Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 65
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Food Chem ; 304: 125428, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31476548

RESUMO

To protect allergic patients and guarantee correct food labeling, robust, specific and sensitive detection methods are urgently needed. Mass spectrometry (MS)-based methods could overcome the limitations of current detection techniques. The first step in the development of an MS-based method is the identification of biomarkers, which are, in the case of food allergens, peptides. Here, we implemented a strategy to identify the most salient peptide biomarkers in peanuts. Processed peanut matrices were prepared and analyzed using an untargeted approach via high-resolution MS. More than 300 identified peptides were further filtered using selection criteria to strengthen the analytical performance of a future, routine quantitative method. The resulting 16 peptides are robust to food processing, specific to peanuts, and satisfy sequence-based criteria. The aspect of multiple protein isoforms is also considered in the selection tree, an aspect that is essential for a quantitative method's robustness but seldom, if ever, considered.

2.
Food Chem ; : 125679, 2019 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-31718834

RESUMO

The interest of using LC-MS/MS as a method for detection of allergens in food is growing. In such methods, peptides are used as biomarkers for the detection and quantification of the allergens. The selection of good biomarker peptides is of high importance to develop a specific, universal and sensitive method. Biomarkers should, for example, be robust to food processing. To evaluate robustness, test material incurred with hazelnut having undergone different food processing techniques was produced. Proteins of these materials were extracted, digested and further analyzed using HRMS. After peptide identification, selection was carried out using several criteria such as hazelnut specificity and amino acid composition. Further selection was done by comparing peptide MS intensities in the different food matrices. Only peptides showing processing robustness were retained. Eventually, eight peptides coming from three major hazelnut proteins were selected as the best biomarkers for hazelnut detection in processed foods.

3.
PLoS One ; 14(2): e0201424, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794540

RESUMO

The reliquary of Jacques de Vitry, a prominent clergyman and theologian in the early 13th century, has experienced several transfers over the last centuries, which seriously question the attribution of the remains to the late Cardinal. Uncertainty about the year of his birth poses an additional question regarding his age at death in 1240. The reliquary, located in the Saint Marie d'Oigines church, Belgium, was reopened in 2015 for an interdisciplinary study around his relics as well as the Treasure of Oignies, a remarkable cultural heritage notably built from Jacques de Vitry's donation. Anthropological, isotopic and genetic analyses were performed independently on the remains found in the reliquary. Results of the analyses provided evidence that the likelihood that these remains are those of Jacques de Vitry is very high: the remains belong to the same human male individual and the historical tradition about his age is confirmed. In addition, a separate relic (left tibia) was analysed and found to match with the remains of the reliquary (right tibia). The unique Jacques de Vitry's mitre, made of parchment, was sampled non-destructively and the extracted parchment collagen was analysed by a proteomic method in order to determine the animal species. The results showed that, surprisingly, not all parts of the mitre were made from the same species. All together, these findings are expected to fertilize knowledge carried by historical tradition around the relics of Jacques de Vitry and his related cultural heritage.


Assuntos
Autopsia/métodos , Clero , Proteômica/métodos , Religião e Ciência , Teologia/história , Animais , Antropologia Cultural , Bélgica , Cromossomos Humanos Y/genética , Clero/história , Testes Genéticos , História Medieval , Humanos , Estudos Interdisciplinares , Masculino , Datação Radiométrica
4.
Sci Rep ; 9(1): 1825, 2019 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755703

