Production of secondary metabolites in cell suspension cultures of Ocimum sanctum L.

Dissertação de mestrado em Plant Molecular Biology, Biotechnology and Bioentrepreneurship

New bio-strategies for ochratoxin A detoxification using lactic acid bacteria

The occurrence of mycotoxigenic moulds such as Aspergillus, Penicillium and Fusarium in food and feed has an important impact on public health, by the appearance of acute and chronic mycotoxicoses in humans and animals, which is more severe in the developing countries due to lack of food security, poverty and malnutrition. This mould contamination also constitutes a major economic problem due the lost of crop production. A great variety of filamentous fungi is able to produce highly toxic secondary metabolites known as mycotoxins. Most of the mycotoxins are carcinogenic, mutagenic, neurotoxic and immunosuppressive, being ochratoxin A (OTA) one of the most important. OTA is toxic to animals and humans, mainly due to its nephrotoxic properties. Several approaches have been developed for decontamination of mycotoxins in foods, such as, prevention of contamination, biodegradation of mycotoxins-containing food and feed with microorganisms or enzymes and inhibition or absorption of mycotoxin content of consumed food into the digestive tract. Some group of Gram-positive bacteria named lactic acid bacteria (LAB) are able to release some molecules that can influence the mould growth, improving the shelf life of many fermented products and reducing health risks due to exposure to mycotoxins. Some LAB are capable of mycotoxin detoxification. Recently our group was the first to describe the ability of LAB strains to biodegrade OTA, more specifically, Pediococcus parvulus strains isolated from Douro wines. The pathway of this biodegradation was identified previously in other microorganisms. OTA can be degraded through the hydrolysis of the amide bond that links the L-β-phenylalanine molecule to the ochratoxin alpha (OTα) a non toxic compound. It is known that some peptidases from different origins can mediate the hydrolysis reaction like, carboxypeptidase A an enzyme from the bovine pancreas, a commercial lipase and several commercial proteases. So, we wanted to have a better understanding of this OTA degradation process when LAB are involved and identify which molecules where present in this process. For achieving our aim we used some bioinformatics tools (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV). We also designed specific primers and realized gene specific PCR. The template DNA used came from LAB strains samples of our previous work, and other DNA LAB strains isolated from elderberry fruit, silage, milk and sausages. Through the employment of bioinformatics tools it was possible to identify several proteins belonging to the carboxypeptidase family that participate in the process of OTA degradation, such as serine type D-Ala-D-Ala carboxypeptidase and membrane carboxypeptidase. In conclusions, this work has identified carboxypeptidase proteins being one of the molecules present in the OTA degradation process when LAB are involved.

Fatôres que afetam a determinação da vitamina C na goiaba (Psidium guajava L.)

In the present paper the authors deal with the content of ascorbic acid in guavas (Psidium guajava L.) and the methods of sampling used. Ascorbic acid was determined directly in the photoeletric colorimeter (EEL), after extraction with a 4 per cent solution of oxalic acid. Guavas from various parts of State of S.Paulo were used and the general mean found was around 100 mgm of ascorbic acid per 100 gm fresh weight of the material used for extraction. It was found that there is great variation in the ascorbic acid content according to the condition of the fruit: Ascorbic acid Condition content in Mean mgm/100 gm Green and hard 93,36 - 119,54 102,43 Small green 57,34 - 92,30 83,40 Firm ripe 71,28 - 115,98 90,32 Overripe 63,22 - 85,98 77,36 Of the diferent parts of the fruit, the skin has the highest content; the pulp between the skin and the parts containing the seeds, the "inner pulp" contains little and finallw the central parts, formed by the pulp between seeds, the "inter pulp" a engligible amount. The proportion of ascorbic acid found in the skin, inner pulp and inter pulp may be as high as 1.6: 1: 0. Furthermore, the section near the peduncule and the sepals are richer than equatorial crossections. It was proved that the amount and intensity of sun-light is at least one important factant factor determining differences in the ascorbic acid content of the fruit, which is higer in the parts which have received more light. A sharp decrease was found in the vitamin C content of ripe fruite stored in a home refrigerator. The periods of the preservation were 12-24-48-96 hours and the decrease of the ascorbic acid content m per cent was 23.4 - 42.0 - 66.8 - 76.4 of the initial content of 144.28 mgm/100 gm. The following five different methods of sampling in the determination of the amount of vitamin C were tested, with extraction in a Waring blendor: 1) whole fruit; 2,) sample taken form fruits cut into many small pieces; 3) half of a fruit divided by a crossection at equal distance from both ends; 4) half of fruit divided lenghtwise; 5) a transversal slice of about 1 cm. It was found that the two first methods geve the most reliable results.

