Respuesta fisiológica y agronómica del olivar en seto (cv. Arbequina) a la orientación de filas y al ancho de calle: relación entre la producción y calidad del aceite y la radiación interceptada

En las últimas dos décadas, los productores han plantado olivares en seto para lograr la mecanización de la poda y en especial de la cosecha, reducir los costes de mano de obra y permitir intervenciones de manejo rápidas y oportunas. Los olivares se desarrollaron en ausencia del conocimiento científico, sobre el diseño óptimo de la estructura de la copa, necesario para incrementar la producción y calidad del aceite. En contraste, con los árboles muy espaciados y distribuidos uniformemente de las plantaciones tradicionales, en el olivar en seto hay una marcada variabilidad espacial y temporal de la radiación disponible en función del diseño de la plantación. Así, conocer la respuesta fisiológica y productiva del olivo a la radiación resulta fundamental en el olivar en seto. La orientación de las filas y el ancho de calle son aspectos que se deciden en el diseño de las plantaciones en seto. Ambos aspectos modifican la radiación interceptada por la canopia y, por lo tanto, pueden incidir en la productividad y calidad del aceite. Una vez realizada la plantación no pueden ser modificados, y así las ventajas o desventajas permanecerán fijas durante toda la vida productiva del olivar. A pesar de esto, el impacto de la orientación de las filas y el ancho de calle han recibido poca atención en olivos y en la mayoría de los frutales conducidos en seto. Por todo ello, los objetivos principales de esta tesis fueron, (i) evaluar el efecto de la orientación del seto y del ancho de calle, sobre la productividad y calidad del aceite, (ii) evaluar un modelo que estime la radiación dentro de la canopia. Este modelo permitirá cuantificar las relaciones entre la radiación y los componentes del rendimiento y calidad del aceite de olivares en setos con un amplio rango de estructuras y (iii) conocer la variabilidad en las características de las hojas (morfológicas y fisiológicas) y de los tejidos del fruto (tamaño y composición) en diferentes posiciones de la copa de los setos. Para ello, se dispuso de 3 ensayos de olivar en seto (cv. Arbequina) implantados en 2008 en el municipio de La Puebla de Montalbán, Toledo. La primera cosecha fue en 2010 y a partir del 2012 los setos formaron una copa continua. A partir de ese año, los setos se mantuvieron mediante poda, con similar ancho (~1 m) y altura (~2,5 m), acordes a las dimensiones de la cosechadora vendimiadora. En los años 2012 y 2013 se estudió en profundidad la respuesta de las plantas de estos ensayos. En el ensayo 1, los setos fueron plantados con cuatro orientaciones de filas: N–S, NE–SO, NO–SE y E–O y el mismo ancho de calle (4 m). En los otros dos ensayos, los setos fueron plantados con tres anchos de calle (5,0, 4,0 y 2,5 m), y con filas orientadas N–S (ensayo 2) y E–O (ensayo 3). La respuesta de la orientación de las filas se evaluó a nivel de seto y de estratos del seto (alturas y caras), a través de mediciones del crecimiento de brotes, componentes reproductivos, características y temperatura del fruto, estado hídrico del suelo y de las plantas, fotosíntesis neta de las hojas y contenido de ácidos grasos. Los setos orientados NE–SO (2,7 t/ha) lograron la mayor producción de aceite, que fue significativamente más alta que la de los setos E–O (2,3 t/ha). La producción de aceite de los setos E–O no se diferenció estadísticamente de los setos N–S (2,5 t/ha). Las diferencias productivas entre orientaciones fueron explicadas por el número de frutos en cosecha, a su vez la variación en el número de frutos estuvo asociada al efecto de la orientación de las filas sobre el número de yemas desarrolladas y el porcentaje de inflorescencias fértiles. Las hojas en las caras iluminadas de los setos NE–SO y N–S presentaron mayor tasa fotosintética a la mañana (~10.0 h) que los setos E–O, en el año 2012, pero no en 2013. La orientación de las filas no tuvo un efecto significativo en el contenido de ácidos grasos de los aceites extraídos, esto ocurrió a pesar de variaciones en la temperatura interna de los frutos (3 °C) y de la radiación (40%) entre las distintas caras de los setos. La orientación del seto afectó significativamente al contenido relativo de agua del suelo, donde setos E–O presentaron valores más altos (12%) que setos N–S durante el verano y otoño. Sin embargo, el potencial hídrico de tallo fue similar entre orientaciones. En los ensayos 2 y 3, se evaluó el efecto que produce, a nivel de seto y de estratos (caras y alturas), reducir el ancho de calle de 5,0 a 4,0 y 2,5 m, en un seto orientado N–S y otro E–O, respectivamente. La relación entre altura/ancho de calle libre aumentó 0,6 a 0,8 y 1,6, al reducir 5,0, 4,0 y 2,5 m el ancho de calle, mientras la longitud de seto y el volumen de copa por hectárea incrementó 100% al reducir de 5,0 a 2,5 m, el ancho de calle. En los setos orientados N–S, la producción de aceite por ha acumulada en 4 campañas, incrementó significativamente un 52 %, al reducir de 5,0 a 2,5 m el ancho de calle. Los setos N–S con calle más estrecha (2,5 m) tuvieron un 19% menos frutos que los setos con calle más ancha (5,0 m) y a su vez el 60% de los mismos se localizaron los estratos altos de la canopia de los setos con calles estrecha en comparación al 40% en setos con calle de 5,0 m. En los estratos más bajos de los setos con calles de 2,5m hubo menor crecimiento de los brotes y los frutos tuvieron menor peso seco, contenido