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1.
Food Chem ; 304: 125415, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31479995

RESUMO

The aim of our study was to characterize the proteolytic activity of 170 Lactobacillus strains isolated from traditional Mongolian dairy products (yogurt and fermented milk), and to investigate their capacity to generate bioactive peptides during milk fermentation. All isolates were screened for proteolytic activity using skim milk agar-well diffusion test. Fifteen strains (9 Lactobacillus helveticus and 6 Lactobacillus delbrueckii subsp. bulgaricus) were then selected and further evaluated using an original strategy based on multiparametric analysis, taking into account growth rate, acidification capacity, proteolytic activity, cell envelope associated peptidase (CEP) profile and LC-MS/MS analysis of peptides. All parameters were analyzed using principal component analysis (PCA). Results showed that strain growth and acidification correlate with peptide production and that Mongolian L. helveticus strains differ from Western strains in terms of CEP distribution. The PCA revealed that CEP profiles are major determinants of ß-casein hydrolysis patterns. Strains with distinctive proteolytic activities were identified.

2.
Artigo em Inglês | MEDLINE | ID: mdl-31659720

RESUMO

The identification of known (dereplication) or unknown nonribosomal peptides (NRPs) produced by microorganisms is a time consuming, expensive, and challenging task where mass spectrometry and nuclear magnetic resonance play a key role. The first step of the identification process always involves the establishment of a molecular formula. Unfortunately, the number of potential molecular formulae increases significantly with higher molecular masses and the lower precision of their measurements. In the present article, we demonstrate that molecular formula assignment can be achieved by a combined approach using the regular Kendrick mass defect (RKMD) and NORINE, the reference curated database of NRPs. We observed that irrespective of the molecular formula, the addition and subtraction of a given atom or atom group always leads to the same RKMD variation and nominal Kendrick mass (NKM). Graphically, these variations translated into a vector mesh can be used to connect an unknown molecule to a known NRP of the NORINE database and establish its molecular formula. We explain and illustrate this concept through the high-resolution mass spectrometry analysis of a commercially available mixture composed of four surfactins. The Kendrick approach enriched with the NORINE database content is a fast, useful, and easy-to-use tool for molecular mass assignment of known and unknown NRP structures.

3.
Chemosphere ; 220: 505-513, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30594803

RESUMO

We evaluated the acute toxicities of metals cadmium (Cd), copper (Cu) and nickel (Ni) to a widely-distributed copepod Eurytemora affinis isolated from the Seine estuary. Both sexes of adult E. affinis were exposed separately to the three metals at concentration gradients to determine its 50% lethal concentration (LC50). After 4 days of exposure, both males and females showed a higher sensitivity to Cu (male LC50: 25.0 µg.L-1 and female LC50: 38.0 µg.L-1) than to Ni (male LC50: 90.0 µg.L-1 and female 161.0 µg.L-1) and Cd (male LC50: 127.8 µg.L-1 and female LC50: 90.0 µg.L-1). To assess for the first time, the extend of metal bioaccumulation and its effect at population scale, late stages (>200 µm) were collected and exposed to each metal at the concentration of 1/3 LC50, and to their mixture during 144 h without feeding. The Cd concentration consistently increased with time until the end of the experiment, whereas the Ni and Cu concentrations reached a plateau after 24 h and 72 h exposure, respectively. The results revealed that the copepods could accumulate Cu faster than Ni and Cd either in the treatment alone (0.58 L g-1.d-1) or in the three-metal mixture (0.72 L g-1.d-1) after 50% of exposure time (72 h). The number of individuals decreased in copepod populations except for the Cd treatment, where the number of nauplii increased. In addition, all treatments of metal exposure negatively affected bacterial densities in the copepod cultures, where the Cu treatment showed a negative remarkable effect compared with Cd and Ni treatment did.


