Twiddler's Syndrome Causing Lead Fracture in Sacral Neuromodulation: A Case Report.

Twiddler's syndrome is the voluntary or involuntary manipulation of an implanted device, most described in cardiac literature. Lead coiling may result in device malfunction due to lead migration or, less commonly, lead fracture. There are few but increasing reports of Twiddler's syndrome resulting in lead migration in sacral neuromodulation, but lead fracture has not yet been described. A 57-year-old Latina female presented with fecal incontinence and refractory overactive bladder. She underwent successful implantation of a sacral neuromodulation device with the resolution of symptoms. Following significant weight loss and two falls, she developed a recurrence of symptoms and was found to have lead migration on pelvic radiographs. At the time of surgical intervention, radiographs demonstrated worsened Twiddler's syndrome with complete lead fracture despite no further trauma. She subsequently underwent partial lead removal and replacement with additional measures to prevent Twiddler's syndrome and its sequelae. Twiddler's syndrome resulting in lead fracture can occur in sacral neuromodulation. Preventive techniques may be applied for patients with known risk factors for Twiddler's syndrome, especially generator anchoring and lead replacement.

[CAPTURE Study: Argentine results on prevalence of cardiovascular disease in type 2 diabetes mellitus]. / Estudio CAPTURE: Resultados argentinos sobre prevalencia de enfermedad cardiovascular en diabetes mellitus tipo 2.

Cardiovascular disease (CVD) is the main cause of morbidity and mortality in patients with type 2 diabetes mellitus (DM2). However, scarce specific data are available about the prevalence of CVD in that population in daily clinical practice. The CAPTURE study investigated the contemporary prevalence of CVD in a representative sample of DM2 population in 13 countries, distributed over 5 continents (n = 9823). Among the 834 Argentinian participants, the CVD prevalence was 41.5% (n = 347) (95% confidence interval [95%CI]: 38.4-44.7%), while worldwide rate was 34.8% (n = 3582) (95%CI: 32.7-36.8%). In most cases, it was categorized as atherosclerotic CVD 35.3% (n = 295) (95%CI: 32.3; -38.4%). Likewise, the rate of use of antidiabetic drugs with proven cardiovascular benefits was low, both in Argentina (12.8%: n = 107) and global data (21.9%; n = 2151).

La enfermedad cardiovascular (ECV) es la principal causa de morbimortalidad en los pacientes con diabetes mellitus tipo 2 (DM2). Sin embargo, se dispone de escasos datos específicos sobre la prevalencia de ECV en esta población en la práctica clínica cotidiana. El estudio CAPTURE investigó la prevalencia contemporánea de ECV en una muestra representativa de la población con diagnóstico de DM2 en 13 países, distribuidos en 5 continentes (n = 9823). Entre los 834 participantes de Argentina, la prevalencia de ECV fue de 41.5% (n = 347) (intervalo de confianza del 95% [IC95%]: 38.4-44.7%), mientras que a nivel mundial la prevalencia fue 34.8% (n = 3582) (IC95%: 32.7-36.8%). La mayoría de los casos fue categorizada como ECV aterosclerótica: 35.3% (n = 295) (IC95%:32.3-38.4%). Asimismo, la utilización de medicamentos antidiabéticos con beneficios cardiovasculares comprobados fue baja: 12.8% (n = 107) y 21.9% (n = 2151) en Argentina y a nivel global respectivamente.

The influence of the interaction between staging, grading and extent on tooth loss due to periodontitis.

