Person:
Puyet Catalina, Antonio

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First Name
Antonio
Last Name
Puyet Catalina
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Veterinaria
Department
Bioquímica y Biología Molecular
Area
Bioquímica y Biología Molecular
Identifiers
UCM identifierORCIDScopus Author IDWeb of Science ResearcherIDDialnet IDGoogle Scholar ID

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Now showing 1 - 10 of 11
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Implementación de un sistema de aprendizaje autónomo en bioinformática para el análisis de datos en biología molecular

2022-06-29, Reyes Palomares, Armando, Coronado Brieva, Montserrat, Abad González, Paloma, Puyet Catalina, Antonio, Baustista Santa Cruz, José Manuel

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Proteomic Approaches to Identifying Carbonylated Proteins in Brain Tissue

2011-01-15, Marín-García, Patricia , Méndez, Darío , Linares Gómez, María, Puyet Catalina, Antonio, Díez Martín, Amalia, Bautista Santa Cruz, José Manuel

Oxidative stress plays a critical role in the pathogenesis of a number of diseases. The carbonyl end products of protein oxidation are among the most commonly measured markers of oxidation in biological samples. Protein carbonyl functional groups may be derivatized with 2,4-dinitrophenylhydrazine (DNPH) to render a stable 2,4-dinitrophenylhydrazone-protein (DNP-protein) and the carbonyl contents of individual proteins then determined by two-dimensional electrophoresis followed by immunoblotting using specific anti-DNP antibodies. Unfortunately, derivatization of proteins with DNPH could affect their mass spectrometry (MS) identification. This problem can be overcome using nontreated samples for protein identification. Nevertheless, derivatization could also affect their mobility, which might be solved by performing the derivatization step after the initial electrophoresis. Here, we compare two-dimensional redox proteome maps of mouse cerebellum acquired by performing the DNPH derivatization step before or after electrophoresis and detect differences in protein patterns. When the same approach is used for protein detection and identification, both methods were found to be useful to identify carbonylated proteins. However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH reaction was performed after transblotting. Comparative data on protein identification using both methods are provided.

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Synchronous culture of Plasmodium falciparum at high parasitemia levels

2009-11-19, Azar Radfar, Darío Méndez, Carlos Moneriz, Patricia Marín-García, Linares Gómez, María, Puyet Catalina, Antonio, Díez Martín, Amalia, Bautista Santa Cruz, José Manuel

This protocol describes a method for preparing cultures of Plasmodium falciparum synchronized at any intraerythrocytic stage. Using this method, around 60% parasitized cells may be obtained. On the basis of Trager and Jensen's original continuous culture method, our approach relies on the use of fresh human blood not older than 2 weeks, a low hematocrit between 0.8 and 1.5%, a starting frozen inoculum of 10% ring-stage parasitemia, human serum replaced with AlbuMAX I and alternating sorbitol and Percoll synchronization methods to shorten the cycle window to 4–6 h and reduce sorbitol toxicity. From our synchronized high parasite density cultures, 3–5 ml of infected red blood cells can be obtained in 1 week, corresponding to 1.2 mg of total parasite protein per ml of harvested culture. On the basis of the variables parasitemia and packed cell volume, we provide an equation to accurately calculate the amount of complete medium required every 24 h corrected for the cycle stage and capacity of the culture flask. Ten days suffice to complete the protocol from a frozen stock of parasites.

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Malaria Hidden in a Patient with Diffuse Large-B-Cell Lymphoma and Sickle-Cell Trait

2011-12, Enriqueta Albizua, Darío Méndez, José M. Rubio, Alejandra Martínez Serna, Miguel A. Martínez, Efren Salto, Joaquin Martinez-López, Linares Gómez, María, Puyet Catalina, Antonio, Díez Martín, Amalia, Bautista Santa Cruz, José Manuel

We report a case of an African patient with sickle cell trait who was diagnosed in Spain with B-cell lymphoma. Blood smears were negative for malaria, and no plasmodium antigens were detected in the blood. To treat his lymphoma, the patient underwent chemotherapy and autologous stem cell transplantation. Following a splenectomy due to a worsening condition, he developed clinical malaria with detectable parasitemia. This case suggests that the humoral response and parasite removal by the spleen may afford protection from overt disease and may even help maintain subclinical human reservoirs of the disease.