Application of chemicals on Ficus carica L. transplanted

This research deals with the effects of chemicals on, transplantation of Ficus carica L. cv. Roxo de Valinhos. Fig plants were sprayed with Transplantone (naphthaleneacetamide plusnaphtaleneacetic acid) 10 g/1 , Oed green (oxyethylene docosanol) 40 ml/1, Good-rite peps (polyethylene polysulfite) 0,6 ml/1, and Mobileaf 200 ml/1. A check treatment and aereduction to half of leaf area were also established. Reduction of leaf area and application of oxyethylene docosanol promoted higher plant survival. The chemicals did not affect the growth of transplanted plants. Auxins constituents of Transplantone increased leaf number of fig plants.

Effects of ethephon and urea on ripening of fruits and leaf abscission of coffee (Coffea arábica L.)

A field experiment was carried out to investigate the effects of ethephon and urea on ripening of fruits and leaf abscission of coffee plant. Ethephon (2-chloroethane phosphonic acid) sprays were applied to green Coffea arábica berries 26 days before counting date in concentrations of 0.5 and 0.25 ml/1 from Ethrel (240 a.i./l). The chemical accelerated the onset of fruit ripening at both concentrations. The efficacy of ethephon was increased adding urea. Ethephon 0.5 ml/1 promoted abscission of leaves and low concentration reduced shedding of leaves. The treatments did not affect the growth and production on the next harvest.

Uric acid is a danger signal activating NALP3 inflammasome in lung injury inflammation and fibrosis.

RATIONALE: Lung injury leads to pulmonary inflammation and fibrosis through myeloid differentiation primary response gene 88 (MyD88) and the IL-1 receptor 1 (IL-1R1) signaling pathway. The molecular mechanisms by which lung injury triggers IL-1beta production, inflammation, and fibrosis remain poorly understood. OBJECTIVES: To determine if lung injury depends on the NALP3 inflammasome and if bleomycin (BLM)-induced lung injury triggers local production of uric acid, thereby activating the NALP3 inflammasome in the lung. Methods: Inflammation upon BLM administration was evaluated in vivo in inflammasome-deficient mice. Pulmonary uric acid accumulation, inflammation, and fibrosis were analyzed in mice treated with the inhibitor of uric acid synthesis or with uricase, which degrades uric acid. MEASUREMENTS AND MAIN RESULTS: Lung injury depends on the NALP3 inflammasome, which is triggered by uric acid locally produced in the lung upon BLM-induced DNA damage and degradation. Reduction of uric acid levels using the inhibitor of uric acid synthesis allopurinol or uricase leads to a decrease in BLM-induced IL-1beta production, lung inflammation, repair, and fibrosis. Local administration of exogenous uric acid crystals recapitulates lung inflammation and repair, which depend on the NALP3 inflammasome, MyD88, and IL-1R1 pathways and Toll-like receptor (TLR)2 and TLR4 for optimal inflammation but are independent of the IL-18 receptor. CONCLUSIONS: Uric acid released from injured cells constitutes a major endogenous danger signal that activates the NALP3 inflammasome, leading to IL-1beta production. Reducing uric acid tissue levels represents a novel therapeutic approach to control IL-1beta production and chronic inflammatory lung pathology.

NEUROPATHOPHYSIOLOGICAL MECHANISMS IN GLUTARIC ACIDURIA TYPE I: 3-HYDROXYGLUTARIC ACID LEADS TO AMMONIA INCREASE AND NON-APOPTOTIC CELL DEATH

A 3D in vitro model of rat organotypic brain cell cultures in aggregates was used to investigate neurotoxicity mechanisms in glutaric aciduria type I (GA-I). 1 mM glutarate (GA) or 3-hydroxyglutarate (3OHGA) were repeatedly added to the culture media at two different time points. In cultures treated with 3OHGA, we observed an increase in lactate in the medium, pointing to a possible inhibition of Krebs cycle and respiratory chain. We further observed that 3OHGA and to a lesser extend GA induced an increase in ammonia production with concomitant decrease of glutamine concentrations, which may suggest an inhibition of the astrocytic enzyme glutamine synthetase. These previously unreported findings may uncover a pathogenic mechanism in this disease which has deleterious effects on early stages of brain development. By immunohistochemistry we showed that 3OHGA increased non-apoptotic cell death. On the cellular level, 3OHGA and to a lesser extend GA led to cell swelling and loss of astrocytic fibers whereas a loss of oligodendrocytes was only observed for 3OHGA. We conclude that 3OHGAwas the most toxic metabolite in our model for GA-I. 3OHGA induced deleterious effects on glial cells, an increase of ammonia production, and resulted in accentuated cell death of non-apoptotic origin.