AIM: To assess the ability of two-way interactions between baseline stage, grade and extent to predict tooth loss due to periodontitis (TLP) over a long-term follow-up period. MATERIALS AND METHODS: Patients treated for periodontal disease with a complete medical history, baseline periodontal chart, full mouth radiographs and a minimum of ≥10 years follow-up were recruited. Supportive periodontal therapy (SPT) visits were recorded during the entire follow-up period. Patients were categorized according to their stage, grade and extent. The absolute survival at 10-, 20-, and 30-year follow-up was calculated for TLP. Kaplan-Meier survival curves were plotted at the tooth-level and multilevel Cox regression frailty models were constructed in order to assess the association among predictive variables and TLP by taking into account the hierarchical patient-teeth structure. RESULTS: 442 patients (11,125 teeth) with a mean follow-up of 23 years met the inclusion criteria and were included in this study. The most prevalent diagnosis at baseline was stage III grade B (30.3%), followed by stage II grade B (23.5%). Among the parameters analysed, stage and grade were found to be the best predictors of TLP. Statistically significant differences were observed for extent only in patients with severe disease (stage IV or grade C). The multilevel Cox regression analysis demonstrated that patients with higher concomitant baseline staging and grading developed greater TLP over the follow-up period. CONCLUSIONS: Higher concomitant staging and grading corresponded to greater risk for TLP and generalized extent only became a significant predictor in patients with stage IV or grade C disease.

Short-Term Low Temperature Induces Nitro-Oxidative Stress that Deregulates the NADP-Malic Enzyme Function by Tyrosine Nitration in Arabidopsis thaliana.

Low temperature (LT) negatively affects plant growth and development via the alteration of the metabolism of reactive oxygen and nitrogen species (ROS and RNS). Among RNS, tyrosine nitration, the addition of an NO2 group to a tyrosine residue, can modulate reduced nicotinamide-dinucleotide phosphate (NADPH)-generating systems and, therefore, can alter the levels of NADPH, a key cofactor in cellular redox homeostasis. NADPH also acts as an indispensable electron donor within a wide range of enzymatic reactions, biosynthetic pathways, and detoxification processes, which could affect plant viability. To extend our knowledge about the regulation of this key cofactor by this nitric oxide (NO)-related post-translational modification, we analyzed the effect of tyrosine nitration on another NADPH-generating enzyme, the NADP-malic enzyme (NADP-ME), under LT stress. In Arabidopsis thaliana seedlings exposed to short-term LT (4 °C for 48 h), a 50% growth reduction accompanied by an increase in the content of superoxide, nitric oxide, and peroxynitrite, in addition to diminished cytosolic NADP-ME activity, were found. In vitro assays confirmed that peroxynitrite inhibits cytosolic NADP-ME2 activity due to tyrosine nitration. The mass spectrometric analysis of nitrated NADP-ME2 enabled us to determine that Tyr-73 was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. The in silico analysis of the Arabidopsis NADP-ME2 protein sequence suggests that Tyr73 nitration could disrupt the interactions between the specific amino acids responsible for protein structure stability. In conclusion, the present data show that short-term LT stress affects the metabolism of ROS and RNS, which appears to negatively modulate the activity of cytosolic NADP-ME through the tyrosine nitration process.

Lessons learned from a single-surgeon series of paediatric robot-assisted laparoscopic urological procedures: predictors of high-grade postoperative complications.