de aceite y madurez, que los frutos en los estratos bajos de los setos a 5,0 m. Los componentes del rendimiento y características de los frutos (agua y madurez) fueron similares entre la caras E y O, independientemente del ancho de calle. En los setos orientados E–O, la producción de aceite por ha acumulada en 4 campañas, no respondió significativamente al ancho de calle, debido a una disminución significativa en el número de frutos y producción de aceite por m de seto, al reducir de 5,0 a 2,5 m, el ancho de calle. En los setos orientados E–O, con calles de 5,0 m, los frutos presentaron similar peso seco, contenido de aceite y agua, en las caras S y N, sin embargo, cuando la calle fue reducida a 2,5, los frutos de la cara S fueron más pesado y maduros que en la cara N. Independientemente del ancho de calle y de la orientación del seto, el aceite presentó mayor contenido de ácidos palmitoleico, palmítico, esteárico y linoleico en los frutos del estrato más alto de la canopia disminuyendo hacia la base. En contraste, el contenido de ácido oleico aumentó desde el estrato más alto hacia la base de los setos. Las diferencias en el contenido de ácidos grasos entre la parte alta y baja de los setos, incrementó al reducir el ancho de calle en los setos N–S, pero no en los E-O. En conclusión, en olivares en seto, reducir el ancho de calle permite incrementar la producción de aceite, en setos orientados N–S, pero no en E–O. Un modelo que estima la cantidad y distribución de la radiación en toda la copa del seto, fue utilizado para estimar la radiación interceptada en distintos estratos del seto. El modelo requiere un valor del coeficiente de extinción (k) para estimar la transmisión de radiación a través de la copa, el cual fue obtenido experimentalmente (k=1,2). Utilizando los datos del ensayo 1, un único modelo lineal relacionó el peso seco y el rendimiento graso de setos con la radiación interceptada por los distintos estratos de setos con cuatro orientaciones de filas. La densidad de frutos fue también relacionada con la radiación, pero más débilmente. En los setos orientados N–S, plantados con tres anchos de calles, (ensayo 2) el contenido de ácidos palmitoleico y linoleico del aceite incrementó linealmente con el incremento de la radiación interceptada, mientras el contenido ácido oleico disminuyó linealmente con el incremento de la radiación. El contenido de ácidos grasos del aceite no estuvo relacionado con la radiación interceptada en setos orientados E–O (Ensayo 3). En los setos N–S y E–O, plantados con anchos de calle de 2,5 m, se estudiaron las interacciones entre la radiación y características de las hojas, número de fruto, tamaño y composición de los frutos a nivel de órgano, tejido y células. Independientemente de la orientación del seto, el área y el contenido de clorofila de las hojas incrementaron significativamente en los estratos más bajos de los setos. Mientras, las hojas de los estratos medios del seto presentaron mayor capacidad fotosintética que en los estratos bajos y alto de los setos. Los estratos del seto que interceptaron más radiación produjeron frutos con mayor tamaño y contenido de aceite en el mesocarpo, sin efectos sobre el tamaño y composición del endocarpo. A nivel celular, los frutos expuestos a mayor nivel de radiación desarrollaron en el mesocarpo células de mayor tamaño en comparación a frutos menos expuestos, mientras el número de células no fue afectado. Adicionalmente, el número y tamaño de las células estuvo relacionado con la composición del mesocarpo en términos de aceite, agua y peso seco menos aceite. Esta tesis, contribuye, desde una perspectiva integral del cultivo del olivo, a cuantificar el impacto de la orientación y ancho de calle sobre la producción y calidad del aceite en olivares conducidos en setos. El análisis y discusión de la relación entre la radiación y los componentes del rendimiento y calidad del aceite, puede ayudar a diseñar plantaciones en seto con dimensiones óptimas para la intercepción de la radiación. ABSTRACT In the last two decades, olive hedgerow system has been established by commercial growers to allow continuous mechanized pruning and especially harvest, reduce costs of manual labour and allow more rapid and timely management interventions. The adoption of hedgerow was done in the absence of adequate scientific knowledge of the impact of this orchard structure and associated mechanization on tree response, yield and quality, after centuries in low-density orchards and open-formed trees. The row orientation and width alley are fundamental aspects in the hedgerow design and have been scarcely studied in olive. Both aspects modify the radiation intercepted by the canopy, and consequently the productivity and oil quality, and once defined in orchard planting cannot be changed, so advantages and disadvantages remain fixed for the lifespan of the orchard. The main objectives of this thesis were to (i) evaluate the impact of the row orientation and width alley on productivity and oil quality by the measurements of profile of the determining processes of shoot growth, fruit temperature, yield components and fruit and oil characteristics on opposite sides of olive hedgerows. Additionally, the effect of row orientation on the plant water status was also evaluated; (ii) evaluate a mathematical model for estimating the radiation within the canopy and quantify the relationships between the radiation estimated and yield components and oil quality in olive hedgerows under wide range of structures and; (iii) determine