Assuntos
Cádmio/toxicidade , Copépodes/metabolismo , Cobre/toxicidade , Níquel/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Copépodes/efeitos dos fármacos , Estuários
4.
Front Microbiol ; 9: 2354, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30386307

RESUMO

To compensate for their amino acid auxotrophy, lactobacilli have developed the ability to hydrolyze proteins present in their environment. This proteolytic activity not only generates the free amino acids needed by the bacteria, but also a large variety of peptides, some of which are endowed with biological activities. These so-called "bioactive peptides" (BAPs) are interesting from a nutrition and healthcare perspective. The use of lactic acid bacteria (LAB) such as lactobacilli is an effective strategy for production and valorization of new BAPs. The proteolytic activity of lactobacilli is exerted in a strain- and species-dependent manner: each species exhibits different proteinase content, leading to a large variety of proteolytic activities. This underlines the high potential of Lactobacillus strains to produce novel hydrolysates and BAPs of major interest. This review aims at discussing the potential of different Lactobacillus species to release BAPs from fermentation media and processes. Strategies used for peptide production are presented. Additionally, we propose a methodology to select the most promising Lactobacillus strains as sources of BAPs. This methodology combines conventional approaches and in silico analyses.

5.
Artigo em Inglês | MEDLINE | ID: mdl-30229513

RESUMO

Here we show that Bacillus pumilus ICVB403 recently isolated from copepod eggs is able to produce, after 48-72 h of growth in Landy medium, extracellular inhibitory compounds, which are active against Staphylococcus aureus ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 43300, MRSA-S1, Staphylococcus epidermidis 11EMB, Staphylococcus warneri 27EMB, and Staphylococcus hominis 13EMB. Moreover, these extracellular inhibitory compound(s) were able to potentiate erythromycin against the aforementioned staphylococci. The minimum inhibitory concentration (MIC) of erythromycin was reduced from 32 µg/mL to 8 µg/mL for MRSA ATCC 43300 and MRSA SA-1 strains, and from 32-64 µg/mL to 4 µg/mL for S. epidermidis 11EMB and S. hominis 13EMB strains.The genome sequencing and analysis of B. pumilus ICVB403 unveiled 3.666.195 nucleotides contained in 22 contigs with a G + C ratio of 42.0%, 3.826 coding sequences, and 73 RNAs. In silico analysis guided identification of two putative genes coding for synthesis of surfactin A, a lipopeptide with 7 amino acids, and for a circular bacteriocin belonging to the circularin A/uberolysin family, respectively.

6.
Front Microbiol ; 8: 1919, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29085344

RESUMO

Copepods represent a major source of food for many aquatic species of commercial interest for aquaculture such as mysis shrimp and early stages of fishes. For the purpose of this study, the culturable mesophilic bacterial flora colonizing Acartia tonsa copepod eggs was isolated and identified. A total of 175 isolates were characterized based on their morphological and biochemical traits. The majority of these isolates (70%) were Gram-negative bacteria. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was used for rapid identification of bacterial isolates. Here, 58% of isolates were successfully identified at the genus level and among them, 54% were identified at the species level. These isolates belong to 12 different genera and 29 species. Five strains, identified as Bacillus pumilus, named 18 COPS, 35A COPS, 35R COPS, 38 COPS, and 40A COPS, showed strong antagonisms against several potential fish pathogens including Vibrio alginolyticus, V. anguillarum, Listeria monocytogenes, and Staphylococcus aureus. Furthermore, using a differential approach, we show that the antimicrobial activity of the 35R COPS strain is linked primarily to the production of antimicrobial compounds of the amicoumacin family, as demonstrated by the specific UV-absorbance and the MS/MS fragmentation patterns of these compounds.

