46 results on '"Amalia, Diez"'
Search Results
2. Plasmodium falciparum immunodominant IgG epitopes in subclinical malaria
- Author
-
Estela Paz-Artal, José M. Bautista, Paloma Abad, Antonio Puyet, Patricia Marín-García, Pedro A. Reche, Julius N. Fobil, Amalia Diez, Susana Pérez-Benavente, Isabel G. Azcárate, José M. Rubio, Ministerio de Economía y Competitividad (España), Complutense University of Madrid (España), and Universidad Complutense de Madrid (España)
- Subjects
Adult ,Male ,0301 basic medicine ,Adolescent ,Science ,Plasmodium falciparum ,030231 tropical medicine ,Protozoan Proteins ,Antibodies, Protozoan ,Antigens, Protozoan ,Immunodominance ,Parasitemia ,Microbiology ,Ghana ,Immunoglobulin G ,Article ,Epitope ,Epitopes ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,parasitic diseases ,medicine ,Humans ,Malaria, Falciparum ,Child ,Subclinical infection ,Multidisciplinary ,biology ,Malaria vaccine ,biology.organism_classification ,medicine.disease ,030104 developmental biology ,Epitope mapping ,Immunology ,biology.protein ,Medicine ,Infectious diseases ,Female ,Antibody ,Epitope Mapping ,Malaria - Abstract
Incomplete non-sterile immunity to malaria is attained in endemic regions after recurrent infections by a large percentage of the adult population, who carry the malaria parasite asymptomatically. Although blood-stagePlasmodium falciparumrapidly elicits IgG responses, the target antigens of partially protective and non-protective IgG antibodies as well as the basis for the acquisition of these antibodies remain largely unknown. We performed IgG-immunomics to screen forP. falciparumantigens and to identify epitopes associated with exposure and clinical disease. Sera from malaria cases identified five prevalent antigens recognized by all analyzed patients’ IgGs. For further epitope mapping, peptide microarrays designed to cover their sequences were probed with a set of 38 sera samples from adult individuals of an endemic malaria region in Ghana. Eight 20-mer peptides with the highest affinity and frequency of recognition among the population were subsequently validated with 16 sera from the same region, segregated into patients with positive or negative subclinical detection ofP. falciparum. Significant binding specificity for two immunodominant antigenic regions was uncovered within the START-related lipid transfer protein and the protein disulfide isomerase PDI8. These 20-mer peptides challenged with sera samples from children under 5 years old displayed specific IgG binding in those with detectable parasitemia, even at subclinical level. These results suggest that the humoral response against START and PDI8 antigens may be triggered even at submicroscopic parasitemia levels in children and may eventually be used to differentially diagnose subclinical malaria in children.SignificanceMalaria in Africa is a leading cause of morbidity and mortality. The reservoirs of the malaria parasite are asymptomatic patients who carry it subclinically. Identifying the parasite antigens and its fragments that trigger the most common immunity response by immunoglobulin G that partially protect people can have profound implications for both, development of a malaria vaccine and diagnosis of the subclinical parasite carriers. Antigen discovery and mapping, validated with sera from subclinical carriers, showed that immunoglobulin G responses in children against parasite’s START and PDI8 may eventually be used to differentially diagnose non-infected from subclinical cases. Furthermore, anti-START and anti-PDI8 endemic immunodominance provides association of these antigens with long-term acquired immunity and immune evasion to malaria.
- Published
- 2020
3. Iron supplementation in mouse expands cellular innate defences in spleen and defers lethal malaria infection
- Author
-
Marta García-Sánchez, Javier Uceda, María-Josefa Morán-Jiménez, José M. Bautista, Amalia Diez, Antonio Puyet, Ali N. Kamali, Susana Pérez-Benavente, Isabel G. Azcárate, María Linares, Patricia Marín-García, and Sandra Sánchez-Jaut
- Subjects
0301 basic medicine ,Iron ,Spleen ,Parasitemia ,Biology ,Lymphocyte Activation ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Immune system ,Superoxide Dismutase-1 ,parasitic diseases ,medicine ,Animals ,030212 general & internal medicine ,RNA, Messenger ,Antigen-presenting cell ,Molecular Biology ,Hemochromatosis ,Mice, Inbred BALB C ,Macrophages ,Membrane Proteins ,Dendritic Cells ,Plasmodium yoelii ,medicine.disease ,biology.organism_classification ,Immunity, Innate ,Malaria ,Disease Models, Animal ,Oxidative Stress ,030104 developmental biology ,medicine.anatomical_structure ,Hereditary hemochromatosis ,Immunology ,CD4 Antigens ,Dietary Supplements ,Molecular Medicine ,Female ,Heme Oxygenase-1 - Abstract
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.
- Published
- 2017
4. Oxidative Stress and Protein Carbonylation in Malaria
- Author
-
Antonio Puyet, María Linares, Amalia Diez, and José M. Bautista
- Subjects
0301 basic medicine ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Biochemistry ,Protein Carbonylation ,030231 tropical medicine ,medicine ,Biology ,medicine.disease ,medicine.disease_cause ,Malaria ,Oxidative stress - Published
- 2017
- Full Text
- View/download PDF
5. Malaria proteomics: Insights into the parasite–host interactions in the pathogenic space
- Author
-
José M. Bautista, Antonio Puyet, Amalia Diez, Isabel G. Azcárate, and Patricia Marín-García
- Subjects
Proteomics ,Plasmodium ,biology ,Biophysics ,Plasmepsin ,Parasitism ,Computational biology ,Bioinformatics ,biology.organism_classification ,medicine.disease ,Biochemistry ,Malaria ,Immunomics ,Host-Pathogen Interactions ,Proteome ,medicine ,Animals ,Humans ,Identification (biology) - Abstract
Proteomics is improving malaria research by providing global information on relevant protein sets from the parasite and the host in connection with its cellular structures and specific functions. In the last decade, reports have described biologically significant elements in the proteome of Plasmodium, which are selectively targeted and quantified, allowing for sensitive and high-throughput comparisons. The identification of molecules by which the parasite and the host react during the malaria infection is crucial to the understanding of the underlying pathogenic mechanisms. Hence, proteomics is playing a major role by defining the elements within the pathogenic space between both organisms that change across the parasite life cycle in association with the host transformation and response. Proteomics has identified post-translational modifications in the parasite and the host that are discussed in terms of functional interactions in malaria parasitism. Furthermore, the contribution of proteomics to the investigation of immunogens for potential vaccine candidates is summarized. The malaria-specific technological advances in proteomics are particularly suited now for identifying host-parasite interactions that could lead to promising targets for therapy, diagnosis or prevention. In this review, we examine the knowledge gained on the biology, pathogenesis, immunity and diagnosis of Plasmodium infection from recent proteomic studies. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.
- Published
- 2014
- Full Text
- View/download PDF
6. Glutathione peroxidase contributes with heme oxygenase-1 to redox balance in mouse brain during the course of cerebral malaria
- Author
-
Antonio Puyet, Amalia Diez, Gabriela Martínez-Chacón, José M. Bautista, Patricia Marín-García, María Linares, and Susana Pérez-Benavente
- Subjects
Male ,Blotting, Western ,Malaria, Cerebral ,Oxidative phosphorylation ,Redox proteomics ,Biology ,medicine.disease_cause ,Antioxidants ,Protein Carbonylation ,Superoxide dismutase ,Mice ,Redox balance ,chemistry.chemical_compound ,Experimental cerebral malaria ,medicine ,Animals ,Molecular Biology ,chemistry.chemical_classification ,Glutathione Peroxidase ,Mice, Inbred BALB C ,Reactive oxygen species ,Superoxide Dismutase ,Glutathione peroxidase ,Brain ,Glutathione ,Catalase ,Reactive Nitrogen Species ,Molecular biology ,Mice, Inbred C57BL ,Heme oxygenase ,Disease Models, Animal ,chemistry ,Oxidative stress ,biology.protein ,Molecular Medicine ,Reactive Oxygen Species ,Oxidation-Reduction ,Heme Oxygenase-1 ,Transcription Factors - Abstract
Oxidative stress has been attributed both a key pathogenic and rescuing role in cerebral malaria (CM). In a Plasmodium berghei ANKA murine model of CM, host redox signaling and functioning were examined during the course of neurological damage. Host antioxidant defenses were early altered at the transcriptional level indicated by the gradually diminished expression of superoxide dismutase-1 (sod-1), sod-2, sod-3 and catalase genes. During severe disease, this led to the dysfunctional activity of superoxide dismutase and catalase enzymes in damaged brain regions. Vitagene associated markers (heat shock protein 70 and thioredoxin-1) also showed a decaying expression pattern that paralleled reduced expression of the transcription factors Parkinson disease 7, Forkhead box O 3 and X-box binding protein 1 with a role in preserving brain redox status. However, the oxidative stress markers reactive oxygen/nitrogen species were not accumulated in the brains of CM mice and redox proteomics and immunohistochemistry failed to detect quantitative or qualitative differences in protein carbonylation. Thus, the loss of antioxidant capacity was compensated for in all cerebral regions by progressive upregulation of heme oxygenase-1, and in specific regions by early glutathione peroxidase-1 induction. This study shows for the first time a scenario of cooperative glutathione peroxidase and heme oxygenase-1 upregulation to suppress superoxide dismutase, catalase, heat shock protein-70 and thioredoxin-1 downregulation effects in experimental CM, counteracting oxidative damage and maintaining redox equilibrium. Our findings reconcile the apparent inconsistency between the lack of oxidative metabolite build up and reported protective effect of antioxidant therapy against CM.
- Published
- 2013
- Full Text
- View/download PDF
7. Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin
- Author
-
Noelia Camacho, Amalia Diez, Susana Pérez-Benavente, L Ribas de Pouplana, Isabel G. Azcárate, Patricia Marín-García, Antonio Puyet, and José M. Bautista
- Subjects
Pharmacology ,0303 health sciences ,030306 microbiology ,medicine.drug_class ,Antibiotics ,Parasitemia ,Biology ,medicine.disease ,biology.organism_classification ,3. Good health ,03 medical and health sciences ,Immune system ,Antigen ,Immunity ,parasitic diseases ,Immunology ,medicine ,Avidity ,Malaria ,Plasmodium yoelii ,030304 developmental biology - Abstract
Background and Purpose Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria. Experimental Approach Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed. Key Results Borrelidin protected against lethal malaria at 0.25 mg·kg−1·day−1. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts. Conclusions and Implications Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.
