Biomedical subjects

Montserrat Carrascal

Publications and source records attributed to Montserrat Carrascal.

14 recordsLinked to original sources

PubMed2026

Proteomic characterization of acidic aqueous extracts from Vicia faba L. pod valves identifies chitinase as a major co-extracted protein macromolecule.

Naturally acidic aqueous extracts from Vicia faba L. pod valves are being explored as sustainable, L-DOPA-oriented plant preparations. Pod valves represent an underutilized processing by-product reported to contain L-DOPA, a compound widely used in Parkinson's disease therapy, while acidic aqueous media may help preserve its physicochemical stability. However, the protein macromolecules co-extracted from V. faba pod valves under these conditions remain poorly characterized. This information is relevant because persistent plant proteins may influence extract composition, stability, susceptibility to degradation, and downstream processing requirements. Here, we characterized co-extracted V. faba protein macromolecules in aqueous pod-valve extracts prepared in ultrapure water or naturally acidic media, including 2% Phyllanthus emblica, 5% Punica granatum, and 2% Ribes rubrum. Protein profiles were first evaluated by SDS-PAGE and subsequently analyzed by nanoflow liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-MS/MS). Protein identifications were complemented with Gene Ontology annotation and a descriptive semi-quantitative assessment of relative protein representation across extraction media. Chitinase was the most represented V. faba-assigned protein macromolecule across the extracts, with additional highly represented proteins including glucan endo-1,3-beta-D-glucosidase, pathogenesis-related proteins, and polyphenol oxidase A1. These co-extracted proteins are mainly associated with plant defense, stress responses, cell-wall remodeling, and oxidative processing, suggesting that they may be relevant for extract quality attributes during handling and storage. This study provides a compositional proteomic reference for the co-extracted protein macromolecules present in acidic aqueous extracts from V. faba pod valves, supporting future studies on extract stability, processing optimization, and the development of standardized plant-based preparations.

Vicia faba↗

PubMed2005

Transcriptomic and proteomic analysis of liver and muscle alterations caused by surgical stress in rats.

The metabolic response to injury includes major alterations in protein metabolism; however, little is known about alterations in the synthesis of individual proteins and their role in the stress response. Our aim was to study how individual proteins in liver and muscle are altered by abdominal surgery. Changes produced in mRNA and proteins by abdominal surgery were studied in rats using RAP (random arbitrary priming)-PCR, to investigate mRNA alterations, and standard or isotopic (with in vivo radioactive labelling of proteins) two-dimensional electrophoresis/MS proteomic analyses, to study differential expression of proteins. Many of the differentially expressed proteins identified in blood were specifically synthesized by the liver to participate in the stress response. The hepatic proteins (antioxidant proteins, serine protease inhibitors, acute-phase proteins and transport proteins) were secreted into the bloodstream to produce a systemic action, indicating the central role of the liver in the stress response. Overexpressed proteins identified in liver were associated with the glycolytic processes and the folding of nascent proteins, confirming the high metabolic activity of the liver after surgery. The role of skeletal muscle protein as an amino acid donor to fuel the processes involved in the stress response was shown by the decrease in high-molecular-mass myofibrillar proteins. Combined use of the three techniques studied, differential RAP-PCR and standard and isotopic proteome analysis, provided complementary information on the differentially expressed proteins in a rat model of surgical stress.

Abdomen↗

PubMed2004

Dissection of the HLA-DR4 peptide repertoire in endocrine epithelial cells: strong influence of invariant chain and HLA-DM expression on the nature of ligands.