7.
J Colloid Interface Sci ; 508: 488-499, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-28865343

RESUMO

Polypeptide/solid charged surface interactions are omnipresent in the biomedical and biochemical fields. The present study aimed to understand the adsorption mechanisms of a cation-exchange membrane (CEM) by a well-characterized peptide mixture at three different pH values. Results demonstrated that fouling was important at pH 6, twice lower at pH 2 and negligible at pH 10. At pH 6, ALPMHIR and TKIPAVFK sequences firstly established electrostatic interactions with the negative CEM charges (SO3-) through their positive K and R residues (NH3+) creating a first nanolayer. Secondly, peptide/peptide interactions occurred through their respective hydrophobic residues creating a second nanolayer. At pH 2, VLVLDTDYK and IDALNENK sequences interacted only electrostatically and that in a lower proportion since at acidic pH values, most of the CEM charges would be protonated and uncharged (HSO3) and then limit the potential electrostatic interactions. In addition, the sequences of peptides interacting at pH 2 and 6 were different. This was explained by their structure in terms of residue nature and position in the sequence. At pH 10, no fouling was observed due to the lack of positive peptide charges. To the best of our knowledge, it is the first in-depth study concerning the fouling of CEMs by peptides from a complex mixture.

8.
Int J Antimicrob Agents ; 49(3): 282-289, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28104423

RESUMO

Enterococcus faecalis 14, a strain previously isolated from meconium, displayed activity against four Clostridium perfringens isolates when co-cultured on agar plates. The anti-Clostridium activity was ascribed to the production of enterocin DD14, which was subsequently purified. The minimum inhibitory concentration (MIC) of enterocin DD14 against one collection strain and one clinical C. perfringens strain was determined at 50 µg/mL. Furthermore, using the intestinal epithelial cell line IPEC-1, it was shown that E. faecalis 14 was not cytotoxic after 24 h of contact, and no cytotoxicity was observed when IPEC-1 cells were incubated with pure enterocin DD14 for 4 h. Enterocin DD14 was characterised using mass spectrometry and was shown to consist of two small proteins of 5200.74 Da and 5206.41 Da, respectively. The two peptides (DD14A and DD14B) have highly similar amino acid sequences and no signal peptide, which classifies enterocin DD14 as a class IIb leaderless two-peptide bacteriocin. The genes encoding DD14A and DD14B were sequenced and were shown to be 100% identical to other previously described enterocins MR10A and MR10B, in contrast to the producing strains, which are different. Consequently, the present in vitro study supports the potential of this E. faecalis 14 strain and/or its purified enterocin DD14 as putative anti-C. perfringens compounds in chickens.


Assuntos
Antibacterianos/farmacologia , Clostridium perfringens/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Antibacterianos/isolamento & purificação , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/isolamento & purificação , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular , Sobrevivência Celular , Enterococcus faecalis/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Testes de Sensibilidade Microbiana , Peso Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência , Suínos
9.
Bioprocess Biosyst Eng ; 40(2): 161-180, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27738757

RESUMO

Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes. These technological innovations are also accompanied by the development and implementation of clean and sustainable integrated processes to meet the growing needs of chemical, pharmaceutical, environmental and biorefinery industries. This review gives an overview of the benefits of high-throughput screening approach from the discovery and engineering of biocatalysts to cell culture for optimizing their production in integrated processes and their extraction/purification.


Assuntos
Enzimas/biossíntese , Enzimas/química , Enzimas/genética , Engenharia de Proteínas/métodos , Catálise
10.
Int J Food Microbiol ; 247: 2-8, 2017 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-27423415

RESUMO

In this study we identified the culturable population of mesophilic lactic acid bacteria (LAB) from a French cheese Maroilles made either with raw or pasteurized milk using MALDI-TOF mass spectrometry (MS). Samples from rind and heart of Maroilles cheese were used, the LAB were selected on MRS agar at 30°C and 197 Gram-positive and catalase-negative strains were subjected to identification by MALDI-TOF MS profiling. All strains were unambiguously identified: 105 strains from Maroilles made with raw milk (38 on the rind and 67 in the heart) and 92 strains from Maroilles made with pasteurized milk (39 on the rind and 53 in the heart). MALDI-TOF MS identification allowed identification of three genera belonging to LAB including Lactobacillus, Enterococcus and Leuconostoc. Lactobacillus was the most represented genus with seven species: Lactobacillus plantarum (L. plantarum), L. paracasei, L. curvatus, L. rhamnosus, L. fructivorans, L. parabuchneri, L. brevis found in Maroilles made with both kind of milk. The correlation between the 16S rDNA-based identification performed on selected strains and those obtained by MALDI-TOF-MS demonstrates that this fast, economically affordable, robust and reliable method for bacteria characterisation stands as an attractive alternative to the commonly-used methods and its application in food industry is discussed.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Queijo/microbiologia , Lactobacillales/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Bovinos , Ácido Láctico/metabolismo , Lactobacillales/química , Lactobacillales/genética , Lactobacillales/metabolismo , Leite/microbiologia
11.
Electrophoresis ; 37(13): 1814-22, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26990205

