Left atrial strain in adults after the arterial switch operation for transposition of the great arteries.

BACKGROUND: No study has focused on left atrial (LA) function assessed by echocardiography in adult patients with simple D-TGA after arterial switch operation (ASO). We aimed to describe LA strain parameters in these patients. METHODS: A prospective cohort study including 42 adult patients with simple D-TGA after ASO and 33 aged-matched controls. Phasic LA and LV global longitudinal strain (GLS) were obtained by transthoracic 2D-speckle tracking echocardiography (STE). Volumetric and functional analysis of LA and LV were also evaluated by 2D and 3D analysis. A multivariable model was performed to investigate the variables that best differentiate patients with D-TGA from healthy controls. RESULTS: LA strain parameters in D-TGA patients were within the normal range described for healthy subjects. However, the three LA strain parameters (Reservoir, Conduit, and Contraction) were lower in patients (LASr: 31.13 ± 7.67 vs. 49.71 ± 8.38; LAS cd: -22.91 ± 5.69 vs. -34.55 ± 6.54; LASct: -8.14 ± 4.93 vs. -15.15 ± 6.07, p < .001 for all three comparisons). LA volumes were similar between patients and controls. LV-GLS remained significantly lower in the D-TGA group than in controls (-17.29 ± 2.68 vs. -21.98 ± 1.84, p < .001). D-TGA patients had evidence of worse LV ejection fraction measured by the Teichholz method (63.38 ± 8.23 vs. 69.28 ± 5.92, p = .001) and 3D analysis (57.97% ± 4.16 vs. 60.67 ± 3.39, p = .011) and diastolic dysfunction as compared to healthy controls. LV-GLS and conduit LAS were the variables best differentiating patients with D-TGA from healthy controls. CONCLUSIONS: LA strain is impaired in young adults with simple D-TGA late after the ASO, probably in agreement with some degree of LV dysfunction previously described.

Insights into ROS-dependent signalling underlying transcriptomic plant responses to the herbicide 2,4-D.

The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) functions as an agronomic weed control herbicide. High concentrations of 2,4-D induce plant growth defects, particularly leaf epinasty and stem curvature. Although the 2,4-D triggered reactive oxygen species (ROS) production, little is known about its signalling. In this study, by using a null mutant in peroxisomal acyl CoA oxidase 1 (acx1-2), we identified acyl-coenzyme A oxidase 1 (ACX1) as one of the main sources of ROS production and, in part, also causing the epinastic phenotype following 2,4-D application. Transcriptomic analyses of wild type (WT) plants after treatment with 2,4-D revealed a ROS-related peroxisomal footprint in early plant responses, while other organelles, such as mitochondria and chloroplasts, are involved in later responses. Interestingly, a group of 2,4-D-responsive ACX1-dependent transcripts previously associated with epinasty is related to auxin biosynthesis, metabolism, and signalling. We found that the auxin receptor auxin signalling F-box 3 (AFB3), a component of Skp, Cullin, F-box containing complex (SCF) (ASK-cullin-F-box) E3 ubiquitin ligase complexes, which mediates auxin/indole acetic acid (AUX/IAA) degradation by the 26S proteasome, acts downstream of ACX1 and is involved in the epinastic phenotype induced by 2,4-D. We also found that protein degradation associated with ubiquitin E3-RING and E3-SCF-FBOX in ACX1-dependent signalling in plant responses to 2,4-D is significantly regulated over longer treatment periods.

Nitric oxide is essential for cadmium-induced peroxule formation and peroxisome proliferation.

