Olive Polyphenol Oxidase Gene Family.

The phenolic compounds containing hydroxytyrosol are the minor components of virgin olive oil (VOO) with the greatest impact on its functional properties and health benefits. Olive breeding for improving the phenolic composition of VOO is strongly dependent on the identification of the key genes determining the biosynthesis of these compounds in the olive fruit and also their transformation during the oil extraction process. In this work, olive polyphenol oxidase (PPO) genes have been identified and fully characterized in order to evaluate their specific role in the metabolism of hydroxytyrosol-derived compounds by combining gene expression analysis and metabolomics data. Four PPO genes have been identified, synthesized, cloned and expressed in Escherichia coli, and the functional identity of the recombinant proteins has been verified using olive phenolic substrates. Among the characterized genes, two stand out: (i) OePPO2 with its diphenolase activity, which is very active in the oxidative degradation of phenols during oil extraction and also seems to be highly involved in the natural defense mechanism in response to biotic stress, and (ii) OePPO3, which codes for a tyrosinase protein, having diphenolase but also monophenolase activity, which catalyzes the hydroxylation of tyrosol to form hydroxytyrosol.

Comparison of GreenLight laser and transurethral resection of the prostate baseline characteristics and outcomes: lessons learned from the Clinical Research Office of the Endourological Society GreenLight Laser Study.

BACKGROUND: To compare baseline characteristics and outcomes of patients undergoing GreenLight laser vaporization (GL) or transurethral resection of the prostate (TURP) in a real life setting. METHODS: In this prospective observational cohort, the Clinical Research Office of the Endourological Society (CROES) collected data of consecutive GL or TURP treated patients. Treatment involved one of three GL laser powers (80 W, 120 W or 180 W) based on availability in each participating centre, or TURP. Data on baseline characteristics as well as functional measures were collected at three time points: 6-12 weeks, 6, and 12months after surgery. Functional measures included urinary flow parameters, perceived prostate function (IPSS), perceived erectile function (IIEF-5) and complications. RESULTS: Seven hundred thirteen patients underwent GL, and 234 patients underwent TURP. Overall, patients treated with GL show higher BMI, IIEF and medication use, together with lower urinary function (voided volume, incontinence, urinary retention) at baseline. After the procedure, despite higher antibiotic and antimuscarinic use and shorter hospital stay, readmission rates, PVR, PSA were higher, but Qmax, and IIEF were lower in the GL group. The rate of post-operative complications was 10.3% and 5.2% for the TURP and GL group, respectively (P=0.006). CONCLUSIONS: We were unable to categorically state which procedure is superior. This observational study confirms that treatment decision for either TURP or GL is not based on patient characteristics.

Monitoring endogenous enzymes during olive fruit ripening and storage: correlation with virgin olive oil phenolic profiles.

The ability of olive endogenous enzymes ß-glucosidase, polyphenol oxidase (PPO) and peroxidase (POX), to determine the phenolic profile of virgin olive oil was investigated. Olives used for oil production were stored for one month at 20 °C and 4 °C and their phenolic content and enzymatic activities were compared to those of ripening olive fruits. Phenolic and volatile profiles of the corresponding oils were also analysed. Oils obtained from fruits stored at 4 °C show similar characteristics to that of freshly harvested fruits. However, the oils obtained from fruits stored at 20 °C presented the lowest phenolic content. Concerning the enzymatic activities, results show that the ß-glucosidase enzyme is the key enzyme responsible for the determination of virgin olive oil phenolic profile as the decrease in this enzyme activity after 3 weeks of storage at 20 °C was parallel to a dramatic decrease in the phenolic content of the oils.

Variability of virgin olive oil phenolic compounds in a segregating progeny from a single cross in Olea europaea L. and sensory and nutritional quality implications.

Virgin olive oil phenolic compounds are responsible for its nutritional and sensory quality. The synthesis of phenolic compounds occurs when enzymes and substrates meet as olive fruit is crushed during the industrial process to obtain the oil. The genetic variability of the major phenolic compounds of virgin olive oil was studied in a progeny of the cross of Picual x Arbequina olive cultivars (Olea europaea L.). They belong to four different groups: compounds that included tyrosol or hydroxytyrosol in their molecules, lignans, flavonoids, and phenolic acids. Data of phenolics in the oils showed that the progeny displayed a large degree of variability, widely transgressing the genitor levels. This high variability can be of interest on breeding programs. Thus, multivariate analysis allowed to identify genotypes within the progeny particularly interesting in terms of phenolic composition and deduced organoleptic and nutritional quality. The present study has demonstrated that it is possible to obtain enough degree of variability with a single cross of olive cultivars for compounds related to the nutritional and organoleptic properties of virgin olive oil.