RESUMO

Consumers and governments have become aware how the daily diet may affect the human health. All proteins from both plant and animal origins are potential sources of a wide range of bioactive peptides and the large majority of those display health-promoting effects. In the meat production food chain, the slaughterhouse blood is an inevitable co-product and, today, the blood proteins remain underexploited despite their bioactive potentiality. Through a comparative food peptidomics approach we illustrate the impact of resolving power, accuracy, sensitivity, and acquisition speed of low-resolution (LR)- and high-resolution (HR)-LC-ESI-MS/MS on the obtained peptide mappings and discuss the limitations of MS-based peptidomics. From in vitro gastrointestinal digestions of partially purified bovine hemoglobin, we have established the peptide maps of each hemoglobin chain. LR technique (normal bore C18 LC-LR-ESI-MS/MS) allows us to identify without ambiguity 75 unique peptides while the HR approach (nano bore C18 LC-HR-ESI-MS/MS) unambiguously identify more than 950 unique peptides (post-translational modifications included). Herein, the food peptidomics approach using the most performant separation methods and mass spectrometers with high-resolution capabilities appears as a promising source of information to assess the health potentiality of proteins.


Assuntos
Cromatografia Líquida/métodos , Digestão , Análise de Alimentos , Hemoglobinas/metabolismo , Peptídeos/metabolismo , Proteômica , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Técnicas In Vitro , Mapeamento de Peptídeos
12.
Food Res Int ; 89(Pt 1): 382-390, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28460928

RESUMO

Dietary proteins have been reported to induce a strong feeling of satiety that has been partially explained by gut hormone level increase. Up to date, various protein hydrolysates have demonstrated in vitro and in vivo their potential to stimulate gut hormone secretion related to food intake decrease and their mechanisms of action have just started to be resolved. In this context, this study aimed at identifying new peptide sequences involved in gut hormone secretion released by protein in vitro gastrointestinal digestion. Targeted gut hormones were Cholecystokinin (CCK) and Glucagon-Like Peptide 1 (GLP-1). The activity of DPP-IV was also considered as it strongly modulates GLP-1 action. In a previous study, simulated digestion of dietary protein has generated hydrolysates with enhancing effect on CCK and GLP-1 secretion in STC-1 cells as well as DPP-IV inhibitory properties. Successive purification steps were performed to isolate peptide fractions involved in these bioactivities whose sequence was determined by LC-MS-MS. Three peptide sequences ANVST, TKAVEH and KAAVT were pointed out for their stimulating effects on GLP-1 secretion. The sequence VAAA was isolated for its DPP-IV inhibitory properties. Two peptide groups were strongly involved in CCK release sharing a certain occurrence of aromatic amino acid residues.

13.
Probiotics Antimicrob Proteins ; 7(4): 233-41, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26459296

RESUMO

We studied here the yeast content of poultry feces, collected randomly from a French farm located in the north of the country. Thus, 81 yeast colonies were isolated and clustered into 22 distinct groups using the rep-PCR method. A single colony was taken from each group and identified using biochemical (ID 32C system) and molecular (sequencing of the D1/D2 domain of 26S rDNA and ITS1-5.8-ITS2 rDNA region) methods. Both methods led to the identification of Candida famata species. One isolate of C. famata strains, named strain Y5, was further studied for its cytotoxicity, adhesion, and surface properties, hemolytic activity, and its survival in simulated gastric and intestine environments. The data obtained advocate the probiotic potential of this isolate.