- Published
- 2013
- Full Text
- View/download PDF
8. Differential carbonylation of cytoskeletal proteins in blood group O erythrocytes: Potential role in protection against severe malaria
- Author
-
Ali N. Kamali, Antonio Puyet, María Luisa Hernáez, Darío Méndez, Amalia Diez, and José M. Bautista
- Subjects
Proteomics ,Microbiology (medical) ,Erythrocytes ,Plasmodium falciparum ,Oxidative phosphorylation ,Protein oxidation ,Microbiology ,ABO Blood-Group System ,Protein Carbonylation ,ABO blood group system ,Genetics ,Humans ,Ankyrin ,Genetic Predisposition to Disease ,Malaria, Falciparum ,Molecular Biology ,Lipid raft ,Ecology, Evolution, Behavior and Systematics ,chemistry.chemical_classification ,Aldehydes ,biology ,Membrane Proteins ,biology.organism_classification ,Molecular biology ,Cytoskeletal Proteins ,Infectious Diseases ,chemistry ,Biochemistry ,Membrane protein ,Case-Control Studies ,Oxidation-Reduction - Abstract
The molecular basis for the prevalence of blood group O in regions where malaria is endemic remains unclear. In some genetic backgrounds oxidative modifications have been linked to a reduced susceptibility to severe malaria disease. Through redox proteomics, we detected differences in carbonylated membrane proteins among the different blood groups, both in Plasmodium-infected and uninfected erythrocytes (RBC). Carbonylation profiles of RBC membrane proteins revealed that group O blood shows a reduced protein oxidation pattern compared to groups A, B and AB. Upon infection with Plasmodium falciparum Dd2, erythrocytes of all blood groups showed increased oxidation of membrane proteins. By examining 4-hydroxy-2-nonenal (4-HNE) modified proteins by LC-MS/MS (liquid chromatography/mass spectrometry) we observed that, upon malaria infection, the protein components of lipid rafts and cytoskeleton were the main targets of 4-HNE carbonylation in all blood groups. Ankyrins and protein bands 4.2 and 4.1 were differentially carbonylated in group O as compared to A and B groups. During trophozoite maturation in group O erythrocytes, a steady increase was observed in the number of 4-HNE-modified proteins, suggesting a parasite-driven 4-HNE-carbonylation process. Our findings indicate a possible correlation between the protection against severe malaria in blood group O individuals and a specific pattern of 4-HNE-carbonylation of cytoskeleton proteins.
- Published
- 2012
- Full Text
- View/download PDF
9. Plasmodium yoelii blood-stage antigens newly identified by immunoaffinity using purified IgG antibodies from malaria-resistant mice
- Author
-
Antonio Puyet, Ali N. Kamali, Patricia Marín-García, Amalia Diez, Isabel G. Azcárate, and José M. Bautista
- Subjects
Proteomics ,Eukaryotic Initiation Factor-3 ,Blotting, Western ,Immunology ,Protein Disulfide-Isomerases ,Inmunología ,Plasmepsin ,Antibodies, Protozoan ,Antigens, Protozoan ,Biology ,Chromatography, Affinity ,Mice ,Immune system ,Antigen ,Heat shock protein ,Malaria Vaccines ,parasitic diseases ,Animals ,Aspartic Acid Endopeptidases ,Humans ,Immunology and Allergy ,HSP70 Heat-Shock Proteins ,Protein disulfide-isomerase ,Disease Resistance ,Mice, Inbred ICR ,Malaria vaccine ,Plasmodium yoelii ,Hematology ,biology.organism_classification ,Virology ,Malaria ,Immunoglobulin G ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Female ,Antibody - Abstract
As the search for an effective human malaria vaccine continues, understanding immune responses to Plasmodium in rodent models is perhaps the key to unlocking new vaccine strategies. The recruitment of parasite-specific antibodies is an important component of natural immunity against infection in blood-stage malaria. Here, we describe the use of sera from naturally surviving ICR mice after infection with lethal doses of Plasmodium yoelii yoelii 17XL to identify highly immunogenic blood-stage antigens. Immobilized protein A/G was used for the affinity-chromatography purification of the IgGs present in pooled sera from surviving mice. These protective IgGs, covalently immobilized on agarose columns, were then used to isolate reactive antigens from whole P. yoelii yoelii 17XL protein extracts obtained from the blood-stage malaria infection. Through proteomics analysis of the recovered parasite antigens, we were able to identify two endoplasmic reticulum lumen proteins: protein disulfide isomerase and a member of the heat shock protein 70 family. Also identified were the digestive protease plasmepsin and the 39 kDa-subunit of eukaryotic translation initiation factor 3, a ribosome associated protein. Of these four proteins, three have not been previously identified as antigenic during blood-stage malaria infection. This procedure of isolating and identifying parasite antigens using serum IgGs from malaria-protected individuals could be a novel strategy for the development of multi-antigen-based vaccine therapies. 2.814 JCR (2012) Q3, 70/137 Immunology
- Published
- 2012
- Full Text
- View/download PDF
10. Multi-targeted activity of maslinic acid as an antimalarial natural compound
- Author
-
Jordi Mestres, Antonio Puyet, Carlos Moneriz, José M. Bautista, and Amalia Diez
- Subjects
Apicoplast ,Proteases ,In silico ,Plasmodium falciparum ,Cell Biology ,Biology ,Phospholipase ,biology.organism_classification ,Biochemistry ,Apicomplexa ,chemistry.chemical_compound ,chemistry ,Maslinic acid ,Merozoite surface protein ,Molecular Biology - Abstract
Most drugs against malaria that are available or under development target a single process of the parasite infective cycle, favouring the appearance of resistant mutants which are easily spread in areas under chemotherapeutic treatments. Maslinic acid (MA) is a low toxic natural pentacyclic triterpene for which a wide variety of biological and therapeutic activities have been reported. Previous work revealed that Plasmodium falciparum erythrocytic cultures were inhibited by MA, which was able to hinder the maturation from ring to schizont stage and, as a consequence, prevent the release of merozoites and the subsequent invasion. We show here that MA effectively inhibits the proteolytic processing of the merozoite surface protein complex, probably by inhibition of PfSUB1. In addition, MA was also found to inhibit metalloproteases of the M16 family by a non-chelating mechanism, suggesting the possible hindrance of plasmodial metalloproteases belonging to that family, such as falcilysin and apicoplast peptide-processing proteases. Finally, in silico target screening was used to search for other potential binding targets that may have remained undetected. Among the targets identified, the method recovered two for which experimental activity could be confirmed, and suggested several putative new targets to which MA could have affinity. One of these unreported targets, phospholipase A2, was shown to be partially inhibited by MA. These results suggest that MA may behave as a multi-targeted drug against the intra-erythrocytic cycle of Plasmodium, providing a new tool to investigate the synergistic effect of inhibiting several unrelated processes with a single compound, a new concept in antimalarial research.
- Published
- 2011
- Full Text
- View/download PDF
11. Early transcriptional response to chloroquine of the Plasmodium falciparum antioxidant defence in sensitive and resistant clones
- Author
-
José M. Bautista, Amalia Diez, Susana Pérez-Benavente, Antonio Puyet, Azar Radfar, Fátima Nogueira, and Virgílio E. do Rosário
- Subjects
Time Factors ,Veterinary (miscellaneous) ,Plasmodium falciparum ,Drug Resistance ,Clone (cell biology) ,Drug resistance ,Models, Biological ,Microbiology ,Antimalarials ,Inhibitory Concentration 50 ,Downregulation and upregulation ,Stress, Physiological ,Chloroquine ,Gene expression ,medicine ,Animals ,Gene ,biology ,Gene Expression Profiling ,biology.organism_classification ,Oxidative Stress ,Infectious Diseases ,Glutathione S-transferase ,Insect Science ,Immunology ,biology.protein ,Parasitology ,medicine.drug - Abstract
Resistance to chloroquine (CQ) in Plasmodium falciparum has a major impact on malaria control worldwide. To gain insight into early parasite stress response, mRNA expression profiles were determined for a set of 10 antioxidant defence genes in synchronized CQ-sensitive (3D7) and CQ-resistant (Dd2) clones under transient IC50 CQ-exposure (Dd2, 200 nM; 3D7, 14 nM). Upon 2-h CQ challenge, the mRNA upregulation detected was greater in 3D7 (six genes overexpressed at 1/3 of the intraerythrocytic cycle) than in Dd2 clone (three genes responding), providing evidence of an early transcriptional response to CQ-induced oxidative stress which might underlie some of the parasite's metabolic adaptation to the drug.
- Published
- 2010
- Full Text
- View/download PDF
12. Altered Nucleotide Receptor Expression in a Murine Model of Cerebral Malaria
- Author
-
Miriam León-Otegui, María Linares, José M. Bautista, Pilar García-Palencia, Patricia Marín-García, María Teresa Miras-Portugal, Amalia Diez, and Jesús Sánchez-Nogueiro
- Subjects
Male ,Plasmodium berghei ,Receptor expression ,Malaria, Cerebral ,Protein Array Analysis ,Parasitemia ,Biology ,P2 receptor ,Neuroprotection ,Mice ,medicine ,Animals ,Immunology and Allergy ,RNA, Messenger ,Receptor ,Messenger RNA ,Receptors, Purinergic P2 ,Purinergic receptor ,Brain ,medicine.disease ,Immunohistochemistry ,Molecular biology ,Mice, Inbred C57BL ,Infectious Diseases ,Gene Expression Regulation ,Cerebral Malaria ,Immunology - Abstract
In cerebral malaria, the most severe complication of malaria, both neurotransmission mechanisms and energy metabolism are affected. To understand how metabolic changes modify neurotransmission, we examined P2 receptor expression in a murine model of cerebral malaria. Quantitative polymerase chain reaction experiments revealed that parasite deposition was greatest in the cerebellum, compared with other areas of the brain, suggesting a correlation between brain parasitemia and loss of control of movement. Infected mice showed modified patterns of expression of P2 receptor subtype messenger RNA (mRNA), depending on both the specific purinergic receptor and the cerebral region analyzed. Immunohistochemical studies indicated altered levels of protein expression by these receptors in infected brains and, in some cases, a pattern of expression different from that noted in control mice. These differences in both the amount of mRNA and the protein distribution of P2 receptors observed in the different brain sites in infected mice suggest an important role for P2 receptors in either provoking cerebral damage or conferring neuroprotection.