Thermal inactivation kinetics of recombinant proteins of the lipoxygenase pathway related to the synthesis of virgin olive oil volatile compounds.

The aim of this work was to characterize the thermal inactivation parameters of recombinant proteins related to the biosynthesis of virgin olive oil (VOO) volatile compounds through the lipoxygenase (LOX) pathway. Three purified LOX isoforms (Oep2LOX1, Oep1LOX2, and Oep2LOX2) and a hydroperoxide lyase (HPL) protein (OepHPL) were studied. According to their thermal inactivation parameters, recombinant Oep1LOX2 and Oep2LOX2 could be identified as the two LOX isoforms active in olive fruit crude preparations responsible for the synthesis of 13-hydroperoxides, the main substrates for the synthesis of VOO volatile compounds. Recombinant Oep2LOX1 displayed a low thermal stability, which suggests a weak actuation during the oil extraction process considering the current thermal conditions of this industrial process. In addition, recombinant OepHPL could be identified as the HPL activity in crude preparations. The thermal stability was the highest among the recombinant proteins studied, which suggests that HPL activity is not a limiting factor for the synthesis of VOO volatile compounds.

Factors limiting the synthesis of virgin olive oil volatile esters.

The aim of the present work was to establish the limiting factors affecting the biosynthesis of volatile esters present in virgin olive oil (VOO). Oil volatile fractions of the main Spanish olive cultivars, Arbequina and Picual, were analyzed. It was observed that acetate esters were the most abundant class of volatile esters in the oils, in concordance with the high content of acetyl-CoA found in olive fruit, and that the content of C6 alcohols is limited for the synthesis of volatile esters during the production of VOO. Thus, the increase of C6 alcohol availability during VOO production produced a significant increase of the corresponding ester in the oils in both cultivars at two different maturity stages. However, the increase of acetyl-CoA availability had no effect on the VOO volatile fraction. The low synthesis of these C6 alcohols seems not to be due to a shortage of precursors or cofactors for alcohol dehydrogenase (ADH) activity because their increase during VOO production had no effect on the C6 alcohol levels. The experimental findings are compatible with a deactivation of ADH activity during olive oil production in the cultivars under study. In this sense, a strong inhibition of olive ADH activity by compounds present in the different tissues of olive fruit has been observed.

Molecular cloning, functional characterization and transcriptional regulation of a 9-lipoxygenase gene from olive.

A lipoxygenase (LOX) cDNA clone (Oep2LOX1) has been isolated from olive fruit (Olea europaea cv. Picual). The deduced amino acid sequence displayed significant similarity to known plant LOX1 sequences. Genomic Southern blot analysis suggests that only one copy of Oep2LOX1 is present in the olive genome. Linolenic acid was the preferred substrate for the recombinant Oep2LOX1, which produced almost exclusively 9-hydroperoxide when linolenic acid was used as substrate, whereas a mixture of 9- and 13-hydroperoxides in a ratio 4:1 was formed from linoleic acid. Expression levels were measured in different tissues of Picual and Arbequina cultivars, including the mesocarp and seed during development and ripening of olive fruit. The results showed that Oep2LOX1 transcript level is spatially and temporally regulated. Besides, the transcriptional regulation of the Oep2LOX1 gene in response to different abiotic stresses was also investigated. Temperature, light and wounding regulate Oep2LOX1 gene expression in olive fruit mesocarp. The physiological role of the Oep2LOX1 gene during olive fruit ripening and in the stress response is discussed.

Synthesis of volatile compounds of virgin olive oil is limited by the lipoxygenase activity load during the oil extraction process.

