Felix Gil - Academia.edu (original) (raw)

Papers by Felix Gil

Journal of Industrial Microbiology & Biotechnology, 2008

A recombinant form of the peptide N-terminally positioned from proSP-B (SP-B N) has been produced... more A recombinant form of the peptide N-terminally positioned from proSP-B (SP-B N) has been produced in Escherichia coli as fusion with the Maltose Binding Protein, separated from it by Factor Xa cleavage and puriWed thereafter. This protein module is thought to control assembly of mature SP-B, a protein essential for respiration, in pulmonary surfactant as it progress through the progressively acidiWed secretory pathway of pneumocytes. Selfaggregation studies of the recombinant propeptide have been carried out as the pH of the medium evolved from neutral to moderately acid, again to neutral and Wnally basic. The proWle of aggregation versus subsequent changes in pH showed diVerences depending on the ionic strength of the medium, low or moderate, and the presence of additives such as L-arginine (a known aggregation suppressor) and Ficoll 70 (a macromolecular crowder). Circular dichroism studies of SP-B N samples along the aggregation process showed a decrease in-helical content and a concomitant increase in-sheet. Intrinsic Xuorescence emission of SP-B N was dominated by the emission of Trp residues in neutral medium, being its emission maximum shifted to red at low pH, suggesting that the protein undergoes a pH-dependent conformational change that increases the exposure of their Trp to the environment. A marked increase in the Xuorescence emission of the extrinsic probe bis-ANS indicated the exposure of hydrophobic regions of SP-B N at pH 5. The Xuorescence of bis-ANS decreased slightly at low ionic strength, but to a great extent at moderate ionic strength when the pH was reversed to neutrality, suggesting that self-aggregation properties of the SP-B N module could be tightly modulated by the conditions of pH and the ionic environment encountered by pulmonary surfactant during assembly and secretion.

International Archives of Allergy and Immunology, 2010

Background: Plant profilins have been reported as minor allergens. They are a well-known pan-alle... more Background: Plant profilins have been reported as minor allergens. They are a well-known pan-allergen family responsible for cross-reactivity between plant-derived foods and pollens. Watermelon profilin has been reported to be a major allergen in watermelon (Citrullus lanatus). The aim of this study was to characterize recombinant watermelon profilin, confirming its reactivity for diagnostic purposes and the development of immunotherapy. Methods: Native profilin was purified from watermelon extract by affinity chromatography using poly-L-proline. Recombinant His-tagged profilin was produced in Pichia pastoris yeast using pPICZ ␣ A vector and purified by metal chelate affinity chromatography. ELISA and immunoblot were carried out with sera from 17 watermelonallergic patients. Biological activity was tested by the basophil activation test. Results: Native profilin and recombinant profilin were purified and identified by mass spectrometry. Both show similar IgE reactivity in vitro and are biologically active. Conclusions: Similarities were found in the IgE-binding patterns and biological activity of recombinant profilin and native profilin. Recombinant profilin may be a powerful tool for specific diagnosis.

Expert Review of Proteomics, 2009

Atherosclerosis is a disease with higher levels of mortality in developed countries. Comprehensio... more Atherosclerosis is a disease with higher levels of mortality in developed countries. Comprehension of the molecular mechanisms can yield very useful information in clinics for prevention, diagnosis and recovery monitoring. Proteomics represents an ideal methodology for this purpose, as proteins constitute the effectors of the different biological processes running during pathogenesis. To date, studies in atherosclerosis have been mainly focused on the search for plasma biomarkers. However, tissue proteomics allows going deeper into tissue secretomes, arterial layers or particular cells of interest, which, in turn, constitutes a more direct approximation to in vivo operating mechanisms. The aim of this review is to report latest advances in tissue proteomics in atherosclerosis and related diseases (e.g., aortic stenosis and ischemic injury).