RESUMO

Recently, historical and conservation studies have attached an increasing importance to investigating the materials used in historic documents. In particular, the identification of the animal species from which parchments are made is of high importance and is currently performed by either genetic or proteomic methods. Here, we introduce an innovative, non-invasive optical method for identifying animal species based on light-parchment interaction. The method relies on conservation of light energy through reflection, transmission and absorption from the sample, as well as on statistical processing of the collected optical data. Measurements are performed from ultraviolet (UV) to near-infrared (NIR) spectral ranges by a standard spectrophotometer and data are processed by Principal Component Analysis (PCA). PCA data from modern parchments, made of sheep, calf and goat skins, are used as a database for PCA analysis of historical parchments. Using only the first two principal components (PCs), the method confirmed visual diagnostics about parchment appearance and aging, and was able to recognise the origin species of historical parchment of among database clusters. Furthermore, taking into account the whole set of PCs, species identification was achieved, with all results matching perfectly their proteomic counterparts used for method assessment. The validated method compares favourably with genetic and proteomic methods used for the same purpose. In addition to animals' proteomic and genetic signatures, a unique "optical fingerprint" of the parchments' origin species is revealed here. This new method is non-invasive, straightforward to implement, potentially cheap and accessible to scholars and conservators, with minimal training. In the context of cultural heritage, the method could help solving questions related to parchment production and, more generally, medieval writing production.

5.
J Biol Chem ; 293(17): 6374-6386, 2018 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-29496995

RESUMO

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

6.
Artigo em Inglês | MEDLINE | ID: mdl-29223774

RESUMO

The comparative effects of cortisol and 11-deoxycorticosterone (DOC), two major corticosteroids in fish, have yet received little attention in teleosts. We evaluated the proteomic and immune responses of Eurasian perch to chronic corticosteroid treatments. We implanted immature perch with cortisol (80mg/kg) or DOC (4mg/kg) and measured the proportions of blood leucocytes, immune indices in the plasma, spleen and liver (complement and lysozyme activity, total immunoglobulin and immune gene expression in the tissues) and differential proteome expression (corticosteroid versus control) in the liver and the spleen on days 2, 4 and 14 post-treatment. Implantation of cortisol decreased the ratio of blood leucocytes and depressed Ig levels in both organs while DOC modulated the proportion of leucocyte sub-populations (increase in lymphocytes and decrease in granulocytes). In contrast, the innate humoral immunity was not strongly influenced by any of corticosteroid implants. The only immune parameter that was significantly affected was lysozyme, after DOC treatment. A number of proteins were differentially regulated by these hormones and some were identified in the liver (21 for cortisol and 8 for DOC) and in the spleen (10 for cortisol and 10 for DOC). None of the proteins was directly linked to immunity, except the natural killer enhancing factor, which was repressed by cortisol in the spleen. Our results also confirm that the proteins involved in energetic and glucose metabolism are affected by corticosteroids. Furthermore, these corticosteroids differently regulate immune status in Eurasian perch and they primarily impact leucocytes, as opposed to innate immune function.

7.
Chem Commun (Camb) ; 53(77): 10632-10635, 2017 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-28905050

RESUMO

In this study, we report a dynamic combinatorial approach along with highly efficient in situ screening to identify inhibitors of UDP-galactopyranose mutase (UGM), an essential enzyme involved in mycobacterial cell wall biosynthesis. These two technologies converged to the identification of a new UGM inhibitor chemotype. Importantly, the best molecule not only displayed high affinity for the target enzyme but also exhibited in vitro growth inhibition against whole Mycobacterium tuberculosis cells. The strategy described here provides an avenue to explore a novel inhibitor class for UGMs and paves the way for further pharmacological studies on tuberculosis treatment.


Assuntos
Parede Celular/metabolismo , Técnicas de Química Combinatória , Hidrazonas/farmacologia , Transferases Intramoleculares/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Antituberculosos/química , Antituberculosos/farmacologia , Hidrazonas/química , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos
8.
Mol Plant Microbe Interact ; 30(11): 855-865, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28726589

RESUMO

Invasive plant pathogens have developed the ability to modify the metabolism of their host, promoting metabolic processes that facilitate the growth of the pathogen at the general expense of the host. The particular enzymatic process SUMOylation, which performs posttranslational modification of target proteins, leading to changes in many aspects of protein activity and, hence, metabolism, has been demonstrated to be active in many eukaryotic organisms, both animals and plants. Here, we provide experimental evidence that indicates that, in leaves of Solanum tuberosum that have been infected by Phytophthora infestans, the SUMO (small ubiquitin-like modifier) pathway enzymes of the host are partially under transcriptional control exerted by the oomycete. Using a recently developed approach that employs three-dimensional gels, we show that, during the infection process, the abundances of most of the known SUMO conjugates of S. tuberosum change significantly, some decreasing, but many increasing in abundance. The new proteomic approach has the potential to greatly facilitate investigation of the molecular events that take place during the invasion by a pathogen of its host plant.


