Biomedical subjects

Jesús Vázquez

Publications and source records attributed to Jesús Vázquez.

At least 19 recordsLinked to original sources

PubMed2007

Proteomic and transcriptomic analyses of differential stress/inflammatory responses in mandibular lymph nodes and oropharyngeal tonsils of European wild boars naturally infected with Mycobacterium bovis.

Differential stress/inflammatory responses were characterized at the mRNA and protein levels in mandibular lymph nodes (MLN) and oropharyngeal tonsils of European wild boars (Sus scrofa), naturally infected with Mycobacterium bovis. Suppression-subtractive hybridization combined with immunohistochemistry and/or quantitative real-time RT-PCR were used to identify and characterize abundant stress/inflammatory gene sequences differentially expressed in tuberculous (TB+) wild boars. Genes identified in MLN and tonsils corresponded to serum amyloid A, arginase I, osteopontin, lysozyme, annexin I, and heat shock proteins, respectively. Global protein patterns in MLN and tonsils were compared between TB+ and nontuberculous (TB-) boars by 2-DE and MALDI-TOF MS. Five proteins, including stress/inflammatory proteins annexin V, serum albumin, and apolipoprotein A1 were found at lower levels in MLN of TB+ boars. Manganese superoxide dismutase was found up-regulated in MLN of TB+ boars. Five proteins, including creatine kinase and MHC class II antigens were found up-regulated in tonsils of TB+ boars. These results demonstrated differential stress/inflammatory responses in wild boars naturally infected with M. bovis and suggest possible markers of tuberculosis in this species that may prove useful for future studies of host-pathogen interactions and for diagnostics and vaccine development.

Amino Acid Sequence↗

PubMed2006

Advanced breast cancer: anti-progressive immunotherapy using a thermostable autologous hemoderivative.

INTRODUCTION: Advanced breast cancer patients, acquired-chemotherapy resistant and in progression, are therapeutically terminal. We tested a recently described medical procedure using a thermostable autohemoderivative purported to inhibit tumor growth possibly through an immunological mechanism of action. PATIENTS AND METHODS: Metastatic breast cancer patients, chemotherapy-resistant, high CEA and CA 15-3 plasma levels of tumor markers, in progression, were 2-group randomized. Group 1 received the test procedure and Group 2 adequate measures to be comparable control. From 121 included patients, 108 could be evaluated. During 8-month follow-up period, tumor growth, number of cases attaining clinical non-progressive status and mortality were monthly assessed. Immunologic effect was assessed by delayed type hypersensitivity test and lymphocyte proliferation assay. Responding-tumors histopathologies were studied. The proteome of the autologous immunogen was characterized by 2-D electrophoresis. RESULTS AND DISCUSSION: In a significant number of cases, the test procedure promoted inhibition of tumor growth, non-progressive disease status, and lower cumulative mortality. These clinical results were associated with polyvalent immunization against several tested antigens: the hemoderivative used for treatment, the blood tumor markers and the derivative obtained from a regulatory lymphocyte population (CD4+CD25+). Interference with this regulatory activity could explain the selective autoimmunity suggested by the histopathology findings in responding tumors. The thermostability could be an essential property of the immunogen hemoderivative. CONCLUSION: The thermostable autohemoderivative tested is antigenically polyvalent and promoted a polytargeted immune response associated to a tumor anti-progressive effect, consequently, acting as an autohemoderivative cancer vaccine.

Adult↗

PubMed2006

Blockade of NFAT activation by the second calcineurin binding site.

Activation of NFAT transcription factors requires their dephosphorylation by the phosphatase calcineurin (CN). NFATs contain two CN binding motifs: PxIxIT and CnBP-B/CNBR2 (which we call LxVP). Here we carry out a detailed comparative analysis of the CN binding activity displayed by the PxIxIT and LxVP sites from different NFATs. Dose-response CN binding experiments with GST fusion proteins of NFATc1 and NFATc2 showed that NFATc1 binds CN in vitro more efficiently than does NFATc2. This difference in binding appears to be caused by the different CN binding potencies of the corresponding LxVP sites; thus while the LxVPc2 peptide fused to GST did not bind CN, GST-LxVPc1 bound it more efficiently than did GST-PxIxITc1 or GST-PxIxITc2. Furthermore, an NFATc2 chimera protein containing the LxVP motif from NFATc1 interacted with CN much more potently than did wild-type NFATc2. Free peptides spanning the LxVP motifs from NFATc1, c3 or c4 displaced CN from GST-NFATc1 and GST-NFATc2 more efficiently than any PxIxIT peptide. PxIxITc2 and LxVPc1 peptides were each able to cross-compete GST-LxVPc1-CN and GST-PxIxITc2-CN binding. In contrast with PxIxITc2, the LxVP peptide not only blocked CN-NFAT binding but also inhibited CN phosphatase activity in vitro. Furthermore, exogenous LxVPc1 blocked NFATc2 phosphorylation and nuclear translocation in vivo. These results suggest a model in which the different CN binding characteristics of the PxIxIT and LxVP sites enable different NFAT members to influence each others activities in cells where they are co-expressed.