- Published
- 2009
- Full Text
- View/download PDF
13. Chloroquine mediates specific proteome oxidative damage across the erythrocytic cycle of resistant Plasmodium falciparum
- Author
-
Azar Radfar, José M. Bautista, and Amalia Diez
- Subjects
Erythrocytes ,Proteome ,Plasmodium falciparum ,Protozoan Proteins ,Antigens, Protozoan ,medicine.disease_cause ,Protein oxidation ,Proteomics ,Biochemistry ,Antimalarials ,Chloroquine ,Physiology (medical) ,medicine ,Animals ,Humans ,Parasite hosting ,biology ,biology.organism_classification ,Phenylhydrazines ,Cell biology ,Signal transduction ,Oxidation-Reduction ,Oxidative stress ,medicine.drug - Abstract
Resistance of Plasmodium falciparum to chloroquine hinders malaria control in endemic areas. Current hypotheses on the action mechanism of chloroquine evoke its ultimate interference with the parasite's oxidative defence systems. Through carbonyl derivatization by 2,4-dinitrophenylhydrazine and proteomics, we compared oxidatively modified proteins across the parasite's intraerythrocytic stages in untreated and transiently IC 50 chloroquine-treated cultures of the chloroquine-resistant P. falciparum strain Dd2. Functional plasmodial protein groups found to be most oxidatively damaged were among those central to the parasite's physiological processes, including protein folding, proteolysis, energy metabolism, signal transduction, and pathogenesis. While an almost constant number of oxidized proteins was detected across the P. falciparum life cycle, chloroquine treatment led to increases in both the extent of protein oxidation and the number of proteins oxidized as the intraerythrocytic cycle progressed to mature stages. Our data provide new insights into early molecular effects produced by chloroquine in the parasite, as well as into the normal protein-oxidation modifications along the parasite cycle. Oxidized proteins involved in the particular parasite drug-response suggest that chloroquine causes specific oxidative stress, sharing common features with eukaryotic cells. Targeting these processes might provide ways of combating chloroquine-resistance and developing new antimalarial drugs.
- Published
- 2008
- Full Text
- View/download PDF
14. Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes
- Author
-
RAFAEL G. SEVILLA, AMALIA DIEZ, MICHAEL NORÉN, OLIVIER MOUCHEL, MARC JÉRÔME, VÉRONIQUE VERREZ-BAGNIS, HILDE VAN PELT, LAURENCE FAVRE-KREY, GRIGORIOS KREY, THE FISHTRACE CONSORTIUM, and JOSÉ M. BAUTISTA
- Subjects
0106 biological sciences ,Rhodopsin ,PCR primers ,Teleost ,010603 evolutionary biology ,01 natural sciences ,Biochemistry ,DNA barcoding ,General Biochemistry, Genetics and Molecular Biology ,law.invention ,03 medical and health sciences ,law ,patterns ,14. Life underwater ,Clade ,Gene ,Polymerase chain reaction ,030304 developmental biology ,Genetics ,0303 health sciences ,Ecology ,Phylogenetic tree ,biology ,clades ,Haplotype ,phylogenies ,Mitochondrial cytochrome b ,Wageningen Marine Research ,Fish ,biology.protein ,Microsatellite - Abstract
This report describes a set of 21 polymerase chain reaction primers and amplification conditions developed to barcode practically any teleost fish species according to their mitochondrial cytochrome b and nuclear rhodopsin gene sequences. The method was successfully tested in more than 200 marine fish species comprising the main Actinopterygii family groups. When used in phylogenetic analyses, its combination of two genes with different evolutionary rates serves to identify fish at the species level. We provide a flow diagram indicating our validated polymerase chain reaction amplification conditions for barcoding and species identification applications as well as population structure or haplotyping analyses, adaptable to high-throughput analyses.
- Published
- 2007
- Full Text
- View/download PDF
15. Conjugated Linoleic Acid Affects Lipid Composition, Metabolism, and Gene Expression in Gilthead Sea Bream (Sparus aurata L)3
- Author
-
Michael J. Leaver, Laurence Favre-Krey, Josep A. Calduch-Giner, José M. Bautista, Evridiki Boukouvala, Silvia Vega-Rubı́n de Celis, David Menoyo, Alex Obach, Douglas R. Tocher, Susana Pérez-Benavente, Jaume Pérez-Sánchez, Amalia Diez, and Grigorios Krey
- Subjects
medicine.medical_specialty ,Nutrition and Dietetics ,integumentary system ,Triglyceride ,Conjugated linoleic acid ,Linoleic acid ,Fish farming ,Dietary lipid ,food and beverages ,Medicine (miscellaneous) ,Biology ,Fish oil ,chemistry.chemical_compound ,Endocrinology ,Postprandial ,chemistry ,Internal medicine ,Lipogenesis ,medicine ,lipids (amino acids, peptides, and proteins) - Abstract
To maximize growth, farmed fish are fed high-fat diets, which can lead to high tissue lipid concentrations that have an impact on quality. The intake of conjugated linoleic acid (CLA) reduces body fat in mammals and this study was undertaken to determine the effects of dietary CLA on growth, composition, and postprandial metabolic variables in sea bream. Fish were fed 3 diets containing 48 g/100 g protein and 24 g/100 g fat, including fish oil supplemented with 0 (control), 2, or 4% CLA for 12 wk. Feed intake, specific growth rate, total body fat, and circulating somatolactin concentration were lower in fish fed CLA than in controls. Feed efficiency was greater in fish fed 2% CLA than in controls. Liver triglyceride concentrations were higher in fish fed 4% CLA and muscle triglyceride concentrations were lower in fish fed both CLA diets than in controls. Hepatic fatty acyl desaturase and elongase mRNA levels in fish fed CLA were lower than in controls. Metabolic differences between controls and CLA-fed fish were observed at 6 h but not at 24 h after the last meal, including lower postprandial circulating triglyceride concentrations, higher hepatic acyl-CoA-oxidase, and lower L-3-hydroxyacyl-CoA dehydrogenase activities in CLA-fed fish than in controls. Dietary CLA did not affect enzymes involved in lipogenesis including hepatic fatty acid synthase and malic enzyme, but it decreased glucose 6-phosphate dehydrogenase activity at 24 h, but not at 6 h after feeding. The data suggest that CLA intake in sea bream has little effect on hepatic lipogenesis, channels dietary lipid from adipose tissue to the liver, and switches hepatic mitochondrial to peroxisomal beta-oxidation.
- Published
- 2007
- Full Text
- View/download PDF
16. In VitroandIn VivoExpression of Human Erythrocyte Pyruvate Kinase in Erythroid Cells: A Gene Therapy Approach
- Author
-
Juan A. Bueren, Paula Río, Amalia Diez, José M. Bautista, José C. Segovia, Nestor W. Meza, Susana Navarro, Antonio Puyet, and Oscar Quintana-Bustamante
- Subjects
Male ,Erythrocytes ,Genetic enhancement ,Cellular differentiation ,Blotting, Western ,Genetic Vectors ,Pyruvate Kinase ,Gene Expression ,Antigens, CD34 ,In Vitro Techniques ,Biology ,Transfection ,Mice ,Genetics ,medicine ,Animals ,Humans ,Transgenes ,Molecular Biology ,Cells, Cultured ,Erythroid Precursor Cells ,Reverse Transcriptase Polymerase Chain Reaction ,Hematopoietic Stem Cell Transplantation ,Cell Differentiation ,Genetic Therapy ,Flow Cytometry ,medicine.disease ,Molecular biology ,Mice, Inbred C57BL ,Haematopoiesis ,Retroviridae ,Mice, Inbred DBA ,Molecular Medicine ,Female ,Stem cell ,Pyruvate kinase ,Pyruvate kinase deficiency - Abstract
Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin(-)Sca-1(+) mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.
- Published
- 2007
- Full Text
- View/download PDF
17. Nnal‐like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily
- Author
-
Julia Lorenzo, Lloyd D. Fricker, Rafael G. Sevilla, Mónica Rodríguez de la Vega, Antoni Hermoso, José M. Bautista, Sebastian Tanco, Francesc X. Avilés, and Amalia Diez
- Subjects
chemistry.chemical_classification ,Subfamily ,biology ,Serine-Type D-Ala-D-Ala Carboxypeptidase ,biology.organism_classification ,Biochemistry ,Carboxypeptidase ,Amino acid ,Protein structure ,chemistry ,Genetics ,biology.protein ,Binding site ,Molecular Biology ,Gene ,Caenorhabditis elegans ,Biotechnology - Abstract
Nna1 has some sequence similarity to metallocarboxypeptidases, but the biochemical characterization of Nna1 has not previously been reported. In this work we performed a detailed genomic scan and found >100 Nna1 homologues in bacteria, Protista, and Animalia, including several paralogs in most eukaryotic species. Phylogenetic analysis of the Nna1-like sequences demonstrates a major divergence between Nna1-like peptidases and the previously known metallocarboxypeptidases subfamilies: M14A, M14B, and M14C. Conformational modeling of representative Nna1-like proteins from a variety of species indicates an unusually open active site, a property that might facilitate its action on a wide variety of peptide and protein substrates. To test this, we expressed a recombinant form of one of the Nna1-like peptidases from Caenorhabditis elegans and demonstrated that this protein is a fully functional metallocarboxypeptidase that cleaves a range of C-terminal amino acids from synthetic peptides. The enzymatic activity is activated by ATP/ADP and salt-inactivated, and is preferentially inhibited by Z-Glu-Tyr dipeptide, which is without precedent in metallocarboxypeptidases and resembles tubulin carboxypeptidase functioning; this hypothesis is strongly reinforced by the results depicted in Kalinina et al. published as accompanying paper in this journal. Our findings demonstrate that the M14 family of metallocarboxypeptidases is more complex and diverse than expected, and that Nna1-like peptidases are functional variants of such enzymes, representing a novel subfamily (we propose the name M14D) that contributes substantially to such diversity.