Assuntos
Candida/isolamento & purificação , Fezes/microbiologia , Aves Domésticas/microbiologia , Probióticos , Animais , Candida/classificação , Candida/fisiologia , França , Humanos
14.
Subcell Biochem ; 76: 125-51, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26219710

RESUMO

The microvessels of the brain represent around 3-4 % of the brain compartment but constitute the most important length (400 miles) and surface of exchange (20 m(2)) between the blood and the parenchyma of brain. Under influence of surrounding tissues, the brain microvessel endothelium expresses a specific phenotype that regulates and restricts the entry of compounds and cells from blood to brain, and defined the so-called blood-brain barrier (BBB). Evidences that alkaline phosphatase (AP) is a characteristic feature of the BBB phenotype that allows differentiating capillary endothelial cells from brain to those of the periphery have rapidly emerge. Thenceforth, AP has been rapidly used as a biomarker of the blood-brain barrier phenotype. In fact, brain capillary endothelial cells (BCECs) express exclusively tissue non-specific alkaline phosphatase (TNAP). There are several lines of evidence in favour of an important role for TNAP in brain function. TNAP is thought to be responsible for the control of transport of some compounds across the plasma membrane of the BCECs. Here, we report that levamisole-mediated inhibition of TNAP provokes an increase of the permeability to Lucifer Yellow of the endothelial monolayer. Moreover, we illustrate the disruption of the cytoskeleton organization. Interestingly, all observed effects were reversible 24 h after levamisole removal and correlated with the return of a full activity of the TNAP. This reversible effect remains to be studied in details to evaluate the potentiality of a levamisole treatment to enhance the entry of drugs in the brain parenchyma.


Assuntos
Fosfatase Alcalina/fisiologia , Vasos Sanguíneos/enzimologia , Encéfalo/irrigação sanguínea , Animais , Biomarcadores/metabolismo , Vasos Sanguíneos/metabolismo , Barreira Hematoencefálica/enzimologia , Barreira Hematoencefálica/metabolismo , Encéfalo/enzimologia , Encéfalo/metabolismo , Permeabilidade Capilar/genética , Circulação Cerebrovascular/genética , Humanos
15.
Ann Clin Biochem ; 51(Pt 2): 248-57, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23982266

RESUMO

BACKGROUND: The soluble form of the receptor for advanced glycation end-products (sRAGE) has been studied in various diseases. It is not clear why sRAGE levels vary between studies, with controversial results. What also remains to be determined is whether receptor for advanced glycation end-products (RAGE) ligands could affect sRAGE assessment by epitope masking. Recently described anti-sRAGE autoantibodies may play an interfering role. The aim of this study was therefore to investigate the influence of RAGE ligands and anti-sRAGE autoantibodies on sRAGE quantification. METHODS: The RAGE ligands carboxymethyllysine (CML; AGEs with a high affinity for RAGE), S100 proteins, high-mobility group protein B1 (HMGB1) and ß-amyloid peptide (aß) were tested by enzyme-linked immunosorbent assay (ELISA) with recombinant sRAGE (rHu-sRAGE) or serum from healthy controls. Using ELISA, anti-sRAGE autoantibodies (IgGs) were identified in haemodialysis (HD) patients, then purified and incubated with rHu-sRAGE or serum to investigate their effects on sRAGE levels. RESULTS: RAGE ligands, either alone at three different concentrations (CML was also tested at different glycation levels) or a mixture of all these ligands, did not affect sRAGE levels when incubated with rHu-sRAGE or control serum. Compared with healthy controls, HD patients had higher levels of sRAGE (P < 0.001) and anti-sRAGE IgGs (P < 0.05). However, incubation of rHu-sRAGE with purified IgGs from HD patients had no effect on sRAGE quantification. CONCLUSIONS: RAGE ligands or anti-sRAGE autoantibodies did not interfere with sRAGE quantification. Further studies are required to elucidate the variability in sRAGE levels reported in the literature and to define the potential of sRAGE for use as a reliable biomarker.


Assuntos
Artefatos , Autoanticorpos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Receptores Imunológicos/análise , Receptores Imunológicos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imunoglobulina G/imunologia , Ligantes , Masculino , Pessoa de Meia-Idade , Estrutura Terciária de Proteína , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/química , Receptores Imunológicos/imunologia , Diálise Renal , Solubilidade
16.
Proteomics ; 13(7): 1185-99, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23436736

RESUMO

In the neurovascular unit, brain microvascular endothelial cells develop characteristic barrier features that control the molecular exchanges between the blood and the brain. These characteristics are partially or totally lost when the cells are isolated for use in in vitro blood-brain barrier (BBB) models. Hence, the re-induction of barrier properties is crucial for the relevance of BBB models. Although the role of astrocyte promiscuity is well established, the molecular mechanisms of re-induction remain largely unknown. Here, we used a DIGE-based proteomics approach to study endothelial cellular proteins showing significant quantitative variations after BBB re-induction. We confirm that quantitative changes mainly concern proteins involved in cell structure and motility. Furthermore, we describe the possible involvement of the asymmetric dimethylarginine pathway in the BBB phenotype re-induction process and we discuss asymmetric dimethylarginine's potential role in regulating endothelial function (in addition to its role as a by-product of protein modification). Our results also suggest that the intracellular redox potential is lower in the in vitro brain capillary endothelial cells displaying re-induced BBB functions than in cells with limited BBB functions.


Assuntos
Barreira Hematoencefálica/metabolismo , Eletroforese em Gel Bidimensional/métodos , Células Endoteliais/metabolismo , Neuroglia/metabolismo , Animais , Arginina/análogos & derivados , Barreira Hematoencefálica/citologia , Bovinos , Meios de Cultura , Immunoblotting , Fenótipo , Ratos , Reprodutibilidade dos Testes
17.
Proteomes ; 1(3): 180-218, 2013 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-28250403

RESUMO

Proteomics became a key tool for the study of biological systems. The comparison between two different physiological states allows unravelling the cellular and molecular mechanisms involved in a biological process. Proteomics can confirm the presence of proteins suggested by their mRNA content and provides a direct measure of the quantity present in a cell. Global and targeted proteomics strategies can be applied. Targeted proteomics strategies limit the number of features that will be monitored and then optimise the methods to obtain the highest sensitivity and throughput for a huge amount of samples. The advantage of global proteomics strategies is that no hypothesis is required, other than a measurable difference in one or more protein species between the samples. Global proteomics methods attempt to separate quantify and identify all the proteins from a given sample. This review highlights only the different techniques of separation and quantification of proteins and peptides, in view of a comparative and quantitative global proteomics analysis. The in-gel and off-gel quantification of proteins will be discussed as well as the corresponding mass spectrometry technology. The overview is focused on the widespread techniques while keeping in mind that each approach is modular and often recovers the other.

18.
PLoS One ; 7(10): e48428, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23119012

RESUMO

Although the physiological properties of the blood-brain barrier (BBB) are relatively well known, the phenotype of the component brain capillary endothelial cells (BCECs) has yet to be described in detail. Likewise, the molecular mechanisms that govern the establishment and maintenance of the BBB are largely unknown. Proteomics can be used to assess quantitative changes in protein levels and identify proteins involved in the molecular pathways responsible for cellular differentiation. Using the well-established in vitro BBB model developed in our laboratory, we performed a differential nano-LC MALDI-TOF/TOF-MS study of Triton X-100-soluble protein species from bovine BCECs displaying either limited BBB functions or BBB functions re-induced by glial cells. Due to the heterogeneity of the crude extract, we increased identification yields by applying a repeatable, reproducible fractionation process based on the proteins' relative hydrophobicity. We present proteomic and biochemical evidence to show that tissue non-specific alkaline phosphatase (TNAP) and Eps15 homology domain-containing protein 1(EDH1) are over-expressed by bovine BCECs after the re-induction of BBB properties. We discuss the impact of these findings on current knowledge of endothelial and BBB permeability.