Assuntos
Interações Hospedeiro-Patógeno , Phytophthora infestans/fisiologia , Proteômica/métodos , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiologia , Sumoilação , Evolução Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genótipo , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Solanum tuberosum/genética , Fatores de Tempo
9.
J AOAC Int ; 100(4): 1126-1130, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28330529

RESUMO

Food laboratories have developed methods for testing allergens in foods. The efficiency of qualitative and quantitative methods is of prime importance in protecting allergic populations. Unfortunately, food laboratories encounter barriers to developing efficient methods. Bottlenecks include the lack of regulatory thresholds, delays in the emergence of reference materials and guidelines, and the need to detect processed allergens. In this study, ultra-HPLC coupled to tandem MS was used to illustrate difficulties encountered in determining method performances. We measured the major influences of both processing and matrix effects on the detection of egg, milk, soy, and peanut allergens in foodstuffs. The main goals of this work were to identify difficulties that food laboratories still encounter in detecting and quantifying allergens and to sensitize researchers to them.


Assuntos
Alérgenos/análise , Análise de Alimentos/métodos , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida de Alta Pressão , Hipersensibilidade Alimentar , Humanos
10.
J Proteomics ; 150: 268-280, 2017 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-27671789

RESUMO

SUMOylation is a post-translational modification which regulates a number of critical biological processes in, for example mammals, yeast and plants. In order to fully understand the functional effects of SUMOylation an essential first step is the identification of endogenous targets for SUMOylation. Here we report the results of using a recently developed proteomic approach based on the use of 3D gels to identify the endogenous SUMO targets in leaves of Solanum tuberosum. By using 3D gels we avoid the problem of co-migration of proteins, which is a major limitation of 2D gels, and we enable the use of the highly sensitive CyDye DIGE fluor saturation dyes. Using this new method we have identified 39 individual proteins as probable SUMO targets in leaves of Solanum tuberosum. The advantages of this method compared with other approaches are discussed, and possible future developments are outlined. SIGNIFICANCE: The authors have no conflicts of interest to declare. All authors have approved the manuscript and agree with submission to Journal of Proteomics.


Assuntos
Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Solanum tuberosum/metabolismo , Sumoilação , Eletroforese/métodos , Proteínas de Plantas/análise , Solanum tuberosum/química , Espectrometria de Massas em Tandem
11.
Free Radic Biol Med ; 99: 436-450, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27591797

RESUMO

Regulation of ROS metabolism plays a major role in cellular adaptation to oxidative stress in cancer cells, but the molecular mechanism that regulates catalase, a key antioxidant enzyme responsible for conversion of hydrogen peroxide to water and oxygen, remains to be elucidated. Therefore, we investigated the transcriptional regulatory mechanism controlling catalase expression in three human mammary cell lines: the normal mammary epithelial 250MK primary cells, the breast adenocarcinoma MCF-7 cells and an experimental model of MCF-7 cells resistant against oxidative stress resulting from chronic exposure to H2O2 (Resox), in which catalase was overexpressed. Here we identify a novel promoter region responsible for the regulation of catalase expression at -1518/-1226 locus and the key molecules that interact with this promoter and affect catalase transcription. We show that the AP-1 family member JunB and retinoic acid receptor alpha (RARα) mediate catalase transcriptional activation and repression, respectively, by controlling chromatin remodeling through a histone deacetylases-dependent mechanism. This regulatory mechanism plays an important role in redox adaptation to chronic exposure to H2O2 in breast cancer cells. Our study suggests that cancer adaptation to oxidative stress may be regulated by transcriptional factors through chromatin remodeling, and reveals a potential new mechanism to target cancer cells.