Nitric oxide (NO) and nitrosylated derivatives are produced in peroxisomes, but the impact of NO metabolism on organelle functions remains largely uncharacterised. Double and triple NO-related mutants expressing cyan florescent protein (CFP)-SKL (nox1 × px-ck and nia1 nia2 × px-ck) were generated to determine whether NO regulates peroxisomal dynamics in response to cadmium (Cd) stress using confocal microscopy. Peroxule production was compromised in the nia1 nia2 mutants, which had lower NO levels than the wild-type plants. These findings show that NO is produced early in the response to Cd stress and was involved in peroxule production. Cd-induced peroxisomal proliferation was analysed using electron microscopy and by the accumulation of the peroxisomal marker PEX14. Peroxisomal proliferation was inhibited in the nia1 nia2 mutants. However, the phenotype was recovered by exogenous NO treatment. The number of peroxisomes and oxidative metabolism were changed in the NO-related mutant cells. Furthermore, the pattern of oxidative modification and S-nitrosylation of the catalase (CAT) protein was changed in the NO-related mutants in both the absence and presence of Cd stress. Peroxisome-dependent signalling was also affected in the NO-related mutants. Taken together, these results show that NO metabolism plays an important role in peroxisome functions and signalling.

Nitric oxide and phytoglobin PHYTOGB1 are regulatory elements in the Solanum lycopersicum-Rhizophagus irregularis mycorrhizal symbiosis.

The regulatory role of nitric oxide (NO) and phytoglobins in plant response to pathogenic and mutualistic microbes has been evidenced. However, little is known about their function in the arbuscular mycorrhizal (AM) symbiosis. We investigated whether NO and phytoglobin PHYTOGB1 are regulatory components in the AM symbiosis. Rhizophagus irregularis in vitro-grown cultures and tomato plants were used to monitor AM-associated NO-related root responses as compared to responses triggered by the pathogen Fusarium oxysporum. A genetic approach was conducted to understand the role of PHYTOGB1 on NO signaling during both interactions. After a common early peak in NO levels in response to both fungi, a specific NO accumulation pattern was triggered in tomato roots during the onset of the AM interaction. PHYTOGB1 was upregulated by the AM interaction. By contrast, the pathogen triggered a continuous NO accumulation and a strong downregulation of PHYTOGB1. Manipulation of PHYTOGB1 levels in overexpressing and silenced roots led to a deregulation of NO levels and altered mycorrhization and pathogen infection. We demonstrate that the onset of the AM symbiosis is associated with a specific NO-related signature in the host root. We propose that NO regulation by PHYTOGB1 is a regulatory component of the AM symbiosis.

Cadmium induces reactive oxygen species-dependent pexophagy in Arabidopsis leaves.

Cadmium treatment induces transient peroxisome proliferation in Arabidopsis leaves. To determine whether this process is regulated by pexophagy and to identify the mechanisms involved, we analysed time course-dependent changes in ATG8, an autophagy marker, and the accumulation of peroxisomal marker PEX14a. After 3 hr of Cd exposure, the transcript levels of ATG8h, ATG8c, a, and i were slightly up-regulated and then returned to normal. ATG8 protein levels also increased after 3 hr of Cd treatment, although an opposite pattern was observed in PEX14. Arabidopsis lines expressing GFP-ATG8a and CFP-SKL enabled us to demonstrate the presence of pexophagic processes in leaves. The Cd-dependent induction of pexophagy was demonstrated by the accumulation of peroxisomes in autophagy gene (ATG)-related Arabidopsis knockout mutants atg5 and atg7. We show that ATG8a colocalizes with catalase and NBR1 in the electron-dense peroxisomal core, thus suggesting that NBR1 may be an autophagic receptor for peroxisomes, with catalase being possibly involved in targeting pexophagy. Protein carbonylation and peroxisomal redox state suggest that protein oxidation may trigger pexophagy. Cathepsine B, legumain, and caspase 6 may also be involved in the regulation of pexophagy. Our results suggest that pexophagy could be an important step in rapid cell responses to cadmium.

Peroxisomes Extend Peroxules in a Fast Response to Stress via a Reactive Oxygen Species-Mediated Induction of the Peroxin PEX11a.