Amino Acid Motifs↗

PubMed2006

Quantitative proteomics using 16O/18O labeling and linear ion trap mass spectrometry.

Quantitative proteomics using stable isotopic 16O/18O labeling has emerged as a very powerful tool, since it has a number of advantages over other methods, including the simplicity of chemistry, the constant mass tag at the C termini and its general applicability. However, due to the small mass difference between labeled and unlabeled peptide species, this approach has usually been restricted to high-resolution mass spectrometers. In this study we explored whether the high-resolution scanning mode, together with the extremely high scanning speed of the linear IT allows the 16O/18O-labeling method to be used for accurate, large-scale quantitative analysis of proteomes. A protocol, including digestion, desalting, labeling, MS and quantitative analysis was developed and tested using protein standards and whole proteome extracts. Using this method we were able to identify and quantify 140 proteins from only 10 mug of a proteome extract from mesenchymal stem cells. Relative expression changes larger than twofold can be identified with this method at the 95% confidence level. Our results demonstrate that accurate quantitative analysis using 16O/18O labeling can be performed in the practice using linear IT MS, without compromising large-scale peptide identification efficiency.

Cells, Cultured↗

PubMed2006

Systematic characterization of phosphorylation sites in NFATc2 by linear ion trap mass spectrometry.

Members of the nuclear factor of activated T cells (NFAT) family of transcription factors regulate transcription of genes involved in the function of many different cellular systems. Activity of NFAT proteins is regulated by a complex interplay of phosphorylation events that are still poorly understood. In this work, we take advantage of the high scanning speed of the linear ion trap to develop a method to make a systematic characterization of NFATc2 phosphorylation. The method is based on the simultaneous monitoring of all tryptic peptides that can be detected by MS and contain potential phosphorylation sites. By this approach, we detected six NFATc2 phosphorylation sites by c-Jun NH2-terminal kinase (JNK) in vitro in only one experiment; a further site was also identified by performing digestion in solution. Using this approach, we have also characterized five basal phosphorylation sites in NFATc2 protein expressed in HEK cell cultures. Two of these NFATc2 phosphorylation sites in vivo have not been described before. The simplicity and sensitivity of this technique, which can be applied to any potential modification, makes it particularly attractive for the systematic detection of post-translational modifications of specific target proteins both in vitro and in vivo.

Amino Acid Sequence↗

PubMed2006

Identification of commercial hake and grenadier species by proteomic analysis of the parvalbumin fraction.

Analysis of parvalbumin fractions through proteomic methodologies allowed the differential classification of ten commercial, closely related species of the family Merlucciidae. Muscle extracts from nine hake species of the genus Merluccius including two subspecies of Merluccius australis (australis and polylepsis) and one grenadier species Macruronus novaezelandiae with two populations (novaezelandiae and magellanicus) were evaluated by 2-DE and MALDI-TOF MS. 2-DE demonstrated that the species tested displayed a low intra-specific degree of polymorphism and the isoform patterns were noticeably species-specific. MALDI-TOF mass fingerprints showed clear differences in the pattern of peptides produced by tryptic digestion between the Merluccius and the Macruronus, making the genus differentiation possible. In addition, a selective peptide mass present in the spectra from certain hakes allowed its classification in two groups: Euro-African and American hakes. Besides, some specific masses allowed a clearly individual identification for M. bilinearis, M. australis polylepsis, M. australis australis, M. productus, M. paradoxus and M. polli, while the rest of the hake species can be grouped in two clusters, comprising M. hubbsi and M. gayi in one and M. merluccius and M. capensis in the other.

Amino Acid Sequence↗

PubMed2006

Advanced colon cancer: antiprogressive immunotherapy using an autologous hemoderivative.