- Published
- 2007
- Full Text
- View/download PDF
18. Calcium controls smooth muscle TRPC gene transcription via the CaMK/calcineurin-dependent pathways
- Author
-
Cristina Camello-Almaraz, Pedro J. Camello, Antonio Puyet, José M. Bautista, Maria J. Pozo, Sara Morales, and Amalia Diez
- Subjects
Male ,Calcium Channels, L-Type ,Transcription, Genetic ,Physiology ,Calcineurin Pathway ,Guinea Pigs ,Down-Regulation ,Gene Expression ,In Vitro Techniques ,Biology ,TRPC5 ,TRPC1 ,TRPC3 ,Animals ,RNA, Messenger ,Phosphorylation ,Cyclic AMP Response Element-Binding Protein ,Egtazic Acid ,CAMK ,TRPC ,Chelating Agents ,TRPC Cation Channels ,Voltage-dependent calcium channel ,Calcineurin ,Gallbladder ,Muscle, Smooth ,Intracellular Membranes ,Cell Biology ,Cell biology ,Enzyme Activation ,Biochemistry ,Calcium-Calmodulin-Dependent Protein Kinases ,Calcium - Abstract
Transient receptor potential protein family C (TRPC) has been proposed as a candidate for channels involved in capacitative Ca2+ entry (CCE) mechanisms, but the modulation of their gene expression remains unexplored. In this study we show that guinea pig gallbladder smooth muscle contains mRNA encoding TRPC1, TRPC2, TRPC3, and TRPC4 proteins whose abundance depends on cytosolic Ca2+ level ([Ca2+]i). Thus lowering the levels of cellular calcium with the chelators EGTA and BAPTA AM results in a downregulation of TRPC1–TRPC4 gene and protein expression. In contrast, activation of Ca2+ influx through L-type Ca2+ channels and Ca2+ release from intracellular stores induced an increase in TRPC1–TRPC4 mRNA and protein abundance. Activation of Ca2+/calmodulin-dependent kinases (CaMK) and phosphorylation of cAMP-response element binding protein accounts for the increase in TRPC mRNA transcription in response to L-type channel-mediated Ca2+ influx . In addition to this mechanism, activation of TRPC gene expression by intracellular Ca2+ release also involves calcineurin pathway. According to the proposed role for these channels, activation of CCE induced an increase in TRPC1 and TRPC3 mRNA abundance, which depends on the integrity of the calcineurin and CaMK pathways. These findings show for the first time an essential autoregulatory role of Ca2+ in Ca2+ homeostasis at the level of TRPC gene and protein expression.
- Published
- 2007
- Full Text
- View/download PDF
19. Experimental Immunization Based on Plasmodium Antigens Isolated by Antibody Affinity
- Author
-
Amalia Diez, Antonio Puyet, Isabel G. Azcárate, Ali N. Kamali, José M. Bautista, and Patricia Marín-García
- Subjects
lcsh:Immunologic diseases. Allergy ,Plasmodium ,Article Subject ,Immunology ,Antibody Affinity ,Antibodies, Protozoan ,Antigens, Protozoan ,Immunoglobulin G ,Mice ,Immune system ,Antigen ,Adjuvants, Immunologic ,parasitic diseases ,Malaria Vaccines ,medicine ,Immunology and Allergy ,Animals ,biology ,General Medicine ,biology.organism_classification ,medicine.disease ,Virology ,Malaria ,Vaccination ,Disease Models, Animal ,Immunization ,biology.protein ,Female ,Antibody ,lcsh:RC581-607 ,Plasmodium yoelii ,Research Article - Abstract
Vaccines blocking malaria parasites in the blood-stage diminish mortality and morbidity caused by the disease. Here, we isolated antigens from total parasite proteins by antibody affinity chromatography to test an immunization against lethal malaria infection in a murine model. We used the sera of malaria self-resistant ICR mice to lethalPlasmodium yoelii yoelii17XL for purification of their IgGs which were subsequently employed to isolate blood-stage parasite antigens that were inoculated to immunize BALB/c mice. The presence of specific antibodies in vaccinated mice serum was studied by immunoblot analysis at different days after vaccination and showed an intensive immune response to a wide range of antigens with molecular weight ranging between 22 and 250 kDa. The humoral response allowed delay of the infection after the inoculation to high lethal doses ofP. yoelii yoelii17XL resulting in a partial protection against malaria disease, although final survival was managed in a low proportion of challenged mice. This approach shows the potential to prevent malaria disease with a set of antigens isolated from blood-stage parasites.
- Published
- 2015
20. Dietary fat type affects lipid metabolism in Atlantic salmon (Salmo salar L.) and differentially regulates glucose transporter GLUT4 expression in muscle
- Author
-
Susana Casado, Amalia Diez, José M. Bautista, Clemente J. Lopez-Bote, David Menoyo, and Alex Obach
- Subjects
medicine.medical_specialty ,food.ingredient ,Sunflower oil ,Glucose transporter ,Lipid metabolism ,Aquatic Science ,Carbohydrate metabolism ,Biology ,Fish oil ,food ,Endocrinology ,Linseed oil ,Internal medicine ,Lipogenesis ,medicine ,Food science ,Carnitine ,medicine.drug - Abstract
An experiment was conducted to study dietary fat type (fish oil (FO) vs. vegetable oil) effect on lipid and glucose metabolism in post-smolt Atlantic salmon. Duplicate groups of salmon were fed one of eight diets in which the two fat sources FO (long chain n-3 fatty acids, FA) or linseed oil (LO) (short chain n-3 FA) were combined in a 2 × 4 factorial design with sunflower oil (SO) (rich in n-6 FA) at inclusion levels of 0, 25, 50 and 75% of total added fat. The effects of the diets on plasma metabolites, the activity of selected enzymes involved in lipid metabolism, biometric indices and muscle glucose transporter GLUT4 expression were determined after 12 weeks of feeding. Lower viscero-somatic indices (VSI) and fatty livers were observed in fish fed LO based diets. Increasing inclusion levels of SO affected plasma glucose concentration in fish fed FO based diets, and plasma triglycerides, which decreased in a linear and quadratic pattern in fish fed FO based diets, but increased linearly in fish fed LO based diets. Specific activity of liver carnitine palmitoyl transferase I (CPT I) and glucose-6-phosphate dehydrogenase (G6PD) and plasma nonesterified fatty acids (NEFA) concentration was higher in fish fed LO based diets. Two GLUT4 isoforms I and II have been described in muscle and proved to be differentially expressed related to dietary fatty acids. In summary, dietary fat type affects lipid metabolism in post-smolted Atlantic salmon. In addition, a possibility to interfere on glucose metabolism by means of dietary fat type is discussed.
- Published
- 2006
- Full Text
- View/download PDF
21. Transient silencing of Plasmodium falciparum bifunctional glucose-6-phosphate dehydrogenase- 6-phosphogluconolactonase
- Author
-
Almudena Crooke, José M. Bautista, Philip J. Mason, and Amalia Diez
- Subjects
Erythrocytes ,Thioredoxin reductase ,Plasmodium falciparum ,Protozoan Proteins ,Glucosephosphate Dehydrogenase ,Biochemistry ,Antioxidants ,parasitic diseases ,Animals ,Humans ,Gene silencing ,Gene Silencing ,RNA, Messenger ,Molecular Biology ,Gene ,6-phosphogluconolactonase ,Regulation of gene expression ,biology ,Cell Biology ,Transfection ,Oxidants ,biology.organism_classification ,Molecular biology ,Oxidative Stress ,RNA silencing ,Gene Expression Regulation ,Carboxylic Ester Hydrolases - Abstract
The bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (G6PD-6PGL) found in Plasmodium falciparum has unique structural and functional characteristics restricted to this genus. This study was designed to examine the effects of RNA-mediated PfG6PD-6PGL gene silencing in cultures of P. falciparum on the expression of parasite antioxidant defense genes at the transcription level. The highest degree of G6PD-6PGL silencing achieved was 86% at the mRNA level, with a recovery to almost normal levels within 24 h, indicating only transient diminished expression of the PfG6PD-6PGL gene. PfG6PD-6PGL silencing caused arrest of the trophozoite stage and enhanced gametocyte formation. In addition, an immediate transcriptional response was shown by thioredoxin reductase suggesting that P. falciparum G6PD-6PGL plays a physiological role in the specific response of the parasite to intracellullar oxidative stress. P. falciparum transfection with an empty DNA vector also promoted intracellular stress, as determined by mRNA up-regulation of antioxidant genes. Collectively, our findings point to an important role for this enzyme in the parasite's infection cycle. The different characteristics of G6PD-6PGL with respect to its homologue in the host make it an ideal target for therapeutic strategies.
- Published
- 2006
- Full Text
- View/download PDF
22. Functional analysis of gammaretroviral vector transduction by quantitative PCR
- Author
-
Juan A. Bueren, José C. Segovia, Susana Pérez-Benavente, José M. Bautista, Nestor W. Meza, Oscar Quintana-Bustamante, Amalia Diez, and Antonio Puyet
- Subjects
Male ,Genetic enhancement ,Transgene ,Genetic Vectors ,Gene Expression ,Mice, Transgenic ,Biology ,Polymerase Chain Reaction ,Cell Line ,law.invention ,Mice ,Transduction (genetics) ,Proviruses ,Genes, Reporter ,Transduction, Genetic ,law ,Drug Discovery ,Gene expression ,Genetics ,Animals ,Humans ,RNA, Messenger ,Molecular Biology ,Gene ,Genetics (clinical) ,Polymerase chain reaction ,Bone Marrow Transplantation ,Reporter gene ,Base Sequence ,3T3 Cells ,Virology ,Molecular biology ,Leukemia Virus, Murine ,Mice, Inbred C57BL ,Real-time polymerase chain reaction ,Molecular Medicine ,HeLa Cells - Abstract
Background In a clinical setting of gene therapy, quantitative methods are required to determine recombinant viral titres and transgene mRNA expression, avoiding the use of reporter genes. Methods We describe procedures based on quantitative polymerase chain reaction (qPCR) designed to assess functional titres of murine leukaemia virus (MLV) vectors, determine proviral copy numbers in transduced cells, and estimate retroviral transgene expression in both target cell lines and mice with transduced chimeric haematopoiesis. Results Compared to EGFP titration, proviral DNA detection by qPCR was more accurate in assessing the number of infective particles in supernatants, such that average viral titres in terms of proviral copies per cell were two-fold higher. Transgene mRNA expression was directly determined from the vectors used without the need for reporter assays. A new parameter, defined here as the ‘transcription index’ (TI), served to establish the association between transcribed transgenic mRNA and each proviral insertion. The TI represents the potential expression of every vector or insertion in each cell type, and is thus useful as a control parameter for monitoring preclinical or clinical protocols. Conclusions The practical use of qPCR is demonstrated as a valuable alternative to reporter genes for the assessment and surveillance of insertion numbers and transgene expression. In combination with protein expression, this approach should be capable of establishing safer therapeutic gene doses, avoiding the potential side effects of high transduction and expression levels. Copyright © 2006 John Wiley & Sons, Ltd.