Peroxisomes are highly dynamic and metabolically active organelles that play an important role in cellular functions, including reactive oxygen species (ROS) metabolism. Peroxisomal dynamics, such as the proliferation, movement, and production of dynamic extensions called peroxules, have been associated with ROS in plant cells. However, the function and regulation of peroxules are largely unknown. Using confocal microscopy, we have shown that treatment of Arabidopsis leaves with the heavy metal cadmium produces time course-dependent changes in peroxisomal dynamics, starting with peroxule formation, followed by peroxisome proliferation, and finally returning to the normal morphology and number. These changes during Cd treatment were regulated by NADPH oxidase (C and F)-related ROS production. Peroxule formation is a general response to stimuli such as arsenic and is regulated by peroxin 11a (PEX11a), as Arabidopsis pex11a RNAi lines are unable to produce peroxules under stress conditions. The pex11a line showed higher levels of lipid peroxidation content and lower expression of genes involved in antioxidative defenses and signaling, suggesting that these extensions are involved in regulating ROS accumulation and ROS-dependent gene expression in response to stress. Our results demonstrate that PEX11a and peroxule formation play a key role in regulating stress perception and fast cell responses to environmental cues.

Epigenetic changes accompany developmental programmed cell death in tapetum cells.

The tapetum, the nursing tissue inside anthers, undergoes cellular degradation by programmed cell death (PCD) during late stages of microspore-early pollen development. Despite the key function of tapetum, little is known about the molecular mechanisms regulating this cell death process in which profound nuclear and chromatin changes occur. Epigenetic features (DNA methylation and histone modifications) have been revealed as hallmarks that establish the functional status of chromatin domains, but no evidence on the epigenetic regulation of PCD has been reported. DNA methylation is accomplished by DNA methyltransferases, among which DNA methyl transferase 1 (MET1) constitutes one of the CG maintenance methyltransferase in plants, also showing de novo methyltransferase activity. In this work, the changes in epigenetic marks during the PCD of tapetal cells have been investigated by a multidisciplinary approach to reveal the dynamics of DNA methylation and the pattern of expression of MET1 in relation to the main cellular changes of this PCD process which have also been characterized in two species, Brassica napus and Nicotiana tabacum. The results showed that tapetum PCD progresses with the increase in global DNA methylation and MET1 expression, epigenetic changes that accompanied the reorganization of the nuclear architecture and a high chromatin condensation, activity of caspase 3-like proteases and Cyt c release. The reported data indicate a relationship between the PCD process and the DNA methylation dynamics and MET1 expression in tapetal cells, suggesting a possible new role for the epigenetic marks in the nuclear events occurring during this cell death process and providing new insights into the epigenetic control of plant PCD.

Cadmium induces two waves of reactive oxygen species in Glycine max (L.) roots.

Cadmium (Cd) is a non-essential heavy metal that may be toxic or even lethal to plants as it can be easily taken up by the roots and loaded into the xylem to the leaves. Using soybean roots (Glycine max L.) DM 4800, we have analysed various parameters related to reactive oxygen metabolism and nitric oxide (NO) during a 6 day Cd exposure. A rise in H(2)O(2) and NO, and to a lesser extent O(2)(·-) content was observed after 6 h exposure with a concomitant increase in lipid peroxidation and carbonyl group content. Both oxidative markers were significantly reduced after 24 h. A second, higher wave of O(2)(·-) production was also observed after 72 h of exposure followed by a reduction until the end of the treatment. NOX and glicolate oxidase activity might be involved in the initial Cd-induced reactive oxygen species (ROS) production and it appears that other sources may also participate. The analysis of antioxidative enzymes showed an increase in glutathione-S-transferase activity and in transcript levels and activity of enzymes involved in the ascorbate-glutathione cycle and the NADPH-generating enzymes. These results suggest that soybean is able to respond rapidly to oxidative stress imposed by Cd by improving the availability of NADPH necessary for the ascorbate-glutathione cycle.

Does the calcineurin inhibitor have influence on cytomegalovirus infection in heart transplantation?