INTRODUCTION: Advanced colon cancer patients, acquired-chemotherapy resistant and in progression, are therapeutically terminal. We tested a recently described medical procedure using a thermostable autologous hemoderivative purported to inhibit tumor growth possibly through an immunological mechanism of action. PATIENTS AND METHODS: Metastatic colon cancer patients chemotherapy-resistant, high CEA plasma levels, in progression, were 2-group randomized. Group 1 received the test procedure and Group 2 adequate control measures. During an 8-mo follow-up period (n = 101), tumor growth, number of cases attaining clinical nonprogressive status, and mortality were assessed monthly. Immunological effect was assessed by delayed-type hypersensitivity test and lymphocyte proliferation assay. Responding-tumors histopathologies were studied. RESULTS AND DISCUSSION: In a significant number of cases, the test procedure promoted inhibition of tumor growth, nonprogressive disease status, and lower cumulative mortality. These clinical results were associated with polyvalent immunization against several tested antigens: the hemoderivative used for treatment, the blood tumor markers, and the regulatory lymphocyte population (CD4+CD25+). Interference with this regulatory activity could explain the selective autoimmunity suggested by the histopathology findings in responding tumors. CONCLUSION: The autologous hemoderivative tested is antigenically polyvalent and promotes a polytargeted immune response associated with a tumor antiprogressive effect, consequently, acting as an autologous hemoderivative cancer vaccine.

Aged↗

PubMed2005

S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities.

Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated with S-nitroso-l-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation.

Adenosine Triphosphatases↗

PubMed2005

c-Jun N-terminal kinase (JNK) positively regulates NFATc2 transactivation through phosphorylation within the N-terminal regulatory domain.

The nuclear factor of activated T cells (NFAT) family of transcription factors regulates the transcription of cytokine genes and other genes involved in the regulation and function of the immune system. NFAT activity is regulated by the phosphatase calcineurin, which binds and dephosphorylates the NFAT N-terminal regulatory domain, a critical step required for nuclear translocation and transcriptional activity. Here we show that the mitogen-activated protein kinase (MAPK) JNK activates NFATc2-dependent transcription. Mass spectrometry revealed that JNK phosphorylates at least six residues within the NFATc2 regulatory domain in vitro. Transfection of cells with a chimeric construct encoding the GAL-4 DNA binding domain linked to wild-type NFATc2 showed that JNK stimulates the NFATc2 transactivation domain in activated Jurkat T lymphocytes, an effect that is inhibited by dominant-negative versions of JNK. Likewise, the mutation of the phosphorylation sites identified revealed that Thr(116) and Ser(170) are critical for the transactivation of NFATc2 by JNK. In addition, clustered mutation of the SP-conserved motifs of NFATc2 showed that SP1 and SP2, but not SP3, are also important for the inducible transactivation of NFATc2. Furthermore, mass spectrometry analysis of NFATc2-transfected cells indicated that the activation of the JNK pathway results in the in vivo phosphorylation of Thr(116). Our results indicate that, unlike other NFAT members, the transcriptional activity of NFATc2 is up-regulated by JNK. JNK-mediated phosphorylation of NFATs thus appears to play a differential physiological role among NFAT family members.

Amino Acid Sequence↗

PubMed2004

Statistical model for large-scale peptide identification in databases from tandem mass spectra using SEQUEST.

Recent technological advances have made multidimensional peptide separation techniques coupled with tandem mass spectrometry the method of choice for high-throughput identification of proteins. Due to these advances, the development of software tools for large-scale, fully automated, unambiguous peptide identification is highly necessary. In this work, we have used as a model the nuclear proteome from Jurkat cells and present a processing algorithm that allows accurate predictions of random matching distributions, based on the two SEQUEST scores Xcorr and DeltaCn. Our method permits a very simple and precise calculation of the probabilities associated with individual peptide assignments, as well as of the false discovery rate among the peptides identified in any experiment. A further mathematical analysis demonstrates that the score distributions are highly dependent on database size and precursor mass window and suggests that the probability associated with SEQUEST scores depends on the number of candidate peptide sequences available for the search. Our results highlight the importance of adjusting the filtering criteria to discriminate between correct and incorrect peptide sequences according to the circumstances of each particular experiment.

Chromatography, Liquid↗

PubMed2003

Insulin-induced enhancement of antitumoral response to methotrexate in breast cancer patients.

PURPOSE: It has been reported that insulin increases the cytotoxic effect in vitro of methotrexate by as much as 10,000-fold. The purpose of this study was to explore the clinical value of insulin as a potentiator of methotrexate. PATIENTS AND METHODS: Included in this prospective, randomized clinical trial were 30 women with metastatic breast cancer resistant to fluorouracil + Adriamycin + cyclophosphamide and also resistant to hormone therapy with measurable lesions. Three groups each of ten patients received two 21-day courses of the following treatments: insulin + methotrexate, methotrexate, and insulin, respectively. In each patient, the size of the target tumor was measured before and after treatment according to the Response Evaluation Criteria In Solid Tumors. The changes in the size of the target tumor in the three groups were compared statistically. RESULTS: Under the trial conditions, the methotrexate-treated group and the insulin-treated group responded most frequently with progressive disease. The group treated with insulin + methotrexate responded most frequently with stable disease. The median increase in tumor size was significantly lower with insulin + methotrexate than with each drug used separately. DISCUSSION: Our results confirmed in vivo the results of previous in vitro studies showing clinical evidence that insulin potentiates methotrexate under conditions where insulin alone does not promote an increase in tumor growth. Therefore, the chemotherapy antitumoral activity must have been enhanced by the biochemical events elicited in tumor cells by insulin. CONCLUSIONS: In multidrug-resistant metastatic breast cancer, methotrexate + insulin produced a significant antitumoral response that was not seen with either methotrexate or insulin used separately.

Adult↗

PubMed2003

Specific interaction of heterogeneous nuclear ribonucleoprotein A1 with the -219T allelic form modulates APOE promoter activity.

The polymorphic -219T/G variant in the APOE promoter has been associated with variations in basal transcriptional activity as well as with the risk of developing Alzheimer's disease, myocardial infarction and early-onset coronary heart disease. The molecular mechanisms underlying these effects are presently unknown. In this report, we show that nuclear extracts from Jurkat cells form a T-specific complex with a motif including the -219 site within the APOE promoter. By DNA-affinity chromatography and mass spectrometry, the human heterogeneous nuclear ribonucleoprotein hnRNPA1(A1) was identified as one component of the complex. In vitro binding analysis indicated that a fragment of A1 had a marked binding specificity for the T form. Interaction of A1 with this region is driven by an adjacent telomeric-like sequence; however, the presence of G, but not T, at -219 position inhibited this interaction. The differences in transcriptional activity between the -219T and -219G promoter allelic forms correlated with the expression levels of A1 in several cell lines; also, over-expression of A1 increased the activity of the T form relative to that of the G form. These results indicate that A1 transactivates APOE promoter activity by direct and specific interaction with the -219T site.

Alleles↗

PubMed2003

Peptide rearrangement during quadrupole ion trap fragmentation: added complexity to MS/MS spectra.

The emergence of proteomics has placed great interest in the understanding of the mechanisms of MS/MS fragmentation of peptides under low-energy collision-induced dissociation. In this work, we describe the presence of anomalous fragments, which correspond to neutral loss elimination of internal amino acids from ions of the b series in quadrupole ion trap MS/MS spectra from naturally occurring peptides. Internal amino acid elimination occurred preferentially with aliphatic amino acids. The phenomenon was more apparent when doubly charged precursors were fragmented and was inhibited when peptides were N-acetylated at the N-terminus. Fragmentation of isomeric peptides where some internal amino acids were relocated in N-terminal position produced MSn spectra indistinguishable from those of the original peptides, indicating that some b ions underwent a structural rearrangement process. Formation of anomalous fragments required a minimum activation time. Our data are consistent with a nucleophile attack of the N-terminal nitrogen over the electrophilic carbonyl carbon at one peptide bond, forming a cyclic b ion intermediate that, by reopening at preferential sites, exposes internal amino acids to the C-terminal side.

Cloning, Molecular↗

PubMed2003

Transcriptional activation by AP-2alpha is modulated by the oncogene DEK.

Cell differentiation and development are highly regulated processes at the transcriptional level. One of the main transcription factors that regulate these processes is AP-2alpha, a cell-type specific protein required for vertebrate development and embryogenesis. AP-2alpha also regulates apoptosis and cell-cycle specific events by interacting with the oncogene c-Myc. In searching for novel AP-2alpha- interacting factors, using an affinity chromatography approach, we have observed that oncoprotein DEK interacts with AP-2alpha in vitro. The existence of an interaction between AP-2alpha and DEK in cellular cultures was demonstrated by expression of a tagged AP-2alpha form followed by immunodetection. By transient co-expression experiments using a reporter for APOE promoter activity we have found that DEK stimulates the transactivation activity of AP-2alpha over APOE promoter. Finally, electrophoretic mobility shift assays suggested that DEK enhances the DNA-binding activity of AP-2alpha. Our data suggest a novel cellular function of DEK as a transcriptional co-activator.

Apolipoproteins E↗

PubMed2003

Qualitative colorimetric tests for solid phase synthesis.