Clinical Medicine …, 2010

Calcified aortic valve disease is a slowly progressive disorder that ranges from mild valve thick... more Calcified aortic valve disease is a slowly progressive disorder that ranges from mild valve thickening with no obstruction of blood flow, known as aortic sclerosis, to severe calcification with impaired leaflet motion or aortic stenosis. In the present work we describe a rapid, reproducible ...

Rev Esp Cardiol, 2010

... b ; Luis F. López-Almodovar c ; Rocío Juárez-Tosina d ; Fernando de la Cuesta e ; Gloria Álva... more ... b ; Luis F. López-Almodovar c ; Rocío Juárez-Tosina d ; Fernando de la Cuesta e ; Gloria Álvarez-Llamas f ; Sergio Alonso-Orgaz g ... A continuación los geles se tiñeron con plata (Silver staining protein with Ampholine PAGplate de Pharmacia Biotech, GE Healthcare) o con azul ...

Revista Española de …, 2010

For many years, degenerative aortic stenosis was thought to be a passive process secondary to cal... more For many years, degenerative aortic stenosis was thought to be a passive process secondary to calcium deposition in aortic valves. Although its etiology remains unknown, several authors have pointed out that degenerative aortic stenosis is associated with the same risk factors as coronary artery disease. Furthermore, histological similarities have been found between aortic valve stenosis and atherosclerotic plaque, giving rise to the hypothesis that degenerative aortic stenosis is an inflammatory process similar to atherosclerosis. Nevertheless, some data do not fit with this hypothesis and, consequently, greater understanding of the condition is needed. The main aim of this study was to develop a practical protocol for extracting protein for use in proteomic analysis from both stenotic and healthy aortic valves. The study was carried out using a number of different proteomic methods: two-dimensional electrophoresis, mass spectrometry and additional techniques. We developed a simple and reproducible methodology in the laboratory for carrying out the proteomic analysis of human aortic valves and for identifying their component proteins. We developed a simple and reproducible method for extracting protein that can be used with mass spectrometry and that makes it possible to carry out large-scale proteomic analysis of stenotic aortic valves. Furthermore, the methodology will significantly increase our understanding of the valve proteome.

Archives of Virology, 2000

Transgenic plants represent an inexpensive alternative to classical fermentation systems for prod... more Transgenic plants represent an inexpensive alternative to classical fermentation systems for production of recombinant subunit vaccines. Transgenic potato plants were created that express the N-terminal domain of the glycoprotein S (N-gS) from Transmissible gastroenteritis coronavirus (TGEV), containing the major antigenic sites of the protein. Extracts from potato tubers expressing N-gS were inoculated intraperitoneally to mice, and the vaccinated mice developed serum IgG specific for TGEV. Furthermore, when potato tubers expressing N-gS were fed directly to mice, they developed serum antibodies specific for gS protein, demonstrating the oral immunogenicity of the plant derived spike protein from TGEV. * Transmissible gastroenteritis coronavirus (TGEV) is the causative agent of acute diarrhea of newborn piglets that provokes high rate mortalities in affected farms. Protective immunity against this disease has to be developed in pregnant sows in order to confer passive protection to the piglets trough colostrum and milk. Neutralizing antibodies against the virus are directed mainly to glycoprotein S , and relevant epitopes in neutralization have been mapped into the Nterminal domain of this protein . Four major antigenic sites have been described in the globular part of glycoprotein S (gS), of which site A is the immunodominant .

FEBS letters, 2001

1. FEBS Lett. 2001 Jan 12;488(1-2):13-7. High-yield expression of a viral peptide vaccine in tran... more 1. FEBS Lett. 2001 Jan 12;488(1-2):13-7. High-yield expression of a viral peptide vaccine in transgenic plants. Gil F, Brun A, Wigdorovitz A, Catalá R, Martínez-Torrecuadrada JL, Casal I, Salinas J, Borca MV, Escribano JM. Departamento ...