- Published
- 2006
- Full Text
- View/download PDF
23. Dietary protein source affects lipid metabolism in the European seabass (Dicentrarchus labrax)
- Author
-
Sadasivam Kaushik, Geneviève Corraze, Amalia Diez, Marcelino Álvarez, J Arzel, José M. Bautista, and Jorge Dias
- Subjects
Fish Proteins ,Glutens ,Physiology ,Biochemistry ,03 medical and health sciences ,Fish meal ,Animals ,Food science ,Molecular Biology ,Soy protein ,Triglycerides ,030304 developmental biology ,2. Zero hunger ,chemistry.chemical_classification ,0303 health sciences ,Meal ,biology ,Fatty acid ,04 agricultural and veterinary sciences ,Lipid Metabolism ,biology.organism_classification ,Cholesterol ,Liver ,chemistry ,Plant protein ,Lipogenesis ,Soybean Proteins ,040102 fisheries ,0401 agriculture, forestry, and fisheries ,Bass ,Digestion ,Dicentrarchus ,Dietary Proteins ,Corn gluten meal ,Edible Grain - Abstract
The study was undertaken to evaluate the effects of dietary protein sources on lipogenesis and fat deposition in a marine teleost, the European seabass (Dicentrarchus labrax). Four isonitrogenous (crude protein (CP, Nx6.25), 44% DM) and isoenergetic (22-23 kJ/g DM) diets were formulated to contain one of the following as the major protein source: fish meal (FM), one of two soy protein concentrates (SPC) and corn gluten meal (CGM). Apparent digestibility coefficients of the diets and raw ingredients, as well as soluble nitrogen (ammonia and urea) and phosphorus excretion were measured. Growth rates of seabass fed plant protein-based diets were significantly lower than those fed fish meal based diet. The protein utilisation was strongly correlated to the dietary essential amino acids index. Measurements of N excretion (ammonia and urea nitrogen) confirmed these data. Daily fat gain at the whole body level ranged between 1.1 to 1.7 g/kg BW, with the highest values being recorded in fish fed the fish meal based diet. Levels of plasma triglycerides and cholesterol were lower in fish fed soy protein diets than in those fed the diet solely based on fish meal. Soy protein rich diets decreased the activities of selected hepatic lipogenic enzymes (glucose 6-phosphate dehydrogenase, malic enzyme, ATP-citrate lysase, acetylcoenzyme A carboxylase and fatty acid synthetase). Highest lipogenic enzyme activities where found in fish fed the fish meal diet, except for fatty acid synthetase which was increased in seabass fed the corn-gluten meal based diets. Overall data suggest that dietary protein sources affects fat deposition and the lipogenic potential in European seabass.
- Published
- 2005
- Full Text
- View/download PDF
24. Dual-function stem molecular beacons to assess mRNA expression in AT-rich transcripts of Plasmodium falciparum
- Author
-
Almudena Crooke, Amalia Diez, Leyla Y. Bustamante, José M. Bautista, and Joaquín Martínez
- Subjects
Genetics ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Profiling ,Plasmodium falciparum ,RNA Probes ,biology.organism_classification ,AT Rich Sequence ,Online Systems ,Genome ,General Biochemistry, Genetics and Molecular Biology ,Reverse transcription polymerase chain reaction ,Apicomplexa ,Molecular beacon ,parasitic diseases ,Gene expression ,Animals ,Protozoa ,Parasite hosting ,RNA, Messenger ,Transcription Factors ,Biotechnology - Abstract
The genome of the human malaria parasite Plasmodium falciparum is extremely AT-rich such that it is particularly difficult to design standard probes to identify and quantify specific transcripts. Biased AT genome contents (70%–80%) lead to a high proportion of short repetitions and a low free energy of binding between target sequences and their specific probes during hybridization. This causes nonspecific annealing and high background noise. We constructed molecular beacon probes with dual-function stems to avoid nonspecific detection and establish identical melting patterns for use with several fluorescent probes for the analysis of mRNA expression in P. falciparum in real-time reverse transcription PCR (RT-PCR) assays. The method proved highly efficient at detecting low transcript levels in P. falciparum microcultures. Conditions were established for two types of real-time instruments, demonstrating that molecular beacons with dual-function stems are a useful tool for the functional analysis of high AT genomes. The procedure could be adapted to high-throughput gene expression protocols for the biomolecular screening of the P. falciparum and other AT-rich genomes.
- Published
- 2004
- Full Text
- View/download PDF
25. Early and late B cell immune responses in lethal and self-cured rodent malaria
- Author
-
José M. Bautista, Amalia Diez, Isabel G. Azcárate, Antonio Puyet, Susana Pérez-Benavente, and Patricia Marín-García
- Subjects
Immunology ,Remission, Spontaneous ,B-Lymphocyte Subsets ,Spleen ,Parasitemia ,Lymphocyte Activation ,Peritoneal cavity ,Mice ,Immune system ,Species Specificity ,Immunity ,parasitic diseases ,medicine ,Immunology and Allergy ,Animals ,Humans ,B cell ,B-Lymphocytes ,Mice, Inbred ICR ,biology ,Hematology ,Plasmodium yoelii ,medicine.disease ,biology.organism_classification ,Malaria ,Disease Models, Animal ,medicine.anatomical_structure ,Disease Progression ,Immunologic Memory - Abstract
ICR mice have heterogeneous susceptibility to lethal Plasmodium yoelii yoelii 17XL from the first days of experimental infection as evidenced by the different parasitemia levels and clinical outcomes. This mouse model has revealed specific immune responses on peripheral blood correlating with the infection fate of the animals. To search for immune-markers linked to parasitemia we examined B lymphocytes in organs of the immune system as key effectors of rodent immunity against malaria. To determine changes in immune cellularity fostered by the different prognostic parasitemia we examined B cell subsets in low (15%) and high (50%) parasitized mice during the first days of the infection. In the case of surviving mice, we studied the preservation of memory immune response 500 days after the primary P. yoelii challenge. Correlating with the parasitemia level, it was observed an increase in total cellularity of spleen during the first week of infection which remained after 16 months of the infection in surviving animals. B cell subsets were also modified across the different infection fates. Subpopulation as follicular B cells and B-1 cells proportions behaved differently depending on the parasitemia kinetics. In addition, peritoneal cavity cells proliferated in response to high parasitemia. More significantly, P. yoelii -specific memory B cells remained in the spleen 500 days after the primo-infection. This study demonstrates that B cell kinetics is influenced by the different parasitemia courses which are naturally developed within a same strain of untreated mice. We show that high levels of parasitemia at the beginning of infection promote an extremely fast and exacerbate response of several cell populations in spleen and peritoneal cavity that, in addition, do not follow the kinetics observed in peripheral blood. Furthermore, our results describe the longest persistence of memory B cells long time upon a single malaria infection in mice.
- Published
- 2014
26. Human Hexose-6-phosphate Dehydrogenase (Glucose 1-Dehydrogenase) Encoded at 1p36: Coding Sequence and Expression
- Author
-
Philip J. Mason, Deborah A. Scopes, Tom Vulliamy, David Stevens, Amalia Diez, and Stuart W. Knight
- Subjects
DNA, Complementary ,Glucose Dehydrogenases ,Molecular Sequence Data ,Biology ,Homology (biology) ,Exon ,Protein sequencing ,Glucose dehydrogenase ,parasitic diseases ,Gene duplication ,Animals ,Humans ,Coding region ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Gene ,Genetics ,Base Sequence ,Genome, Human ,Intron ,Chromosome Mapping ,Glucose 1-Dehydrogenase ,Cell Biology ,Hematology ,Biochemistry ,Chromosomes, Human, Pair 1 ,Molecular Medicine ,Rabbits ,Sequence Analysis - Abstract
ABSTRACT: Using the published protein sequence from a rabbit microsomal glucose-6-phosphate dehydrogenase G6PD we have isolated and sequenced a cDNA clone coding for its human equivalent, which is also known as hexose-6-phosphate dehydrogenase (H6PD) and glucose dehydrogenase. The corresponding genomic sequence is in the databases enabling its localization to chromosome 1p36. The gene spans 37 kb and consists of 5 exons, the fifth of which codes for more than half of the 89kDa protein. The first intron is a 10kb insertion in the 5′ untranslated sequence. The predicted mRNA has an exceptionally long (6.5kb) 3′ untranslated sequence. The predicted protein shows extensive homology with X-linked G6PD, suggesting the two genes share a common ancestor but no intron positions are conserved between the two genes suggesting the gene duplication was an ancient event. The C-terminal portion of the protein is not homologous with G6PD but shows limited homology with proteins of unknown function found throughout evolution and encoded next to G6PD in various micro-organisms. Intriguingly this C-terminal portion has some homology with the N-terminal sequence of Plasmodium falciparum G6PD.
- Published
- 1999
- Full Text
- View/download PDF
27. Regulation of hepatic lipogenesis by dietary protein/energy in juvenile European seabass (Dicentrarchus labrax)
- Author
-
Geneviève Corraze, José M. Bautista, J Arzel, Marcelino Álvarez, Sadasivam Kaushik, Amalia Diez, and Jorge Dias
- Subjects
medicine.medical_specialty ,ATP citrate lyase ,Starch ,Malic enzyme ,Aquatic Science ,03 medical and health sciences ,chemistry.chemical_compound ,Internal medicine ,medicine ,Food science ,030304 developmental biology ,2. Zero hunger ,chemistry.chemical_classification ,0303 health sciences ,biology ,Fatty acid ,04 agricultural and veterinary sciences ,Metabolism ,biology.organism_classification ,Enzyme ,Endocrinology ,chemistry ,Lipogenesis ,040102 fisheries ,0401 agriculture, forestry, and fisheries ,Dicentrarchus - Abstract
A growth trial was conducted with groups of European seabass having an initial weight of 6 g to study the lipogenic action of dietary protein and non-protein energy supplies. Six experimental diets were formulated to contain one of two crude protein levels (43 and 52%) with digestible protein (DP) to digestible energy (DE) ratios ranging from 19 to 26 mg/kJ. At the end of the growth trial (12 weeks), the activities of liver glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), malic enzyme (ME, EC 1.1.1.40), ATP citrate lyase (ACL, EC 4.1.3.8), acetyl-CoA carboxylase (ACoAC, EC 6.4.1.2) and fatty acid synthetase (FAS, EC 2.3.1.38) were measured. Digestibility of main dietary components was also determined over a three-week period. At each protein level, an increase in dietary DE led to improved growth performance, protein efficiency, daily N deposition and to a reduction of N loss. Best results were achieved at 40% DP and a DP/DE ratio of 19–20 mg/kJ. G6PD, ME and ACoAC were found to be the key regulatory enzymes in the lipogenic pathway, with G6PD being the main NADPH-generating enzyme. Activities of G6PD, ME, ACL and FAS were reduced with increasing fat intake. Activities of G6PD, ME and ACL were increased with increasing starch intake. ACoAC activity was negatively correlated with starch intake and positively with fat intake.
- Published
- 1998
- Full Text
- View/download PDF
28. Differential Immune Response Associated to Malaria Outcome Is Detectable in Peripheral Blood following Plasmodium yoelii Infection in Mice
- Author
-
Patricia Marín-García, Amalia Diez, Antonio Puyet, Isabel G. Azcárate, Ali N. Kamali, José M. Bautista, and Susana Pérez-Benavente
- Subjects
CD4-Positive T-Lymphocytes ,Adoptive cell transfer ,Anatomy and Physiology ,Mouse ,medicine.medical_treatment ,lcsh:Medicine ,Antibodies, Protozoan ,Parasitemia ,CD8-Positive T-Lymphocytes ,Cardiovascular System ,Monocytes ,Mice ,lcsh:Science ,Immune Response ,Mice, Inbred BALB C ,Mice, Inbred ICR ,Multidisciplinary ,biology ,Forkhead Transcription Factors ,Animal Models ,Acquired immune system ,Adoptive Transfer ,Cytokine ,medicine.anatomical_structure ,Infectious Diseases ,Treatment Outcome ,Circulatory Physiology ,Medicine ,Cytokines ,Female ,Plasmodium yoelii ,Research Article ,Clinical Research Design ,T cell ,Immunology ,Immunoglobulins ,Microbiology ,Immune system ,Model Organisms ,Immunity ,medicine ,Parasitic Diseases ,Animals, Outbred Strains ,Animals ,Humans ,Animal Models of Disease ,Biology ,lcsh:R ,Histocompatibility Antigens Class II ,Tropical Diseases (Non-Neglected) ,Dendritic Cells ,biology.organism_classification ,medicine.disease ,Virology ,Malaria ,Immunity, Humoral ,Leukocyte Common Antigens ,lcsh:Q ,Parasitology ,Infectious Disease Modeling - Abstract
Malaria infection in humans elicits a wide range of immune responses that can be detected in peripheral blood, but we lack detailed long-term follow-up data on the primary and subsequent infections that lead to naturally acquired immunity. Studies on antimalarial immune responses in mice have been based on models yielding homogenous infection profiles. Here, we present a mouse model in which a heterogeneous course of Plasmodium yoelii lethal malaria infection is produced in a non-congenic ICR strain to allow comparison among different immunological and clinical outcomes. Three different disease courses were observed ranging from a fatal outcome, either early or late, to a self-resolved infection that conferred long-term immunity against re-infection. Qualitative and quantitative changes produced in leukocyte subpopulations and cytokine profiles detected in peripheral blood during the first week of infection revealed that monocytes, dendritic cells and immature B cells were the main cell subsets present in highly-parasitized mice dying in the first week after infection. Besides, CD4(+)CD25(high) T cells expanded at an earlier time point in early deceased mice than in surviving mice and expressed higher levels of intracellular Foxp3 protein. In contrast, survivors showed a limited increase of cytokines release and stable circulating innate cells. From the second week of infection, mice that would die or survive showed similar immune profiles, although CD4(+)CD25(high) T cells number increased earlier in mice with the worst prognosis. In surviving mice the expansion of activated circulating T cell and switched-class B cells with a long-term protective humoral response from the second infection week is remarkable. Our results demonstrate that the follow-up studies of immunological blood parameters during a malaria infection can offer information about the course of the pathological process and the immune response.
- Published
- 2014
29. Antiplasmodial Activity and Mechanism of Action of RSM-932A, a Promising Synergistic Inhibitor of Plasmodium falciparum Choline Kinase
- Author
-
Amalia Diez, Juan Carlos Lacal, Carlos Moneriz, José M. Bautista, Tahl Zimmerman, Arancha Cebrián, and Teresa Gómez del Pulgar
- Subjects
Choline kinase ,Erythrocytes ,Plasmodium falciparum ,Protozoan Proteins ,Antineoplastic Agents ,Pyridinium Compounds ,Pharmacology ,Choline ,chemistry.chemical_compound ,Antimalarials ,Adenosine Triphosphate ,Parasitic Sensitivity Tests ,medicine ,Escherichia coli ,Choline Kinase ,Humans ,Pharmacology (medical) ,Experimental Therapeutics ,Trophozoites ,Enzyme Inhibitors ,Phosphorylation ,Phosphocholine ,chemistry.chemical_classification ,Aniline Compounds ,biology ,Dose-Response Relationship, Drug ,Quinolinium Compounds ,In vitro toxicology ,Chloroquine ,Drug Synergism ,biology.organism_classification ,In vitro ,Recombinant Proteins ,Kinetics ,Infectious Diseases ,Enzyme ,Biochemistry ,Mechanism of action ,chemistry ,Butanes ,medicine.symptom - Abstract
We have investigated the mechanism of action of inhibition of the choline kinase of P. falciparum ( p.f. -ChoK) by two inhibitors of the human ChoKα, MN58b and RSM-932A, which have previously been shown to be potent antitumoral agents. The efficacy of these inhibitors against p.f. -ChoK is investigated using enzymatic and in vitro assays. While MN58b may enter the choline/phosphocholine binding site, RSM-932A appears to have an altogether novel mechanism of inhibition and is synergistic with respect to both choline and ATP. A model of inhibition for RSM-932A in which this inhibitor traps p.f. -ChoK in a phosphorylated intermediate state blocking phosphate transfer to choline is presented. Importantly, MN58b and RSM-932A have in vitro inhibitory activity in the low nanomolar range and are equally effective against chloroquine-sensitive and chloroquine-resistant strains. RSM-932A and MN58b significantly reduced parasitemia and induced the accumulation of trophozoites and schizonts, blocking intraerythrocytic development and interfering with parasite egress or invasion, suggesting a delay of the parasite maturation stage. The present data provide two new potent structures for the development of antimalarial compounds and validate p.f. -ChoK as an accessible drug target against the parasite.
- Published
- 2013
30. Monochloroacetate dehalogenase activities of bacterial strains isolated from soil
- Author
-
Amando Garrido-Pertierra, Amalia Diez, José M. Bautista, M.I. Prieto, and Marcelino Álvarez
- Subjects
Azotobacter ,biology ,Immunology ,Pseudomonas ,General Medicine ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Microbiology ,Biochemistry ,Arthrobacter ,Genetics ,Haloacetate dehalogenase ,Alcaligenes ,Molecular Biology ,Azotobacteraceae ,Pseudomonadaceae ,Dehalogenase - Abstract
Seven bacterial strains capable of utilizing monochloroacetate (MCA) at a concentration of 50 mM as the sole carbon source were isolated from soil and displayed MCA dehalogenase activity. Three of them were identified as Pseudomonas spp., and the remaining four strains as Alcaligenes sp., Agrobacterium sp., Arthrobacter sp., and Azotobacter sp. This latter is the first reported example of a bacterium fixing atmospheric nitrogen under aerobic conditions that also uses a chloro-organic compound as sole source of carbon and energy. MCA dehalogenase activity in these strains was found to be inducible under different growth conditions. Crude extracts from all seven isolated strains also displayed dehalogenating activity with a relatively wide range of halogenated organic compounds (aliphatic acids, ketones, alcohols, alkanes, and aromatics), which, depending on the strain, were dehalogenated to different extents. The estimated Kmvalues for MCA were used to classify the dehalogenase activities into three groups: high affinity (30–40 μM) in Alcaligenes and Agrobacterium species, medium affinity (100–180 μM) in Pseudomonas and Azotobacter species, and low affinity (100 mM) in Arthrobacter sp. Both the optimal pH range for MCA dehalogenase activity (between pH 8 and 10) and the pH profile of stability (in the neutral–basic range) were found to be similar in all strains, whereas the thermal stability profiles were variable.Key words: dehalogenase, halohydrolase, monochloroacetate, soil.
- Published
- 1995
- Full Text
- View/download PDF
31. Brain-derived neurotrophic factor and the course of experimental cerebral malaria
- Author
-
José M. Bautista, Susana Pérez-Benavente, Amalia Diez, Antonio Puyet, Patricia Marín-García, María Linares, and Jesús Sánchez-Nogueiro
- Subjects
Male ,Cerebellum ,Proteasome Endopeptidase Complex ,Plasmodium berghei ,Central nervous system ,Blotting, Western ,Malaria, Cerebral ,Fluorescent Antibody Technique ,Polymerase Chain Reaction ,Parasite Load ,Pathogenesis ,Mice ,Downregulation and upregulation ,Neurotrophic factors ,medicine ,Animals ,Molecular Biology ,Neural Cell Adhesion Molecules ,Brain-derived neurotrophic factor ,Brain Chemistry ,biology ,Behavior, Animal ,General Neuroscience ,Brain-Derived Neurotrophic Factor ,biology.organism_classification ,Mice, Inbred C57BL ,medicine.anatomical_structure ,nervous system ,Gene Expression Regulation ,Cerebral Malaria ,Immunology ,Disease Progression ,Cytokines ,RNA ,Neurology (clinical) ,Developmental Biology - Abstract
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.