the variability in the characteristics of the leaves (morphological and physiological) and fruit tissues (size and composition) in different positions of the hedgerows canopy. Three plots of olive hedgerows (cv. Arbequina) planted in 2008 in La Puebla de Montalbán, Toledo were evaluated during the 2012 and 2013 seasons. The hedgerows were maintained by lateral pruning and topping with the same width (1 m) and height (2.5 m) compatible with the intended harvester. In a plot (experiment 1), the hedgerows were planted with the same width alley (4 m) and four row orientations: N–S, NE–SW, NW–SE and E–W. Other two plots (Experiments 2 and 3) separated by approximately 100 m were planted with N–S and E–O oriented rows and three alley widths in each orientation: 5.0, 4.0 and 2.5 m. In the exp. 1, maximum fruit yield were achieved by NE–SW and NW–SW (15.7 t/ha). Of these, NE–SW achieved the highest oil yield (2.7 t/ha). There were no differences in fruit or oil yield between N–S (2.5 t oil/ha) and E–W (2.3 t oil/ha) orientations. Fruit number was the most important component to explain these differences, by previous influence on number of bud developed and percentage of fertile inflorescences. Fruit maturity and oil quality on both sides of the hedgerows were not affected by row orientation. This occurred despite significant variations in the internal fruit temperature, which was closely related to the irradiance received by the canopy and the time of day. Additionally, row orientation significantly affected the relative water content of the soil, where E–W oriented hedgerows showed consistently higher values than N–S during summer-autumn season. The stem water potential at midday, however, was similar between orientations, revealing possible lower water consumption of E–W than N–S oriented hedgerows. In the exp. 2, regardless of row orientation, reduction of row spacing from 5.0 to 4.0 and 2.5 m increases the ratio of canopy depth to free alley width (Al/An) from 0.6 to 0.8 and 1.6, respectively, and ads 25 and 100 % more hedgerow length per ha. In N–S oriented hedgerows, oil production per ha increased significantly by 14 and 52 % in 4.0 m and 2.5 m relative to 5.0 m row spacing, the effect being proportionally less than the increase in hedgerow length per ha. Hedgerows spaced 2.5 m with Al/An = 1.6 produced relatively fewer fruits per unit length than did wider spacings and were preferentially distributed in upper layers. Fruits located at the bottom of the canopy were smaller, with lower oil content and were less mature. In E–W oriented hedgerows, oil production per ha did not respond significantly to row spacing, despite the doubling of row length from the 5.0 to the 2.5 m row spacing. The explanation was found in fewer fruit per unit length of hedgerow and smaller oil content at 2.5 m than 5.0 m row spacing, averaged over the experimental period. In E–W hedgerows spaced at 5.0 m with Al/An = 0.6, the vertical profiles of fruit characteristics (mass, oil and water contents, and maturity) were similar between opposing sides, but at 4.0 m (Al/An= 0.8) and 2.5 m (Al/An=1.6) spacings, fruits on the S side were heavier and more mature than on N side. The oil extracted from fruits harvested at different heights of N–S and E–W oriented hedgerows showed higher palmitoleic, palmitic, stearic and linoleic contents at the canopy top decreasing toward base. The oleic content was reverse, increased from top to base. In N–S hedgerows, vertical gradients increased by reducing the alley width, but not in the E–W oriented hedgerows. The simulation of internal canopy irradiance was related in a single relationship (R2 = 0.63) to the vertical profiles of fruit weight and oil content of olive hedgerows with wide range of structures. The density of fruits was also associated with the irradiance but more weakly (R2 = 0.27), and revealed a more complex response involving changes in the vegetative structure by canopy management (topping) and the effect of radiation on the previous sequence that defines the number of fruits. The vertical profiles of oil quality traits were closely associated to canopy irradiance, but only when the N–S oriented hedgerows were considered. The contents of palmitoleic and linoleic acid in the oil increased linearly when intercepted irradiance increased from 9 to 19 mol PAR/m2. In contrast, oleic content decreased linearly in this irradiance range. Additionally, we advanced knowledge regarding the interactions among irradiance and leaf, fruit number, size and composition at organ-, tissue- and cellular- levels. The irradiance received at different positions in the canopy strongly affected the leaf area and chlorophyll content, and mesocarp size and composition (water and oil), without effects on endocarp size and composition. At the cellular level, light-exposed fruit developed larger mesocarp cells than shaded fruits, but cell number was not affected. Our results indicate that cell number and size are related to mesocarp composition in term of oil, water, and dry weight menus oil, although the specific manner in which they interact remains to be determined. This research contributes from an integral perspective of olive growing to quantify the impact of row orientation and width alley on productivity and oil quality in hedgerows systems. The analysis and discussion of the relationships between radiation and yield components and oil quality can help understand the impact of design olive hedgerows in general and in a wide range of environmental conditions.