Vaccine, 2002

The expression of antigens in transgenic plants has been increasingly used as an alternative to t... more The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the ...

Aortic Stenosis - Etiology, Pathophysiology and Treatment, 2011

Expert Review of Proteomics, 2012

Stroke is one of the most common causes of death worldwide and a major cause of acquired disabili... more Stroke is one of the most common causes of death worldwide and a major cause of acquired disability in adults. Despite advances in research during the last decade, prevention and treatment strategies still suffer from significant limitations, and therefore new theoretical and technical approaches are required. Technological advances in the proteomic and metabolomic areas, during recent years, have permitted a more effective search for novel biomarkers and therapeutic targets that may allow for effective risk stratification and early diagnosis with subsequent rapid treatment. This review provides a comprehensive overview of the latest candidate proteins and metabolites proposed as new potential biomarkers in stroke.

Journal of Proteome Research, 2012

Degenerative aortic stenosis is the most common worldwide cause of valve replacement. While it sh... more Degenerative aortic stenosis is the most common worldwide cause of valve replacement. While it shares certain risk factors with coronary artery disease, it is not delayed or reversed by reducing exposure to risk factors (e.g., therapies that lower lipids). Therefore, it is necessary to better understand its pathophysiology for preventive measures to be taken. In this work, aortic valve samples were collected from 20 patients that underwent aortic valve replacement (55% males, mean age of 74 years) and 20 normal control valves were obtained from necropsies (40% males, mean age of 69 years). The proteome of the samples was analyzed by quantitative differential electrophoresis (2D-DIGE) and mass spectrometry, and 35 protein species were clearly increased in aortic valves, including apolipoprotein AI, alpha-1-antitrypsin, serum albumin, lumican, alfa-1-glycoprotein, vimentin, superoxide dismutase Cu−Zn, serum amyloid P-component, glutathione S-transferase-P, fatty acid-binding protein, transthyretin, and fibrinogen gamma. By contrast, 8 protein species were decreased (transgelin, haptoglobin, glutathione peroxidase 3, HSP27, and calreticulin). All of the proteins identified play a significant role in cardiovascular processes, such as fibrosis, homeostasis, and coagulation. The significant changes observed in the abundance of key cardiovascular proteins strongly suggest that they can be involved in the pathogenesis of degenerative aortic stenosis. Further studies are warranted to better understand this process before we can attempt to modulate it.

Journal of Biotechnology, Feb 20, 2007

Previous literature addressing the production of recombinant proteins in heterologous systems has... more Previous literature addressing the production of recombinant proteins in heterologous systems has consistently shown that proteins capable of forming complex structures tend to accumulate within host cells at relatively higher levels than monomeric forms. In this report, we translationally fused a 21-aminoacids long highly immunogenic peptide (2L21), derived from canine parvovirus (CPV) VP2 protein to a 41-aminoacid long tetramerization domain (TD) from the transcriptional factor p53. The chimerical DNA construction 2L21-TD was cloned in a binary plant transformation vector and used to transform Arabidopsis thaliana plants. Fifteen of the 25 transgenic lines obtained in the experiment showed detectable 2L21-TD RNA accumulation and from these we chose 4 to study 2L21-TD protein accumulation. Non-denaturing immunoblotting assays revealed that 2L21-TD chimeras effectively formed tetrameric complexes with yields reaching up to 12 g/mg of soluble protein. Mice immunized by oral or intraperitoneal routes with crude protein extracts containing 2L21-TD protein were able to detect both 2L21-synthetic peptide and CPV VP2 proteins, with titers similar to those elicited by a previously reported fusion between 2L21 and the ␤-glucuronidase protein. These results demonstrate that multimerization directed by the small TD domain contributed to the stabilization and consequently to the accumulation of the 2L21 peptide in transgenic plants, without altering its native antigenicity and immunogenicity.