- Published
- 2012
32. Population proteomics of the European Hake (Merluccius merluccius)
- Author
-
Amalia Diez, Montserrat Espiñeira, Antonio Puyet, Elena G. Gonzalez, José M. Bautista, and Grigorios Krey
- Subjects
Fish Proteins ,Proteomics ,Population ,Population Dynamics ,Zoology ,Biochemistry ,Protein expression ,Mass Spectrometry ,03 medical and health sciences ,Mediterranean sea ,Hake ,Mediterranean Sea ,Animals ,Cluster Analysis ,Protein Isoforms ,Electrophoresis, Gel, Two-Dimensional ,14. Life underwater ,education ,Atlantic Ocean ,030304 developmental biology ,0303 health sciences ,education.field_of_study ,Principal Component Analysis ,biology ,Geography ,030302 biochemistry & molecular biology ,Brain ,Merluccius merluccius ,General Chemistry ,biology.organism_classification ,Fishery ,Europe ,Gadiformes ,Protein variation ,Liver ,Multivariate Analysis - Abstract
We report the novel use of proteomics to investigate protein variation among populations of the European hake (Merluccius merluccius). The liver and brain extracts of 18 hake (N = 36) captured in the Mediterranean Sea, Cantabrian Sea, and Atlantic Ocean were examined by 2D/DIGE and mass spectrometry. Significant differences in protein expression among populations were revealed by 84 spots obtained in the gels for the liver and 145 spots for the brain. Population groups of samples were defined by multivariate analysis (PCA and hierarchical clustering). According to protein expression levels and the functions of the 55 candidate protein spots identified, which showed significant expression differences, highest population discrimination was rendered by brain proteins involved in cell signaling and metabolism/energy and by liver proteins involved in protein fate. Finally, we present a statistically robust framework to accurately classify individuals according to their population of origin. Thus, purposely identified protein isoforms were found to be competent at discriminating populations. These results suggest the possibility of identifying protein biomarkers related to environmental changes in a nonmodel species such as the hake and pave the way for more extensive research on protein variation among populations of marine fishes.
- Published
- 2010
33. Stress response and cytoskeletal proteins involved in erythrocyte membrane remodeling upon Plasmodium falciparum invasion are differentially carbonylated in G6PD A- deficiency
- Author
-
Amalia Diez, Antonio Puyet, Darío Méndez, José M. Bautista, and María Linares
- Subjects
Male ,Protein Carbonylation ,Plasmodium falciparum ,Oxidative phosphorylation ,medicine.disease_cause ,Proteomics ,Biochemistry ,Models, Biological ,Physiology (medical) ,parasitic diseases ,medicine ,Humans ,Cytoskeleton ,biology ,Red Cell ,Erythrocyte Membrane ,biology.organism_classification ,Cell biology ,Cytoskeletal Proteins ,Oxidative Stress ,Glucosephosphate Dehydrogenase Deficiency ,Membrane protein ,Oxidation-Reduction ,Oxidative stress - Abstract
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.
- Published
- 2010
34. Combined proteomic approaches for the identification of specific amino acid residues modified by 4-hydroxy-2-nonenal under physiological conditions
- Author
-
Antonio Puyet, María Luisa Hernáez, Amalia Diez, Darío Méndez, and José M. Bautista
- Subjects
Proteomics ,Peptide ,medicine.disease_cause ,Mass spectrometry ,Biochemistry ,Protein sequencing ,Affinity chromatography ,medicine ,Animals ,Humans ,Histidine ,Bovine serum albumin ,Amino Acids ,chemistry.chemical_classification ,Aldehydes ,biology ,Lysine ,Erythrocyte Membrane ,Membrane Proteins ,Spectrin ,Serum Albumin, Bovine ,General Chemistry ,Oxidative Stress ,chemistry ,biology.protein ,Cattle ,Post-translational protein modification ,Quantitative analysis (chemistry) ,Protein Processing, Post-Translational ,Oxidative stress ,Biomarkers - Abstract
Proteins modified by 4-hydroxy-2-nonenal (HNE) are cellular markers of oxidative stress in health and disease. HNE is generated by free radical chain reactions during oxidative stress as a major end-product of the oxidative fatty acid metabolism. Identification and quantitative analysis of HNE-modified proteins are readily performed by using specific antibodies raised against them. Further on, the identification of the amino acid residues involved in the HNE-modification is an additional step in proteomic post-transcriptional modification analysis to explain the nature of the specificity underlying oxidative stress mechanisms. For this purpose, a combined protocol of immune-detection, peptide enrichment, mass spectrometry, and de novo protein sequencing has been developed. The methodology was first examined in the model protein bovine serum albumin (BSA), allowing the comparison of matrix-assisted laser desorption/ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) performance and sensitivity. Peptide enrichment was optimized by affinity chromatography on HNE-BSA resulting in increased sensitivity. Identification of amino acid residues modified by HNE was finally ascertained by de novo sequencing analysis. The improved methodology was demonstrated on human erythrocyte membrane proteins allowing the identification of HNE-lysine and HNE-histidine Michael adducts in the β-spectrin under physiological conditions.
- Published
- 2010
35. Synchronous culture of Plasmodium falciparum at high parasitemia levels
- Author
-
Carlos Moneriz, Antonio Puyet, Darío Méndez, José M. Bautista, Amalia Diez, Azar Radfar, María Linares, and Patricia Marín-García
- Subjects
Erythrocytes ,Plasmodium falciparum ,Cell Culture Techniques ,Protozoan Proteins ,Histology ,Parasitemia ,Biology ,medicine.disease ,biology.organism_classification ,General Biochemistry, Genetics and Molecular Biology ,Synchronous culture ,Culture Media ,Andrology ,chemistry.chemical_compound ,Biochemistry ,chemistry ,Cell culture ,Toxicity ,medicine ,Humans ,Sorbitol ,Percoll - Abstract
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.
- Published
- 2009
36. Haemoglobin interference and increased sensitivity of fluorimetric assays for quantification of low-parasitaemia Plasmodium infected erythrocytes
- Author
-
Antonio Puyet, Amalia Diez, Carlos Moneriz, Patricia Marín-García, and José M. Bautista
- Subjects
Lysis ,Erythrocytes ,lcsh:Arctic medicine. Tropical medicine ,lcsh:RC955-962 ,Plasmodium falciparum ,Parasitic Sensitivity Tests ,Parasitemia ,Cell Separation ,Diamines ,Sensitivity and Specificity ,lcsh:Infectious and parasitic diseases ,Hemoglobins ,Inhibitory Concentration 50 ,Chloroquine ,Limit of Detection ,parasitic diseases ,medicine ,Animals ,Humans ,lcsh:RC109-216 ,Benzothiazoles ,Malaria, Falciparum ,Organic Chemicals ,Fluorescent Dyes ,Detection limit ,biology ,Staining and Labeling ,Methodology ,DNA, Protozoan ,biology.organism_classification ,medicine.disease ,Virology ,Molecular biology ,Infectious Diseases ,Parasitology ,Quinolines ,Cytophotometry ,Quantitative analysis (chemistry) ,medicine.drug - Abstract
Background Improvements on malarial diagnostic methods are currently needed for the correct detection in low-density Plasmodium falciparum infections. Microfluorimetric DNA-based assays have been previously used for evaluation of anti-malarial drug efficacy on Plasmodium infected erythrocytes. Several factors affecting the sensitivity of these methods have been evaluated, and tested for the detection and quantification of the parasite in low parasitaemia conditions. Methods Parasitaemia was assessed by measuring SYBRGreen I® (SGI) and PicoGreen® (PG) fluorescence of P. falciparum Dd2 cultures on human red blood cells. Different modifications of standard methods were tested to improve the detection sensitivity. Calculation of IC50 for chloroquine was used to validate the method. Results Removal of haemoglobin from infected red-blood cells culture (IRBC) increased considerably the fluorescent signal obtained from both SGI and PG. Detergents used for cell lysis also showed to have an effect on the fluorescent signal. Upon depletion of haemoglobin and detergents the fluorescence emission of SGI and PG increased, respectively, 10- and 60-fold, extending notably the dynamic range of the assay. Under these conditions, a 20-fold higher PG vs. SGI fluorescent signal was observed. The estimated limits of detection and quantification for the PG haemoglobin/detergent-depleted method were 0.2% and 0.7% parasitaemia, respectively, which allow the detection of ~10 parasites per microliter. The method was validated on whole blood-infected samples, displaying similar results as those obtained using IRBC. Removal of white-blood cells prior to the assay allowed to increase the accuracy of the measurement, by reducing the relative uncertainty at the limit of detection from 0.5 to 0.1. Conclusion The use of PG microassays on detergent-free, haemoglobin-depleted samples appears as the best choice both for the detection of Plasmodium in low-density infections and anti-malarial drugs tests.