Influence of castration on growth performance and carcass and meat quality of heavy female pigs

The main objetive of this Doctoral Thesis was to study the influence of female castration and pig sex on growth performance and carcass and meat quality of white pigs slaughtered at different final weights. Three experiments (Exp.) were conducted. In Exp. 1, a total of 200 (Landrace * Large White dam x Pietrain * Large White sire) gilts of 50 ± 3 days of age (23.3 ± 1.47 kg BW) was used to investigate the effects of castration (intact females, IF vs. castrated feamles, CF) and slaughter weight (106 vs. 122 kg BW) on productive performance and carcass and meat quality. There were four experimental treatments arranged as a 2 x 2 factorial and 5 replicates of 10 pigs each per treatment. Half of the gilts were ovariectomized at 58 d of age (8 days after the beginning of the trial; 29.8 ± 1.64 kg BW) whereas the other half remained intact. Meat samples were taken at m. Longissimus thoracis at the level of the last rib and subcutaneous fat samples were taken at the tail insertion. For the entire experiment period, CF had higher BW gain (P<0.05) and backfat and m. Gluteus medius (GM) fat thickness (P<0.001) than IF. However, IF had higher loin and trimmed primal cut yields (P<0.05) than CF. Meat quality was similar for IF and CF but the proportion of linoleic acid in subcutaneous fat was higher (P<0.001) for IF. Pigs slaughtered at 122 kg BW had higher (P<0.001) feed intake and poorer feed efficiency than pigs slaughtered at 106 kg BW. An increase in slaughter weight (SW) improved (P<0.001) carcass yield but decreased (P<0.05) trimmed primal cut yield. Meat from females slaughtered at the heavier BW was redder (a*; P<0.001) and had more (P<0.01) intramuscular fat and less thawing (P<0.05) and cooking (P<0.10) loss than meat from females slaughtered at the lighter BW. Also, females slaughtered at 122 kg BW had less (P<0.01) linoleic acid content in the subcutaneous fat than pigs slaughtered at 106 kg BW. Castration of gilts and slaughtering at heavier BW might be useful practices for the production of heavy pigs destined to the dry cured industry in which a certain amount of fat in the carcass is required. In contrast, when the carcasses are destined to fresh meat production, IF slaughtered at 106 kg BW are a more efficient alternative. In Exp. 2, crossbred pigs (n=240) from Pietrain*Large White sires mated to Landrace*Large White dams with an average of 100 d of age (60.5 ± 2.3 kg) were used to investigate the effects of gender and slaughter weight (SW) on growth performance and carcass and meat quality characteristics. There were 6 treatments arranged factorially with 3 genders (IF vs. CF vs.castrated males, CM) and 2 SW (114 vs. 122 kg BW). Each of the 6 combinations of treatments was replicated 4 times and the experimental unit was a pen with 10 pigs. Castrated males and CF ate more feed, grew faster and had more carcass backfat depth and fat thickness at the GM muscle, but lower loin yield than IF (P<0.05). In addition, CF and CM had more intramuscular fat (P<0.05) and less linoleic acid content in the subcutaneous fat (P<0.01) than IF. Pigs slaughtered at 122 kg BW had lower ADG (P<0.05), poor gain-to-feed ratio (P<0.05), and more GM fat than pigs slaughtered at 114 kg BW (P < 0.05). It is concluded that CF and CM had similar productive performance and meat quality characteristics when slaughtered at the same age, and that the castration of females improved daily gains and increased weight and fat content of primal cuts with respect to IF. Therefore, castration of females is recommended in pigs destined to the dry-cured industry because of the beneficial effects on the quality of the primal cuts. In Exp. 3, the effects of gender and castration of females (IF vs. CF vs. CM) on performance and carcass and meat quality were studied in crossbred pigs (Landrace x Large White dams x Duroc sires) slaughtered at 119.2 (trial 1) or 131.6 (trial 2) kg BW. Intact females had better feed conversion and less carcass fat than CF and CM. Trimmed shoulder yield was higher for CM than for CF with IF being intermediate. Primal cut yield and meat quality, however were similar for all treatments. Proportion of linoleic acid in backfat was lower for CF than for IF or CM, and the differences were significant in pigs slaughtered witn 131.6 kg BW. The higher fat content and the fatty acid profile favour the use of CF and CM over IF for the production of heavy pigs destined to the dry-cured industry.

A maternal diet high in n − 6 polyunsaturated fats alters mammary gland development, puberty onset, and breast cancer risk among female rat offspring

We hypothesized that feeding pregnant rats with a high-fat diet would increase both circulating 17β-estradiol (E2) levels in the dams and the risk of developing carcinogen-induced mammary tumors among their female offspring. Pregnant rats were fed isocaloric diets containing 12% or 16% (low fat) or 43% or 46% (high fat) of calories from corn oil, which primarily contains the n − 6 polyunsaturated fatty acid (PUFA) linoleic acid, throughout pregnancy. The plasma concentrations of E2 were significantly higher in pregnant females fed a high n − 6 PUFA diet. The female offspring of these rats were fed with a laboratory chow from birth onward, and when exposed to 7,12-dimethylbenz(a)anthracene had a significantly higher mammary tumor incidence (60% vs. 30%) and shorter latency for tumor appearance (11.4 ± 0.5 weeks vs. 14.2 ± 0.6 weeks) than the offspring of the low-fat mothers. The high-fat offspring also had puberty onset at a younger age, and their mammary glands contained significantly higher numbers of the epithelial structures that are the targets for malignant transformation. Comparable changes in puberty onset, mammary gland morphology, and tumor incidence were observed in the offspring of rats treated daily with 20 ng of E2 during pregnancy. These data, if extrapolated to humans, may explain the link among diet, early puberty onset, mammary parenchymal patterns, and breast cancer risk, and indicate that an in utero exposure to a diet high in n − 6 PUFA and/or estrogenic stimuli may be critical for affecting breast cancer risk.

Cyanide-Resistant, ATP-Synthesis-Sustained, and Uncoupling-Protein-Sustained Respiration during Postharvest Ripening of Tomato Fruit1

Tomato (Lycopersicon esculentum) mitochondria contain both alternative oxidase (AOX) and uncoupling protein as energy-dissipating systems that can decrease the efficiency of oxidative phosphorylation. We followed the cyanide (CN)-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration of isolated mitochondria, as well as the immunologically detectable levels of uncoupling protein and AOX, during tomato fruit ripening from the mature green stage to the red stage. The AOX protein level and CN-resistant respiration of isolated mitochondria decreased with ripening from the green to the red stage. The ATP-synthesis-sustained respiration followed the same behavior. In contrast, the level of uncoupling protein and the total uncoupling-protein-sustained respiration of isolated mitochondria decreased from only the yellow stage on. We observed an acute inhibition of the CN-resistant respiration by linoleic acid in the micromolar range. These results suggest that the two energy-dissipating systems could have different roles during the ripening process.

Phosphoglycerylethanolamine Posttranslational Modification of Plant Eukaryotic Elongation Factor 1α1

Eukaryotic elongation factor 1α (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carota L.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [3H]glycerol, [32P]Pi, [14C]myristic acid, and [14C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell.

Soybean Lipoxygenase-1 Oxidizes 3Z-Nonenal : A Route to 4S-Hydroperoxy-2E-Nonenal and Related Products

In previous work with soybean (Glycine max), it was reported that the initial product of 3Z-nonenal (NON) oxidation is 4-hydroperoxy-2E-nonenal (4-HPNE). 4-HPNE can be converted to 4-hydroxy-2E-nonenal by a hydroperoxide-dependent peroxygenase. In the present work we have attempted to purify the 4-HPNE-producing oxygenase from soybean seed. Chromatography on various supports had shown that O2 uptake with NON substrate consistently coincided with lipoxygenase (LOX)-1 activity. Compared with oxidation of LOX's preferred substrate, linoleic acid, the activity with NON was about 400- to 1000-fold less. Rather than obtaining the expected 4-HPNE, 4-oxo-2E-nonenal was the principal product of NON oxidation, presumably arising from the enzyme-generated alkoxyl radical of 4-HPNE. In further work a precipitous drop in activity was noted upon dilution of LOX-1 concentration; however, activity could be enhanced by spiking the reaction with 13S-hydroperoxy-9Z,11E-octadecadienoic acid. Under these conditions the principal product of NON oxidation shifted to the expected 4-HPNE. 4-HPNE was demonstrated to be 83% of the 4S-hydroperoxy-stereoisomer. Therefore, LOX-1 is also a 3Z-alkenal oxygenase, and it exerts the same stereospecificity of oxidation as it does with polyunsaturated fatty acids. Two other LOX isozymes of soybean seed were also found to oxidize NON to 4-HPNE with an excess of 4S-hydroperoxy-stereoisomer.

Evidence that free polyunsaturated fatty acids modify Na+ channels by directly binding to the channel proteins.

The effects of free polyunsaturated fatty acids (PUFA) on the binding of ligands to receptors on voltage-sensitive Na+ channels of neonatal rat cardiac myocytes were assessed. The radioligand was [benzoyl-2,5-(3)H] batrachotoxinin A 20alpha-benzoate ([(3)H]BTXB), a toxin that binds to the Na+ channel. The PUFA that have been shown to be antiarrhythmic, including eicosapentaenoic acid (EPA; C20:5n-3), docosahexaenoic acid (DHA; C22:6n-3), eicosatetraynoic acid (ETYA), linolenic acid (C18:3n-3), and linoleic acid (C18:2n-6), inhibited [(3)H]BTXB binding in a dose-dependent fashion with IC50 values of 28-35 microM, whereas those fatty acids that have no antiarrhythmic effects including saturated fatty acid (stearic acid, C18:0), monounsaturated fatty acid (oleic acid; C18:1n-9), and EPA methyl ester did not have a significant effect on [(3)H]BTXB binding. Enrichment of the myocyte membrane with cholesterol neither affected [(3)H]BTXB binding when compared with control cells nor altered the inhibitory effects of PUFA on [(3)H]BTXB binding. Scatchard analysis of [(3)H]BTXB binding showed that EPA reduced the maximal binding without altering the Kd for [(3)H]BTXB binding, indicating allosteric inhibition. The inhibition by EPA of [(3)H]BTXB binding was reversible (within 30 min) when delipidated bovine serum albumin was added. The binding of the PUFA to this site on the Na+ channel is reversible and structure-specific and occurs at concentrations close to those required for apparent antiarrhythmic effects and a blocking effect on the Na+ current, suggesting that binding of the PUFA at this site relates to their antiarrhythmic action.

Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants.