Genetic diversity and differentiation processes in the ploidy series of Olea europaea L.: a multiscale approach from subspecies to insular populations.

Geographical isolation and polyploidization are central concepts in plant evolution. The hierarchical organization of archipelagos in this study provides a framework for testing the evolutionary consequences for polyploid taxa and populations occurring in isolation. Using amplified fragment length polymorphism and simple sequence repeat markers, we determined the genetic diversity and differentiation patterns at three levels of geographical isolation in Olea europaea: mainland-archipelagos, islands within an archipelago, and populations within an island. At the subspecies scale, the hexaploid ssp. maroccana (southwest Morocco) exhibited higher genetic diversity than the insular counterparts. In contrast, the tetraploid ssp. cerasiformis (Madeira) displayed values similar to those obtained for the diploid ssp. guanchica (Canary Islands). Geographical isolation was associated with a high genetic differentiation at this scale. In the Canarian archipelago, the stepping-stone model of differentiation suggested in a previous study was partially supported. Within the western lineage, an east-to-west differentiation pattern was confirmed. Conversely, the easternmost populations were more related to the mainland ssp. europaea than to the western guanchica lineage. Genetic diversity across the Canarian archipelago was significantly correlated with the date of the last volcanic activity in the area/island where each population occurs. At the island scale, this pattern was not confirmed in older islands (Tenerife and Madeira), where populations were genetically homogeneous. In contrast, founder effects resulted in low genetic diversity and marked genetic differentiation among populations of the youngest island, La Palma.

Aplicació de la termoquimiolisi - cromatografia de gasos - espectrometria de masses a l'estudi de la matèria orgànica del sòl

Estudi realitzat a partir d’una estada a la Université de Poitiers, França, entre 2007 i 2009. El treball s'ha centrat en dues activitats bàsiques. El treball realitzat s’ha centrat en dues activitats bàsiques. D’una banda, la posada a punt d'un protocol de fraccionament de la matèria orgànica del sòl, per extraccions successives amb solvents alcalins després d'una seqüència de pretractaments al sòl: cap pretractament, atac amb àcid (per destruir els carbonats), atac amb ditionit (per reduir els òxids de Fe i Al i facilitar l'extracció de la matèria orgànica associada a aquests compostos). El protocol dóna una visió de conjunt de la situació de la matèria orgànica del sòl, combinant aspectes físics (protecció, precipitació, oclusió per carbonats) i químics (grau d'humificació). D’altra banda, l'aprenentatge de la tècnica de termoquimiolisi-cromatografia de gasos-espectrometria de masses. Aquest era l'objectiu de l'estada a Poitiers, al qual hem donat prioritat. Ens hem centrat en l'estudi de fraccions físiques (densimètriques) obtingudes en estudis anteriors sobre sòls forestals. Les fraccions considerades són: fracció lleugera (FL), tres fraccions ocluïdes (OC1, OC2 i OC3) i fracció densa (FD). L’aplicació de la termoquimiolisi permet de caracteritzar diversos grups de substàncies, de les quals ens hem centrat en alguns indicadors bioquímics: àcids grasos, alcohols, diàcids, productes fenòlics i altres productes aromàtics, derivats de carbohidrats. L’estudi de conjunt d’aquests productes indica que és a les fraccions ocluïdes (que solen ser minoritàries a tots els horitzons) on la matèria orgànica d’origen microbià és dominant, mentre que a les fraccions lleugera (FL) i densa (FD) la matèria orgànica d’origen vegetal sembla dominant. Es preveu aplicar aquesta tècnica a l’estudi de les fraccions obtingudes a la primera part del treball, actualment congelades i a l’espera de ser processades.