OBJECTIVES: To describe postoperative complications after robot-assisted laparoscopic urological surgery in children, and identify potential predictors of these complications by analysing the outcomes of a large-volume single-surgeon experience. PATIENTS AND METHODS: We reviewed our institutional database to identify all robot-assisted laparoscopy (RAL) cases performed between December 2007 and December 2017. Patients were grouped into three cohorts based on the anatomical location of the procedure: upper urinary tract (kidney and renal pelvis); lower urinary tract (ureter); and lower urinary tract reconstruction with bowel (bladder reconstruction). A descriptive analysis of baseline characteristics, intra-operative variables and postoperative outcomes was carried out. All complications were graded using the Clavien-Dindo scale, and grouped based on type and time of occurrence (<30, 30-90, >90 days). Multivariable logistic regression analysis was performed to identify predictors of high-grade complications (Clavien-Dindo grade ≥ III). We also measured complication rates based on year of surgery and surgical caseload. RESULTS: Our database included a total of 326 patients, of whom 57% (n = 186) underwent upper urinary tract procedures, 30% (n = 97) ureteric procedures, and 13% bladder reconstruction. The median follow-up for each procedure was 13, 11 and 57 months, respectively. Of the total, 10 cases were converted to an open approach and excluded from further analysis. The most common types of complication in all groups were infections (urinary tract infections) and urinary complications (urine leaks and urolithiasis). Bladder reconstructive procedures, which require the use of bowel, presented the highest rate of high-grade complications (32%). Length of hospital stay (LOS; odds ratio [OR] 1.33, confidence interval [CI] 1.16-1.53), estimated blood loss (EBL) in surgery (OR 1.01, CI 1.002-1.019) and operating time (OR 1.004, CI 1.002-1.006) were all associated with increased odds of high-grade complications on multivariate analysis (P < 0.05). CONCLUSIONS: In this single-surgeon series, we have described the most commonly encountered complications after RAL in paediatric urology, finding rates similar to the complication rates reported in the current literature on other surgical approaches. In addition, LOS, operating time and EBL, which are probable surrogates of case complexity, were associated with increased odds of high-grade complications.

Anatomical and Biochemical Changes Induced by Gluconacetobacter diazotrophicus Stand Up for Arabidopsis thaliana Seedlings From Ralstonia solanacearum Infection.

Nowadays, fertilization and pest control are carried out using chemical compounds that contaminate soil and deteriorate human health. Plant growth promoting bacteria endophytes (PGPBEs), are a well-studied group of bacteria that offers benefits to the host plant, such as phytostimulation, biofertilization, and protection against other microorganisms. The study of Gluconacetobacter diazotrophicus-which belongs to PGPBEs-aids the development of alternative strategies of an integrated approach for crop management practices. Ralstonia solanacearum is responsible for bacterial wilt disease. This phytopathogen is of great interest worldwide due to the enormous economic losses it causes. In this study the action of G. diazotrophicus as a growth promoting bacterium in Arabidopsis thaliana seedlings is analyzed, evaluating the antagonistic mechanisms of this beneficial endophytic bacterium during biotic stress produced by R. solanacearum. Effective colonization of G. diazotrophicus was determined through bacterial counting assays, evaluation of anatomical and growth parameters, and pigments quantification. Biocontrol assays were carried out with Ralstonia pseudosolanacearum GMI1000 model strain and R. solanacearum A21 a recently isolated strain. Inoculation of A. thaliana (Col 0) with G. diazotrophicus Pal 5 triggers a set of biochemical and structural changes in roots, stems, and leaves of seedlings. Discrete callose deposits as papillae were observed at specific sites of root hairs, trichomes, and leaf tissue. Upon R. pseudosolanacearum GMI1000 infection, endophyte-treated plants demonstrated being induced for defense through an augmented callose deposition at root hairs and leaves compared with the non-endophyte-treated controls. The endophytic bacterium appears to be able to prime callose response. Roots and stems cross sections showed that integrity of all tissues was preserved in endophyte-treated plants infected with R. solanacearum A21. The mechanisms of resistance elicited by the plant after inoculation with the endophyte would be greater lignification and sclerosis in tissues and reinforcement of the cell wall through the deposition of callose. As a consequence of this priming in plant defense response, viable phytopathogenic bacteria counting were considerably fewer in endophyte-inoculated plants than in not-inoculated controls. Our results indicate that G. diazotrophicus colonizes A. thaliana plants performing a protective role against the phytopathogenic bacterium R. solanacearum promoting the activation of plant defense system.