It is becoming clear that an adequate level of long-chain highly unsaturated fatty acids in the nervous system is required for optimal function and development; however, the ability of infants to biosynthesize long-chain fatty acids is unknown. This study explores the capacity of human infants to convert 18-carbon essential fatty acids to their elongated and desaturated forms, in vivo. A newly developed gas chromatography/negative chemical ionization/mass spectrometry method employing 2H-labeled essential fatty acids allowed assessment of this in vivo conversion with very high sensitivity and selectivity. Our results demonstrate that human infants have the capacity to convert dietary essential fatty acids administered enterally as 2H-labeled ethyl esters to their longer-chain derivatives, transport them to plasma, and incorporate them into membrane lipids. The in vivo conversion of linoleic acid (18:2n6) to arachidonic acid (20:4n6) is demonstrated in human beings. All elongases/desaturases necessary for the conversion of linolenic acid (18:3n3) to docosahexaenoic acid (22:6n3) are also active in the first week after birth. Although the absolute amounts of n-3 fatty acid metabolites accumulated in plasma are greater than those of the n-6 family, estimates of the endogenous pools of 18:2n6 and 18:3n3 indicate that n-6 fatty acid conversion rates are greater than those of the n-3 family. While these data clearly demonstrate the capability of infants to biosynthesize 22:6n3, a lipid that is required for optimal neural development, the amounts produced in vivo from 18:3n3 may be inadequate to support the 22:6n3 level observed in breast-fed infants.

Transformation of mammalian cells by overexpressing H2O2-generating peroxisomal fatty acyl-CoA oxidase.

Peroxisome proliferators induce qualitatively predictable pleiotropic responses, including development of hepatocellular carcinomas in rats and mice despite the inability of these compounds to interact with and damage DNA directly. In view of the nongenotoxic nature of peroxisome proliferators, it has been postulated that hepatocarcinogenesis by this class of chemicals is due to a receptor-mediated process leading to transcriptional activation of H2O2-generating peroxisomal fatty acyl-CoA oxidase (ACOX) in liver. To test this hypothesis, we overexpressed rat ACOX in African green monkey kidney cells (CV-1 cells) under control of the cytomegalovirus promoter. A stably transfected CV-1 cell line overexpressing rat ACOX, designated CV-ACOX4, when exposed to a fatty acid substrate (150 microM linoleic acid) for 2-6 weeks, formed transformed foci, grew efficiently in soft agar, and developed adenocarcinomas when transplanted into nude mice. These findings indicate that sustained overexpression of H2O2-generating ACOX causes cell transformation and provide further support for the role of peroxisome proliferation in hepatocarcinogenesis induced by peroxisome proliferators.

The octadecanoic pathway: signal molecules for the regulation of secondary pathways.

Plant defense against microbial pathogens and herbivores relies heavily on the induction of defense proteins and low molecular weight antibiotics. The signals between perception of the aggression, gene activation, and the subsequent biosynthesis of secondary compounds are assumed to be pentacylic oxylipin derivatives. The rapid, but transient, synthesis of cis-jasmonic acid was demonstrated after insect attack on a food plant and by microbial elicitor addition to plant suspension cultures. This effect is highly specific and not caused by a number of environmental stresses such as light, heavy metals, or cold or heat shock. Elicitation of Eschscholtzia cell cultures also led to a rapid alkalinization of the growth medium prior to jasmonate formation. Inhibition of this alkalinization process by the protein kinase inhibitor staurosporine also inhibited jasmonate formation. The induction of specific enzymes in the benzo[c]phenanthridine alkaloid pathway leading to the antimicrobial sanguinarine was induced to a qualitatively and quantitatively similar extent by fungal elicitor, methyl jasmonate, and its linolenic acid-derived precursor 12-oxophytodienoic acid. It is herein proposed that a second oxylipid cascade may exist in plants starting from linoleic acid via 15,16-dihydro-12-oxophytodienoic acid to 9,10-dihydrojasmonate. Experiments with synthetic trihomojasmonate demonstrated that beta-oxidation is not a prerequisite for biological activity and that 12-oxophytodienoic acid and derivatives are most likely fully active as signal transducers. Octadecanoic acid-derived compounds are essential elements in modulating the synthesis of antibiotic compounds and are thus integral to plant defense.

Free, long-chain, polyunsaturated fatty acids reduce membrane electrical excitability in neonatal rat cardiac myocytes.

Because previous studies showed that polyunsaturated fatty acids can reduce the contraction rate of spontaneously beating heart cells and have antiarrhythmic effects, we examined the effects of the fatty acids on the electrophysiology of the cardiac cycle in isolated neonatal rat cardiac myocytes. Exposure of cardiomyocytes to 10 microM eicosapentaenoic acid for 2-5 min markedly increased the strength of the depolarizing current required to elicit an action potential (from 18.0 +/- 2.4 pA to 26.8 +/- 2.7 pA, P < 0.01) and the cycle length of excitability (from 525 ms to 1225 ms, delta = 700 +/- 212, P < 0.05). These changes were due to an increase in the threshold for action potential (from -52 mV to -43 mV, delta = 9 +/- 3, P < 0.05) and a more negative resting membrane potential (from -52 mV to -57 mV, delta = 5 +/- 1, P < 0.05). There was a progressive prolongation of intervals between spontaneous action potentials and a slowed rate of phase 4 depolarization. Other polyunsaturated fatty acids--including docosahexaenoic acid, linolenic acid, linoleic acid, arachidonic acid, and its nonmetabolizable analog eicosatetraynoic acid, but neither the monounsaturated oleic acid nor the saturated stearic acid--had similar effects. The effects of the fatty acids could be reversed by washing with fatty acid-free bovine serum albumin. These results show that free polyunsaturated fatty acids can reduce membrane electrical excitability of heart cells and provide an electrophysiological basis for the antiarrhythmic effects of these fatty acids.

