RESUMEN
UNLABELLED: Based on recent studies in adult subjects, saliva composition is increasingly considered as a physiological factor contributing to taste sensitivity or acceptance. In order to evaluate a possible link between salivary protein composition and taste acceptance in infants, 73 infants participated longitudinally in taste acceptance tests and donated saliva at the age at 3 and 6 months. Intake ratios, reflecting acceptance of a taste solution relative to water were calculated for the five basic tastes. Salivary proteins were separated by one-dimensional electrophoresis and bands were semi-quantified by image analysis. Partial least square (PLS) regression analyses were performed for each taste at both ages to explain intake ratios by band intensities. Bitterness acceptance in the younger infants was unique in the sense that salivary protein profiles could partly predict bitter taste acceptance. At that age, infants were on average indifferent to the 0.18-M urea solution, but great variability in acceptance was observed. The six bands considered as the best predictors for bitterness acceptance were identified by MALDI-TOF mass spectrometry. Higher abundance of bands containing secretory component, zinc-α-2-glycoprotein and carbonic anhydrase 6 was associated to a lower bitterness acceptance, while higher abundance of bands containing lactoperoxidase, prolactin-inducible protein and S-type cystatins was associated to a higher bitterness acceptance. In a second stage, S-type cystatin abundance was measured by Western blotting in order to tentatively confirm this particular finding in an independent group of 22 infants. Although not reaching statistical significance, probably due to a relatively small sample size, it was again observed that cystatin abundance was higher in infants accepting more readily the bitter solution over water. CONCLUSION: saliva protein composition may contribute to bitter taste acceptance in the younger infants.
Asunto(s)
Proteínas y Péptidos Salivales/fisiología , Gusto/fisiología , Western Blotting , Ácido Cítrico/farmacología , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Lactante , Lactosa/farmacología , Estudios Longitudinales , Masculino , Estudios Prospectivos , Saliva/química , Proteínas y Péptidos Salivales/análisis , Cloruro de Sodio/farmacología , Glutamato de Sodio/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Umbral Gustativo/fisiología , Urea/farmacologíaRESUMEN
Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide. The development of novel diagnostic methods is needed to detect tumours at an early stage when patients are eligible for curative treatments. The purpose of this proof-of-concept study was to determine if micro-Raman spectroscopy applied to the sera of cirrhotic patients may be an alternative method for rapidly discriminating patients with and without HCC. Serum samples were collected from 2 groups of patients: cirrhotic patients with HCC (n = 37) and without HCC (n = 34). Two different approaches were used, dried serum drops and freeze-dried serum, and micro-Raman spectra were acquired in the point-mode with a 785 nm laser excitation in the spectral range of 600-1800 cm(-1). Spectra were quality-tested and pre-processed (smoothing, baseline subtraction, vector normalization). Using principal component analysis, the 2 classes, corresponding to cirrhotic patients with and without HCC, could not be differentiated. In contrast, the support vector machine method using the leave-one-out cross validation procedure was able to correctly classify the two groups of patients with an overall rate of accuracy of 84.5% to 90.2% for dried serum drops and 86% to 91.5% for freeze-dried serum. These results are promising and support the concept that serum micro-Raman spectroscopy may become a useful diagnostic tool to detect biomarkers in the field of cancer, as described here for distinguishing between cirrhotic patients with and without HCC.
Asunto(s)
Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/diagnóstico , Cirrosis Hepática/diagnóstico , Neoplasias Hepáticas/diagnóstico , Espectrometría Raman/métodos , Anciano , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/etiología , Estudios de Casos y Controles , Femenino , Humanos , Cirrosis Hepática/sangre , Cirrosis Hepática/complicaciones , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/etiología , Masculino , Persona de Mediana Edad , Análisis de Componente Principal , Sensibilidad y Especificidad , Máquina de Vectores de SoporteRESUMEN
Protein biomarker discovery and validation are crucial for diagnosis, prognosis, and theranostics of human pathologies; "omics" approaches bring new insights in this field. In particular, the combination of immuno-sensors in array format with mass spectrometry efficiently extends the classical immunoassay format and includes molecular characterization. Here, we coupled surface plasmon resonance imaging (SPRi) with MALDI-TOF mass spectrometry in a hyphenated technique which enables multiplexed quantification of binding by SPRi and molecular characterization of interacting partners by subsequent MS analysis. This adds specificity, because MS enables differentiation of molecules that are difficult to distinguish by use of antibodies, for example truncation variants or protein isoforms. Proof of concept was established for detection, identification, and characterization of a potential breast cancer marker, the LAG3 protein, at ~1 µg mL(-1), added to human plasma. The analytical performance of this new method, dubbed "SUPRA-MS", was established, particularly its specificity (S/N > 10) and reliability (100 % LAG3 identification with high significant mascot score >87.9). The adjusted format for rapid, collective, and automated on-chip MALDI-MS analysis is robust at the femtomole level and has numerous potential applications in proteomics.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias de la Mama/diagnóstico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Resonancia por Plasmón de Superficie/métodos , Secuencia de Aminoácidos , Biomarcadores de Tumor/química , Neoplasias de la Mama/sangre , Femenino , Humanos , Datos de Secuencia MolecularRESUMEN
OBJECTIVE: The objective of this study was to describe the changes in salivary protein profiles in infants between the ages of 3 and 6 months, and to evaluate the impact of teeth eruption and introduction of solid foods on such profiles. DESIGN: 73 infants were followed longitudinally at 3 and 6 months of age. Their whole saliva proteins were separated by SDS-PAGE electrophoresis and semi-quantified by image analysis. Amylase activity was also measured on a sub-sample of the population (n=42 infants). Bands which abundance was significantly different between the two ages according to paired comparisons were identified by mass spectrometry techniques. RESULTS: Out of 21 bands, 13 were significantly different between 3 and 6 months of age. Two short variants of amylase increased in abundance with age, as did amylase activity. Other changes possibly translated developmental physiological events, for example maturation of the adaptive immune system. The balance between S-type cystatins and cystatins A and B was modified, in favour of S-type cystatins at 6 months of age. Teeth eruption resulted in an increase in albumin abundance, whilst introduction of solid foods was associated with higher levels of ß-2 microglobulin and S-type cystatins. CONCLUSIONS: Salivary profiles were modified substantially between the ages of 3 and 6 months. Both teeth eruption and diet had an impact on abundance changes for some proteins, revealing dynamic interactions between saliva proteome, oral physiology and diet.
Asunto(s)
Dieta , Electroforesis en Gel de Poliacrilamida , Proteínas y Péptidos Salivales/análisis , Erupción Dental/fisiología , Amilasas/análisis , Cromatografía Liquida , Cistatina A/análisis , Cistatina B/análisis , Femenino , Estudios de Seguimiento , Humanos , Lactante , Alimentos Infantiles , Fórmulas Infantiles , Estudios Longitudinales , Masculino , Leche Humana , Estudios Prospectivos , Inhibidores de Proteasas/análisis , Cistatinas Salivales/análisis , Componente Secretorio/análisis , Albúmina Sérica/análisis , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem , Destete , Microglobulina beta-2/análisisRESUMEN
Peroxisome proliferators have been extensively studied in rodents and are known to induce liver tumors, whereas the effects of these compounds are not very clearly identified in humans when they are widely exposed to herbicides, plasticizers, solvents or drugs such as the lipid-lowering fibrate bezafibrate (BEZA). We assessed the effect of BEZA on human hepatocyte proteome. Hepatocyte proteins, including those membrane-associated, were successfully extracted and separated using 2D-liquid chromatography (PF2D, Beckman coulter). Proteins that were regulated by ≥ 1.5 fold compared to controls were identified by mass spectrometry (MALDI-TOF, Bruker Daltonics) and SwissProt bank search. BEZA modified the expression of proteins involved in various metabolic pathways as well as in cell homeostasis. No marker of peroxisome proliferation was obtained but surprisingly the expression of proteins involved in liver carcinogenicity was modulated. The co-treatment of cultures with N-acetylcysteine modified the set of proteins regulated by BEZA, either by a potentiation or an inhibition of the effects. Our study points out that the hepatocellular redox environment has to be taken into account when using fibrates in therapeutics.
Asunto(s)
Bezafibrato/farmacología , Cromatografía Liquida/métodos , Hepatocitos/efectos de los fármacos , Proteómica/métodos , Acetilcisteína/farmacología , Células Cultivadas , Hepatocitos/química , Hepatocitos/metabolismo , Humanos , Oxidación-ReducciónRESUMEN
Research of new diagnosis or prognosis biomarkers is a major challenge for the management of patients with complex pathologies like cancer. Clinical proteomics is one of the recent approaches to identify these biomarkers in biological fluids. Over the last five years, many problems related to the variability and the quality control of these analyses have been observed. This was notably related to the different preanalytical status of each sample. A strong need for standardization of the critical preanalytical phases (collection, transport, processing, storage...) has been therefore recognized. With this goal in mind, working groups of the "Institut national du cancer" (INCa) and the "Société française de biologie clinique" (SFBC) proposed here preanalytical proteomics guidelines for the most common biological fluids: plasma, serum, urine and cerebrospinal fluid. To goal is to provide the basis for the harmonization of the procedures in clinical laboratories and biobanks to allow an optimal use of biological collections.
Asunto(s)
Líquidos Corporales/fisiología , Técnicas de Laboratorio Clínico/normas , Técnicas y Procedimientos Diagnósticos/normas , Guías de Práctica Clínica como Asunto , Proteómica/métodos , Análisis Químico de la Sangre/normas , Humanos , Pronóstico , Proteinuria/diagnóstico , Proteómica/normas , Orina/químicaRESUMEN
We present the results of a study in which biomolecular interaction analysis (BIA, Biacoretrade mark 2000) was combined with mass spectrometry (MS) using entire "on-a-chip" procedure. Most BIA-MS studies included an elution step of the analyte prior MS analysis. Here, we report a low-cost approach combining Biacore analysis with homemade chips and MS in situ identification onto the chips without elution step. First experiments have been made with rat serum albumin to determine the sensitivity and validation of the concept has been obtained with an antibody/antigen couple. Our "on-a-chip" procedure allowed complete analysis by MS/MS(2) of the biochip leading to protein identifications at low femtomole to sub-femtomole levels. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the "on-a-chip" processing to complete the BIA-MS approach in the discovery and analysis of protein complexes.
Asunto(s)
Técnicas Biosensibles/instrumentación , Cromatografía Líquida de Alta Presión/instrumentación , Microquímica/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Nanotecnología/instrumentación , Mapeo de Interacción de Proteínas/instrumentación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Técnicas Biosensibles/métodos , Cromatografía Líquida de Alta Presión/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Microquímica/métodos , Técnicas Analíticas Microfluídicas/métodos , Nanotecnología/métodos , Mapeo de Interacción de Proteínas/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Members of the inhibitor of apoptosis protein (IAP) family have demonstrated functions in cell death, cell signalling, cell migration and mitosis. Several of them are E3 enzymes in the ubiquitination of proteins that leads to their degradation by the proteosomal machinery. We previously reported that one of them, cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. Here, we show that c-IAP1 is a client protein of the stress protein HSP90 beta. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm along the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90 beta isoform by siRNA both lead to auto-ubiquitination of c-IAP1 and its degradation by the proteasome machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its beta isoform as specific depletion of HSP90alpha does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90 beta both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90 beta prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Células Epiteliales/citología , Células Epiteliales/fisiología , Proteínas HSP90 de Choque Térmico/genética , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Macrófagos/citología , Macrófagos/fisiología , Isoformas de Proteínas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Members of the inhibitor of apoptosis protein (IAP) family have demonstrated functions in cell death, cell signalling, cell migration and mitosis. Several of them are E3 enzymes in the ubiquitination of proteins that leads to their degradation by the proteosomal machinery. We previously reported that one of them, cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. Here, we show that c-IAP1 is a client protein of the stress protein HSP90ß. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm along the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90ß isoform by siRNA both lead to auto-ubiquitination of c-IAP1 and its degradation by the proteasome machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its ß isoform as specific depletion of HSP90α does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90ß both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90ß prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation.Cell Death and Differentiation advance online publication, 1 February 2008; doi:10.1038/sj.cdd.4402320.