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Microscopic and submicroscopic infection by Plasmodium falciparum: Immunoglobulin M and A profiles as markers of intensity and exposure

2022-09-02, Paloma Abad, Patricia Marín-García, Marcos Heras, Julius N. Fobil, Alfred G. Hutchful, Díez Martín, Amalia, Puyet Catalina, Antonio, Reyes Palomares, Armando Adolfo, Isabel G. Azcárate, Bautista Santa Cruz, José Manuel

Assessment of serological Plasmodium falciparum-specific antibodies in highly endemic areas provides valuable information about malaria status and parasite exposure in the population. Although serological evidence of Plasmodium exposure is commonly determined by Plasmodium-specific immunoglobulin G (IgG) levels; IgM and IgA are likely markers of malaria status that remain relatively unexplored. Previous studies on IgM and IgA responses have been based on their affinity for single antigens with shortage of immune responses analysis against the whole Plasmodium proteome. Here, we provide evidence of how P. falciparum infection triggers the production of specific IgM and IgA in plasma and its relationship with parasite density and changes in hematological parameters. A total of 201 individuals attending a hospital in Breman Asikuma, Ghana, were recruited into this study. Total and P. falciparum-specific IgM, IgA, and IgG were assessed by ELISA and examined in relation to age (0-5, 14-49, and ≥50 age ranges); infection (submicroscopic vs. microscopic malaria); pregnancy and hematological parameters. Well-known IgG response was used as baseline control. P. falciparum-specific IgM and IgA levels increased in the population with the age, similarly to IgG. These data confirm that acquired humoral immunity develops by repeated infections through the years endorsing IgM and IgA as exposure markers in endemic malaria regions. High levels of specific IgA and IgM in children were associated with microscopic malaria and worse prognosis, because most of them showed severe anemia. This new finding shows that IgM and IgA may be used as diagnostic markers in this age group. We also found an extremely high prevalence of submicroscopic malaria (46.27% on average) accompanied by IgM and IgA levels indistinguishable from those of uninfected individuals. These data, together with the observed lack of sensitivity of rapid diagnostic tests (RDTs) compared to PCR, invoke the urgent need to implement diagnostic markers for submicroscopic malaria. Overall, this study opens the potential use of P. falciparum-specific IgM and IgA as new serological markers to predict malaria status in children and parasite exposure in endemic populations. The difficulties in finding markers of submicroscopic malaria are highlighted, emphasizing the need to explore this field in depth.

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Estandarización de un modelo murino de malaria cerebral en fases clínicas para la evaluación de terapias antimaláricas y de rescate

2013, Martínez, Gabriela, Linares Gómez, María, Marín-García, Patricia, Pérez-Benavente, Susana, Puyet Catalina, Antonio, Bautista Santa Cruz, José Manuel, Díez Martín, Amalia

Entre las enfermedades infecciosas más devastadoras del SNC se incluye la MC, debido a la alta mortalidad y las graves secuelas que ocasiona. Actualmente, no existe tratamiento farmacológico específico, ni de rescate de lesiones neurocognitivas residuales, y su desarrollo está limitado por la inexistencia de modelos experimentales bien definidos. En este trabajo se caracterizó fenotípicamente la infección en un modelo murino de MC evaluando parámetros clínicos que permitieron establecer cuatro estadios de la enfermedad. Este protocolo proporciona el marco experimental adecuado para estudiar terapias coadyuvantes neuroprotectoras que puedan prevenir y/o eliminar las secuelas neurológicas presentes en los individuos que sobreviven.

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Iron supplementation in mouse expands cellular innate defences in spleen and defers lethal malaria infection

2017-10-16, Azcárate, I.G., Sánchez-Jaut, S., Marín-García, P., Linares, M., Pérez-Benavente, S., García-Sánchez, M., Uceda, J., Kamali, A.N., Morán-Jiménez, M.-J., Puyet Catalina, Antonio, Díez Martín, Amalia, Bautista Santa Cruz, José Manuel

The co-endemicity of malnutrition, erythrocytopathies, transmissible diseases and iron-deficiency contribute to the prevalence of chronic anaemia in many populations of the developing world. Although iron dietary supplementation is applied or recommended in at risk populations, its use is controversial due to undesirable outcomes, particularly regarding the response to infections, including highly prevalent malaria. We hypothesized that a boosted oxidative stress due to iron supplementation have a similar impact on malaria to that of hereditary anaemias, enhancing innate response and conditioning tissues to prevent damage during infection. Thus, we have analysed antioxidant and innate responses against lethal Plasmodium yoelii during the first five days of infection in an iron-supplemented mouse. This murine model showed high iron concentration in plasma with upregulated expression of hemoxygenase-1. The sustained homeostasis after this extrinsic iron conditioning, delayed parasitemia growth that, once installed, developed without anaemia. This protection was not conferred by the intrinsic iron overload of hereditary hemochromatosis. Upon iron-supplementation, a large increase of the macrophages/dendritic cells ratio and the antigen presenting cells was observed in the mouse spleen, independently of malaria infection. Complementary, malaria promoted the splenic B and T CD4 cells activation. Our results show that the iron supplementation in mice prepares host tissues for oxidative-stress and induces unspecific cellular immune responses, which could be seen as an advantage to promote early defences against malaria infection.