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality in heart transplant (HTx). Our aim was to analyze the rate of CMV infection in HTx patients receiving treatment with cyclosporine (CsA) or tacrolimus (Tac). Ninety-five patients were randomized to receive either CsA (53.7%) or Tac (46.3%). We performed prophylaxis with valganciclovir in patients with the highest risk of CMV infection. We considered CMV infection as an increased viral load or the presence of CMV in histological samples. We analyzed baseline characteristics, CMV infection, and other complications. Event-free rates were calculated using the Kaplan-Meier method. There were no significant differences in baseline characteristics between both groups. CMV infection was detected in 31.6% of patients (in 66.7% due to asymptomatic replication). The group treated with Tac had a lower rate of CMV infection (15.9% vs. 45.1%, p = 0.002) and longer CMV infection-free survival time (1440 vs. 899 d, p = 0.001). No differences were observed in the complications analyzed in both groups. The independent risk factors for infection identified in the multivariate analysis were treatment with CsA and bacterial infections. This was the first study to demonstrate a lower rate of CMV infection in patients treated with Tac vs. those treated with CsA after HTx.

Role of peroxisomes as a source of reactive oxygen species (ROS) signaling molecules.

Peroxisomes are very dynamic and metabolically active organelles and are a very important source of reactive oxygen species (ROS), H2O2, O2 (.-) and · OH, which are mainly produced in different metabolic pathways, including fatty acid ß-oxidation, photorespiration, nucleic acid and polyamine catabolism, ureide metabolism, etc. ROS were originally associated to oxygen toxicity; however, these reactive species also play a central role in the signaling network regulating essential processes in the cell. Peroxisomes have the capacity to rapidly produce and scavenge H2O2 and O2 (.-) which allows to regulate dynamic changes in ROS levels. This fact and the plasticity of these organelles, which allows adjusting their metabolism depending on different developmental and environmental cues, makes these organelles play a central role in cellular signal transduction. The use of catalase and glycolate oxidase loss-of-function mutants has allowed to study the consequences of changes in the levels of endogenous H2O2 in peroxisomes and has improved our knowledge of the transcriptomic profile of genes regulated by peroxisomal ROS. It is now known that peroxisomal ROS participate in more complex signaling networks involving calcium, hormones, and redox homeostasis which finally determine the response of plants to their environment.

Quality of life of patients with rheumatic diseases.

OBJECTIVE: Health-related quality of life (HRQoL) is an important indicator of population health and can measure the impact of medical actions. The main objective of this study was to determine the HRQoL of patients with rheumatic diseases (RD) and compare it with that of the general population. METHODS: Observational, cross-sectional, single-center study, with consecutive inclusion of outpatients over 18 years of age seen at a Rheumatology hospital-based outpatient clinic in Madrid. Sociodemographic, clinical variables and HRQoL were recorded. HRQoL was measured with the 5-dimension, 5-level EuroQoL (EQ-5D-5L), which includes the EQ-Index (0-1 scale) and a visual analog scale (VAS, 0-100 scale). A descriptive analysis and a comparison with the HRQoL of the Spanish general population were performed. RESULTS: 1144 patients were included, 820 (71.68%) women, with a mean age of 56.1 years (range 18-95), of whom 241 (25.44%) were new patients. In patients with RD, the HRQoL measured with the EQ-Index and with the VAS, was 0.186 and 12 points lower, respectively, than in the general population. The decrease in HRQoL affected the 5 health dimensions, especially "pain/discomfort", followed by "daily activities" and "mobility". This reduction in HRQoL was observed in both men and women, and in all age ranges, although it was greater between 18 and 65 years of age. The reduction in HRQoL affected all RD subtypes, especially the "peripheral and axial mechanical pathology" and the "soft tissue pathology" group. CONCLUSIONS: Patients with rheumatic diseases report worse HRQoL when compared to the general population in all dimensions of HRQoL.

Spatiotemporal Complexity of Vena Contracta and Mitral Regurgitation Grading Using Three-Dimensional Echocardiographic Analysis.