The aim of this work was to determine whether the lipoxygenase (LOX) activity is a limiting factor for the biosynthesis of virgin olive oil (VOO) volatile compounds during the oil extraction process. For this purpose, LOX activity load was modified during this process using exogenous LOX activity and specific LOX inhibitors on olive cultivars producing oils with different volatile profiles (Arbequina and Picual). Experimental data suggest that LOX activity is a limiting factor for the synthesis of the oil volatile fraction, this limitation being significantly higher in Picual cultivar than in Arbequina, in line with the lowest content of volatile compounds in the oils obtained from the former. Moreover, there is evidence that this limitation of LOX activity takes place mostly during the milling step in the process of olive oil extraction.

Increasing omega-3 desaturase expression in tomato results in altered aroma profile and enhanced resistance to cold stress.

One of the drawbacks in improving the aroma properties of tomato (Solanum lycopersicum) fruit is the complexity of this organoleptic trait, with a great variety of volatiles contributing to determine specific quality features. It is well established that the oxylipins hexanal and (Z)-hex-3-enal, synthesized through the lipoxygenase pathway, are among the most important aroma compounds and impart in a correct proportion some of the unique fresh notes in tomato. Here, we confirm that all enzymes responsible for the synthesis of these C6 compounds are present and active in tomato fruit. Moreover, due to the low odor threshold of (Z)-hex-3-enal, small changes in the concentration of this compound could modify the properties of the tomato fruit aroma. To address this possibility, we have overexpressed the omega-3 fatty acid desaturases FAD3 and FAD7 that catalyze the conversion of linoleic acid (18:2) to linolenic acid (18:3), the precursor of hexenals and its derived alcohols. Transgenic OE-FAD tomato plants exhibit altered fatty acid composition, with an increase in the 18:3/18:2 ratio in leaves and fruits. These changes provoke a clear variation in the C6 content that results in a significant alteration of the (Z)-hex-3-enal/hexanal ratio that is particularly important in ripe OE-FAD3FAD7 fruits. In addition to this effect on tomato volatile profile, OE-FAD tomato plants are more tolerant to chilling. However, the different behaviors of OE-FAD plants underscore the existence of separate fatty acid fluxes to ensure plant survival under adverse conditions.

Isolation, expression, and characterization of a 13-hydroperoxide lyase gene from olive fruit related to the biosynthesis of the main virgin olive oil aroma compounds.

A full-length cDNA clone (OepHPL) coding for hydroperoxide lyase was isolated from olive fruit ( Olea europaea cv. Picual). The deduced amino acid sequence shows significant similarity to known plant hydroperoxide lyases and contains a N-terminal sequence that displays structural features of a chloroplast transit peptide. Genomic Southern blot analysis indicates that at least one copy of OepHPL is present in the olive genome. The recombinant hydroperoxide lyase was specific for 13-hydroperoxide derivatives of linolenic and linoleic acids but did not use 9-hydroperoxy isomers as substrates. Analyses of reaction products revealed that this enzyme produces primarily (Z)-hex-3-enal, which partially isomerizes to (E)-hex-2-enal, from 13-hydroperoxylinolenic acid and hexanal from 13-hydroperoxylinoleic acid. Expression levels were measured in different tissues of Picual and Arbequina varieties, including mesocarp and seed during development and ripening of olive fruits. The involvement of this olive hydroperoxide lyase gene in the biosynthesis of virgin olive oil aroma compounds is discussed.

Functional characterization of two 13-lipoxygenase genes from olive fruit in relation to the biosynthesis of volatile compounds of virgin olive oil.

Two LOX cDNA clones, Oep1LOX2 and Oep2LOX2, have been isolated from olive ( Olea europaea cv. Picual). Both deduced amino acid sequences showed significant similarity to known plant LOX2, and they contain an N-terminal chloroplastic transit peptide. Genomic Southern blot analyses suggest that at least three copies of Oep1LOX2 and one copy of Oep2LOX2 should be present in the olive genome. Linolenic acid proved to be the preferred substrate for both olive recombinant LOXs, and analyses of reaction products revealed that both enzymes produce primarily 13-hydroperoxides from linoleic and linolenic acids. Expression levels of both genes were measured in the mesocarp and seeds during development and ripening of Picual and Arbequina olive fruit along with the level of volatile compounds in the corresponding virgin olive oils. Biochemical and gene expression data suggest a major involvement of the Oep2LOX2 gene in the biosynthesis of virgin olive oil aroma compounds.

Purification and characterization of an olive fruit beta-glucosidase involved in the biosynthesis of virgin olive oil phenolics.