Atherosclerosis Supplements, 2010

Current Research Topics in Applied Microbiology and Microbial Biotechnology, 2009

Current Research Topics in Applied Microbiology and Microbial Biotechnology, 2009

Biomarker insights, 2009

Since the function of the spinal cord depends on the proteins found there, better defing the norm... more Since the function of the spinal cord depends on the proteins found there, better defing the normal Spinal Cord Proteome is an important and challenging task. Although brain and cerebrospinal fluid samples from patients with different central nervous system (CNS) disorders have been studied, a thorough examination of specific spinal cord proteins and the changes induced by injury or associated to conditions such as neurodegeneration, spasticity and neuropathies has yet to be performed. In the present study, we aimed to describe total protein content in the spinal cord of healthy rats, employing different proteomics tools. Accordingly, we have developed a fast, easy, and reproducible sequential protocol for protein extraction from rat spinal cords. We employed conventional two dimensional electrophoresis (2DE) in different pH ranges (eg. 4-7, 3-11 NL) combined with identification by mass spectrometry (MALDI-TOF/TOF), as well as first dimension protein separation combined with Liquid ...

Journal of proteomics, Jan 23, 2014

Acute myocardial infarction with ST-segment elevation (STEMI) initiates with intraluminal thrombo... more Acute myocardial infarction with ST-segment elevation (STEMI) initiates with intraluminal thrombosis and results in total occlusion of the coronary artery. To date, characterization of the coronary thrombus proteome in STEMI patients has not been yet accomplished. Therefore, we aimed to perform an in-depth proteomic characterization of the human coronary thrombus by means of three different approaches: 2-DE followed by mass spectrometry (MALDI MS/MS), 1-DE combined either with liquid chromatography coupled to mass spectrometry in a MALDI TOF/TOF (LC-MALDI-MS/MS), or in a LTQ-Orbitrap (LC-ESI-MS/MS). This approach allowed us to identify a total of 708 proteins in the thrombus. Expression in coronary thrombi (n=20) of 14 proteins was verified, and the expression of fibrin and 6 cell markers (platelets, monocytes, neutrophils, eosinophils, T-cells and B-cells) quantified by selected reaction monitoring (SRM). A positive correlation of 5 proteins (fermitin homolog 3, thrombospondin-1, m...

Vaccine, 2002

The expression of antigens in transgenic plants has been increasingly used as an alternative to t... more The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the recombinant antigens in the plant tissues, which reduces the possibilities of practical applications. Because the site of insertion of the transferred DNA into the cellular chromosomal DNA is at random, different levels of foreign protein expression in independent transformants is expected. Strategies to allow the evaluation of a high number of the transgenic individuals, usually an expensive and very time consuming process, would permit the selection of those plants presenting the highest levels of recombinant protein expression. Here, we present the development of an experimental immunogen based in the expression of a highly immunogenic epitope from foot and mouth disease virus (FMDV) fused to the glucuronidase (gus A) reporter gene, which allows selection of the transgenic plants by the ß-glucuronidase (ßGUS) enzymatic activity. We produced transgenic plants of alfalfa expressing the immunogenic site between amino acid residues 135-160 of structural protein VP1 (VP135-160), fused to the ßGUS protein. Plants expressing the highest levels of the immunogenic epitope VP135-160, analyzed by Western blot, were efficiently selected based on their levels of ßGUS enzymatic activity. The FMDV epitope expressed in plants was highly immunogenic in mice which developed, after immunization, a strong anti-FMDV antibody response against a synthetic peptide representing the region VP135-160, to native virus VP1, and to purified FMDV particles. Additionally, these mice were completely protected against experimental challenge with the virulent virus. To our knowledge, this constitutes the first report of a peptide-based vaccine produced in transgenic plants that induces a protective immune response when used in experimental hosts. Also, these results demonstrated the possibility of using a novel and simple methodology for obtaining transgenic plants expressing high levels of foreign immunogenic epitopes, which could be directly applied in the development of plant-based vaccines.