Class II MHC (MHC II) expression is restricted to professional APCs and thymic epithelium but it also occurs in the epithelial cells of autoimmune organs which are the unique targets of the CD4 autoreactive T cells in endocrine autoimmune diseases. This specificity is presumably conditioned by an epithelium-specific peptide repertoire associated to MHC II at the cell surface. MHC II expression and function is dependent on the action of two main chaperones, invariant chain (Ii) and DM, whose expression is coregulated with MHC II. However, there is limited information about the in vivo expression levels of these molecules and uncoordinated expression has been demonstrated in class II-positive epithelial cells that may influence the MHC-associated peptide repertoires and the outcome of the autoimmune response. We have examined the pool of peptides associated to DR4 molecules expressed by a neuroendocrine epithelial cell and the consequences of Ii and DM coexpression. The RINm5F rat insulinoma cell line was transfected with HLA-DRB1*0401, Ii, and DM molecules in four different combinations: RIN-DR4, -DR4Ii, -DR4DM, and -DR4IiDM. The analysis of the peptide repertoire and the identification of the DR4 naturally processed ligands in each transfected cell were achieved by mass spectrometry. The results demonstrate that 1) the expression of Ii and DM affected the DR4 peptide repertoires by producing important variations in their content and in the origin of peptides; 2) these restrictions affected the stability and sequence of the peptides of each repertoire; and 3) Ii and DM had both independent and coordinate effects on these repertoires.

Animals↗

PubMed2004

The biliverdin chromophore binds covalently to a conserved cysteine residue in the N-terminus of Agrobacterium phytochrome Agp1.

Phytochromes are widely distributed biliprotein photoreceptors. Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction. Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain. For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20. This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven. We therefore performed mass spectrometry studies on proteolytic holopeptide fragments. For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag. Following proteolysis, the chromophore co-purified with a ca. 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus. Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site. We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis. The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome. We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction.

Amino Acid Sequence↗

PubMed2004

Isoform-specific quantification of endothelins in HUVEC culture supernatants by on-line high-performance liquid chromatography/electrospray mass spectrometry.

A method for the quantitative analysis of endothelin peptides in human umbilical vein endothelial cell (HUVEC) culture supernatants is reported. The analysis is isoform-specific and employs solid-phase extraction and subsequent HPLC fractionation followed by HPLC-ESIMS analysis. The peptide vasoactive-intestinal-contractor (VIC) was used as internal standard for the HPLC-ESIMS analysis. Linearity of calibration curves was from 50 fmol to 25 pmol. The limit of detection of the HPLC-ESIMS step using a buffer matrix was estimated at 50 fmol (S/N > 3). The overall limit of detection for supernatants of HUVEC was 500 fmol/mL. In HUVEC culture supernatants only ions of endothelin-1 (ET1) were observed. Basal levels were determined to be 1.8 +/- 0.3 pmol/mL. Quantitative results obtained for ET1 were in agreement with those obtained by using a standard addition method and by an ELISA method.

Amino Acid Sequence↗

PubMed2004

Determination of protein markers in human serum: Analysis of protein expression in toxic oil syndrome studies.

Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. It affected more than 20 000 people and produced over 300 deaths in the first 2 years. In this paper, a prospective study on the differences in gene expression in sera between a control versus a TOS-affected population, both originally exposed to the toxic oil, is presented. Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Several problems related with serum analysis by 2-DE were addressed in order to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins that can be obscured by the presence of a few families of high abundance proteins (albumin, immunoglobulins). Other factors, such as the use of nonionic reductants or the presence of thiourea in the gels, were also tested. From these optimized images, a group of 329 major gel spots was located, matched and compared in serum samples. Thirty-five of these protein spots were found to be under- or overexpressed in TOS patients (> three-fold increase or decrease). Proteins in the differential spots were identified by matrix-assisted laser desorption/ionization-time of flight peptide map fingerprinting and database search. Several haptoglobin isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS affection. Haptoglobin phenotypes have been previously reported to have important biological and clinical consequences and have been described as risk factors for several diseases.

Blood Proteins↗

PubMed2004

The defense response of germinating maize embryos against fungal infection: a proteomics approach.

Pathogen attack on plants results in numerous host-specific biochemical responses, the activation of some of them being critical for the ability of the plant to withstand disease. We have used high-resolution two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify proteins that are differentially expressed in response to fungal infection in maize embryos. Differential spots corresponding to induced or repressed proteins were apparent in silver stained 2-DE gels of proteins extracted from sterile and fungal-infected germinating embryos. Selected spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and nanospray ion-trap tandem mass spectrometry. Among the proteins induced in response to infection are proteins involved in protein synthesis, or in protein folding and stabilization, as well as proteins involved in oxidative stress tolerance. Additionally, the accumulation of specific pathogenesis-related proteins in tissues of the fungal-infected germinating embryos was studied by 2-DE and immunoblotting.

Amino Acid Sequence↗

PubMed2004

Proteomics-based identification of novel Candida albicans antigens for diagnosis of systemic candidiasis in patients with underlying hematological malignancies.

Systemic candidiasis remains a major cause of disease and death, particularly among patients suffering from hematological malignancies. In an attempt to contribute to the discovery of useful biomarkers for its diagnosis and therapeutic monitoring, we embarked on a mapping of Candida albicans immunogenic proteins specifically recognized by antibodies produced during the natural course of systemic Candida infection in this high-risk population. About 85 immunoreactive protein species were detected with systemic candidiasis patients' serum specimens by using immunoproteomics (i.e., two-dimensional electrophoresis followed by Western blotting), and identified through a combination of peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), de novo peptide sequencing using nano-electrospray ionization-ion trap (ESI-IT) MS, and genomic database searches. This proteomic approach has led to the characterization of 42 different housekeeping enzymes as C. albicans antigens. Their biological significance is also discussed. Furthermore, this study is the first to report that 26 of them exhibit antigenic properties in C. albicans, and 35 of them become targets of the human antibody response to systemic candidiasis. Our findings suggest that the production of antibodies to C. albicans phosphoglycerate kinase and alcohol dehydrogenase during systemic candidiasis could be associated with a differentiation of the human immune response. We also highlight the relationship between changes in maintenance of circulating levels of specific anti-Candida antibodies and patients' outcome. Some of these variations, especially the rise of high anti-enolase antibody concentrations, appear to be related to recovery from systemic candidiasis in these patients, which might serve as markers for predicting their outcome. This approach could therefore provide new challenges for diagnosis and clinical follow-up of these fungal infections, and even for antifungal drug or vaccine design.

Adult↗

PubMed2004

Capillary separations.

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Chemistry Techniques, Analytical↗

PubMed2003

Identification of the autoantigen HB as the barrier-to-autointegration factor.

The HB autoantigen, a 10-kDa DNA-binding protein recognized by autoantibodies only when bound to DNA, was identified by two-dimensional electrophoresis. Silver-stained protein spots corresponding to the antigen were excised from two-dimensional electrophoresis gels, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization-reflectron time of flight and nano-electrospray ionization-ion trap/mass spectrometry. Data base search identified the HB antigen as the barrier-to-autointegration factor, a cellular protein implicated in the cellular cycle that blocks autointegration and promotes intermolecular integration of retrovirus such as the Moloney murine leukemia and the human immunodeficiency type 1 virus. The physicochemical characteristics described for these proteins, their ability to bind double-stranded DNA but not single-stranded DNA, and their nuclear localization confirm that HB and barrier-to-autointegration factor are the same protein.

Amino Acid Sequence↗

PubMed2003

Roles of hnRNP A1, SR proteins, and p68 helicase in c-H-ras alternative splicing regulation.

Human ras genes play central roles in coupling extracellular signals with complex intracellular networks controlling proliferation, differentiation, and apoptosis, among others processes. c-H-ras pre-mRNA can be alternatively processed into two mRNAs due to the inclusion or exclusion of the alternative exon IDX; this renders two proteins, p21H-Ras and p19H-RasIDX, which differ only at the carboxy terminus. Here, we have characterized some of the cis-acting sequences and trans-acting factors regulating IDX splicing. A downstream intronic silencer sequence (rasISS1), acting in concert with IDX, negatively regulates upstream intron splicing. This effect is mediated, at least in part, by the binding of hnRNP A1. Depletion and add-back experiments in nuclear extracts have confirmed hnRNP A1's inhibitory role in IDX splicing. Moreover, the addition of two SR proteins, SC35 and SRp40, can counteract this inhibition by strongly promoting the splicing of the upstream intron both in vivo and in vitro. Further, the RNA-dependent helicase p68 is also associated with both IDX and rasISS1 RNA, and suppression of p68 expression in HeLa cells by RNAi experiments results in a marked increase of IDX inclusion in the endogenous mRNA, suggesting a role for this protein in alternative splicing regulation.

Alternative Splicing↗

PubMed2002

HLA-DR4 molecules in neuroendocrine epithelial cells associate to a heterogeneous repertoire of cytoplasmic and surface self peptides.

Expression of MHC class II genes by epithelial cells is induced in inflammatory conditions such as autoimmunity and organ transplantation. Class II ligands generated by the epithelial cell processing mechanisms are unknown, although some unique epitopes have been described in epithelial cells that B cells could not generate. Epithelial cells are the targets of autoreactive T cell responses in autoimmune diseases and of transplant rejection processes, which may involve recognition of cell type-specific epitopes. In the present report, we have compared the DR4-associated repertoire and the intracellular distribution of class II, invariant chain (Ii), and DM molecules between a human DR4-, Ii-, and DM-transfected rat neuroendocrine epithelial cell line and a homozygous DR4 (DRB1*0401) lymphoblastoid B cell line, by mass spectrometry sequencing techniques, and immunoelectron microscopy. The epithelial cells chosen for transfection, RINm5F, are rat insular cells widely used for human studies of autoimmune diabetes. The results revealed a remarkably heterogeneous pool of self protein-derived peptides from the cell surface and various intracellular compartments, including the cytosol and secretory vesicles in epithelial cells, compared with a very restricted homogeneous repertoire in lymphoblastoid B cell lines, where few epitopes from surface molecules were predominant. The generation of distinct DR4-associated peptide repertoires in these two cell types could be due to the effect of several factors including differences in subcellular location of Ii and DM molecules, differential DO expression, and cell type-specific mechanisms of class II ligand generation. This is specially relevant to processes involving epithelial T cell interactions such as organ-specific autoimmunity and transplant rejection.

Amino Acid Sequence↗

PubMed2002

Identification of p21Cip1 binding proteins by gel electrophoresis and capillary liquid chromatography microelectrospray tandem mass spectrometry.

Proteins bound to a glutathione-S-transferase-p21Cip1 affinity column were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified using tandem mass spectrometry. Capillary liquid chromatography coupled to microelectrospray tandem mass spectrometry (capLC-microESI MS/MS) in an ion trap allowed identification of the proteins present in the gel bands. Of eleven bands analyzed, fifty-three proteins were identified. More than one hundred tryptic peptides were detected on-line, automatically fragmented and used for protein characterization in databases. Samples were also analyzed by off-line nanospray and matrix-assisted laser desorption/ionization mass spectrometry. CapLC-microESI MS/MS was the most efficient technique for the analysis of these protein mixtures.

Amino Acid Sequence↗

PubMed2002

rBAT-b(0,+)AT heterodimer is the main apical reabsorption system for cystine in the kidney.

Mutations in the rBAT and b(0,+)AT genes cause type I and non-type I cystinuria, respectively. The disulfide-linked rBAT-b(0,+)AT heterodimer mediates high-affinity transport of cystine and dibasic amino acids (b(0,+)-like activity) in heterologous cell systems. However, the significance of this heterodimer for cystine reabsorption is unknown, as direct evidence for such a complex in vivo is lacking and the expression patterns of rBAT and b(0,+)AT along the proximal tubule are opposite. We addressed this issue by biochemical means. Western blot analysis of mouse and human kidney brush-border membranes showed that rBAT and b(0,+)AT were solely expressed as heterodimers of identical size and that both proteins coprecipitated. Moreover, quantitative immunopurification of b(0,+)AT followed by SDS-PAGE and mass spectrometry analysis established that b(0,+)AT heterodimerizes exclusively with rBAT. Together with cystine reabsorption data, our results demonstrate that a decreasing expression gradient of heterodimeric rBAT-b(0,+)AT along the proximal tubule is responsible for virtually all apical cystine reabsorption. As a corollary of the above, there should be an excess of rBAT expression over that of b(0,+)AT protein in the kidney. Indeed, complete immunodepletion of b(0,+)AT did not coprecipitate >20-30% of rBAT. Therefore, another rBAT-associated subunit may be present in latter parts of the proximal tubule.

Absorption↗