del processo accompagnata da un aumento di attivazione di ERK. Su questa base, é stato ipotizzato un possibile meccanismo per spiegare la fosforilazione di ERK indotta da NO, basato sulla nitrazione di ERK, come suggerito da esperimenti paralleli di immunoprecipitazione, che hanno mostrato la correlazione tra il livello di fosforilazione di ERK e la sua nitrazione. Un altro dato importante emerso da questa tesi è la dimostrazione che l'NO media anche una altra via di trasduzione durante la metamorfosi di ciona che comporta la nitrazione delle proteine. Il trattamento con l'agente nitrante, il perossinitrito, determina una accelerazione della metamorfosi, mentre agenti che legano l'NO provocano un effetto opposto. Un notevole aumento di nitrazione è stato rivelato allo stadio larvale rispetto a quello embrionale ed inoltre ERK e P-ERK sono risultate essere nitrate mediante metodologie immunochimiche. La scoperta di elevati livelli di proteine nitrate durante un processo fisiologico, quale lo sviluppo, riveste particolare importanza alla luce di dati sempre più numerosi in letteratura sulla nitrazione delle proteine quale una nuova via di trasduzione capace di agire direttamente o indirettamente su altre vie. Ulteriori esperimenti sono stati condotti per indagare la risposta allo stress in larve di ciona indotto dalla aldeide prodotta dalle diatomee, la decadienale (DD). Larve trattate con questa aldeide mostrano un ritardo della metamorfosi dipendente dalla concentrazione dell'aldeide e dallo stadio larvale al quale si fa l'incubazione. È interessante notare che il trattamento con DD determina una riduzione della fosforilazione di ERK analogamente a quanto osservato quando i livelli endogeni di NO vengono dimunuiti. Ulteriori studi saranno necessari per delineare il meccanismo con il quale DD influenzi la metamorfosi ed il coinvolgimento dell'NO.Università della Calabri

ERK independent mkp expression by NO regulates ERK activation.

<p>(A) Hatched larvae were treated with 0.25 µM of dusp 1/6 I (+) in the absence or presence of TRIM (250 µM). Samples collected after 6 h of treatment were examined for ERK activation. Representative western blot analyzed with anti-P-ERK and anti-ERK antibodies. (B) Data of densitometric analyses of 3 independent experiments are reported as means ± SEM and assessed by unpaired <i>t</i>-test. Asterisk represents the significance respect to the control *<i>P</i><0.05 (0.049), **<i>P</i><0.01 (0.007), ***<i>P</i><0.001 (0.0002). # represents the significance respect to the TRIM alone (<i>P</i> = 0.0008). (C) Gene regulation in response to MEK inhibitor U0126. Histogram shows the values of gene expression at 6 h treatment respect to the untreated larvae, followed by Real Time qPCR. Data are reported as a fold difference compared to the control (means ± SD). Values equal or greater than ±2 were considered significant. The experiments were repeated at least 3 times.</p

Nitric Oxide Affects ERK Signaling through Down-Regulation of MAP Kinase Phosphatase Levels during Larval Development of the Ascidian <i>Ciona intestinalis</i>

<div><p>In the ascidian <i>Ciona intestinalis</i> larval development and metamorphosis require a complex interplay of events, including nitric oxide (NO) production, MAP kinases (ERK, JNK) and caspase-3 activation. We have previously shown that NO levels affect the rate of metamorphosis, regulate caspase activity and promote an oxidative stress pathway, resulting in protein nitration. Here, we report that NO down-regulates MAP kinase phosphatases (mkps) expression affecting positively ERK signaling. By pharmacological approach, we observed that the reduction of endogenous NO levels caused a decrease of ERK phosphorylation, whereas increasing levels of NO induced ERK activation. We have also identified the ERK gene network affected by NO, including <i>mpk1</i>, <i>mpk3</i> and some key developmental genes by quantitative gene expression analysis. We demonstrate that NO induces an ERK-independent down-regulation of <i>mkp1</i> and <i>mkp3</i>, responsible for maintaining the ERK phosphorylation levels necessary for transcription of key metamorphic genes, such as the hormone receptor <i>rev-erb</i> and the van willebrand protein <i>vwa1c</i>. These results add new insights into the role played by NO during larval development and metamorphosis in <i>Ciona</i>, highlighting the cross-talk between different signaling pathways.</p></div

Increase of ERK phosphorylation by NO donor sperNO.

<p>Hatched larvae were treated with sperNO (250 µM) and samples at different times were examined for ERK activation. (A) Representative experiment showing the western blot analyzed with anti-P-ERK and anti-ERK antibodies. (B) Histogram showing densitometric analysis of immunopositive P-ERK respect to ERK bands. White bars, without sperNO; grey bars, with sperNO. Results are representative of 3 independent experiments. Data, expressed as means ± SEM, are assessed by unpaired <i>t</i>-test. Asterisk represents the significance respect to the control **<i>P</i><0.01 (0.005).</p

Multiple sequence alignment of <i>C. intestinalis</i> mkp1 with homologous counterparts.

<p><i>Ciona</i> sequence ci0100138796 was aligned using ClustalW with the following sequences retrieved from Uniprot database: zebrafish (<i>Danio rerio</i>) Q6IQU5, <i>Xenopus laevis</i> Q91790, chicken (<i>Gallus gallus</i>) F1NPN1, human P28562 and mouse P28563. The MAPK binding domain (MKB) or N-terminal domain is underlined in blue (21–136 aa in human sequence), the dual specific phosphatase domain (DSP) or C-terminal domain is underlined in red (175–367 aa in human sequence). The highly conserved catalytic motif I/VHCXAGXXR in DSP domain is highlighted in yellow, and the motif -ΨΨXRRΨXG- in the MKB domain is highlighted in green. Ψ is a hydrophobic residue and X is any residue. In red are the highly conserved positions including the catalytic cysteine (C 258 in human).</p

Features of transcripts used for gene expression analysis: accession numbers, primer sequences, length of PCR amplified fragments and data on gene regulation and localization in larvae.

<p>*This work.</p

Gene regulation in response to NO levels during larval development.

<p>Histogram shows the differences in expression levels of analyzed genes, followed by Real Time qPCR. Hatched larvae (18 hpf) incubated with 250 µM TRIM (A) or 250 µM sperNO (B) were collected at 22 hpf (blue), 24 hpf (yellow) and 26 hpf (green). Data are reported as a fold difference compared to the control, larvae in sea water without TRIM or sperNO (means ± SD). Values equal or greater than ±2 were considered significant. The experiments were repeated at least 3 times.</p

Protein nitration as footprint of oxidative stress-related nitric oxide signaling pathways in developing Ciona intestinalis

Developmental processes in the ascidian Ciona intestinalis depend on a complex interplay of events including, during metamorphosis, a caspase-dependent apoptosis which is regulated by the nitric oxide (NO)-cGMP signaling pathway. Herein we disclose an alternate NO-mediated signaling pathway during Ciona development which appears to be critically dependent on local redox control. Evidence in support of this conclusion includes: (a) inhibitors of NO synthase (NOS) and scavengers of NO-derived nitrating agents markedly decrease the rate of Ciona metamorphosis; (b) an NO donor or peroxynitrite caused an opposite effect; (c) increased protein nitration is observed at larva stage. Integrated proteomic and immunochemical methodologies identified nitrated tyrosine residues in ERK and snail. Overall, these results point to protein nitration as a hitherto overlooked NO-dependent regulatory mechanism in Ciona which is specifically triggered by elevated ROS production during developmental processes