Journal of Industrial Microbiology & Biotechnology, 2008

A recombinant form of the peptide N-terminally positioned from proSP-B (SP-B N) has been produced... more A recombinant form of the peptide N-terminally positioned from proSP-B (SP-B N) has been produced in Escherichia coli as fusion with the Maltose Binding Protein, separated from it by Factor Xa cleavage and puriWed thereafter. This protein module is thought to control assembly of mature SP-B, a protein essential for respiration, in pulmonary surfactant as it progress through the progressively acidiWed secretory pathway of pneumocytes. Selfaggregation studies of the recombinant propeptide have been carried out as the pH of the medium evolved from neutral to moderately acid, again to neutral and Wnally basic. The proWle of aggregation versus subsequent changes in pH showed diVerences depending on the ionic strength of the medium, low or moderate, and the presence of additives such as L-arginine (a known aggregation suppressor) and Ficoll 70 (a macromolecular crowder). Circular dichroism studies of SP-B N samples along the aggregation process showed a decrease in-helical content and a concomitant increase in-sheet. Intrinsic Xuorescence emission of SP-B N was dominated by the emission of Trp residues in neutral medium, being its emission maximum shifted to red at low pH, suggesting that the protein undergoes a pH-dependent conformational change that increases the exposure of their Trp to the environment. A marked increase in the Xuorescence emission of the extrinsic probe bis-ANS indicated the exposure of hydrophobic regions of SP-B N at pH 5. The Xuorescence of bis-ANS decreased slightly at low ionic strength, but to a great extent at moderate ionic strength when the pH was reversed to neutrality, suggesting that self-aggregation properties of the SP-B N module could be tightly modulated by the conditions of pH and the ionic environment encountered by pulmonary surfactant during assembly and secretion.

International Archives of Allergy and Immunology, 2010

Background: Plant profilins have been reported as minor allergens. They are a well-known pan-alle... more Background: Plant profilins have been reported as minor allergens. They are a well-known pan-allergen family responsible for cross-reactivity between plant-derived foods and pollens. Watermelon profilin has been reported to be a major allergen in watermelon (Citrullus lanatus). The aim of this study was to characterize recombinant watermelon profilin, confirming its reactivity for diagnostic purposes and the development of immunotherapy. Methods: Native profilin was purified from watermelon extract by affinity chromatography using poly-L-proline. Recombinant His-tagged profilin was produced in Pichia pastoris yeast using pPICZ ␣ A vector and purified by metal chelate affinity chromatography. ELISA and immunoblot were carried out with sera from 17 watermelonallergic patients. Biological activity was tested by the basophil activation test. Results: Native profilin and recombinant profilin were purified and identified by mass spectrometry. Both show similar IgE reactivity in vitro and are biologically active. Conclusions: Similarities were found in the IgE-binding patterns and biological activity of recombinant profilin and native profilin. Recombinant profilin may be a powerful tool for specific diagnosis.

Expert Review of Proteomics, 2009

Atherosclerosis is a disease with higher levels of mortality in developed countries. Comprehensio... more Atherosclerosis is a disease with higher levels of mortality in developed countries. Comprehension of the molecular mechanisms can yield very useful information in clinics for prevention, diagnosis and recovery monitoring. Proteomics represents an ideal methodology for this purpose, as proteins constitute the effectors of the different biological processes running during pathogenesis. To date, studies in atherosclerosis have been mainly focused on the search for plasma biomarkers. However, tissue proteomics allows going deeper into tissue secretomes, arterial layers or particular cells of interest, which, in turn, constitutes a more direct approximation to in vivo operating mechanisms. The aim of this review is to report latest advances in tissue proteomics in atherosclerosis and related diseases (e.g., aortic stenosis and ischemic injury).