Composición en ácidos grasos de huevos y larvas de dentón (Dentex dentex L.) de hasta 40 días en condiciones de cultivo

Total lipid content and fatty acid (FA) composition of common dentex eggs spawned at different times and larvae reared under different culture conditions until 40 days post hatch (dph) were analysed in order to get a general pattern of lipid composition during larval development. Results were grouped according to the developmental stage of the larvae instead of age in dph. Saturated and monounsaturated fatty acids decreased along larval development, while polyunsaturated fatty acid (PUFA) content increased. The ratio of docosahexaenoic acid (DHA) / eicosapentaenoic acid (EPA) shifted from 4 – 5 in early developmental stages to lower than 1 after metamorphosis. Results suggest a subdivision of the larval development into two stages of opposite FA requirements.

Pharmacological screening of Ageratum conyzoides L. (mentrasto)

The pharmacological activities of a water extract (WE) of Ageratum conyzoides L, a plant populary known for its analgesic and anti-inflamatory properties, were studied in vivo and in vitro preparations. Oral administration (p.o.) of the water extract (WE, 0.1 to 5 g/Kg) to rats and mice induced quietness and reduced the spontaneous motility. the sleeping time induced by sodium pentobarbital (50 mg/Kg, i.p.) in mice was not altered by previous treatment with We (2 g/Kg, p.o.). The same treatment did not influence the paw edema induced by carrageenan or dextran, nor did it reduce the chronic paw edema induced by complete Freund's adjuvant or formaldehyde in rats. The tail flick response in immersion test and writhings induced by 0.8%acetic acid in mice were not altered by WE either. In isolated guinea-pig ilea WE (0.4 to 4 mg/ml) did not alter the EC50 values of histamine or acetylcholine, but reduced the maximal response to the agonists by 20 to 50%. We (0.01 to 10 mg/ml) produced tonic contractions of the ileal smooth muscle proportional to the doses, reaching a maximum of 75% relatively to the maximum obtained with histamine. Those contractions were blocked by diphenhydramine (10 nM) and reduced by 32% in presence of atropine (10 nM). The results indicated that oral treatment of rodents with A. conyzoides L neither reduced the inflammatory edema nor did it decrease the reaction to pain stimuli. In vitro the extract presented an unexpected histamine-like activity characteristic of a partial agonist. The results did not confirm the popular medicinal indications of the plant.

Analgesic activity of a triterpene isolated from Scoparia dulcis L. (vassourinha)

Analgesic and anti-inflammatory activities of water (WE) and ethanolic (EE) extracts of Scoparia dulcis L. were investigated in rats and mice, and compared to the effects induced by Glutinol, a triterpene isolated by purification of EE. Oral adminsitration (p.o.) of either WE or EE (up to 2 g/Kg) did not alter the normal spontaneous activity of mice and rats. The sleeping time induced by sodium pentobarbital (50 mg/Kg, i.p.) was prolonged by 2 fold in mice pretreated with 0.5 g/Kg EE, p.o. Neither extract altered the tail flick response of mice in immersion test, but previous administration of EE (0.5 g/Kg, p.o.) reduced writhings induced by 0.8% acetic acid (0.1 ml/10 g, i.p.) in mice by 47% EE (0.5 and 1 g/Kg, p.o.) inhibited the paw edema induced by carrageenan in rats by respectively 46% and 58% after 2 h, being ineffective on the paw edema induced by dextran. No significant analgesic or anti-edema effects were detected in animals pretreated with WE (1 g/Kg, p.o.). Administration of Glutinol (30 mg/Kg, p.o.) reduced writhing induced by acetic acid in mice by 40% and the carrageenan induced paw edema in rats by 73%. The results indicate that the analgesic activity of S dulcis L. may be explained by explained by an anti-inflammatory activity probably related to the triterpene Glutinol.

Simultaneous Evaluation of Intact Glucosinolates and Phenolic Compounds by UPLC-DAD-MS/MS in Brassica oleracea L. var. botrytis

A method for the simultaneous determination of intact glucosinolates and main phenolic compounds (flavonoids and sinapic acid derivatives) in Brassica oleracea L. var. botrytis was proposed. A simplified sample extraction procedure and a UPLC separation were carried out to reduce the total time of analysis. Brassica oleracea samples were added with internal standards (glucotropaeolin and rutin), and extracted with boiling methanol. Crude extracts were evaporated under nitrogen, redissolved in mobile phase and analyzed by UPLC with double detection (ESI--MRM for glucosinolates and flavonoids, and DAD for main sinapic acid derivatives). The proposed method allowed a satisfactory quantification of main native sinapic acid derivatives, flavonoids and glucosinolates with a reduced time of analysis.