BACKGROUND: Spatiotemporal complexity of the color Doppler vena contracta challenging the assumption of a circular and constant orifice may lead to mitral regurgitation (MR) grading inconsistencies. Using 3D transesophageal echocardiography, we characterized spatiotemporal vena contracta complexity and its impact on MR severity grading. METHODS: In 192 patients with suspected moderate or severe MR (100 primary MR [PMR]; 92 secondary MR [SMR]), we performed three-dimensional vena contracta area (VCA) quantification using single-frame (midsystolic or VCAmid, maximum or VCAmax) and multiframe (VCAmean) methods, as well as measures of orifice shape (shape index) and systolic variation of VCA. Vena contracta complexity and intermethod discrepancies were analyzed and correlated with functional class and pulmonary vein flow (PVF) patterns and with cardiac magnetic resonance (CMR) in a subset of cases (n = 20). RESULTS: The vena contracta was noncircular (shape index > 1.5) in 90% of patients. Severe noncircularity (shape index > 3) was more prevalent in SMR than in PMR (32.4% vs 14.6%). Variations of the VCA were more prominent in SMR than in PMR. VCAmid showed a low grading agreement with VCAmax (62%) and high grading agreement with VCAmean (83.3%). Pulmonary vein flow systolic reversal was associated with MR severity by VCA in SMR but not in PMR. VCAmid and VCAmean showed a stronger association with systolic flow reversal than VCAmax (area under the curve, 0.88, 0.86, and 0.79, respectively). In the subset of patients with CMR quantification, severe MR by VCAmax was graded as nonsevere by CMR more frequently compared with VCAmid and VCAmean. CONCLUSIONS: Highly prevalent spatiotemporal vena contracta complexity features in MR challenge the assumption of a circular and constant orifice. VCAmid seems the best single-frame approximation to multiframe quantification, and VCAmax may lead to severity overestimation.

Incidence and Risk Factors for Development of Cardiac Toxicity in Adult Patients with Newly Diagnosed Acute Myeloid Leukemia.

The incidence of cardiac morbimortality in acute myeloid leukemia (AML) is not well known. We aim to estimate the cumulative incidence (CI) of cardiac events in AML patients and to identify risk factors for their occurrence. Among 571 newly diagnosed AML patients, 26 (4.6%) developed fatal cardiac events, and among 525 treated patients, 19 (3.6%) experienced fatal cardiac events (CI: 2% at 6 months; 6.7% at 9 years). Prior heart disease was associated with the development of fatal cardiac events (hazard ratio (HR) = 6.9). The CI of non-fatal cardiac events was 43.7% at 6 months and 56.9% at 9 years. Age ≥ 65 (HR = 2.2), relevant cardiac antecedents (HR = 1.4), and non-intensive chemotherapy (HR = 1.8) were associated with non-fatal cardiac events. The 9-year CI of grade 1-2 QTcF prolongation was 11.2%, grade 3 was 2.7%, and no patient had grade 4-5 events. The 9-year CI of grade 1-2 cardiac failure was 1.3%, grade 3-4 was 15%, and grade 5 was 2.1%; of grade 1-2, arrhythmia was 1.9%, grade 3-4 was 9.1%, and grade 5 was 1%. Among 285 intensive therapy patients, median overall survival decreased in those experiencing grade 3-4 cardiac events (p < 0.001). We observed a high incidence of cardiac toxicity associated with significant mortality in AML.

CA125: a new biomarker in patients with Fontan circulation.