Clinical Medicine …, 2010

Calcified aortic valve disease is a slowly progressive disorder that ranges from mild valve thick... more Calcified aortic valve disease is a slowly progressive disorder that ranges from mild valve thickening with no obstruction of blood flow, known as aortic sclerosis, to severe calcification with impaired leaflet motion or aortic stenosis. In the present work we describe a rapid, reproducible ...

Rev Esp Cardiol, 2010

... b ; Luis F. López-Almodovar c ; Rocío Juárez-Tosina d ; Fernando de la Cuesta e ; Gloria Álva... more ... b ; Luis F. López-Almodovar c ; Rocío Juárez-Tosina d ; Fernando de la Cuesta e ; Gloria Álvarez-Llamas f ; Sergio Alonso-Orgaz g ... A continuación los geles se tiñeron con plata (Silver staining protein with Ampholine PAGplate de Pharmacia Biotech, GE Healthcare) o con azul ...

Revista Española de …, 2010

For many years, degenerative aortic stenosis was thought to be a passive process secondary to cal... more For many years, degenerative aortic stenosis was thought to be a passive process secondary to calcium deposition in aortic valves. Although its etiology remains unknown, several authors have pointed out that degenerative aortic stenosis is associated with the same risk factors as coronary artery disease. Furthermore, histological similarities have been found between aortic valve stenosis and atherosclerotic plaque, giving rise to the hypothesis that degenerative aortic stenosis is an inflammatory process similar to atherosclerosis. Nevertheless, some data do not fit with this hypothesis and, consequently, greater understanding of the condition is needed. The main aim of this study was to develop a practical protocol for extracting protein for use in proteomic analysis from both stenotic and healthy aortic valves. The study was carried out using a number of different proteomic methods: two-dimensional electrophoresis, mass spectrometry and additional techniques. We developed a simple and reproducible methodology in the laboratory for carrying out the proteomic analysis of human aortic valves and for identifying their component proteins. We developed a simple and reproducible method for extracting protein that can be used with mass spectrometry and that makes it possible to carry out large-scale proteomic analysis of stenotic aortic valves. Furthermore, the methodology will significantly increase our understanding of the valve proteome.

Archives of Virology, 2000

Transgenic plants represent an inexpensive alternative to classical fermentation systems for prod... more Transgenic plants represent an inexpensive alternative to classical fermentation systems for production of recombinant subunit vaccines. Transgenic potato plants were created that express the N-terminal domain of the glycoprotein S (N-gS) from Transmissible gastroenteritis coronavirus (TGEV), containing the major antigenic sites of the protein. Extracts from potato tubers expressing N-gS were inoculated intraperitoneally to mice, and the vaccinated mice developed serum IgG specific for TGEV. Furthermore, when potato tubers expressing N-gS were fed directly to mice, they developed serum antibodies specific for gS protein, demonstrating the oral immunogenicity of the plant derived spike protein from TGEV. * Transmissible gastroenteritis coronavirus (TGEV) is the causative agent of acute diarrhea of newborn piglets that provokes high rate mortalities in affected farms. Protective immunity against this disease has to be developed in pregnant sows in order to confer passive protection to the piglets trough colostrum and milk. Neutralizing antibodies against the virus are directed mainly to glycoprotein S , and relevant epitopes in neutralization have been mapped into the Nterminal domain of this protein . Four major antigenic sites have been described in the globular part of glycoprotein S (gS), of which site A is the immunodominant .

FEBS letters, 2001

1. FEBS Lett. 2001 Jan 12;488(1-2):13-7. High-yield expression of a viral peptide vaccine in tran... more 1. FEBS Lett. 2001 Jan 12;488(1-2):13-7. High-yield expression of a viral peptide vaccine in transgenic plants. Gil F, Brun A, Wigdorovitz A, Catalá R, Martínez-Torrecuadrada JL, Casal I, Salinas J, Borca MV, Escribano JM. Departamento ...

Vaccine, 2002

The expression of antigens in transgenic plants has been increasingly used as an alternative to t... more The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the ...