Multivariate statistical analysis of morpho-anatomical data of nine sect. Caulopterae species (Baccharis - Asteraceae) used in folk medicine

Abstract Baccharis species belonging to sect. Caulopterae are difficult to identify. Most countries are controlling the quality of herbal medicines destined for the internal market or export. "Carquejas" are used arbitrarily for the same medicinal purposes and only three species of sect. Caulopterae are official herbal medicines. In the present study, a morpho-anatomical and statistical analysis was performed with nine species of sect. Caulopterae: Baccharis articulata, B. crispa, B. gaudichaudiana, B. microcephala, B. penningtonii, B. phyteumoides, B. sagittalis, B. triangularis and B. trimera, emphasizing the importance of anatomy as a taxonomic tool. A total of 114 populations of these nine species were examined. The first three principal components of morphoanatomical data provided relevant information to classify the species (75.04% of the total variability). The most discriminatory variable in this issue was the stomatal index (1.0530). We determined the qualitative and quantitative variables in order to differentiate the species by using principal components analysis and ANOVA tests. Stomata type, uniseriate trichome type and presence/absence of collenchyma in the wing margin are the qualitative variables that should be analyzed. Regarding quantitative variables, the epidermal ones in superficial view are more important and discriminatory than those of alate stem cross section and they must be considered for proper quality control of the species of this work.

Pre-harvest Sprouting and Grain Dormancy in Sorghum bicolor: What Have We Learned?

The possibility of obtaining sorghum grains with quality to match the standards for a diversity of end-uses is frequently hampered by the susceptibility to pre-harvest sprouting (PHS) displayed by many elite genotypes. For these reasons, obtaining resistance to PHS is considered in sorghum breeding programs, particularly when the crop is expected to approach harvest maturity under rainy or damp conditions prevalence. As in other cereals, the primary cause for sprouting susceptibility is a low dormancy prior to crop harvest; in consequence, most research has focused in understanding the mechanisms through which the duration of dormancy is differentially controlled in genotypes with contrasting sprouting behavior. With this aim two tannin-less, red-grained inbred lines were used as a model system: IS9530 (sprouting resistant) and Redland B2 (sprouting susceptible). Redland B2 grains are able to germinate well before reaching physiological maturity (PM) while IS9530 ones can start to germinate at 40-45 days after pollination, well after PM. Results show that the anticipated dormancy loss displayed by Redland B2 grains is related reduced embryo sensitivity to abscisic acid (ABA) and increased levels of GA upon imbibition. In turn, transcriptional data showed that ABA signal transduction is impaired in Redland B2, which appears to have an impact on GA catabolism, thus affecting the overall GA/ABA balance that regulates germination. QTL analyses were conducted to test whether previous candidate genes were located in a dormancy QTL, but also to identify new genes involved in dormancy. These analyses yielded several dormancy QTL and one of them located in chromosome 9 (qGI-9) was consistently detected even across environments. Fine mapping is already in progress to narrow down the number of candidate genes in qGI-9.

National Surgical Quality Improvement Program surgical risk calculator poorly predicts complications in patients undergoing radical cystectomy with urinary diversion.

PURPOSE: To evaluate the accuracy of the American College of Surgeons National Surgical Quality Improvement Programs (ACS-NSQIP) surgical risk calculator in patients undergoing radical cystectomy (RC) with urinary diversion. MATERIALS AND METHODS: Preoperative characteristics of patients who underwent RC with ileal conduit or orthotropic neobladder (ONB) between 2007 and 2016 were entered into the proprietary online ACS-NSQIP calculator to generate 30-day predicted risk profiles. Predicted and observed outcomes were compared by measuring Brier score (BS) and area under the receiver operating characteristic curve (AUC). RESULTS: Of 954 patients undergoing RC, 609 (64%) received ileal conduit and 345 (36%) received ONB. The calculator underestimated most risks by 10%-81%. The BSs exceeded the acceptable threshold of 0.01 and AUC were less than 0.8 for all outcomes in the overall cohort. The mean (standard deviation) predicted vs. observed length of stay was 9 (1.5) vs. 10.6 (7.4) days (Pearson's r = 0.09). Among patients who received ONB, adequate BS (<0.01) was observed for pneumonia, cardiac complications, and death. The receiver operating characteristic curve analysis revealed moderate accuracy of calculator for cardiac complications (AUC = 0.69) and discharge to rehab center (AUC = 0.75) among patients who underwent RC with ONB. CONCLUSIONS: The universal ACS-NSQIP calculator poorly predicts most postoperative complications among patients undergoing RC with urinary diversion. A procedure-specific risk calculator is required to better counsel patients in the preoperative setting and generate realistic quality measures.

