Proteomic analysis of peach fruit mesocarp softening and chilling injury using difference gel electrophoresis (DIGE)

Background: Peach fruit undergoes a rapid softening process that involves a number of metabolic changes. Storing fruit at low temperatures has been widely used to extend its postharvest life. However, this leads to undesired changes, such as mealiness and browning, which affect the quality of the fruit. In this study, a 2-D DIGE approach was designed to screen for differentially accumulated proteins in peach fruit during normal softening as well as under conditions that led to fruit chilling injury. Results:The analysis allowed us to identify 43 spots -representing about 18% of the total number analyzed- that show statistically significant changes. Thirty-nine of the proteins could be identified by mass spectrometry. Some of the proteins that changed during postharvest had been related to peach fruit ripening and cold stress in the past. However, we identified other proteins that had not been linked to these processes. A graphical display of the relationship between the differentially accumulated proteins was obtained using pairwise average-linkage cluster analysis and principal component analysis. Proteins such as endopolygalacturonase, catalase, NADP-dependent isocitrate dehydrogenase, pectin methylesterase and dehydrins were found to be very important for distinguishing between healthy and chill injured fruit. A categorization of the differentially accumulated proteins was performed using Gene Ontology annotation. The results showed that the 'response to stress', 'cellular homeostasis', 'metabolism of carbohydrates' and 'amino acid metabolism' biological processes were affected the most during the postharvest. Conclusions: Using a comparative proteomic approach with 2-D DIGE allowed us to identify proteins that showed stage-specific changes in their accumulation pattern. Several proteins that are related to response to stress, cellular homeostasis, cellular component organization and carbohydrate metabolism were detected as being differentially accumulated. Finally, a significant proportion of the proteins identified had not been associated with softening, cold storage or chilling injury-altered fruit before; thus, comparative proteomics has proven to be a valuable tool for understanding fruit softening and postharvest.

I. Identification and characterisation of epigenetically altered tumor supressor genes by proteomic and genomic approches / II. Studies of genes regualted by MeCP2 union to its promoter regions by proteomic and genmomic approches

DNA methylation has an important impact on normal cell physiology, thus any defects in this mechanism may be related to the development of various diseases In this project we are interested in identifying epigeneticaliy modified genes, in general controlled by processes related to the DNA methylation, by means of a new strategy combining protomic and genomic analyses. First, the two Dimensional-Difference Gel Electrophoresis (2-DIGE) protein analyses of extracts obtained from HCT-116 wt and double knockout for DNMT1 and DNMT3b (DKO) cells revealed 34 proteins overexpressed in the condition of DNMTs depletion. From five genes with higher transcript lavels in DKO cells, comparing with HCT-116 wt. oniy AKR1B1, UCHLl and VIM are melhylated in HCT-116. As expected. the DNA methvlation 1s lost in DKO cells. The rneth,vl ation of VIM and UCHLl promoters in some cancer samples has already been repaired, thus further studies has been focused on AKRlBI. AKR1B1 expression due lo DNA methyiaton of promoter region seems to occur specilfically in the colon cancer cell Iines. which was confirmed in the DNA rnethylation status and expression analyses. performed on 32 different cancer cell lines (including colon, breast, lymphoma, leukemia, neuroblastoma, glioma and lung cancer cell Iines) as well as normal colon and normal lymphocytes samples. AKRIBI expression after treatments with DNA demethvlating agent (AZA) was rescued in 5 coloncancer cell lines (including genetic regulation of the candidate gene. The methylation status of the rest of the genes identified in proteomic analysis was checked by methylation specific PCR (MSP) experiment and all appeared to be unmethylated. The similar research has been done also bv means of Mecp2-null mouse model For 14 selected candidate genes the analyses of expression leveis, methylation Status and MeCP2 interaction with promoters are currently being performed.