Assuntos
Fosfatase Alcalina/genética , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Expressão Gênica , Proteínas de Transporte Vesicular/genética , Fosfatase Alcalina/metabolismo , Animais , Barreira Hematoencefálica/química , Bovinos , Células Cultivadas , Células Endoteliais/química , Ativação Enzimática/efeitos dos fármacos , Levamisol/farmacologia , Neuroglia/metabolismo , Proteômica , Ratos , Proteínas de Transporte Vesicular/metabolismo
19.
J Proteomics ; 75(2): 628-41, 2011 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-21982828

RESUMO

When in the vicinity of astrocytes, brain capillary endothelial cells (BCECs) develop the characteristic structural and functional features of the blood-brain barrier (BBB). The latter has low cellular permeability and restricts various compounds from entering the brain. We recently reported that the cytoskeleton-related proteins actin, gelsolin and filamin-A undergo the largest quantitative changes in bovine BCECs after re-induction of BBB functions by co-culture with glial cells. In the present study, we used an in-depth, proteomic approach to quantitatively compare differences in Triton-X-100-solubilized proteins from bovine BCECs with limited or re-induced BBB functions (i.e. cultured in the absence or presence of glial cells, respectively). The 81 protein spots of differing abundance were linked to 55 distinct genes. According to the Protein ANalysis THrough Evolutionary Relationships classification system and an Ingenuity Pathway Analysis, these quantitative changes mainly affected proteins involved in (i) cell structure and motility and (ii) protein metabolism and modification. The fold-changes affecting HSPB1, moesin and ANXA5 protein levels were confirmed by western blot analysis but were not accompanied by changes in the corresponding mRNA expression levels. Our results reveal that the bovine BCECs' phenotype adaptation to variations in their environment involves the reorganization of the actin cytoskeleton.


Assuntos
Barreira Hematoencefálica/fisiologia , Encéfalo/irrigação sanguínea , Diferenciação Celular/fisiologia , Células Endoteliais/citologia , Actinas/genética , Animais , Anexinas/genética , Barreira Hematoencefálica/citologia , Bovinos , Técnicas de Cocultura , Eletroforese em Gel Bidimensional , Células Endoteliais/fisiologia , Proteínas de Choque Térmico HSP27/genética , Proteínas dos Microfilamentos , Neuroglia/citologia , Mapas de Interação de Proteínas , Proteômica/métodos , RNA Mensageiro/metabolismo , Ratos , Vimentina/genética
20.
Proteomics Clin Appl ; 5(7-8): 405-14, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21751410

RESUMO

PURPOSE: Universal newborn screening for sickle cell diseases (SCDs) is not currently performed in many countries concerned by this public health problem. Owing to the technical and financial limitations of standard profiling methods (IEF coupled to subsequent HPLC), ethnically targeted neonatal screening is often preferred. Here, we demonstrate that MALDI-MS-based SCD newborn screening could be considered as a potential method for a strategy to universal screening because of its high throughput, cost-effectiveness, sensitivity and ability to automatically discriminate sickle haemoglobin. EXPERIMENTAL DESIGN: We carried out a retrospective study of dried blood spots from 844 Guthrie cards. Four determinations of 1000 mass spectra were performed from each tested dried blood spot. RESULTS: The MALDI-MS-based screening was highly correlated with the reference method. Only 2.3% of the samples presented a poor spectral quality. CONCLUSIONS AND CLINICAL RELEVANCE: Given that the overall acquisition, data reprocessing and software-assisted classification (ClinProTools™) time for processing four mass determinations (corresponding to one sample) was around 1 min, 1000 samples can be analysed per day. Rather than seeking to detect as many different haemoglobinopathies as possible, it would become possible to use MALDI-TOF-MS to screen (at a constant cost) as many samples as possible for sickle cell disease.


Assuntos
Anemia Falciforme/diagnóstico , Hemoglobina Falciforme/análise , Ensaios de Triagem em Larga Escala/métodos , Triagem Neonatal/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Anemia Falciforme/economia , Anemia Falciforme/patologia , Estudos de Casos e Controles , Análise Custo-Benefício , Feminino , Ensaios de Triagem em Larga Escala/economia , Humanos , Recém-Nascido , Masculino , Triagem Neonatal/economia , Saúde Pública , Controle de Qualidade , Estudos Retrospectivos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economia
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