Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers

Background Trypanosomatids of the genera Angomonas and Strigomonas live in a mutualistic association characterized by extensive metabolic cooperation with obligate endosymbiotic Betaproteobacteria. However, the role played by the symbiont has been more guessed by indirect means than evidenced. Symbiont-harboring trypanosomatids, in contrast to their counterparts lacking symbionts, exhibit lower nutritional requirements and are autotrophic for essential amino acids. To evidence the symbiont’s contributions to this autotrophy, entire genomes of symbionts and trypanosomatids with and without symbionts were sequenced here. Results Analyses of the essential amino acid pathways revealed that most biosynthetic routes are in the symbiont genome. By contrast, the host trypanosomatid genome contains fewer genes, about half of which originated from different bacterial groups, perhaps only one of which (ornithine cyclodeaminase, EC:4.3.1.12) derived from the symbiont. Nutritional, enzymatic, and genomic data were jointly analyzed to construct an integrated view of essential amino acid metabolism in symbiont-harboring trypanosomatids. This comprehensive analysis showed perfect concordance among all these data, and revealed that the symbiont contains genes for enzymes that complete essential biosynthetic routes for the host amino acid production, thus explaining the low requirement for these elements in symbiont-harboring trypanosomatids. Phylogenetic analyses show that the cooperation between symbionts and their hosts is complemented by multiple horizontal gene transfers, from bacterial lineages to trypanosomatids, that occurred several times in the course of their evolution. Transfers occur preferentially in parts of the pathways that are missing from other eukaryotes. Conclusion We have herein uncovered the genetic and evolutionary bases of essential amino acid biosynthesis in several trypanosomatids with and without endosymbionts, explaining and complementing decades of experimental results. We uncovered the remarkable plasticity in essential amino acid biosynthesis pathway evolution in these protozoans, demonstrating heavy influence of horizontal gene transfer events, from Bacteria to trypanosomatid nuclei, in the evolution of these pathways.

A proteomic analysis of the functional effects of fatty acids in NIH 3T3 fibroblasts

Previous studies have demonstrated that long chain fatty acids influence fibroblast function at sub-lethal concentrations. This study is the first to assess the effects of oleic, linoleic or palmitic acids on protein expression of fibroblasts, as determined by standard proteomic techniques. The fatty acids were not cytotoxic at the concentration used in this work as assessed by membrane integrity, DNA fragmentation and the MTT assay but significantly increased cell proliferation. Subsequently, a proteomic analysis was performed using two dimensional difference gel electrophoresis (2D-DIGE) and MS based identification. Cells treated with 50 μM oleic, linoleic or palmitic acid for 24 h were associated with 24, 22, 16 spots differentially expressed, respectively. Among the identified proteins, α-enolase and far upstream element binding protein 1 (FBP-1) are of importance due to their function in fibroblast-associated diseases. However, modulation of α-enolase and FBP-1 expression by fatty acids was not validated by the Western blot technique.

Mechanisms underlying skeletal muscle insulin resistance induced by fatty acids: importance of the mitochondrial function

Insulin resistance condition is associated to the development of several syndromes, such as obesity, type 2 diabetes mellitus and metabolic syndrome. Although the factors linking insulin resistance to these syndromes are not precisely defined yet, evidence suggests that the elevated plasma free fatty acid (FFA) level plays an important role in the development of skeletal muscle insulin resistance. Accordantly, in vivo and in vitro exposure of skeletal muscle and myocytes to physiological concentrations of saturated fatty acids is associated with insulin resistance condition. Several mechanisms have been postulated to account for fatty acids-induced muscle insulin resistance, including Randle cycle, oxidative stress, inflammation and mitochondrial dysfunction. Here we reviewed experimental evidence supporting the involvement of each of these propositions in the development of skeletal muscle insulin resistance induced by saturated fatty acids and propose an integrative model placing mitochondrial dysfunction as an important and common factor to the other mechanisms.

Potential therapeutic effects of branched-chain amino acids supplementation on resistance exercise-based muscle damage in humans

Branched-chain amino acids (BCAA) supplementation has been considered an interesting nutritional strategy to improve skeletal muscle protein turnover in several conditions. In this context, there is evidence that resistance exercise (RE)-derived biochemical markers of muscle soreness (creatine kinase (CK), aldolase, myoglobin), soreness, and functional strength may be modulated by BCAA supplementation in order to favor of muscle adaptation. However, few studies have investigated such effects in well-controlled conditions in humans. Therefore, the aim of this short report is to describe the potential therapeutic effects of BCAA supplementation on RE-based muscle damage in humans. The main point is that BCAA supplementation may decrease some biochemical markers related with muscle soreness but this does not necessarily reflect on muscle functionality.