RESUMEN
Direct analysis of tissue by MALDI-MS allows the acquisition of its biomolecular profile while maintaining the integrity of the tissue, giving cellular localization, and avoiding tedious extraction and purification steps. However, direct tissue analysis generally leads to some extent to a lowered spectral quality due to variation in thickness, freezing tissue date, and nature of the tissue. We present here new technical developments for the direct tissue analysis of peptides with ionic liquid made of matrix mixtures (alpha-cyano-4-hydroxycinnamic acid (CHCA)/2-amino-4-methyl-5-nitropyridine and alpha-cyano-4-hydroxycinnamic acid/N,N-dimethylaniline (CHCA/DANI)). The properties of these direct tissue analysis matrixes, especially CHCA/aniline when compared to CHCA, 2,5-dihydroxybenzoic acid, and sinapinic acid, are as follows: (1) better spectral quality in terms of resolution, sensitivity, intensity, noise, number of compounds detected, and contaminant tolerance, (2) better crystallization on tissues, i.e., coverage capacity, homogeneity of crystallization, homogeneity of crystal sizes, and time of crystallization, (3) better analysis duration in term of vacuum stability, (4) better resistance to laser irradiation especially for high-frequency lasers, (5) better ionic yield in negative mode, and (6) enough fragmentation yield to use the PSD mode on sections to get structural information. Applied to MALDI imaging on a MALDI LIFT-TOF with a 50-Hz laser frequency, these ionic matrixes have allowed the realization of a new type of image in both polarities and reflector mode using the same tissue section. These results give a new outlook on peptide tissue profiling by MS, characterization of compounds from tissue slices, and MALDI-MS high-quality imaging.
Asunto(s)
Derivados del Benceno/química , Membranas Artificiales , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Química Encefálica , Líquidos Iónicos/química , Masculino , Ratas , Ratas Wistar , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Propiedades de SuperficieAsunto(s)
Artritis Experimental/prevención & control , Guanidinas/farmacología , Inmunosupresores/farmacología , Interleucina-10/sangre , Animales , Formación de Anticuerpos , Artritis Experimental/inmunología , Colágeno , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Isotipos de Inmunoglobulinas/sangre , Interleucina-10/biosíntesis , Masculino , Ratones , Ratones Endogámicos DBAAsunto(s)
Artritis Experimental/inmunología , Artritis Experimental/prevención & control , Colágeno/inmunología , Guanidinas/farmacología , Inmunoglobulina G/sangre , Inmunosupresores/farmacología , Análisis de Varianza , Animales , Formación de Anticuerpos/efectos de los fármacos , Artritis Experimental/fisiopatología , Bovinos , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G/biosíntesis , Idiotipos de Inmunoglobulinas/biosíntesis , Idiotipos de Inmunoglobulinas/sangre , Masculino , Ratones , Ratones Endogámicos DBA , Factores de TiempoRESUMEN
Anticancer drugs can induce tumour cell death by apoptosis. The main pathway from specific damage induced by the drug to apoptosis involves activation of caspases in the cytosol by pro-apoptotic molecules such as cytochrome c released from the mitochondria. At least in some cell types, anticancer drugs also upregulate the expression of death receptors and sensitize tumour cells to their cognate ligands, which could be used to amplify the response to cytotoxic drugs. The Bcl-2 family of proteins, which includes anti- and pro-apoptotic molecules, regulates cell sensitivity at the mitochondrial level. Chemotherapeutic drugs modulate their expression (e.g. through p53-dependent gene transcription), their activity (e.g. by phosphorylation) and their subcellular localization (e.g. by translocation of pro-apoptotic proteins from the cytosol to the mitochondria). When interacting with tumour cells, anticancer drugs also activate lipid- and kinase-dependent signalling pathways that modulate the death response to specific damage. Protective pathways include activation of NF kappa B transcription factor, accumulation of heat shock proteins and activation of proteins involved in cell cycle regulation. The recent identification on these pathways to cell death has suggested several new strategies to improve the therapeutic efficacy of currently used anticancer drug regimens.