An olive beta-glucosidase was purified to apparent homogeneity from mature fruits ( Olea europaea cv. Picual) by selective extraction and successive anion exchange and hydrophobic interaction chromatographic procedures. The enzyme was shown to be a homodimer made up of two identical subunits of 65.4 kDa. Optimum activity was recorded at pH 5.5 and 45 degrees C. The enzyme was active on the main olive phenolic glycosides, with maximum activity toward oleuropein (100%), followed by ligstroside (65%) and demethyloleuropein (21%). The enzyme showed very low activity with apigenin and luteolin glucosides and was not active on verbascoside and rutin. Kinetic values show that olive beta-glucosidase is 200-fold more active against oleuropein than against the synthetic substrate p-nitrophenyl-beta-d-glucopyranoside (pNPG). According to its catalytic properties, the implication of the purified olive beta-glucosidase on the synthesis of virgin olive oil phenolics is discussed.

Oxygen concentration affects volatile compound biosynthesis during virgin olive oil production.

The effect of O 2 concentration on oil volatile compounds synthesized during the process to obtain virgin olive oil (VOO) was established. The study was carried out either on the whole process or within the main steps (milling and malaxation) of this process with two olive cultivars, Picual and Arbequina, at two ripening stages. Data show that O 2 control during milling has a negative impact on VOO volatile synthesis. This effect seems to depend on cultivar and on the ripening stage in cultivar Picual. Because most VOO volatiles are synthesized during olive fruit crushing at the milling step, O 2 control during malaxation seems to affect just slightly the volatile synthesis. The highest effect was observed when control of O 2 concentration was performed over the whole process. In this case, the content of volatile compounds of oils obtained from both cultivars and ripening stages showed quite similar trends.

Cultivar differences on nonesterified polyunsaturated fatty acid as a limiting factor for the biogenesis of virgin olive oil aroma.

The relationship between the content of nonesterified polyunsaturated fatty acids and the contents of oil aroma compounds that arise during the process to obtain virgin olive oil (VOO) was studied in two olive cultivars, Picual and Arbequina, producing oils with distinct aroma profiles and fatty acid compositions. Results suggest that the biosynthesis of VOO aroma compounds depends mainly on the availability of nonesterified polyunsaturated fatty acids, especially linolenic acid, during the process and then on the enzymatic activity load of the lipoxygenase/hydroperoxide lyase system. Both availability of substrates and enzymatic activity load seem to be cultivar-dependent.

Thermal stability of lipoxygenase and hydroperoxide lyase from olive fruit and repercussion on olive oil aroma biosynthesis.

Thermal stabilities of main enzymes involved in the biosynthesis of virgin olive oil (VOO) aroma through the lipoxygenase (LOX) pathway were studied in crude enzymatic preparations. Kinetic parameters of thermal inactivation for LOX were determined graphically and were shown to be compatible with the presence of two LOX isoenzymes (LOXlab and LOXres) having different thermal stabilities and displaying relative activities of 88 and 12% each. Data on hydroperoxide lyase (HPL) suggest the existence of just one HPL isoform. Thermal stabilities of LOX and HPL enzymatic activities in crude preparations seem to explain the observed decrease of volatile contents in VOO aroma as a consequence of heat treatments of olive fruit. Moreover, differences in thermal stability of LOXlab and LOXres would justify the distinct pattern of reduction of C6 and C5 compound contents observed in the aroma of these oils.

Differential distribution of the lipoxygenase pathway enzymes within potato chloroplasts.