Absorption and metabolism of volatile fatty acids by rumen and omasum

Volatile fatty acids (VFA) absorption and metabolic capacity of rumen and omasum were compared, in vitro. Fragments of rumen wall and omasum laminae were taken from eight adult crossbred bovines. An isolated fragment of the mucosa was fitted in a tissue diffusion chamber. Valeric acid and CrEDTA were added to ruminal fluid and placed on the mucosal side and buffer solution was placed on the serosal side. Fractional absorption rates were measured by exponential VFA:Cr ratio decay over time. Metabolism rate was determined as the difference between VFA absorbed and VFA which appeared on the serosal side over time. Mitotic index was higher in omasum (0.52%) than in rumen epithelium (0.28%). VFA fractional absorption rate was higher in omasum (4.6%/h.cm²) than in rumen (0.4%/h.cm²). Acetate, propionate, butyrate, and valerate showed similar fractional absorption rates in both fragments. Percentage of metabolized acetate and propionate was lower than butyrate and valerate in both stomach compartments. In the rumen, individual VFA metabolism rates were similar (mean of 7.7 , but in the omasum, valerate (90.0 was more metabolized than butyrate (59.6 propionate (69.8 and acetate (51.7 . Correlation between VFA metabolism and mitotic index was positive in the rumen and in the omasum. In conclusion, VFA metabolism and absorption potential per surface of the omasum is higher than that of the rumen. Variations on rumen and omasum absorption capacities occur in the same way, and there are indications that factors capable of stimulating rumen wall proliferation are similarly capable of stimulating omasum walls.

Importance of the proportions of dietary polyunsaturated fatty acids and antioxoidants in larval development of marine fish

La acuicultura es uno de los sectores con mayor crecimiento en la producción animal mundial, con una tasa superior al 5% anual en la última década (Izquierdo et al., 2008). En la acuicultura, el éxito del cultivo de cualquier especie de peces marinos está limitado por la cantidad y la calidad de la producción en masa de sus alevines (Izquierdo y Fernández - Palacios, 1997). Por lo tanto, para cubrir las crecientes demandas de la producción de semillas, es necesario mejorar la calidad nutritiva de sus larvas, lo cual todavía constituye una de las principales limitaciones para el desarrollo del cultivo de especies de peces marinos (Watanabe et al., 1983; Yúfera y Pascual, 1984; Sargent et al. 1997; Izquierdo et al., 2000). Aunque la producción Mediterránea del cultivo de peces marinos se ha incrementado en varias especies, la dorada Sparus aurata continúa siendo la especie mas cultivada (Izquierdo, 2005), con una producción anual de alevines que supera los 120 millones/ año. La demanda de alevines de buena calidad está aumentando a un ritmo del 10% anual, pero el éxito de la producción de los juveniles se ve muy afectado por la eficacia de la primera alimentación y la calidad nutricional de la dieta de arranque (Kolkovski et al., 1993; Sargent et al., 1997; Izquierdo et al., 2000). En general, la dorada y la lubina europea (Dicentrarchus labrax) son las especies más importantes de peces marinos criados en la región mediterránea y han caracterizado el desarrollo de la acuicultura marina en esta región en las últimas tres décadas (FAO, 1999). Además, en la producción, de ambas especies, se prevee una mayor expansión (Basurco y Abellán, 1999). Sin embargo, a pesar de que el engorde de estas especies está bien controlado, el conocimiento de sus necesidades nutricionales, en comparación con otras especies como salmónidos y carpas, sigue siendo incompleto (NRC, 1993). Por lo tanto, para obtener un mejor crecimiento y una mayor tasa de supervivencia, es esencial una dieta que responda a las necesidades nutricionales de las larvas, tanto cualitativas como cuantitativas (Kolkovski et al., 1993; Sargent et al., 1997). Además, incluso antes de que comience el desarrollo larvario, los huevos de los peces deben contener todos los nutrientes que cubran los requerimientos adecuados para el desarrollo del embrión (Izquierdo y Fernández - Palacios, 1997). En los últimos años, las investigaciones han prestado gran atención a la importancia de los lípidos dietéticos para larvas de peces marinos (Izquierdo et al., 2003), puesto que son esenciales para el crecimiento y el desarrollo de los mismos (Watanabe, 1982; Sargent et al., 1999a). Como reflejo de esta importancia, la dieta para dorada y lubina se ha convertido en una dieta altamente energética (25% de lípidos) en comparación con la década anterior (12% de lípidos) (Izquierdo et al., 2003). Los lípidos dietéticos proporcionan una fuente rica de energía y fosfolípidos y son fundamentales para la estructura de las biomembranas. Los lípidos dietéticos también sirven como vehículos para la absorción de otros nutrientes, como las vitaminas liposolubles A, D, E, y K, y pigmentos naturales o sintéticos. Los lípidos son componentes de las hormonas y precursores para la síntesis de diversos metabolitos funcionales, como las prostaglandinas y otros eicosonoides. Además, los lípidos dietéticos son reconocidos como uno de los factores nutricionales más importantes que afectan el crecimiento y la supervivencia de las larvas (Watanabe et al., 1983), porque constituyen materiales esenciales para la formación normal de célula, las membranas de los tejidos y el desarrollo de órganos (Izquierdo et al., 1998, 2003; Pousaò et al., 2003). Sin embargo, la utilización de los lípidos dietéticos por las larvas puede verse afectada directa o indirectamente por varios cambios morfológicos y fisiológicos que ocurren durante el desarrollo larvario. En los últimos años ha habido más interés en todos estos aspectos nutricionales de los lípidos en las larvas de peces, debido a la importancia de utilización de los lípidos dieteticos para el óptimo crecimiento y supervivencia larvaria (Izquierdo et al., 2000).