Aortic Stenosis - Etiology, Pathophysiology and Treatment, 2011

Expert Review of Proteomics, 2012

Stroke is one of the most common causes of death worldwide and a major cause of acquired disabili... more Stroke is one of the most common causes of death worldwide and a major cause of acquired disability in adults. Despite advances in research during the last decade, prevention and treatment strategies still suffer from significant limitations, and therefore new theoretical and technical approaches are required. Technological advances in the proteomic and metabolomic areas, during recent years, have permitted a more effective search for novel biomarkers and therapeutic targets that may allow for effective risk stratification and early diagnosis with subsequent rapid treatment. This review provides a comprehensive overview of the latest candidate proteins and metabolites proposed as new potential biomarkers in stroke.

Journal of Proteome Research, 2012

Degenerative aortic stenosis is the most common worldwide cause of valve replacement. While it sh... more Degenerative aortic stenosis is the most common worldwide cause of valve replacement. While it shares certain risk factors with coronary artery disease, it is not delayed or reversed by reducing exposure to risk factors (e.g., therapies that lower lipids). Therefore, it is necessary to better understand its pathophysiology for preventive measures to be taken. In this work, aortic valve samples were collected from 20 patients that underwent aortic valve replacement (55% males, mean age of 74 years) and 20 normal control valves were obtained from necropsies (40% males, mean age of 69 years). The proteome of the samples was analyzed by quantitative differential electrophoresis (2D-DIGE) and mass spectrometry, and 35 protein species were clearly increased in aortic valves, including apolipoprotein AI, alpha-1-antitrypsin, serum albumin, lumican, alfa-1-glycoprotein, vimentin, superoxide dismutase Cu−Zn, serum amyloid P-component, glutathione S-transferase-P, fatty acid-binding protein, transthyretin, and fibrinogen gamma. By contrast, 8 protein species were decreased (transgelin, haptoglobin, glutathione peroxidase 3, HSP27, and calreticulin). All of the proteins identified play a significant role in cardiovascular processes, such as fibrosis, homeostasis, and coagulation. The significant changes observed in the abundance of key cardiovascular proteins strongly suggest that they can be involved in the pathogenesis of degenerative aortic stenosis. Further studies are warranted to better understand this process before we can attempt to modulate it.

Journal of Biotechnology, Feb 20, 2007

Previous literature addressing the production of recombinant proteins in heterologous systems has... more Previous literature addressing the production of recombinant proteins in heterologous systems has consistently shown that proteins capable of forming complex structures tend to accumulate within host cells at relatively higher levels than monomeric forms. In this report, we translationally fused a 21-aminoacids long highly immunogenic peptide (2L21), derived from canine parvovirus (CPV) VP2 protein to a 41-aminoacid long tetramerization domain (TD) from the transcriptional factor p53. The chimerical DNA construction 2L21-TD was cloned in a binary plant transformation vector and used to transform Arabidopsis thaliana plants. Fifteen of the 25 transgenic lines obtained in the experiment showed detectable 2L21-TD RNA accumulation and from these we chose 4 to study 2L21-TD protein accumulation. Non-denaturing immunoblotting assays revealed that 2L21-TD chimeras effectively formed tetrameric complexes with yields reaching up to 12 g/mg of soluble protein. Mice immunized by oral or intraperitoneal routes with crude protein extracts containing 2L21-TD protein were able to detect both 2L21-synthetic peptide and CPV VP2 proteins, with titers similar to those elicited by a previously reported fusion between 2L21 and the ␤-glucuronidase protein. These results demonstrate that multimerization directed by the small TD domain contributed to the stabilization and consequently to the accumulation of the 2L21 peptide in transgenic plants, without altering its native antigenicity and immunogenicity.