Evaluación del uso de exosomas como aloantígenos para la inducción de tolerancia en trasplante

La consecución de tolerancia aloespecífica es de mucha relevancia en trasplante. Las células dendríticas (DC) son las principales responsables de la inducción de la respuesta inmune frente a las moléculas de histocompatibilidad (MHC) del donante, provocando el rechazo del injerto. Sin embargo las DC son también responsables de la inducción de tolerancia. Diversos modelos animales de alotrasplante han mostrado la tolerización del injerto mediante DC diferenciadas in vitro en condiciones tolerogénicas (tDC). En humanos, las fuentes de aloantígenos potencialmente utilizables en terapia son, entre otras, los cuerpos apoptóticos y los exosomas. Éstos expresan antígenos MHC de forma abundante y su composición es relativamente uniforme, lo que supone una ventaja frente a otras fuentes. En este proyecto, se ha evaluado la obtención de exosomas secretados por una línea de linfocitos T y por células dendríticas derivadas de médula ósea. Se ha caracterizado la captura de exosomas derivados de linfocitos T por células dendríticas humanas derivadas de sangre periférica y su presentación a linfocitos T autólogos. Por otra parte, se ha comenzado a desarrollar los experimentos para estudiar la inducción de tolerancia en un modelo de trasplante renal en rata. Se han generado células dendríticas tolerógenicas derivadas de médula ósea (tolDC), en presencia de dexametasona. Las tolDC expresan menos moléculas de histocompatibilidad y de coestimulación e inducen una menor proliferación en reacciones mixtas leucocitaras, comparadas con las células dendríticas maduras. Por último, se han caracterizado los exosomas de plasma humano con el fin de estudiar su posible uso como aloantígenos. El análisis proteómico revela la presencia de proteínas relacionadas con el sistema inmune, la coagulación, la señalización celular y moléculas implicadas en el transporte y metabolismo de nutrientes. El estudio de la captura por diferentes líneas celulares sugiere que deben existir mecanismos específicos para su internalización.

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. Conclusions We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data.

Outer membrane vesicles from cold-adapted antarctic bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter with potential biotechnological applications. Transmission electron microscopy (TEM) analysis after high-pressure freezing and freeze substitution (HPF-FS) showed that this extracellular matter is structurally complex, appearing around cells as a netlike mesh, and composed of an exopolymeric substance (EPS) containing large numbers of outer membrane vesicles (OMVs). Isolation, purification and protein profiling via 1D SDS-PAGE confirmed the outer membrane origin of these Antarctic bacteria OMVs. In an initial attempt to elucidate the role of OMVs in cold-adapted strains of Gram-negative bacteria, a proteomic analysis demonstrated that they were highly enriched in outer membrane proteins and periplasmic proteins associated with nutrient processing and transport, suggesting that the OMVs may be involved in nutrient sensing and bacterial survival. OMVs from Gram-negative bacteria are known to play a role in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. A structural study of Shewanella vesiculosa M7T using TEM and Cryo-TEM revealed that this Antarctic Gram-negative bacterium naturally releases conventional one-bilayer OMVs, together with a more complex type of OMV, previously undescribed, which on formation drags along inner membrane and cytoplasmic content and can therefore also entrap DNA.

Outer membrane vesicles from cold-adapted antarctic bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter with potential biotechnological applications. Transmission electron microscopy (TEM) analysis after high-pressure freezing and freeze substitution (HPF-FS) showed that this extracellular matter is structurally complex, appearing around cells as a netlike mesh, and composed of an exopolymeric substance (EPS) containing large numbers of outer membrane vesicles (OMVs). Isolation, purification and protein profiling via 1D SDS-PAGE confirmed the outer membrane origin of these Antarctic bacteria OMVs. In an initial attempt to elucidate the role of OMVs in cold-adapted strains of Gram-negative bacteria, a proteomic analysis demonstrated that they were highly enriched in outer membrane proteins and periplasmic proteins associated with nutrient processing and transport, suggesting that the OMVs may be involved in nutrient sensing and bacterial survival. OMVs from Gram-negative bacteria are known to play a role in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. A structural study of Shewanella vesiculosa M7T using TEM and Cryo-TEM revealed that this Antarctic Gram-negative bacterium naturally releases conventional one-bilayer OMVs, together with a more complex type of OMV, previously undescribed, which on formation drags along inner membrane and cytoplasmic content and can therefore also entrap DNA.