Avaliação do potencial fotoprotetor e identificação de metabólitos secundários do fungo endofítico Annulohypoxylon stygium associado à alga marinha Bostrychia radicans

Os organismos marinhos constituem uma fonte potencial de metabólitos secundários biologicamente ativos. Neste contexto, os micro-organismos isolados de algas marinhas, dentre eles fungos endofíticos, representam alvos para a pesquisa de novas substâncias com potencial farmacológico pronunciado. Substâncias naturais provenientes de espécies de fungos associados às algas marinhas vêm sendo bastante utilizadas em formulações fotoprotetoras devido à ação antioxidante e ao potencial contra a radiação solar. Deste modo, o presente trabalho teve como objetivo a investigação biológica e química dos fungos endofíticos marinhos pertencentes à família Xylariaceae, o Annulohypoxylon stygium, o Cladosporium sp. e o Acremonium implicatum (Hypocreaceae). A princípio, foi realizado um screening para avaliar a absorção de luz ultravioleta na faixa do UVA e UVB pelos extratos obtidos em escala piloto destes fungos endofíticos associados às algas marinhas. O extrato do fungo A. stygium apresentou intensa absorção na região do UV, mostrando-se promissor para a produção de metabólitos secundários com ação fotoprotetora. Além do ensaio proposto, foi realizada a avaliação do potencial antibacteriano e antifúngico da espécie A. stygium. O estudo químico em escala ampliada deste fungo proporcionou o isolamento e identificação de uma substância inédita da classe derivada da 2,5- dicetopiperazina, 3-benzilideno-2-metil-hexahidro-pirrolo [1,2-?] pirazina-1,4-diona (Sf3), e além desta, foram isolados mais quatro metabólitos como, os diasteroisômeros 1-fenil-1,2- propanediol (Sd2) e 1-fenil-1,2-propanediol (Sd3), 1,3-benzodioxole-5-metanol (Sc1), 1,2- propanodiol-1-(1,3-benzodioxol-5-il) (Se1). Ainda foi possível a desreplicação de substâncias via cromatografia gasosa acoplada à espectrometria de massas (CG-EM), entre elas o ácido palmítico, palmitato de metila, ácido metil linoléico, ácido oléico, álcool benzílico e o piperonal. Quanto ao estudo da atividade biológica, não foi observado potencial antibacteriano e antifúngico para os extratos e frações do fungo. Entretanto, notouse um potencial como fotoprotetor in vitro para as frações n-Hexano/AcOEt (2:3) e n- Hexano/AcOEt (1:4) obtidas a partir do extrato do cultivo de 28 dias do fungo A. stygium, extraído com solventes diclorometano/metanol (CH2Cl2/MeOH 2:1) e para a substância (Sf3) isolada do mesmo. Desta forma, o estudo químico e biológico do fungo Annulohypoxylon stygium demonstrou potencial para a produção de metabólitos secundários com atividade fotoprotetora, visto que uma estrutura inédita com esta atividade foi isolada e identificada como produto natural.

Efeito dos ácidos graxos ômega-3, ômega-6 e ômega-9 sobre o risco cardiovascular de indivíduos adultos: estudo clínico de prevenção primária.