Atherosclerosis Supplements, 2010

Current Research Topics in Applied Microbiology and Microbial Biotechnology, 2009

Current Research Topics in Applied Microbiology and Microbial Biotechnology, 2009

Biomarker insights, 2009

Since the function of the spinal cord depends on the proteins found there, better defing the norm... more Since the function of the spinal cord depends on the proteins found there, better defing the normal Spinal Cord Proteome is an important and challenging task. Although brain and cerebrospinal fluid samples from patients with different central nervous system (CNS) disorders have been studied, a thorough examination of specific spinal cord proteins and the changes induced by injury or associated to conditions such as neurodegeneration, spasticity and neuropathies has yet to be performed. In the present study, we aimed to describe total protein content in the spinal cord of healthy rats, employing different proteomics tools. Accordingly, we have developed a fast, easy, and reproducible sequential protocol for protein extraction from rat spinal cords. We employed conventional two dimensional electrophoresis (2DE) in different pH ranges (eg. 4-7, 3-11 NL) combined with identification by mass spectrometry (MALDI-TOF/TOF), as well as first dimension protein separation combined with Liquid ...

Journal of proteomics, Jan 23, 2014

Acute myocardial infarction with ST-segment elevation (STEMI) initiates with intraluminal thrombo... more Acute myocardial infarction with ST-segment elevation (STEMI) initiates with intraluminal thrombosis and results in total occlusion of the coronary artery. To date, characterization of the coronary thrombus proteome in STEMI patients has not been yet accomplished. Therefore, we aimed to perform an in-depth proteomic characterization of the human coronary thrombus by means of three different approaches: 2-DE followed by mass spectrometry (MALDI MS/MS), 1-DE combined either with liquid chromatography coupled to mass spectrometry in a MALDI TOF/TOF (LC-MALDI-MS/MS), or in a LTQ-Orbitrap (LC-ESI-MS/MS). This approach allowed us to identify a total of 708 proteins in the thrombus. Expression in coronary thrombi (n=20) of 14 proteins was verified, and the expression of fibrin and 6 cell markers (platelets, monocytes, neutrophils, eosinophils, T-cells and B-cells) quantified by selected reaction monitoring (SRM). A positive correlation of 5 proteins (fermitin homolog 3, thrombospondin-1, m...

Vaccine, 2002

The expression of antigens in transgenic plants has been increasingly used as an alternative to t... more The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the recombinant antigens in the plant tissues, which reduces the possibilities of practical applications. Because the site of insertion of the transferred DNA into the cellular chromosomal DNA is at random, different levels of foreign protein expression in independent transformants is expected. Strategies to allow the evaluation of a high number of the transgenic individuals, usually an expensive and very time consuming process, would permit the selection of those plants presenting the highest levels of recombinant protein expression. Here, we present the development of an experimental immunogen based in the expression of a highly immunogenic epitope from foot and mouth disease virus (FMDV) fused to the glucuronidase (gus A) reporter gene, which allows selection of the transgenic plants by the ß-glucuronidase (ßGUS) enzymatic activity. We produced transgenic plants of alfalfa expressing the immunogenic site between amino acid residues 135-160 of structural protein VP1 (VP135-160), fused to the ßGUS protein. Plants expressing the highest levels of the immunogenic epitope VP135-160, analyzed by Western blot, were efficiently selected based on their levels of ßGUS enzymatic activity. The FMDV epitope expressed in plants was highly immunogenic in mice which developed, after immunization, a strong anti-FMDV antibody response against a synthetic peptide representing the region VP135-160, to native virus VP1, and to purified FMDV particles. Additionally, these mice were completely protected against experimental challenge with the virulent virus. To our knowledge, this constitutes the first report of a peptide-based vaccine produced in transgenic plants that induces a protective immune response when used in experimental hosts. Also, these results demonstrated the possibility of using a novel and simple methodology for obtaining transgenic plants expressing high levels of foreign immunogenic epitopes, which could be directly applied in the development of plant-based vaccines.