The lipoxygenase pathway is responsible for the production of oxylipins, which are important compounds for plant defence responses. Jasmonic acid, the final product of the allene oxide synthase/allene oxide cyclase branch of the pathway, regulates wound-induced gene expression. In contrast, C6 aliphatic aldehydes produced via an alternative branch catalysed by hydroperoxide lyase, are themselves toxic to pests and pathogens. Current evidence on the subcellular localization of the lipoxygenase pathway is conflicting, and the regulation of metabolic channelling between the two branches of the pathway is largely unknown. It is shown here that while a 13-lipoxygenase (LOX H3), allene oxide synthase and allene oxide cyclase proteins accumulate upon wounding in potato, a second 13-lipoxygenase (LOX H1) and hydroperoxide lyase are present at constant levels in both non-wounded and wounded tissues. Wound-induced accumulation of the jasmonic acid biosynthetic enzymes may thus commit the lipoxygenase pathway to jasmonic acid production in damaged plants. It is shown that all enzymes of the lipoxygenase pathway differentially localize within chloroplasts, and are largely found associated to thylakoid membranes. This differential localization is consistently observed using confocal microscopy of GFP-tagged proteins, chloroplast fractionation, and western blotting, and immunodetection by electron microscopy. While LOX H1 and LOX H3 are localized both in stroma and thylakoids, both allene oxide synthase and hydroperoxide lyase protein localize almost exclusively to thylakoids and are strongly bound to membranes. Allene oxide cyclase is weakly associated with the thylakoid membrane and is also detected in the stroma. Moreover, allene oxide synthase and hydroperoxide lyase are differentially distributed in thylakoids, with hydroperoxide lyase localized almost exclusively to the stromal part, thus closely resembling the localization pattern of LOX H1. It is suggested that, in addition to their differential expression pattern, this segregation underlies the regulation of metabolic fluxes through the alternative branches of the lipoxygenase pathway.

Tácticas y trucos de endourología en el aparato urinario inferior.Tácticas en uretra / No disponible

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Modification of volatile compound profile of virgin olive oil due to hot-water treatment of olive fruit.

The effect of hot-water treatments of olive fruits before processing on the biosynthesis of virgin olive oil aroma was investigated by quantifying the variation within the major classes of volatile compounds. Data showed that hot-water treatments gave rise to changes in the volatile aroma profile of virgin olive oil from the three olive cultivars under study, Manzanilla, Picual, and Verdial. Different effects by thermal treatments were observed according to cultivar. In general, these changes are mainly due to a decrease in the contents of C(6) aldehydes and C(5) compounds. Contents of C(6) alcohols and esters remained constant or decreased slightly when the temperature of the treatment was increased. Thus, heat treatments seemed to promote a partial deactivation of the lipoxygenase/hydroperoxide lyase enzyme system, whereas other enzymatic activities, within the lipoxygenase pathway, such as alcohol dehydrogenase and alcohol acyltransferase, remained apparently unaffected as a consequence of heat treatments.

Role of olive seed in the biogenesis of virgin olive oil aroma.

Results obtained in a set of experiments point to an effective participation of olive seeds in the biosynthesis of olive oil aroma through the lipoxygenase pathway during the extraction process to produce virgin olive oil. Data showed that olive seeds should contain enzymatic activities metabolizing 13-hydroperoxides other than hydroperoxide lyase, giving rise to a net decrease in the content of C6 unsaturated aldehydes during the olive oil extraction process. Olive seeds seem also to supply this process with alcohol dehydrogenase activity, being more specific for saturated C6 aldehydes and not acting on C5 alcohols. Moreover, olive seeds would be responsible for the biosynthesis of 30-50% esters during the olive oil extraction process of intact fruits. Thus, olive seeds would afford a load of alcohol acyltransferase activity that might be quite unspecific in terms of substrate, producing any kind of esters.

Lipoxygenase H1 gene silencing reveals a specific role in supplying fatty acid hydroperoxides for aliphatic aldehyde production.

Lipoxygenases catalyze the formation of fatty acid hydroperoxide precursors of an array of compounds involved in the regulation of plant development and responses to stress. To elucidate the function of the potato 13-lipoxygenase H1 (LOX H1), we have generated transgenic potato plants with reduced expression of the LOX H1 gene as a consequence of co-suppression-mediated gene silencing. Three independent LOX H1-silenced transgenic lines were obtained, having less than 1% of the LOX H1 protein present in wild-type plants. This depletion of LOX H1 has no effect on the basal or wound-induced levels of jasmonates derived from 13-hydroperoxylinolenic acid. However, LOX H1 depletion results in a marked reduction in the production of volatile aliphatic C6 aldehydes. These compounds are involved in plant defense responses, acting as either signaling molecules for wound-induced gene expression or as antimicrobial substances. LOX H1 protein was localized to the chloroplast and the protein, expressed in Escherichia coli, showed activity toward unesterified linoleic and linolenic acids and plastidic phosphatidylglycerol. The results demonstrate that LOX H1 is a specific isoform involved in the generation of volatile defense and signaling compounds through the HPL branch of the octadecanoid pathway.