Asunto(s)
Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Citotoxinas/toxicidad , Neoplasias/tratamiento farmacológico , Humanos , Neoplasias/patología , Neoplasias/fisiopatología , Células Tumorales CultivadasRESUMEN
The MHC of the urodele amphibian Ambystoma mexicanum consists of multiple polymorphic class I loci linked, so far as yet known, to a single class II B locus. This architecture is very different from that of the anuran amphibian Xenopus. The number of class I loci in the axolotl can vary from 6 to 21 according to the haplotypes as shown by cDNA analysis and Southern blot studies in families. These loci can be classified into seven sequence groups with features ranging from the class Ia to the class Ib type. All individuals express genes from at least three of the seven groups, and all individuals possess the class Ia-like type.
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Ambystoma/genética , Ambystoma/inmunología , Complejo Mayor de Histocompatibilidad/genética , Complejo Mayor de Histocompatibilidad/inmunología , Polimorfismo Genético/inmunología , Secuencia de Aminoácidos , Animales , Southern Blotting , Secuencia Conservada , Evolución Molecular , Genes MHC Clase I/inmunología , Genes MHC Clase II/inmunología , Antígeno HLA-A2/química , Humanos , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
Lymphocytes apoptosis was characterized in a urodele amphibian, the axolotl, by morphology using electron microscopy and by flow cytometry after propidium iodide staining, as well as by biochemical criteria with the detection of DNA ladders after glucocorticoid treatment. The morphological and biochemical features observed in treated axolotls are in accordance with the criteria of apoptosis found in different models of mammalian lymphocyte programmed cell death. The onset of natural apoptosis was then detected by DNA fragmentation in thymus and in spleen during lymphocyte development and ontogenesis. A typical DNA ladder characteristic of apoptosis is detectable in the thymus as early as 5 months; apoptosis increases and peaks at 8 months, and is no longer detected by 10 months or thereafter. The ability of a superantigen, Staphylococcus aureus enterotoxin B (SEB), to induce T lymphocyte apoptosis in larvae was investigated as well. In vivo exposure of young axolotl larvae to SEB induces, as in mammals, thymocyte apoptosis as indicated by the enhancement of DNA fragmentation. These last results, natural programmed cell death and SEB induced apoptosis during thymic ontogeny, are discussed in correlation with what is known during mammalian thymic selection and apoptosis.
Asunto(s)
Ambystoma , Apoptosis , Linfocitos T/citología , Animales , Diferenciación Celular , Enterotoxinas/farmacología , Hidrocortisona/farmacología , Larva , Linfocitos/citología , Linfocitos/efectos de los fármacos , Superantígenos/farmacología , Linfocitos T/efectos de los fármacosRESUMEN
Despite the fact that the axolotl (Ambystoma spp. a urodele amphibian) displays a large T-cell repertoire and a reasonable B-cell repertoire, its humoral immune response is slow (60 days), non-anamnestic, with a unique IgM class. The cytotoxic immune response is slow as well (21 days) with poor mixed lymphocyte reaction stimulation. Therefore, this amphibian can be considered as immunodeficient. The reason for this subdued immune response could be an altered antigenic presentation by major histocompatibility complex (MHC) molecules. This article summarizes our work on axolotl MHC genes. Class I genes have been characterized and the cDNA sequences show a good conservation of non-polymorphic peptide binding positions of the alpha chain as well as a high diversity of the variable amino acids positions, suggesting that axolotl class I molecules can present numerous antigenic epitopes. Moreover, class I genes are ubiquitously transcribed at the time of hatching. These class I genes also present an important polylocism and belong to the same linkage group as the class II B gene; they can be reasonably considered as classical class Ia genes. However, only one class II B gene has been characterized so far by Southern blot analysis. As in higher vertebrates, this gene is transcribed in lymphoid organs when they start to be functional. The sequence analysis shows that the peptide binding region of this class II beta chain is relatively well conserved, but most of all does not present any variability in the beta 1 domain in inbred as well as in wild axolotls, presuming a limited antigenic presentation of few antigenic epitopes. The immunodeficiency of the axolotl could then be explained by an altered class II presentation of antigenic peptides, putting into question the existence of cellular co-operation in this lower vertebrate. It will be interesting to analyze the situation in other urodele species and to determine whether our observations in axolotl represent a normal feature in urodele amphibians. But already two different models in amphibians, Xenopus and axolotl, must be considered in our search for understanding immune system and MHC evolution.