Effects of dietary fatty acids on patterns of early mineralisation and bone formation in the axial skeleton of Sparus aurata (Linnaeus, 1758)

Máster Oficial en Cultivos Marinos. VI Máster Internacional en Acuicultura. Trabajo presentado como requisito parcial para la obtención del Título de Máster Oficial en Cultivos Marinos, otorgado por la Universidad de Las Palmas de Gran Canaria (ULPGC), el Instituto Canario de Ciencias Marinas (ICCM), y el Centro Internacional de Altos Estudios Agronómicos Mediterráneos de Zaragoza (CIHEAM)

Enhancement of gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) larval growth by dietary vitamin E en relation to diffetent levels of essential fatty acids

[EN]Most marine fish larvae require high amounts of n-3 HUFA (highly unsaturated fatty acids) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Watanabe, 1982; Izquierdo, 1996). Fish larvae tissue lipids are also very high in n-3 HUFA, what implies a higher risk of peroxidation (Sargent et al. 1999) and cellular damage (Kanazawa, 1991), requiring then antioxidants to protect them intra- and extra-cellularly from free radical compounds. Vitamin E (Vit E) functions as a chain breaking antioxidant, reacting with the lipid peroxide radical produced and preventing the further reaction with a new PUFA. Hence their requirements are related with the dietary and tissue PUFA contents. The objective of the present study was to determine the effect of dietary Vit E on gilthead sea bream and sea bass survival, growth and stress, at different n-3 HUFA levels.

Effetti del chitosano su composti polifenolici in colture cellulari di vite: aspetti molecolari e metabolici