Assuntos
Catalase/genética , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptor alfa de Ácido Retinoico/genética , Fatores de Transcrição/genética , Adaptação Fisiológica , Sequência de Bases , Catalase/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/química , Cromatina/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Células MCF-7 , Estresse Oxidativo , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Receptor alfa de Ácido Retinoico/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Genética
12.
Nucl Med Biol ; 43(7): 415-23, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27179250

RESUMO

INTRODUCTION: Radiolabeled antibodies directed against endoglin (CD105) are promising tools for imaging and antiangiogenic cancer therapy. To validate iodinated antibodies as reliable tracers, we investigated the influence of the radiolabeling method (direct or indirect) on their in vivo stability. METHODS: Anti-CD105 mAbs were radioiodinated directly using chloramine-T ((125)I-anti-CD105-mAbs) or indirectly using D-KRYRR peptide as a linker ((125)I-KRYRR-anti-CD105-mAbs). The biodistribution was studied in B16 tumor-bearing mice via SPECT/CT imaging. RESULTS: Radioiodinated mAbs were stable in vitro. In vivo, thyroid showed the most important increase of uptake after 24h for (125)I-anti-CD105-mAbs (91.9±4.0%ID/ml) versus(125)I-KRYRR-anti-CD105-mAbs (4.4±0.6%ID/ml). Tumor uptake of (125)I-anti-CD105-mAbs (0.9±0.3%ID/ml) was significantly lower than that of (125)I-KRYRR-anti-CD105-mAbs (4.7±0.2%ID/ml). CONCLUSIONS: An accurate characterization of the in vivo stability of radioiodinated mAbs and the choice of an appropriate method for the radioiodination are required, especially for novel targets. The indirect radioiodination of internalizing anti-CD105 mAbs leads to more stable tracer by decreasing in vivo deiodination and improves the tumor retention of radioiodinated mAbs. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: To date, the only antiangiogenic antibody approved for clinical indications is bevacizumab. There is a need to develop more antibodies that have targets highly expressed on tumor endothelium. CD105 represents a promising marker of angiogenesis, but its therapeutic relevance in cancer needs to be further investigated. In this context, this study suggests the potential use of indirectly iodinated anti-CD105 mAbs for tumor imaging and for therapeutic purposes.


Assuntos
Endoglina/imunologia , Imunoconjugados/química , Imunoconjugados/farmacocinética , Radioisótopos do Iodo , Melanoma Experimental/diagnóstico por imagem , Melanoma Experimental/metabolismo , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Marcação por Isótopo , Melanoma Experimental/patologia , Camundongos , Ratos , Distribuição Tecidual
13.
Plant Sci ; 247: 60-70, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27095400

RESUMO

Plant innate immunity offers considerable opportunities for plant protection but beside flagellin and chitin, not many molecules and their receptors have been extensively characterized and very few have successfully reached the field. COS-OGA, an elicitor that combines cationic chitosan oligomers (COS) with anionic pectin oligomers (OGA), efficiently protected tomato (Solanum lycopersicum) grown in greenhouse against powdery mildew (Leveillula taurica). Leaf proteomic analysis of plants sprayed with COS-OGA showed accumulation of Pathogenesis-Related proteins (PR), especially subtilisin-like proteases. qRT-PCR confirmed upregulation of PR-proteins and salicylic acid (SA)-related genes while expression of jasmonic acid/ethylene-associated genes was not modified. SA concentration and class III peroxidase activity were increased in leaves and appeared to be a cumulative process dependent on the number of sprayings with the elicitor. These results suggest a systemic acquired resistance (SAR) mechanism of action of the COS-OGA elicitor and highlight the importance of repeated applications to ensure efficient protection against disease.