INTRODUCTION AND OBJECTIVES: Patients with Fontan circulation (FC) have a high incidence of clinical complications. However, no biomarker is able to accurately stratify risk. The aim of this study was to analyze the relationship between biomarkers and clinical complications, including carbohydrate antigen 125 (CA125) for the first time, and to propose a risk estimation based on a combination of biomarkers. METHODS: Cross-sectional study of patients with FC. The clinical endpoint was the combination of heart failure, atrial arrhythmias, veno-venous fistulae, protein-losing enteropathy, or plastic bronchitis. Demographic, clinical, and laboratory variables were analyzed, including CA125, NT-proBNP, renal and liver function, and red cell distribution width (RDW). We performed univariate and multivariate analyses of the relationship between these variables and the composite endpoint. Cutoff values were calculated by ROC curves. RESULTS: We included 56 patients (27.4±7.8 years). A total of 34% showed the composite endpoint, with significantly higher CA125 levels (30.1 IU/mL vs 12.6 IU/mL; P=.001). In the multivariate model, the biomarkers related to the endpoint were LnCA125 (OR, 5.1; 95%CI, 1.2-22), RDW (OR, 1.8; 95%CI, 1.1-3.1), and FIB4 (OR, 38, 95%CI, 1.7-855). The cutoff points were CA125 ≥ 20 U/mL, FIB4 ≥ 0.75, and RDW ≥ 14.5%, and the probability of the occurrence of the endpoint was 81% if ≥ 2 biomarkers were elevated. CONCLUSIONS: CA125 elevation is associated with a higher prevalence of complications in patients with Fontan-type circulation. CA125 levels ≥ 20U/mL, FIB4 ≥ 0.75 and RDW ≥ 14.5% identify with a high probability the clinical failure of FC.

Peroxynitrite mediates programmed cell death both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.).

Programmed cell death (PCD) has been found to be induced after pollination both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.). Reactive oxygen species (ROS) and nitric oxide (NO) are known to be produced in the pistil and pollen during pollination but their contribution to PCD has so far remained elusive. The possible role of ROS and NO was investigated in olive pollen-pistil interaction during free and controlled pollination and it was found that bidirectional interaction appears to exist between the pollen and the stigma, which seems to regulate ROS and NO production. Biochemical evidence strongly suggesting that both O(2)(-) and NO are essential for triggering PCD in self-incompatibility processes was also obtained. It was observed for the first time that peroxynitrite, a powerful oxidizing and nitrating agent generated during a rapid reaction between O(2)(-) and NO, is produced during pollination and that this is related to an increase in protein nitration which, in turn, is strongly associated with PCD. It may be concluded that peroxynitrite mediates PCD during pollen-pistil interaction in Olea europaea L. both in self-incompatible pollen and papillar cells.

NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley.

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.

DNA methylation dynamics and MET1a-like gene expression changes during stress-induced pollen reprogramming to embryogenesis.

Stress-induced plant cell reprogramming involves changes in global genome organization, being the epigenetic modifications key factors in the regulation of genome flexibility. DNA methylation, accomplished by DNA methyltransferases, constitutes a prominent epigenetic modification of the chromatin fibre which is locked in a transcriptionally inactive conformation. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation. After a stress treatment, in vitro-cultured microspores are reprogrammed and change their gametophytic developmental pathway towards embryogenesis, the process constituting a useful system of reprogramming in isolated cells for applied and basic research. Gene expression driven by developmental and stress cues often depends on DNA methylation; however, global DNA methylation and genome-wide expression patterns relationship is still poorly understood. In this work, the dynamics of DNA methylation patterns in relation to nuclear architecture and the expression of BnMET1a-like DNA methyltransferase genes have been analysed during pollen development and pollen reprogramming to embryogenesis in Brassica napus L. by a multidisciplinary approach. Results showed an epigenetic reprogramming after microspore embryogenesis induction which involved a decrease of global DNA methylation and its nuclear redistribution with the change of developmental programme and the activation of cell proliferation, while DNA methylation increases with pollen and embryo differentiation in a cell-type-specific manner. Changes in the presence, abundance, and distribution of BnMET1a-like transcripts highly correlated with variations in DNA methylation. Mature zygotic and pollen embryos presented analogous patterns of DNA methylation and MET1a-like expression, providing new evidence of the similarities between both developmental embryogenic programmes.

S-Nitrosylated proteins in pea (Pisum sativum L.) leaf peroxisomes: changes under abiotic stress.