Polyphenols, including flavonoids and stilbenes, are an essential part of human diet and constitute one of the most abundant and ubiquitous group of plant secondary metabolites. The level of these compounds is inducible by stress or fungal attack, so attempts are being made to identify likely biotic and abiotic elicitors and to better understand the underlying mechanism. Resveratrol (3,5,4’-trihydroxystilbene), which belongs to the stilbene family, is a naturally occurring polyphenol, found in several fruits, vegetables and beverages including red wine. It is one of the most important plant polyphenols with proved benefic activity on animal health. In the last two decades, the potential protective effects of resveratrol against cardiovascular and neurodegenerative diseases, as well as the chemopreventive properties against cancer, have been largely investigated. The most important source of polyphenols and in particular resveratrol for human diet is grape (Vitis vinifera). Since stilbenes and flavonoids play a very important role in plant defence responses and enviromental interactions, and their effects on human health seem promising, the aim of the research of this Thesis was to study at different levels the activation and the regulation of their biosynthetic pathways after chitosan treatment. Moreover, the polyphenol production in grape cells and the optimisation of cultural conditions bioreactor scale-up, were also investigated. Cell suspensions were obtained from cv. Barbera (Vitis vinifera L.) petioles and were treated with a biotic elicitor, chitosan (50 μg/mL, dissolved in acetic acid) to promote phenylpropanoid metabolism. Chitosan is a D-glucosamine polymer from fungi cell wall and therefore mimes fungal pathogen attack. Liquid cultures have been monitored for 15 days, measuring cell number, cell viability, pH and grams of fresh weight. The endogenous and released amounts of 7 stilbenes (trans and cis isomers of resveratrol, piceid and resveratroloside, and piceatannol), gallic acid, 6 hydroxycinnamic acids (trans-cinnamic, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acids), 5 catechines (catechin, epicatechin, epigallocatechin-gallate (EGCG), epigallocatechin and epicatechin-gallate) and other 5 flavonoids (chalcon, naringenin, kaempferol, quercetin and rutin) in cells and cultural medium, were measured by HPLC-DAD analysis and total anthocyanins were quantified by spectrophotometric analysis. Chitosan was effective in stimulating trans-resveratrol endogenous accumulation with a sharp peak at day 4 (exceeding acetic acid and water controls by 36% and 63%, respectively), while it did not influence the production of the cis-isomer. Compared to both water and acetic acid controls, chitosan decreased the release of both trans- and cis-resveratrol respect to controls. No effect was shown on the accumulation of single resveratrol mono-glucoside isomers, but considering their total amount, normalized for the relative water control, it was possible to evidence an increase in both accumulation and release of those compounds, in chitosan-treated cells, throughout the culture period and particularly during the second week. Many of the analysed flavonoids and hydroxycinnamic acids were not present or detectable in trace amounts. Catechin, epicatechin and epigallocatechin-gallate (EGCG) were detectable both inside the cells and in the culture media, but chitosan did not affect their amounts. On the contrary, total anthocyanins have been stimulated by chitosan and their level, from day 4 to 14, was about 2-fold higher than in both controls, confirming macroscopic observations that treated suspensions showed an intense brown-red color, from day 3 onwards. These elicitation results suggest that chitosan selectively up-regulates specific biosynthetic pathways, without modifying the general accumulation pattern of other flavonoids. Proteins have been extracted from cells at day 4 of culture (corresponding to the production peak of trans-resveratrol) and separated by bidimensional electrophoresis. The 73 proteins that showed a consistently changed amount between untreated, chitosan and acetic acid (chitosan solvent) treated cells, have been identified by mass spectrometry. Chitosan induced an increase in stilbene synthase (STS, the resveratrol biosynthetic enzyme), chalcone-flavanone isomerase (CHI, that switches the pathway from chalcones to flavones and anthocyanins), pathogenesis-related proteins 10 (PRs10, a large family of defence proteins), and a decrease in many proteins belonging to primary metabolisms. A train of six distinct spots of STS encoded by the same gene and increased by chitosan, was detected on the 2-D gels, and related to the different phosphorylation degree of STS spots. Northern blot analyses have been performed on RNA extracted from cells treated with chitosan and relative controls, using probes for STS, PAL (phenylalanine ammonia lyase, the first enzyme of the biosynthetic pathway), CHS (chalcone synthase, that shares with STS the same precursors), CHI and PR-10. The up-regulation of PAL, CHS and CHI transcript expression levels correlated with the accumulation of anthocyanins. The strong increase of different molecular weight PR-10 mRNAs, correlated with the 11 PR-10 protein spots identified in proteomic analyses. The sudden decrease in trans-resveratrol endogenous accumulation after day 4 of culture, could be simply explained by the diminished resveratrol biosynthetic activity due to the lower amount of biosynthetic enzymes. This might be indirectly demonstrated by northern blot expression analyses, that showed lower levels of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) mRNAs starting from day 4. Other possible explanations could be a resveratrol oxidation process and/or the formation of other different mono-, di-glucosides and resveratrol oligomers such as viniferins. Immunolocalisation experiments performed on grape protoplasts and the subsequent analyses by confocal microscope, showed that STS, and therefore the resveratrol synthetic site, is mostly associated to intracellular membranes close to the cytosolic side of plasma membrane and in a smaller amount is localized in the cytosol. STS seemed not to be present inside vacuole and nucleus. There were no differences in the STS intracellular localisation between the different treatments. Since it was shown that stilbenes are largely released in the culture medium and that STS is a soluble protein, a possible interaction of STS with a plasma membrane transporter responsible for the extrusion of stilbenes in the culture medium, might be hypothesized. Proteomic analyses performed on subcellular fractions identified in the microsomial fraction 5 proteins taking part in channel complexes or associated with channels, that significantly changed their amount after chitosan treatment. In soluble and membrane fractions respectively 3 and 4 STS and 6 and 3 PR-10 have been identified. Proteomic results obtained from subcellular fractions substantially confirmed previous result obtained from total cell protein extracts and added more information about protein localisation and co-localisation. The interesting results obtained on Barbera cell cultures with the aim to increase polyphenol (especially stilbenes) production, have encouraged scale up tests in 1 litre bioreactors. The first trial fermentation was performed in parallel with a normal time-course in 20 mL flasks, showing that the scale-up (bigger volume and different conditions) process influenced in a very relevant way stilbenes production. In order to optimise culture parameters such as medium sucrose amount, fermentation length and inoculum cell concentration, few other fermentations were performed. Chitosan treatments were also performed. The modification of each parameter brought relevant variations in stilbenes and catechins levels, so that the production of a certain compound (or class of compounds) could be hypothetically promoted by modulating one or more culture parameters. For example the catechin yield could be improved by increasing sucrose content and the time of fermentation. The best results in stilbene yield were obtained in a 800 mL fermentation inoculated with 10.8 grams of cells and supplemented with chitosan. The culture was fed with MS medium added with 30 g/L sucrose, 25 μg/mL rifampicin and 50 μg/mL of chitosan, and was maintained at 24°C, stirred by marine impeller at 100 rpm and supplied of air at 0.16 L/min rate. Resveratroloside was the stilbene present in the larger amount, 3-5 times more than resveratrol. Because resveratrol glucosides are similarly active and more stable than free resveratrol, their production using a bioreactor could be a great advantage in an hypothetical industrial process. In my bioreactor tests, stilbenes were mainly released in the culture medium (60-80% of the total) and this fact could be another advantage for industrial applications, because it allows recovering the products directly from the culture medium without stopping the fermentation and/or killing the cells. In my best cultural conditions, it was possible to obtain 3.95 mg/L of stilbenes at day 4 (maximum resveratrol accumulation) and 5.13 mg/L at day 14 (maximum resveratroloside production). In conclusion, chitosan effect in inducing Vitis vinifera defense mechanisms can be related to its ability to increase the intracellular content of a large spectrum of antioxidants, and in particular of resveratrol, its derivates and anthocyanins. Its effect can be observed at transcriptional, proteomic (variation of soluble and membrane protein amounts) and metabolic (polyphenols production) level. The chitosan ability to elicit specific plant matabolisms can be useful to produce large quantities of antioxidant compounds from cell culture in bioreactor.