Assuntos
Quitosana/farmacologia , Lycopersicon esculentum/imunologia , Pectinas/farmacologia , Doenças das Plantas/imunologia , Imunidade Vegetal/efeitos dos fármacos , Ácido Salicílico/metabolismo , Ascomicetos/fisiologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Lycopersicon esculentum/efeitos dos fármacos , Lycopersicon esculentum/genética , Lycopersicon esculentum/fisiologia , Peroxidase/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Proteômica
14.
J Agric Food Chem ; 64(11): 2405-14, 2016 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-26943838

RESUMO

The outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom in 1986, with processed animal proteins (PAPs) as the main vector of the disease, has led to their prohibition in feed. The progressive release of the feed ban required the development of new analytical methods to determine the exact origin of PAPs from meat and bone meal. We set up a promising MS-based method to determine the species and the source (legal or not) present in PAPs: a TCA-acetone protein extraction followed by a cleanup step, an in-solution tryptic digestion of 5 h (with a 1:20 protein/trypsin ratio), and mass spectrometry analyses, first without any a priori, with a Q-TOF, followed by a targeted triple-quadrupole analysis. Using this procedure, we were able to overcome some of the major limitations of the official methods to analyze PAPs, detecting and identifying prohibited animal products in feedstuffs by the monitoring of peptides specific for cows, pigs, and sheep in PAPs.


Assuntos
Ração Animal/análise , Biomarcadores/análise , Espectrometria de Massas/métodos , Carne/análise , Minerais/análise , Proteínas/análise , Sequência de Aminoácidos , Animais , Produtos Biológicos/análise , Bovinos , Encefalopatia Espongiforme Bovina/prevenção & controle , Contaminação de Alimentos/análise , Manipulação de Alimentos , Legislação sobre Alimentos , Peptídeos/análise , Peptídeos/química , Ovinos , Suínos , Reino Unido
15.
J Proteomics ; 137: 83-96, 2016 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-26785284

RESUMO

UNLABELLED: Using proteomic data as biomarkers of environmental pollution has the potential to be of a great interest in ecological risk assessment as they constitute early warning indicators of ecologically relevant effects on biological systems. To develop such specific and sensitive biomarkers, the use of a set of proteins is required and the identification of protein expression signatures (PES) may reflect the exposure to specific classes of pollutants. Using 2D-DIGE (Differential in Gel Electrophoresis) methodology, this study aimed at identifying specific PES on European eel (Anguilla anguilla) peripheral blood mononuclear cells (PBMC) after 48 h in vitro exposure to two sublethal concentrations of dichlorodiphenyltrichloroethane (p,p'-DDT) (10 µg/L and 1mg/L) or cadmium (Cd) (1 µg/L and 100 µg/L). The present results have been supplemented with data of a first in vitro study on cells exposed to perfluorooctane sulfonate (PFOS) (10 µg/L and 1mg/L). A total of thirty-four protein spots, belonging to 18 different identified proteins found in all conditions, have been selected as possible biomarkers to develop a synthetic Integrated Biomarker Proteomic (IBP) index. IBP follows a dose-response relationship with higher values at the highest tested concentration for each pollutant (Cd: 9.96; DDT: 7.44; PFOS: 7.94) compared to the lowest tested concentration (Cd: 3.81; DDT: 2.91; PFOS: 2.06). In a second step, star plot graphs have been applied to proteomic data in order to allow visual integration of a set of early warning responses measured with protein biomarkers. Such star plots permit to discriminate the type of pollutant inducing a proteomic response. We conclude that using IBP is relevant in environmental risk assessment, giving to this index the potential to be applied as a global index of proteome alteration in endangered species such as the European eel. BIOLOGICAL SIGNIFICANCE: In this study, 34 protein spots have been selected as possible biomarkers to develop a synthetic Integrated Biomarker Proteomic index (IBP). Results show that IBP follows a dose-response relationship with higher values at the highest tested concentration for each pollutant compared to the lowest tested concentration. Star plot graphs have also been applied to proteomic data in order to allow visual integration of a set of early warning responses measured with protein biomarkers. Such star plots permit to discriminate the type of pollutant inducing a proteomic response. IBP is relevant in environmental risk assessment, giving to this index the potential to be applied as a global index of proteome alteration in endangered species such as the European eel.