Interspecific Potato Breeding Lines Display Differential Colonization Patterns and Induced Defense Responses after Ralstonia solanacearum Infection.

Potato (Solanum tuberosum L.) is one of the main hosts of Ralstonia solanacearum, the causative agent of bacterial wilt. This plant pathogen bacteria produce asymptomatic latent infections that promote its global spread, hindering disease control. A potato breeding program is conducted in Uruguay based on the introgression of resistance from the wild native species S. commersonii Dun. Currently, several backcrosses were generated exploiting the high genetic variability of this wild species resulting in advanced interspecific breeding lines with different levels of bacterial wilt resistance. The overall aim of this work was to characterize the interaction of the improved potato germplasm with R. solanacearum. Potato clones with different responses to R. solanacearum were selected, and colonization, dissemination and multiplication patterns after infection were evaluated. A R. solanacearum strain belonging to the phylotype IIB-sequevar 1, with high aggressiveness on potato was genetically modified to constitutively generate fluorescence and luminescence from either the green fluorescence protein gene or lux operon. These reporter strains were used to allow a direct and precise visualization of fluorescent and luminescent cells in plant tissues by confocal microscopy and luminometry. Based on wilting scoring and detection of latent infections, the selected clones were classified as susceptible or tolerant, while no immune-like resistance response was identified. Typical wilting symptoms in susceptible plants were correlated with high concentrations of bacteria in roots and along the stems. Tolerant clones showed a colonization pattern restricted to roots and a limited number of xylem vessels only in the stem base. Results indicate that resistance in potato is achieved through restriction of bacterial invasion and multiplication inside plant tissues, particularly in stems. Tolerant plants were also characterized by induction of anatomical and biochemical changes after R. solanacearum infection, including hyperplasic activity of conductor tissue, tylose production, callose and lignin deposition, and accumulation of reactive oxygen species. This study highlights the potential of the identified tolerant interspecific potato clones as valuable genetic resources for potato-breeding programs and leads to a better understanding of resistance against R. solanacearum in potato.

A 13th-century description of uterine prolapse: causes, symptoms and treatment.

OBJECTIVE: To describe and discuss Gilbertus Anglicus' contribution to the diagnosis and management of uterine prolapse in the Middle Ages. STUDY DESIGN: Anglicus'Compendium medicinae (ca. 1240) is generally considered a well-documented, systematic epitome of classical and early medieval medical knowledge. The British Library's Sloane manuscript 3486 contains a 15th-century English translation of Anglicus' work, and devotes folios 140(v)-147(v) to a small treatise on gynecology and obstetrics (the so-called 'sekenesse of wymmen'). RESULTS: Chapter six, in particular, deals with uterine prolapse by first approaching disease etiology, female physiology and symptom complex, and ending with a proposal of different therapeutic options to effectively treat or prevent the disorder. CONCLUSIONS: The 13th-century English physician Gilbertus Anglicus identified three different types of uterine prolapse, taking into account the obstetrical and gynecological writings of classical and early medieval authors. Anglicus systematically compiled some of the relevant information available to provide an accurate description of the etiology, symptom complex and treatment of this female disorder.

Parámetros micrográficos para la identificación de leño, corteza y hoja de Quassia amara L. (Simaroubaceae) / Micrographic parameters for identification of Quassia amara L., wood, bark and leaf

Quassia amara L. popularly known as “quasia”, is a shrubby plant from Tropical America. The wood, bark and leave are used in either folk medicine or in procuring phytotherapeutic drugs. The aim of the present work was to analize morphoanatomical and micrographic features which might provide assistance in the identification, analysis and standardization of Quasia amara L wood, bark and leaves. Results. Anatomical study showed white yellowish and diffuse porous wood, confluent paratracheal parenchyma. Rays width 1 cell wide and 8-30 cells high. CaOx crystals are absent. Cortex, 1-4 mm thick, a periderm up to 12 layers phellem cells. Leaf, hipostomatic with dorsiventral mesophyll and high number of sclerosed idioblasts.

Quassia amara L. popularmente conocida como “quasia” es un planta arbustiva de América Tropical. El leño, corteza y hojas son usadas tanto en medicina popular como en la obtención de drogas fitoterapéuticas. El objetivo del presente trabajo es analizar características morfoanatómicas y micrográficas las cuales provean asistencia en la identificación, análisis y estandarización de la madera, corteza y hojas de Quassia amara L. Resultados. El estudio anatómico mostró leño, blanco amarillento, de porosidad difusa. Parénquima paratraqueal confluente. Radios de 1 célula de ancho y 8-30 hileras de alto. Faltan cristales CaOx. Corteza, 1-4 mm de espesor, una peridermis de hasta 12 estratos de células de súber. Hoja, hipoestomática, con mesófilo dorsiventral, con elevado número de idioblastos esclerosados.

Antifungal activity of eugenol analogues. Influence of different substituents and studies on mechanism of action.

Twenty one phenylpropanoids (including eugenol and safrole) and synthetic analogues, thirteen of them new compounds, were evaluated for antifungal properties, first with non-targeted assays against a panel of human opportunistic pathogenic fungi. Some structure-activity relationships could be observed, mainly related to the influence of an allyl substituent at C-4, an OH group at C-1 and an OCH(3) at C-2 or the presence of one or two NO(2) groups in different positions of the benzene ring. All active compounds were tested in a second panel of clinical isolates of C. albicans and non-albicans Candida spp., Cryptococcus neoformans and dermatophytes. The eugenol derivative 4-allyl-2-methoxy-5-nitrophenol (2) was the most active structure against all strains tested, and therefore it was submitted to targeted assays. These studies showed that the antifungal activity of 2 was not reversed in the presence of an osmotic support such as sorbitol, suggesting that it does not act by inhibiting the fungal cell wall synthesis or assembly. On the other hand, the Ergosterol Assay showed that 2 did not bind to the main sterol of the fungal membrane up to 250 µg mL-1. In contrast, a 22% of fungal membrane damage was observed at concentrations = 1 × MIC and 71% at 4× MIC, when 2 was tested in the Cellular Leakage assay. The comparison of log P and MICs for all compounds revealed that the antifungal activity of the eugenol analogues would not to be related to lipophilicity.

High temperature triggers the metabolism of S-nitrosothiols in sunflower mediating a process of nitrosative stress which provokes the inhibition of ferredoxin-NADP reductase by tyrosine nitration.

High temperature (HT) is considered a major abiotic stress that negatively affects both vegetative and reproductive growth. Whereas the metabolism of reactive oxygen species (ROS) is well established under HT, less is known about the metabolism of reactive nitrogen species (RNS). In sunflower (Helianthus annuus L.) seedlings exposed to HT, NO content as well as S-nitrosoglutathione reductase (GSNOR) activity and expression were down-regulated with the simultaneous accumulation of total S-nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO). However, the content of tyrosine nitration (NO(2) -Tyr) studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and by confocal laser scanning microscope was induced. Nitroproteome analysis under HT showed that this stress induced the protein expression of 13 tyrosine-nitrated proteins. Among the induced proteins, ferredoxin-NADP reductase (FNR) was selected to evaluate the effect of nitration on its activity after heat stress and in vitro conditions using 3-morpholinosydnonimine (SIN-1) (peroxynitrite donor) as the nitrating agent, the FNR activity being inhibited. Taken together, these results suggest that HT augments SNOs, which appear to mediate protein tyrosine nitration, inhibiting FNR, which is involved in the photosynthesis process.

Functional analysis of superoxide dismutases (SODs) in sunflower under biotic and abiotic stress conditions. Identification of two new genes of mitochondrial Mn-SOD.

Superoxide dismutases (SODs) are a family of metalloenzymes that catalyse the disproportionation of superoxide radicals into hydrogen peroxide and oxygen. In sunflower (Helianthus annuus L.) seedlings, two new Mn-SOD isozymes, designated as I and II, were identified. However, no evidence for a Fe-SOD was found. Both Mn-SOD I and Mn-SOD II have a cleaved sequence of 14 residues that target the mitochondrion with a probability of 81% and 95%, respectively. The gene expression of these new mitochondrial Mn-SODs as well as the previously reported cytosolic and chloroplastic CuZnSODs was analyzed by real-time quantitative reverse transcription-PCR. This was done in the main organs (roots, hypocotyls, and cotyledons) of sunflower seedlings and also under biotic (infection by the pathogen Plasmopara halstedii) and abiotic stress conditions, including high and low temperature and mechanical wounding. Both CuZn-SODs had a gene expression of 1000-fold higher than that of mitochondrial Mn-SODs. And the expression of the Mn-SOD I was approximately 12-fold higher than that of Mn-SOD II. The Mn-SOD I showed a significant modulation in response to the assayed biotic and abiotic stresses even when it had no apparent oxidative stress, such as low temperature. Thus, it is proposed that the mitochondrial Mn-SOD I gene could act as an early sensor of adverse conditions to prevent potential oxidative damage.

Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings.

Nitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-arginine-dependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO(2)-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO(2)-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a down-regulation of GSNOR activity, while NO(2)-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants.

Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls.

Tyrosine nitration is recognized as an important post-translational protein modification in animal cells that can be used as an indicator of a nitrosative process. However, in plant systems, there is scant information on proteins that undergo this process. In sunflower hypocotyls, the content of tyrosine nitration (NO(2)-Tyr) and the identification of nitrated proteins were studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic approaches, respectively. In addition, the cell localization of nitrotyrosine proteins and peroxynitrite were analysed by confocal laser-scanning microscopy (CLSM) using antibodies against 3-nitrotyrosine and 3'-(p-aminophenyl) fluorescein (APF) as the fluorescent probe, in that order. The concentration of Tyr and NO(2)-Tyr in hypocotyls was 0.56 micromol mg(-1) protein and 0.19 pmol mg(-1) protein, respectively. By proteomic analysis, a total of 21 nitrotyrosine-immunopositive proteins were identified. These targets include proteins involved in photosynthesis, and in antioxidant, ATP, carbohydrate, and nitrogen metabolism. Among the proteins identified, S-adenosyl homocysteine hydrolase (SAHH) was selected as a model to evaluate the effect of nitration on SAHH activity using SIN-1 (a peroxynitrite donor) as the nitrating agent. When the hypocotyl extracts were exposed to 0.5 mM, 1 mM, and 5 mM SIN-1, the SAHH activity was inhibited by some 49%, 89%, and 94%, respectively. In silico analysis of the barley SAHH sequence, characterized Tyr448 as the most likely potential target for nitration. In summary, the present data are the first in plants concerning the content of nitrotyrosine and the identification of candidates of protein nitration. Taken together, the results suggest that Tyr nitration occurs in plant tissues under physiological conditions that could constitute an important process of protein regulation in such a way that, when it is overproduced in adverse circumstances, it can be used as a marker of nitrosative stress.

Involvement of reactive nitrogen and oxygen species (RNS and ROS) in sunflower-mildew interaction.

Nitric oxide (.NO) has been shown to participate in plant response against pathogen infection; however, less is known of the participation of other NO-derived molecules designated as reactive nitrogen species (RNS). Using two sunflower (Helianthus annuus L.) cultivars with different sensitivity to infection by the pathogen Plasmopara halstedii, we studied key components involved in RNS and ROS metabolism. We analyzed the superoxide radical production, hydrogen peroxide content, l-arginine-dependent nitric oxide synthase (NOS) and S-nitrosoglutathione reductase (GSNOR) activities. Furthermore, we examined the location and contents of .NO, S-nitrosothiols (RSNOs), S-nitrosoglutathione (GSNO) and protein 3-nitrotyrosine (NO(2)-Tyr) by confocal laser scanning microscopy (CLSM) and biochemical analyses. In the susceptible cultivar, the pathogen induces an increase in proteins that undergo tyrosine nitration accompanied by an augmentation in RSNOs. This rise of RSNOs seems to be independent of the enzymatic generation of .NO because the l-arginine-dependent NOS activity is reduced after infection. These results suggest that pathogens induce nitrosative stress in susceptible cultivars. In contrast, in the resistant cultivar, no increase of RSNOs or tyrosine nitration of proteins was observed, implying an absence of nitrosative stress. Therefore, it is proposed that the increase of tyrosine nitration of proteins can be considered a general biological marker of nitrosative stress in plants under biotic conditions.

Nitrosative stress in plants.

Nitrosative stress has become a usual term in the physiology of nitric oxide in mammalian systems. However, in plants there is much less information on this type of stress. Using olive leaves as experimental model, the effect of salinity on the potential induction of nitrosative stress was studied. The enzymatic l-arginine-dependent production of nitric oxide (NOS activity) was measured by ozone chemiluminiscence. The specific activity of NOS in olive leaves was 0.280nmol NOmg(-1) proteinmin(-1), and was dependent on l-arginine, NADPH and calcium. Salt stress (200mM NaCl) caused an increase of the l-arginine-dependent production of nitric oxide (NO), total S-nitrosothiols (RSNO) and number of proteins that underwent tyrosine nitration. Confocal laser scanning microscopy analysis using either specific fluorescent probes for NO and RSNO or antibodies to S-nitrosoglutathione and 3-nitrotyrosine, showed also a general increase of these reactive nitrogen species (RNS) mainly in the vascular tissue. Taken together, these findings show that in olive leaves salinity induces nitrosative stress, and vascular tissues could play an important role in the redistribution of NO-derived molecules during nitrosative stress.

The dehydrogenase-mediated recycling of NADPH is a key antioxidant system against salt-induced oxidative stress in olive plants.

NADPH is an important molecule in the redox balance of the cell. In this paper, using olive tissue cultures as a model of the function of the NADPH-generating dehydrogenases in the mechanism of oxidative stress induced by severe salinity conditions was studied. When olive (Olea europaea) plants were grown with 200 mM NaCl, a 40% reduction in leaf fresh weight was produced. The content of non-enzymatic antioxidants such as ascorbate and glutathione was diminished between 20% to 39%, whereas the H2O2 content was increased threefold. In contrast, the analysis of the activity and protein contents of the main antioxidative enzymes showed a significant increase of catalase, superoxide dismutase and glutathione reductase. Overall, these changes strongly suggests that NaCl induces oxidative stress in olive plants. On the other hand, while the content of glucose-6-phosphate was increased almost eightfold in leaves of plants grown under salt stress, the content of NAD(P)H (reduced and oxided forms) did not show significant variations. Under salt stress conditions, the activity and protein contents of the main NADPH-recycling enzymes, glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (ICDH), malic enzyme (ME) and ferrodoxin-NADP reductase (FNR) showed an enhancement of 30-50%. In leaves of olive plants grown with 200 mM NaCl, analysis of G6PDH by immunocytochemistry and confocal laser scanning microscopy showed a general increase of this protein in epidermis, palisade and spongy mesophyll cells. These results indicate that in olive plants, salinity causes reactive oxygen species (ROS)-mediated oxidative stress, and plants respond to this situation by inducing different antioxidative enzymes, especially the NADPH-producing dehydrogenases in order to recycle NADPH necessary for the protection against oxidative damages. These NADP-dehydrogenases appear to be key antioxidative enzymes in olive plants under salt stress conditions.