Peroxisomes, single-membrane-bounded organelles with essentially oxidative metabolism, are key in plant responses to abiotic and biotic stresses. Recently, the presence of nitric oxide (NO) described in peroxisomes opened the possibility of new cellular functions, as NO regulates diverse biological processes by directly modifying proteins. However, this mechanism has not yet been analysed in peroxisomes. This study assessed the presence of S-nitrosylation in pea-leaf peroxisomes, purified S-nitrosylated peroxisome proteins by immunoprecipitation, and identified the purified proteins by two different mass-spectrometry techniques (matrix-assisted laser desorption/ionization tandem time-of-flight and two-dimensional nano-liquid chromatography coupled to ion-trap tandem mass spectrometry). Six peroxisomal proteins were identified as putative targets of S-nitrosylation involved in photorespiration, ß-oxidation, and reactive oxygen species detoxification. The activity of three of these proteins (catalase, glycolate oxidase, and malate dehydrogenase) is inhibited by NO donors. NO metabolism/S-nitrosylation and peroxisomes were analysed under two different types of abiotic stress, i.e. cadmium and 2,4-dichlorophenoxy acetic acid (2,4-D). Both types of stress reduced NO production in pea plants, and an increase in S-nitrosylation was observed in pea extracts under 2,4-D treatment while no total changes were observed in peroxisomes. However, the S-nitrosylation levels of catalase and glycolate oxidase changed under cadmium and 2,4-D treatments, suggesting that this post-translational modification could be involved in the regulation of H(2)O(2) level under abiotic stress.

Differential response of young and adult leaves to herbicide 2,4-dichlorophenoxyacetic acid in pea plants: role of reactive oxygen species.

In this work the differential response of adult and young leaves from pea (Pisum sativum L.) plants to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) (23 mm) applied by foliar spraying was investigated. The concentration of 2,4-D (23 mm) and the time of treatment (72 h) were previously optimized in order to visualize its toxic effects on pea plants. Under these conditions, the herbicide induced severe disturbances in mesophyll cells structure and proliferation of vascular tissue in young leaves and increased acyl-CoA oxidase (ACX), xanthine oxidase (XOD) and lipoxygenase (LOX) activities in young leaves, and only ACX and LOX in adult leaves. This situation produced reactive oxygen species (ROS) over-accumulation favoured by the absence of significant changes in the enzymatic antioxidants, giving rise to oxidative damages to proteins and membrane lipids. An increase of ethylene took place in both young and adult leaves and the induction of genes encoding the stress proteins, PRP4A and HSP 71,2, was observed mainly in young leaves. These results suggest that ROS overproduction is a key factor in the effect of high concentrations of 2,4-D, and ROS can trigger a differential response in young and adult leaves, either epinasty development in young leaves or senescence processes in adult tissues.

ß2-Adrenoceptors and GRK2 as Potential Biomarkers in Patients With Chronic Pulmonary Regurgitation.

Pulmonary regurgitation (PR) is a frequent complication after repair of congenital heart disease. Three different GRK isoforms (GRK2, GRK5, and GRK3) and two ß-adrenoceptors (ß1-AR and ß2-AR) are present in peripheral blood mononuclear cells (PBMC) and their expression changes as a consequence of the hemodynamic and neurohumoral alterations that occur in some cardiovascular diseases. Therefore, they could be useful as biomarkers in PR. A prospective study was conducted to describe the expression (TaqMan Gene Expression Assays) of ß-ARs and GRKs in PBMC isolated (Ficoll® gradient) from patients with severe PR before and after pulmonary valve replacement and establish if this expression correlates to clinical status. 23 patients with severe PR were included and compared with 22 healthy volunteers (controls). PR patients before the PVR had a significantly lower expression of ß2-AR (513.8 ± 261.2 mRNA copies) vs. controls (812.5 ± 497.2 mRNA copies), so as GRK2 expression (503.4 ± 364.9 copies vs. 858.1 ± 380.3 mRNA copies). The expression of ß2-AR and GRK2 significantly decreases in symptomatic and asymptomatic patients, as well as in patients under treatment with beta-blockers and non-treated patients. The expression of ß2-AR and GRK2 in PR patients recovers the normal values after pulmonary valve replacement (754,8 ± 77,1 and 897,8 ± 87,4 copies, respectively). Therefore, changes in the expression of ß2-AR and GRK2 in PBMC of PR patients, could be considered as potential biomarkers to determine clinical decisions.