Assuntos
Diclorodifenildicloroetano/toxicidade , Enguias/metabolismo , Proteínas de Peixes/metabolismo , Leucócitos Mononucleares/metabolismo , Proteômica , Poluentes da Água/toxicidade , Animais , Biomarcadores/metabolismo , Relação Dose-Resposta a Droga
16.
Carbohydr Polym ; 137: 39-51, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26686103

RESUMO

Modified forms of citrus pectin possess anticancer properties. However, their mechanism of action and the structural features involved remain unclear. Here, we showed that citrus pectin modified by heat treatment displayed cytotoxic effects in cancer cells. A fractionation approach was used aiming to identify active molecules. Dialysis and ethanol precipitation followed by HPLC analysis evidenced that most of the activity was related to molecules with molecular weight corresponding to low degree of polymerization oligogalacturonic acid. Heat-treatment of galacturonic acid also generated cytotoxic molecules. Furthermore, heat-modified galacturonic acid and heat-fragmented pectin contained the same molecule that induced cell death when isolated by HPLC separation. Mass spectrometry analyses revealed that 4,5-dihydroxy-2-cyclopenten-1-one was one cytotoxic molecule present in heat-treated pectin. Finally, we synthesized the enantiopure (4R,5R)-4,5-dihydroxy-2-cyclopenten-1-one and demonstrated that this molecule was cytotoxic and induced a similar pattern of apoptotic-like features than heat-modified pectin.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Ciclopentanos/química , Ciclopentanos/farmacologia , Pectinas/química , Linhagem Celular Tumoral , Células Hep G2 , Temperatura Alta , Humanos , Peso Molecular
17.
Mol Cancer ; 14: 79, 2015 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-25889892

RESUMO

BACKGROUND: Tumor hypoxia is one of the features of tumor microenvironment that contributes to chemoresistance. miRNAs have recently been shown to play important roles in tumorigenesis and drug resistance. Moreover, hypoxia also regulates the expression of a series of miRNAs. However, the interaction between chemoresistance, hypoxia and miRNAs has not been explored yet. The aim of this study is to understand the mechanisms activated/inhibited by miRNAs under hypoxia that induce resistance to chemotherapy-induced apoptosis. METHODS: TaqMan low-density array was used to identify changes in miRNA expression when cells were exposed to etoposide under hypoxia or normoxia. The effects of miR-196b overexpression on apoptosis and cell proliferation were studied in HepG2 cells. miR-196b target mRNAs were identified by proteomic analysis, luciferase activity assay, RT-qPCR and western blot analysis. RESULTS: Results showed that hypoxia down-regulated miR-196b expression that was induced by etoposide. miR-196b overexpression increased the etoposide-induced apoptosis and reversed the protection of cell death observed under hypoxia. By a proteomic approach combined with bioinformatics analyses, we identified IGF2BP1 as a potential target of miR-196b. Indeed, miR-196b overexpression decreased IGF2BP1 RNA expression and protein level. The IGF2BP1 down-regulation by either miR-196b or IGF2BP1 siRNA led to an increase in apoptosis and a decrease in cell viability and proliferation in normal culture conditions. However, IGF2BP1 silencing did not modify the chemoresistance induced by hypoxia, probably because it is not the only target of miR-196b involved in the regulation of apoptosis. CONCLUSIONS: In conclusion, for the first time, we identified IGF2BP1 as a direct and functional target of miR-196b and showed that miR-196b overexpression reverses the chemoresistance induced by hypoxia. These results emphasize that the chemoresistance induced by hypoxia is a complex mechanism.