Synthetic and mechanistic investigation of new radical processes: reaction of organic azides with group-XIII Lewis acids

Dichloroindium hydride revealed to be a valid alternative to tributyltin hydride for radical reduction of organic (alkyl, aryl, acyl, solfonyl) azides. The new approach entails mild reaction conditions and provides high yields of the corresponding amines and amides, also showing high degrees of selectivity. The system dichloroindium hydride / azides can be utilised in fivemembered ring closures of g-azidonitriles, as a new source of aminyl radicals for the attractive synthesis of interesting amidine compounds in the absence of both toxic reagents and tedious purification procedures. Allylindium dichloride seems a good substitute for dichloroindium hydride for generation of indium centred radicals under photolytic conditions, since it allows allylation of electrophilic azides (e.g. phenylsulfonyl azide) and halogen or ester δ-substituted azides, the latter through a 1,5-H transfer rearrangement mechanism. Evidences of the radical nature of the reactions mechanism were provided by ESR spectroscopy, furthermore the same technique, allowed to discover that the reaction of azides with indium trichloride and other group XIII Lewis acids, in particular gallium trichloride, gives rise to strongly coloured, persistent paramagnetic species, whose structure is consistent with the radical cation of the head-to-tail dimer of the aniline corresponding to the starting azide.

First evidences of the effect of polyunsaturated fatty acids in fish neural activity and their implications in behaviour

Programa de doctorado: Acuicultura: producción controlada de animales acuáticos; Grupo de Investigación en Acuicultura (GIA)