Natural abundance of stable isotopes (15N and 13C) and isotopic fractionation of sea bream muscle as markers of food and culture conditions and its relationship with metabolic and proteomic analyses

Els isòtops estables com a traçadors de la cadena alimentària, s'han utilitzat per caracteritzar la relació entre els consumidors i els seus aliments, ja que el fraccionament isotòpic implica una discriminació en contra de certs isòtops. Però les anàlisis d'isòtops estables (SIA), també es poden dur a terme en peixos cultivats amb dietes artificials, com la orada (Sparus aurata), la especie más cultivada en el Mediterráneo. Canvis en l'abundància natural d'isòtops estables (13C i 15N) en els teixits i les seves reserves poden reflectir els canvis en l'ús i reciclatge dels nutrients ja que els enzims catabòlics implicats en els processos de descarboxilació i desaminació mostren una preferència pels isòtops més lleugers. Per tant, aquestes anàlisis ens poden proporcionar informació útil sobre l'estat nutricional i metabòlic dels peixos. L'objectiu d'aquest projecte va ser determinar la capacitat dels isòtops estables per ser utilitzats com a marcadors potencials de la capacitat de creixement i condicions de cria de l'orada. En aquest sentit, les anàlisis d'isòtops estables s'han combinat amb altres metabòlics (activitats citocrom-c-oxidasa, COX, i citrat sintasa, CS) i els paràmetres de creixement (ARN/ADN). El conjunt de resultats obtinguts en els diferents estudis realitzats en aquest projecte demostra que el SIA, en combinació amb altres paràmetres metabòlics, pot servir com una eina eficaç per discriminar els peixos amb millor potencial de creixement, així com a marcador sensible de l'estat nutricional i d'engreix. D'altra banda, la combinació de l'anàlisi d'isòtops estables amb les eines emergents, com ara tècniques de proteòmica (2D-PAGE), ens proporciona nous coneixements sobre els canvis metabòlics que ocorren en els músculs dels peixos durant l‟increment del creixement muscular induït per l'exercici.

Oxidative stress in aging: advances in proteomic approaches

Aging is a gradual, complex process in which cells, tissues, organs, and the whole organism itself deteriorate in a progressive and irreversible manner that, in the majority of cases, implies pathological conditions that affect the individual"s Quality of Life (QOL). Although extensive research efforts in recent years have been made, the anticipation of aging and prophylactic or treatment strategies continue to experience major limitations. In this review, the focus is essentially on the compilation of the advances generated by cellular expression profile analysis through proteomics studies (two-dimensional [2D] electrophoresis and mass spectrometry [MS]), which are currently used as an integral approach to study the aging process. Additionally, the relevance of the oxidative stress factors is discussed. Emphasis is placed on postmitotic tissues, such as neuronal, muscular, and red blood cells, which appear to be those most frequently studied with respect to aging. Additionally, models for the study of aging are discussed in a number of organisms, such as Caenorhabditis elegans, senescence-accelerated probe-8 mice (SAMP8), naked mole-rat (Heterocephalus glaber), and the beagle canine. Proteomic studies in specific tissues and organisms have revealed the extensive involvement of reactive oxygen species (ROS) and oxidative stress in aging.

Oxidative stress in aging: advances in proteomic approaches

Aging is a gradual, complex process in which cells, tissues, organs, and the whole organism itself deteriorate in a progressive and irreversible manner that, in the majority of cases, implies pathological conditions that affect the individual"s Quality of Life (QOL). Although extensive research efforts in recent years have been made, the anticipation of aging and prophylactic or treatment strategies continue to experience major limitations. In this review, the focus is essentially on the compilation of the advances generated by cellular expression profile analysis through proteomics studies (two-dimensional [2D] electrophoresis and mass spectrometry [MS]), which are currently used as an integral approach to study the aging process. Additionally, the relevance of the oxidative stress factors is discussed. Emphasis is placed on postmitotic tissues, such as neuronal, muscular, and red blood cells, which appear to be those most frequently studied with respect to aging. Additionally, models for the study of aging are discussed in a number of organisms, such as Caenorhabditis elegans, senescence-accelerated probe-8 mice (SAMP8), naked mole-rat (Heterocephalus glaber), and the beagle canine. Proteomic studies in specific tissues and organisms have revealed the extensive involvement of reactive oxygen species (ROS) and oxidative stress in aging.