INTRODUÇÃO: As doenças cardiovasculares (DCV) são a principal causa de morte no mundo, sendo muitos dos fatores de risco passíveis de prevenção e controle. Embora as DCV sejam complexas em sua etiologia e desenvolvimento, a concentração elevada de LDL-c e baixa de HDL-c constituem os fatores de risco modificáveis mais monitorados na prática clínica, embora não sejam capazes de explicar todos os eventos cardiovasculares. Portanto, investigar como intervenções farmacológicas e nutricionais podem modular parâmetros oxidativos, físicos e estruturais das lipoproteínas pode fornecer estimativa adicional ao risco cardiovascular. Dentre os diversos nutrientes e compostos bioativos relacionados às DCV, os lipídeos representam os mais investigados e descritos na literatura. Nesse contexto, os ácidos graxos insaturados (ômega-3, ômega-6 e ômega-9) têm sido foco de inúmeros estudos. OBJETIVOS: Avaliar o efeito da suplementação com ômega-3, ômega-6 e ômega-9 sobre os parâmetros cardiometabólicos em indivíduos adultos com múltiplos fatores de risco e sem evento cardiovascular prévio. MATERIAL E MÉTODOS: Estudo clínico, randomizado, duplo-cego, baseado em intervenção nutricional (3,0 g/dia de ácidos graxos) sob a fórmula de cápsulas contendo: ômega-3 (37 por cento de EPA e 23 por cento de DHA) ou ômega-6 (65 por cento de ácido linoleico) ou ômega-9 (72 por cento de ácido oleico). A amostra foi composta por indivíduos de ambos os sexos, com idade entre 30 e 74 anos, apresentando pelo menos um dos seguintes fatores de risco: Dislipidemia, Diabetes Mellitus, Obesidade e Hipertensão Arterial Sistêmica. Após aprovação do Comitê de Ética, os indivíduos foram distribuídos nos três grupos de intervenção. No momento basal, os indivíduos foram caracterizados quanto aos aspectos demográficos (sexo, idade e etnia) e clínicos (medicamentos, doenças atuais e antecedentes familiares). Nos momentos basal e após 8 semanas de intervenção, amostras de sangue foram coletadas após 12h de jejum. A partir do plasma foram analisados: perfil lipídico (CT, LDL-c, HDL-c, TG), apolipoproteínas AI e B, ácidos graxos não esterificados, atividade da PON1, LDL(-) e auto-anticorpos, ácidos graxos, glicose, insulina, tamanho e distribuição percentual da LDL (7 subfrações e fenótipo A e não-A) e HDL (10 subfrações). O efeito do tempo, da intervenção e associações entre os ácidos graxos e aspectos qualitativos das lipoproteínas foram testados (SPSS versão 20.0, p <0,05). RESULTADOS: Uma primeira análise dos resultados baseada em um corte transversal demonstrou, por meio da análise de tendência linear ajustada pelo nível de risco cardiovascular, que o maior tercil plasmático de DHA se associou positivamente com HDL-c, HDLGRANDE e tamanho de LDL e negativamente com HDLPEQUENA e TG. Observou-se também que o maior tercil plasmático de ácido linoleico se associou positivamente com HDLGRANDE e tamanho de LDL e negativamente com HDLPEQUENA e TG. Esse perfil de associação não foi observado quando foram avaliados os parâmetros dietéticos. Avaliando uma subamostra que incluiu indivíduos tabagistas suplementados com ômega-6 e ômega-3, observou-se que ômega-3 modificou positivamente o perfil lipídico e as subfrações da HDL. Nos modelos de regressão linear ajustados pela idade, sexo e hipertensão, o DHA plasmático apresentou associações negativas com a HDLPEQUENA. Quando se avaliou exclusivamente o efeito do ômega-3 em indivíduos tabagistas e não tabagistas, observou-se que fumantes, do sexo masculino, acima de 60 anos de idade, apresentando baixo percentual plasmático de EPA e DHA (<8 por cento ), com excesso de peso e gordura corporal elevada, apresentam maior probabilidade de ter um perfil de subfrações de HDL mais aterogênicas. Tendo por base os resultados acima, foi comparado o efeito do ômega-3, ômega-6 e ômega-9 sobre os parâmetros cardiometabólicos. O ômega-3 promoveu redução no TG, aumento do percentual de HDLGRANDE e redução de HDLPEQUENA. O papel cardioprotetor do ômega-3 foi reforçado pelo aumento na incorporação de EPA e DHA, no qual indivíduos com EPA e DHA acima de 8 por cento apresentaram maior probabilidade de ter HDLGRANDE e menor de ter HDLPEQUENA. Em adição, observou-se também que o elevado percentual plasmático de ômega-9 se associou com partículas de LDL menos aterogênicas (fenótipo A). CONCLUSÃO: Ácidos graxos plasmáticos, mas não dietéticos, se correlacionam com parâmetros cardiometabólicos. A suplementação com ômega-3, presente no óleo de peixe, promoveu redução no TG e melhoria nos parâmetros qualitativos da HDL (mais HDLGRANDE e menos HDLPEQUENA). Os benefícios do ômega-3 foram particularmente relevantes nos indivíduos tabagistas e naqueles com menor conteúdo basal de EPA e DHA plasmáticos. Observou-se ainda que o ômega-9 plasmático, presente no azeite de oliva, exerceu impacto positivo no tamanho e subfrações da LDL.

Novel particulate BCG-loaded delivery system for mucosal immunization against tuberculosis

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016

Fatty acids as haptens: exploring the limits of antigenicity

Fatty acids (FAs) are relatively small, hydrophobic and highly mobile molecular structures with vital biological functions and a ubiquitous distribution. Surprisingly, however, they can be rendered immunogenic. We have synthesised a novel immunogen in which dicarboxylic linoleic acid was conjugated to a carrier protein. Dicarboxylic fatty acids (DCA) differ from their normal counterparts only by their possession of a carboxyl group at each end of the molecule. When conjugated to proteins as haptens, they are, therefore, presented to the immune system with a free carboxyl group at the distal end, instead of a methyl group. Polyclonal IgG antibodies raised in response to this unique immunogen could bind not only conjugated hapten with high affinity, but also the equivalent free FA in mono and dicarboxylic form. Similar conjugates constructed from normal FAs produced much weaker antibody responses and could scarcely be considered antigenic at all. The cross-reactivities of the anti-DCA antibodies with FA variants differing in the number, position and configuration of their double bonds showed that the antibody paratope (binding site) was structured to accommodate the hapten in a way that depended on the precise shape of the acyl chain. We suggest that FAs become much more effective as B-cell epitopes when presented with their hydrophilic carboxyl group exposed on the surface of immunogenic conjugates. This type of epitope is determined by the particular double bond pattern of the unsaturated acyl chain, as well as the polar head group. (C) 2003 Elsevier Ltd. All rights reserved.