Assuntos
Apoptose/genética , Proliferação de Células/genética , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Apoptose/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/genética , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células Hep G2 , Humanos , Proteômica/métodos
18.
Methods Mol Biol ; 1295: 427-40, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25820738

RESUMO

Quantitative 2D-gel-dependent proteomics became feasible with 2D fluorescence difference gel electrophoresis (2D-DIGE), and this technique has gained wide acceptance because it has eliminated the gel to gel variations and greatly facilitated the quantitative comparisons across gels for many different experimental conditions. However, the co-migration of several proteins in the same spot is still a major limitation which detracts from the accuracy of comparative quantification and prevents unambiguous post-translational modifications (PTMs) detection.A protocol based on traditional polyacrylamide gel IEF sample fractionation, and followed by two consecutive SDS-PAGE electrophoreses alleviates co-migration limitations. The use of two different buffer systems for SDS-PAGE is central to the proposed approach.


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Proteoma , Proteômica , Western Blotting , Eletroforese em Gel Bidimensional , Focalização Isoelétrica/métodos , Proteômica/métodos
19.
Mol Cell Biol ; 35(9): 1491-505, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25691661

RESUMO

The oxygen-limiting (hypoxic) microenvironment of tumors induces metabolic reprogramming and cell survival, but the underlying mechanisms involving mitochondria remain poorly understood. We previously demonstrated that hypoxia-inducible factor 1 mediates the hyperfusion of mitochondria by inducing Bcl-2/adenovirus E1B 19-kDa interacting protein 3 and posttranslational truncation of the mitochondrial ATP transporter outer membrane voltage-dependent anion channel 1 in hypoxic cells. In addition, we showed that truncation is associated with increased resistance to drug-induced apoptosis and is indicative of increased patient chemoresistance. We now show that silencing of the tumor suppressor TP53 decreases truncation and increases drug-induced apoptosis. We also show that TP53 regulates truncation through induction of the mitochondrial protein Mieap. While we found that truncation was independent of mitophagy, we observed local microfusion between mitochondria and endolysosomes in hypoxic cells in culture and in patients' tumor tissues. Since we found that the endolysosomal asparagine endopeptidase was responsible for truncation, we propose that it is a readout of mitochondrial-endolysosomal microfusion in hypoxia. These novel findings provide the framework for a better understanding of hypoxic cell metabolism and cell survival through mitochondrial-endolysosomal microfusion regulated by hypoxia-inducible factor 1 and TP53.


Assuntos
Lisossomos/metabolismo , Mitocôndrias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular , Células HeLa , Células Hep G2 , Humanos , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Lisossomos/patologia , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/patologia , Proteínas Proto-Oncogênicas/metabolismo , Canal de Ânion 1 Dependente de Voltagem/análise
20.
Development ; 141(20): 3988-93, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25231762

RESUMO

Heterogeneity within a population of cells of the same type is a common theme in metazoan biology. Dissecting complex developmental and physiological processes crucially relies on our ability to probe the expression profile of these cell subpopulations. Current strategies rely on cell enrichment based on sequential or simultaneous use of multiple intersecting markers starting from a heterogeneous cell suspension. The extensive tissue manipulations required to generate single-cell suspensions, as well as the complexity of the required equipment, inherently complicate these approaches. Here, we propose an alternative methodology based on a genetically encoded system in the model organism Danio rerio (zebrafish). In transgenic fish, we take advantage of the combinatorial biotin transfer system, where polysome-associated mRNAs are selectively recovered from cells expressing both a tagged ribosomal subunit, Rpl10a, and the bacterial biotin ligase BirA. We have applied this technique to skeletal muscle development and identified new genes with interesting temporal expression patterns. Through this work we have thus developed additional tools for highly specific gene expression profiling.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/fisiologia , Transcrição Genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Biotinilação , Coenzima A Ligases/química , Proteínas de Fluorescência Verde/química , Hibridização In Situ , Espectrometria de Massas , Músculo Esquelético/patologia , Polirribossomos/química , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/fisiologia , Ribossomos/metabolismo , Peixe-Zebra
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA