Development of tools for studying Olea europaea: Pseudomonas savastanoi interaction

Poster apresentado no 13th Congress of the Federation of European Societies of Plant Physiology, em 2002, Hersonissos, Creta, GréciaPortugal is one of the main world olive oil producers. Consequently, the culture of olive trees is of considerable importance for our country. In order to maintain and improve our varieties, responses to environmental stresses and pests, which can seriously affect productivity, must be studied, and clones more adapted to challenge stress conditions must be found. One problem that seriously affects Portuguese olive orchards' is olive knot, a disease caused by Pseudomonas savastanoi (Pseudomonas syringae pv savastanoi) which, causing tumours in the stems and leaves of the trees, drastically reduces the production of fruits. Some varieties, like Galega Vulgar, are know to be resistant to the disease, while others, like Cordovil de Serpa, variety are know to be susceptible. With the objective of developing an in vitro system of eliciation of Olea europaea with an avirulent strain of Pseudomonas savastanoi, we have initiated suspension cell cultures of from calli of the variety Galega Vulgar. Cells in middle exponential growth phase were incubated with a suspension of P. savastanoi.Hypersensitive response was studied using XTT (sodium,3’-(1-[phenylamino-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid) by the quantification of the perhydroxyl / superoxide (H2O./O2-) radical acid-base pair associated to the oxidative burst, during the time course of elicitation. The results showed the existence of two bursts (100 and 300 min after eliciation), which are characteristic of the HR that occurs in the incompatible interactions). By screening an elicited O. europaea cDNA library a clone encoding phenylalanine ammonia lyase (PAL) was obtained. The coding region was used as probe for evaluate Pal expression levels. The results will be discussed by comparing to variation of PAL activity, during the time course of elicitation.Fundação para a Ciência e a Tecnologia (FCT) – grant ref. SFRH/BD/6654/2001, Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI) - POCTI / AGR / 36239 / 99-00

The hypersensitive response of Olea europaea L. elicitates by Pseudomonas savastanoi

Poster apresentado no 14th Federation of European Societies for Plant Biology Congress, Cracóvia, Polónia.In plants, the Hypersensitive Response (HR) is an early defense mechanism that is elicited by the recognition of an incompatible pathogen, with the objective of restricting its spread. One of the earliest events in the HR is the rapid and significant increase in Reactive Oxygen Species (ROS) levels in the cells. This Oxidative Burst has various consequences, in particular the induction of the synthesis of secondary metabolites like phythoalexins (compounds with anti-microbial activity) and lignin (to reinforce the cell wall), and the triggering of a programmed cell death. We used a previously established in vitro elicitation system to study the interaction between Olea europaea L. and its pathogen Pseudomonas savastanoi, responsible for the olive knot, a disease that drastically affects olive oil production in Portugal. Material and Methods: The in vitro elicitation system used in this work was composed of suspension cell cultures of the resistant Olea europaea variety Galega Vulgar and by an avirulent strain of the pathogen Pseudomonas savastanoi. ROS detection was performed using a modification of the method described by Parsons et al. (1999), lipid peroxidation was measured using the TBA test and phenylalanine ammonia lyase (PAL) activity was evaluated by the quantification of trans-cinnamic acid determined spectrophotometrically at 290 nm. Results and Conclusions: Evaluation of ROS levels show a significant increase in production by the elicitated O. europaea cells, with a pattern typical of incompatible interactions, indicating that the Galega Vulgar variety is in fact resistant to the pathogen. This OB leads to significant cellular damage, illustrated by increased levels of lipid peroxidation. PAL activity also increased in elicitated cells, pointing toward changes in the phenylpropanoid pathway and, therefore, in the production of secondary metabolites. Quantification of lignin and of the total soluble phenolics allowed us to assess these changes. In order to evaluate the programmed cell death of the challenged cells, the T.U.N.E.L. assay was carried out during the time course of elicitation. References: Parsons HL, Yip YHJ, Vanlerberghe GC. 1999. Increased Respiratory Restriction during Phosphate Limited Growth in Transgenic Tobacco Cells Lacking Alternative Oxidase. Plant Physiol 121; 1309-1320. Since the ROS produced in the OB function mainly as an intracellular signal, we studied the effect of Hydrogen peroxide as a signal molecule. Various concentrations of H2O2 were added to suspension cell cultures of O. europaea and PAL activity was measured. The increase in PAL activity when the added concentrations were low indicates that hydrogen peroxide may in fact function as a transduction signal during the HR. Heath RL, Packer L. 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125(1); 189-98.Projecto Olea - ref. POCTI/AGR/36239.Fundação para a Ciência e a Tecnologia (FCT) - grant ref. SFRH/BD/6654/2001

Pseudomonas savastanoi triggers the hypersensitive response of Olea europaea var. Galega Vulgar suspension cell cultures

Apresentação efectuada no XIV Congresso da Sociedade Portuguesa de Bioquímica, Vilamoura, Portugal, 2004The bacteria Pseudomonas savastanoi is a natural pathogen of Olea europaea L., and is responsible for the olive knot, a disease that seriously affects productivity of Portuguese olive orchards. In previous work, we established an in vitro elicitation system using the bacteria and suspension cell cultures of O. europaea var. Galega Vulgar, which is known to be resistant to this pathogen. This system is now being used to study the interaction between plant and pathogen and evaluate the Hypersensitive Response triggered by the infection. Evaluation of Reactive Oxygen Species (ROS) levels was performed by spectrofluorimetric quantification of the fluorescent probe H2DCFDA, and showed a significant increase in production by the elicitated cells, with a pattern typical of the Oxidative Burst (OB) of incompatible interactions. The levels of lipid peroxidation followed a similar pattern, indicating ROS production in the OB as an origin of this cellular damage. Phenylalanine ammonia lyase (PAL) activity was measured to determine changes in the phenylpropanoid pathway and, therefore, in secondary metabolism. Determination of total lignin content of cell walls allowed us to assess these changes. Challenged cells showed a significant increase in PAL activity during the time course of elicitation, as well as an increase in the lignin content. These results point to changes in the production of secondary metabolites, which are induced by the elicitation with an avirulent pathogen and lead to the reinforcement of the cell wall. The TUNEL assay was performed to evaluate if programmed cell death is triggered in challenged cells.Fundação para a Ciência e a Tecnologia (FCT) - Projecto Olea POCTI/AGR/36239.Fundação para a Ciência e a Tecnologia (FCT) - grant ref. SFRH/BD/6654/2001

Primary Hyperoxaluria type 1 – Two Case Reports

Primary hyperoxaluria type 1 is a rare autosomal recessive inherited disease, caused by mutations in AGXT gene, with an estimated incidence of 1:100.000 live births per year in Europe. Over 50% present with end stage renal disease at diagnosis. Case reports: The first case is a 14‑year‑old boy, second child to consanguineous parents, with history of recurrent lithiasis and ureteral dilatation starting 5 years before. Urine/stone analysis revealed calcium oxalate monohydrate crystals and markedly elevated urine oxalate excretion. Genetic tests confirmed a mutation in AGXT gene, c.1151T>C, in homozygosity. Two years after, nephrocalcinosis was identified and glomerular filtration rate gradually declined. Oxalate deposition in solid organs was excluded and successful orthotopic liver transplantation was performed, with stabilization of glomerular filtration rate. The second case is a 16‑year‑old girl, with recurrent episodes of renal colic. At diagnosis, she had obstructive hydronephrosis, multiple kidney stones and an estimated glomerular filtration of 42.1mL/min/1.73m2. Metabolic study showed hypocitraturia and hyperoxaluria. With dietetic measures and irregular treatment, urine oxalate excretion remained high but renal function improved. Genetic tests confirmed the presence of two pathologic variants in AGXT gene: c.731T>C and c.1151T>C in compound heterozygous. Conclusions: Recurrent urolithiasis and nephrocalcinosis in children along with family history/consanguinity should raise the suspicion of Primary Hyperoxaluria type 1. Conservative treatment may increase renal survival. Effects of systemic oxalosis must be screened when glomerular filtration rate declines below 30‑50mL/ min/1.73m2, and sequential or combined liver and kidney transplantation should be considered.info:eu-repo/semantics/publishedVersio

Design Specifications for an Auxiliary Incision Retractor in Dacryocystorhinostomy Surgeries

It is presented the design specifications for a Retractor Robotic System (RRS) based on the surgical necessities in the incisions procedure for a dacryocystorhinostomy. The specifications are conformed by a mathematical model, the experimental data measured, a modular flexible architecture, energy supplier system, the mechanical group, and the safety system. The specifications suggest that the use of flexible polymeric materials for the RRS provide a mayor adaptability of the system with the biological tissue; so a pneumatic actuator could be a suitable option.This research was funded by CONACYT (Consejo Nacional de Ciencia y Tecnología) Grant No. 86356

UPLC-MS-ESI-QTOF analysis and antifungal activity of the spondias tuberosa arruda leaf and root hydroalcoholic extracts

The aim of this study was to identify and evaluate the chemical compositions and effects of the S. tuberosa leaf and root hydroalcoholic extracts (HELST and HERST) against different strains of Candida. Chemical analysis was performed by Ultra-Performance Liquid Chromatography Coupled to Quadrupole/Time of Flight System (UPLC-MS-ESI-QTOF). The Inhibitory Concentration of 50% of the growth (IC50) as well as the intrinsic and combined action of the extracts with the antifungal fluconazole (FCZ) were determined by the microdilution method while the minimum fungicidal concentrations (MFCs) and the effect on fungal morphological transitions were analyzed by subculture and in humid chambers, respectively. From the preliminary phytochemical analysis, the phenols and flavonoids were the most abundant. The intrinsic IC50 values for HELST ranged from 5716.3 to 7805.8 \ub5g/mL and from 6175.4 to 51070.9 \ub5g/mL for the HERST, whereas the combination of the extracts with fluconazole presented IC50 values from 2.65 to 278.41 \ub5g/mL. The MFC of the extracts, individually, for all the tested strains was 6516384 \ub5g/mL. When fluconazole was combined with each extract, the MFC against CA URM 5974 was reduced (HELST: 2048 and HERST: 4096 \ub5g/mL). Synergism was observed against standard C. albicans (CA) and C. tropicalis (CT) strains and with the root extract against the CT isolate. The leaf extract inhibited the morphological transition of all strains while the root extract inhibited only CT strains

Synthesis of Selenium-Quinone Hybrid Compounds with Potential Antitumor Activity via Rh-Catalyzed C-H Bond Activation and Click Reactions

In continuation of our quest for new redox-modulating catalytic antitumor molecules, selenium-containing quinone-based 1,2,3-triazoles were synthesized using rhodium-catalyzed C-H bond activation and click reactions. All compounds were evaluated against five types of cancer cell lines: HL-60 (human promyelocytic leukemia cells), HCT-116 (human colon carcinoma cells), SF295 (human glioblastoma cells), NCIH-460 (human lung cells) and PC3 (human prostate cancer cells). Some compounds showed good activity with IC50 values below 1 µM. The cytotoxic potential of the naphthoquinoidal derivatives was also evaluated in non-tumor cells, exemplified by L929 cells. Overall, these compounds represent promising new lead derivatives and stand for a new class of chalcogenium-containing derivatives with potential antitumor activity

Sistemas de recuperação de pastagens degradadas em São Luiz, Roraima.

o presente trabalho teve como objetivo avaliar 13 diferentes manejos de recuperação da capacidade produtiva de pastagens, sobretudo, para que pequenos pecuaristas da região sul do Estado de Roraima possam adequar as práticas aos recursos que dispõem.bitstream/item/224766/1/Comunicado-Tecnico-92-C1.pd