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Stress response and cytoskeletal proteins involved in erythrocyte membrane remodeling upon Plasmodium falciparum invasion are differentially carbonylated in G6PD A− deficiency

2011-05-15, Dario Méndez, Linares Gómez, María, Díez Martín, Amalia, Puyet Catalina, Antonio, Bautista Santa Cruz, José Manuel

Multiple glucose-6-phosphate dehydrogenase (G6PD)-deficient alleles have reached polymorphic frequencies because of the protection they confer against malaria infection. A protection mechanism based on enhanced phagocytosis of parasitized G6PD-deficient erythrocytes that are oxidatively damaged is well accepted. Although an association of this phenotype with the impairment of the antioxidant defense in G6PD deficiency has been demonstrated, the dysfunctional pathway leading to membrane damage and modified exposure of the malaria-infected red cell to the host is not known. Thus, in this study, erythrocytes from the common African variant G6PD A− were used to analyze by redox proteomics the major oxidative changes occurring in the host membrane proteins during the intraerythrocytic development of Plasmodium falciparum, the most lethal malaria parasite. Fifteen carbonylated membrane proteins exclusively identified in infected G6PD A− red blood cells revealed selective oxidation of host proteins upon malarial infection. As a result, three pathways in the host erythrocyte were oxidatively damaged in G6PD A−: (1) traffic/assembly of exported parasite proteins in red cell cytoskeleton and surface, (2) oxidative stress defense proteins, and (3) stress response proteins. Additional identification of hemichromes associated with membrane proteins also supports a role for specific oxidative modifications in protection against malaria by G6PD polymorphisms.

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Brain-derived neurotrophic factor and the course of experimental cerebral malaria

2013-01, Patricia Marín-García, Susana Pérez-Benavente, Jesús Sánchez-Nogueiro, Linares Gómez, María, Puyet Catalina, Antonio, Bautista Santa Cruz, José Manuel, Díez Martín, Amalia

The role of neurotrophic factors on the integrity of the central nervous system (CNS) during cerebral malaria (CM) infection remains obscure, but the long-standing neurocognitive sequelae often observed in rescued children can be attributed in part to the modulation of neuronal survival and synaptic plasticity. To discriminate the contribution of key responses in the time-sequence of the pathogenic events that trigger the development of neurocognitive malaria syndrome we defined four stages (I–IV) of the neurological progression of CM in C57BL/6 mice infected with Plasmodium berghei ANKA. Upregulation of ICAM-1, VCAM-1, e-selectin and p-selectin expression was detected in all cerebral regions before parasitized red blood cells (pRBC) accumulation. As the severity of symptoms increased, BDNF mRNA progressively diminished in several brain regions, earliest in the thalamus–hypothalamus, cerebellum, brainstem and cortex, and correlated with a four-stage disease sequence. Immunohistochemical confocal microscopy revealed changes in the BDNF distribution pattern, suggesting altered axonal transport. During CM progression, molecular markers of neurological infection and inflammation in the parasite and the host, respectively, were accompanied by a switch in the brain constitutive proteasome to the immunoproteasome, which could impede normal protein turnover. In parallel with BDNF downregulation, NCAM expression also diminished with increased CM severity. Together, these data suggest that changes in BDNF availability could be involved in the pathogenesis of CM.

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Protein Susceptibility to Peroxidation by 4-Hydroxynonenal in Hereditary Hemochromatosis

2023-02-02, Sandra Sánchez-Jaut, Susana Pérez-Benavente, Paloma Abad, Darío Méndez-Cuadro, Antonio Puyet, Amalia Diez, Gonzalo Galicia-Poblet, Elena Gómez-Domínguez, María J. Moran-Jiménez, José M. Bautista, Isabel G. Azcárate, Puyet Catalina, Antonio

Iron overload caused by hereditary hemochromatosis (HH) increases free reactive oxygen species that, in turn, induce lipid peroxidation. Its 4-hydroxynonenal (HNE) by-product is a wellestablished marker of lipid peroxidation since it reacts with accessible proteins with deleterious consequences. Indeed, elevated levels of HNE are often detected in a wide variety of human diseases related to oxidative stress. Here, we evaluated HNE-modified proteins in the membrane of erythrocytes from HH patients and in organs of Hfe−/− male and female mice, a mouse model of HH. For this purpose, we used one- and two-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF analysis. We identified cytoskeletal membrane proteins and membrane receptors of erythrocytes bound to HNE exclusively in HH patients. Furthermore, kidney and brain of Hfe−/−mice contained more HNE-adducted protein than healthy controls. Our results identified main HNE-modified proteins suggesting that HH favours preferred protein targets for oxidation by HNE.