Results obtained in the application of these tests are summarized in Table II. We have encountered some variations in the reproducibility and accuracy of some tests. Due to the numerous factors that can influence colorimetric test results (e.g., test reagent stability, resin type, functional group interference, and lability of protecting group) we highly recommend performing a positive and a negative control for any test applied to a new synthesis. We also emphasize the importance of reagent solution purity on the outcome of test results, hence we strongly encourage the use of correctly prepared and carefully stored reactants. To minimize false results due to lability of the resin-product bond or the product itself (such as Fmoc-protected amino acids), colorimetric tests should be performed with the utmost immediacy in regards to completion of the step to be monitored. Hence the storage of resin over long periods of time (more than 24 h) before testing is not advisable. When the result of a colorimetric test is in doubt we advise repeating the test a few times until a reproducible result is obtained. The use of multiple tests for the same functional group may elucidate ambiguous or otherwise challenging cases.

Alcohols↗

PubMed2003

Serum markers variation consistent with autoschizis induced by ascorbic acid-menadione in patients with prostate cancer.

In vitro exposure of malignant prostate cell lines to ascorbic acid-menadione showed that tumor cells were killed through a mechanism named autoschizis. Experimental animal studies showed that autoschizis is also evident when ascorbic acid-menadione is administered to nude mice with implanted human prostate tumors. Prostate-specific antigen (PSA) is a known serum marker of prostate tumor cells specific activity. Recently, total serum homocysteine has been identified as a marker of tumor cell numbers with sensitivity for early detection of tumor cell death induced by treatments. A clinical trial with prostate cancer patients submitted to the association of ascorbic acid-menadione was performed and PSA/homocysteine was assessed in the follow- up. The early response in serum PSA and homocysteine levels was reported. The results showed that ascorbic acid-menadione produced an immediate drop in tumor cell numbers as assessed by homocysteine levels. Serum PSA levels showed an early rise and later dropped. These results suggest that autoschizis can also be induced by this pharmacological association at the clinical level in prostate cancer patients. Further studies are being performed in order to research if these results can be found with other primary tumors as it was shown in in vitro and experimental models. Ascorbic acid-menadione could be emerging as a new antitumoral chemotherapy.

Aged↗

PubMed2002

Reconstitution of holin activity with a synthetic peptide containing the 1-32 sequence region of EJh, the EJ-1 phage holin.

Pneumococcal EJ-1 phage holin (EJh) is a hydrophobic polypeptide of 85 amino acid residues displaying lethal inner membrane disruption activity. To get an insight into holin structure and function, several peptides representing the different topological regions predicted by sequence analysis have been synthesized. Peptides were structurally characterized in both aqueous buffer and membrane environments, and their potential to induce membrane perturbation was determined. Among them, only the N-terminal predicted transmembrane helix increased the membrane permeability. This segment, only when flanked by the positive charged residues on its N-terminal side, which are present in the sequence of the full-length protein, folds into a major alpha-helix structure with a transmembrane preferential orientation. Fluorescein quenching experiments of N-terminal-labeled peptide evidenced the formation of oligomers of variable size depending on the peptideto-lipid molar ratio. The self-assembling tendency correlated with the formation of transmembrane pores that permit the release of encapsulated dextrans of various sizes. When analyzed by atomic force microscopy, peptide-induced membrane lesions are visualized as transbilayer holes. These findings are the first evidence for a lytic domain in holins and for the nature of membrane lesions caused by them.

Amino Acid Sequence↗

PubMed2002

Cutting edge: association of the motor protein nonmuscle myosin heavy chain-IIA with the C terminus of the chemokine receptor CXCR4 in T lymphocytes.

The binding of chemokines to their receptors guides lymphocyte migration. However, the precise mechanism that links the chemotactic signals with the energy and traction force generated by the actomyosin complex of the cell has not been elucidated. Using biochemical approaches and mass spectrometry analysis, we found an association between the C-termini of CXCR4 and CCR5 and the motor protein nonmuscle myosin H chain-IIA. Immunoprecipitation experiments revealed that this association also occurs between the endogenous molecules in T lymphocytes. As expected, myosin L chain was also associated with CXCR4. Confocal microscopy analysis showed that CXCR4 and motor protein nonmuscle myosin H chain-IIA colocalize at the leading edge of migrating T lymphocytes, together with filamentous actin and myosin L chain. These results provide the first evidence of a biochemical association between chemokine receptors and motor proteins, a mechanosignaling mechanism that may have a key role in lymphocyte migration.

Actins↗