- Published
- 2009
37. Life-threatening nonspherocytic hemolytic anemia in a patient with a null mutation in the PKLR gene and no compensatory PKM gene expression
- Author
-
José M. Bautista, Nestor W. Meza, Amalia Diez, Susana Pérez-Benavente, Florinda Gilsanz, and Joaquín Martínez
- Subjects
Hemolytic anemia ,Male ,Immunology ,Mutant ,Population ,DNA Mutational Analysis ,Pyruvate Kinase ,Mutation, Missense ,Biology ,Biochemistry ,Frameshift mutation ,Hereditary spherocytosis ,medicine ,Humans ,RNA, Messenger ,education ,Child ,Genetics ,Family Health ,education.field_of_study ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Biology ,Hematology ,Anemia, Hemolytic, Congenital Nonspherocytic ,Exons ,medicine.disease ,Null allele ,Molecular biology ,Spain ,Female ,Pyruvate kinase ,Pyruvate kinase deficiency - Abstract
Human erythrocyte R-type pyruvate kinase (RPK) deficiency is an autosomal recessive disorder produced by mutations in the PKLR gene, causing chronic nonspherocytic hemolytic anemia. Survival of patients with severe RPK deficiency has been associated with compensatory expression in red blood cells (RBCs) of M2PK, an isoenzyme showing wide tissue distribution. We describe a novel homozygous null mutation of the PKLR gene found in a girl with a prenatal diagnosis of PK deficiency. The mutant PK gene revealed an 11-nucleotide (nt) duplication at exon 8, causing frameshift of the PKLR transcript, predicting a truncated protein inferred to have no catalytic activity. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) detected no M2PK expression in the peripheral blood red cell fraction. The expression of mutant RPK mRNA in the RBCs was almost 6 times higher than that detected in a control patient with hereditary spherocytosis. This molecular phenotypic analysis of the null mutation in the PKLR gene provides evidence for a lack of M2PK in the mature RBCs of this patient and suggests that normal red cell functions and survival are achieved through a population of young erythroid cells released into the circulation in response to anemia. (Blood. 2005;106:1851-1856)
- Published
- 2005
38. Three peroxisome proliferator-activated receptor isotypes from each of two species of marine fish
- Author
-
Michael J. Leaver, Efthimia Antonopoulou, Douglas R. Tocher, M Tariq Ezaz, Laurence Favre-Krey, Amalia Diez, Evridiki Boukouvala, Grigorios Krey, and José M. Bautista
- Subjects
Transcriptional Activation ,medicine.medical_specialty ,DNA, Complementary ,Molecular Sequence Data ,Peroxisome Proliferator-Activated Receptors ,Peroxisome proliferator-activated receptor ,Flounder ,Biology ,Response Elements ,Endocrinology ,Internal medicine ,medicine ,Animals ,PPAR alpha ,Tissue Distribution ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Receptor ,Peptide sequence ,Gene ,PPAR-beta ,Phylogeny ,Cloning ,chemistry.chemical_classification ,Promoter ,Anatomy ,Peroxisome ,Sea Bream ,Cell biology ,PPAR gamma ,chemistry ,Function (biology) - Abstract
The cloning and characterization of cDNAs and genes encoding three peroxisome proliferator-activated receptor (PPAR) isotypes from two species of marine fish, the plaice (Pleuronectes platessa) and the gilthead sea bream (Sparus aurata), are reported for the first time. Although differences in the genomic organization of the fish PPAR genes compared with their mammalian counterparts are evident, sequence alignments and phylogenetic comparisons show the fish genes to be homologs of mammalian PPARα, PPARβ/δ, and PPARγ. Like their mammalian homologs, fish PPARs bind to a variety of natural PPAR response elements (PPREs) present in the promoters of mammalian or piscine genes. In contrast, the mRNA expression pattern of PPARs in the two fish species differs from that observed in other vertebrates. Thus, PPARγ is expressed more widely in fish tissues than in mammals, whereas PPARα and β are expressed similarly in profile to mammals. Furthermore, nutritional status strongly influences the expression of all three PPAR isotypes in liver, whereas it has no effect on PPAR expression in intestinal and adipose tissues. Fish PPARα and β exhibit an activation profile similar to that of the mammalian PPAR in response to a variety of activators/ligands, whereas PPARγ is not activated by mammalian PPARγ-specific ligands. Amino acid residues shown to be critical for ligand binding in mammalian PPARs are not conserved in fish PPARγ and therefore, together with the distinct tissue expression profile of this receptor, suggest potential differences in the function of PPARγ in fish compared with mammals.
- Published
- 2005
39. Dietary protein source affects the susceptibility to lipid peroxidation of rainbow trout (Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax) muscle
- Author
-
Geneviève Corraze, Marcelino Álvarez, José M. Bautista, J Arzel, Clemente J. Lopez-Bote, Jorge Dias, Amalia Diez, Sadasivam Kaushik, Station d'hydrobiologie, Institut National de la Recherche Agronomique (INRA), and ProdInra, Migration
- Subjects
biology ,0402 animal and dairy science ,Zoology ,04 agricultural and veterinary sciences ,biology.organism_classification ,040201 dairy & animal science ,ALIMENTATION DES POISSONS ,Fishery ,Lipid peroxidation ,chemistry.chemical_compound ,Animal science ,Dietary protein ,chemistry ,[SDV.SA.SPA]Life Sciences [q-bio]/Agricultural sciences/Animal production studies ,040103 agronomy & agriculture ,0401 agriculture, forestry, and fisheries ,Animal Science and Zoology ,Rainbow trout ,Dicentrarchus ,[SDV.SA.SPA] Life Sciences [q-bio]/Agricultural sciences/Animal production studies ,Sea bass ,ComputingMilieux_MISCELLANEOUS ,BAR COMMUN - Abstract
This study was designed to explore the effect of protein source on muscle susceptibility to lipid peroxidation in two representative species of fish farmed for human consumption: the freshwater rainbow trout and the seawater European sea bass. Four isoproteic diets (digestible protein in the range 366 to 392 for rainbow trout and 391 to 415 g/kg for European sea bass) were formulated to contain one of the following as the main protein source: fish meal, warm water alcohol-extracted or toasted soya protein concentrates or maize gluten meal. Highest daily growth indices were always achieved using the diets based on fish meal as the main source of protein (P< 0·05). Fish of both species given diets containing maize gluten and the toasted soya protein concentrate showed slowest growth. The depressant growth effect of the vegetable protein concentrates was greater in sea bass than in rainbow trout. Dietary treatment was not correlated with any significant effect on whole-body composition or intramuscular fat content except for ash concentration in European sea bass. Under conditions of forced peroxidation in vitro for 240 min, muscle specimens of trout and sea bass given diets containing fish protein as the main source of protein showed the highest peroxidation levels (P< 0·05); while the lowest peroxidation values were found in fish given maize gluten-containing diets (P< 0·05). In the present case, the partial substitution of fish meal with vegetable proteins in diets led to a lower susceptibility of fish flesh to peroxidation. This finding may have applications in the production of fish of improved quality and longer shelf life.
- Published
- 2001
40. The partial substitution of digestible protein with gelatinized starch as an energy source reduces susceptibility to lipid oxidation in rainbow trout (Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax) muscle
- Author
-
Marcelino Álvarez, Geneviève Corraze, Clemente J. Lopez-Bote, Amalia Diez, José M. Bautista, J Arzel, Jorge Dias, and Sadasivam Kaushik
- Subjects
Starch ,Biology ,Lipid peroxidation ,03 medical and health sciences ,chemistry.chemical_compound ,Lipid oxidation ,Genetics ,Dietary Carbohydrates ,Animals ,14. Life underwater ,Food science ,Sea bass ,Salmonidae ,030304 developmental biology ,0303 health sciences ,Muscles ,Fatty Acids ,04 agricultural and veterinary sciences ,General Medicine ,biology.organism_classification ,Lipid Metabolism ,chemistry ,Biochemistry ,Oncorhynchus mykiss ,Lipogenesis ,040102 fisheries ,0401 agriculture, forestry, and fisheries ,Gelatin ,Animal Science and Zoology ,Rainbow trout ,Bass ,Digestion ,Dietary Proteins ,Lipid Peroxidation ,Energy source ,Energy Metabolism ,Oxidation-Reduction ,Food Science - Abstract
We evaluated the influence of dietary gelatinized starch and protein on the fatty acid composition of muscle in rainbow trout and European sea bass and on the susceptibility of flesh to lipid peroxidation. The possibility that flesh peroxidation could be accounted for by lipogenesis and the deposition of fat was also explored. The inclusion of gelatinized starch in the diet of rainbow trout improved growth with respect to that observed in fish fed crude starch (P
- Published
- 2000
41. Dietary fish oil and digestible protein modify susceptibility to lipid peroxidation in the muscle of rainbow trout (Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax)
- Author
-
José M. Bautista, Marcelino Álvarez, J Arzel, Jorge Dias, Amalia Diez, Geneviève Corraze, Sadasivam Kaushik, Clemente J. Lopez-Bote, Station d'hydrobiologie, Institut National de la Recherche Agronomique (INRA), and ProdInra, Migration
- Subjects
030309 nutrition & dietetics ,Medicine (miscellaneous) ,Lipid peroxidation ,03 medical and health sciences ,chemistry.chemical_compound ,Animal science ,medicine ,14. Life underwater ,Sea bass ,Salmonidae ,ComputingMilieux_MISCELLANEOUS ,0303 health sciences ,Nutrition and Dietetics ,biology ,04 agricultural and veterinary sciences ,Fish oil ,biology.organism_classification ,ALIMENTATION DES POISSONS ,[SDV.AEN] Life Sciences [q-bio]/Food and Nutrition ,Trout ,Biochemistry ,chemistry ,040102 fisheries ,0401 agriculture, forestry, and fisheries ,Dicentrarchus ,Rainbow trout ,medicine.symptom ,Weight gain ,[SDV.AEN]Life Sciences [q-bio]/Food and Nutrition ,BAR COMMUN - Abstract
The effects of dietary fish oil and digestible protein (DP) levels on muscle fatty acid composition and susceptibility to lipid peroxidation were studied in two representative fish species for human nutrition, from fresh and seawater, rainbow trout (Oncorhynchus mykiss) and European sea bass (Dicentrarchus labrax). In rainbow trout, higher concentrations of dietary fat and DP led to higher weight gain (g/d) (P = 0.001 and P = 0.043 respectively). Additionally, an interaction effect was observed in this species, since the effect of DP was only evident when the dietary fat concentration was low (P = 0.043). A similar tendency was also observed in European sea bass, although with less marked differences among nutritional treatments. Trout fed on diets with a higher concentration of dietary fat had higher concentrations of intramuscular total and neutral lipids in the dorsal muscle (P = 0.005). Increased levels of dietary DP led to significantly lower concentrations of polar lipids in the dorsal muscle of both rainbow trout (P = 0.005) and European sea bass (P = 0.006). In the neutral fraction of intramuscular lipids of dorsal muscle the concentration of n-3 fatty acids was positively affected by the dietary fat concentration in both rainbow trout (P = 0.04) and sea bass (P = 0.001). Muscle homogenates from trout and sea bass fed on diets rich in fish oil showed a significantly higher susceptibility to oxidation than muscle homogenates from fish fed on low-fat diets (P = 0.001). The higher DP concentration also increased susceptibility to oxidation. Moreover, in rainbow trout an interaction effect was observed where the pro-oxidant effect was of higher magnitude when the dietary concentration of both nutrients, fat and protein, was high (P = 0.004).
- Published
- 1998
42. Interaction of the local anesthetics dibucaine and tetracaine with sarcoplasmic reticulum membranes. Differential scanning calorimetry and fluorescence studies
- Author
-
A. Molina, B. Escudero, José Laynez, Amalia Diez, and Carlos Gutiérrez-Merino
- Subjects
Diphenylhexatriene ,Protein Denaturation ,ATPase ,Analytical chemistry ,Dibucaine ,Calorimetry ,Calcium-Transporting ATPases ,In Vitro Techniques ,Biochemistry ,chemistry.chemical_compound ,Tetracaine ,medicine ,Animals ,Denaturation (biochemistry) ,Binding Sites ,biology ,Calorimetry, Differential Scanning ,Endoplasmic reticulum ,Temperature ,Intracellular Membranes ,Sarcoplasmic reticulum membrane ,Sarcoplasmic Reticulum ,Membrane ,Spectrometry, Fluorescence ,chemistry ,biology.protein ,Biophysics ,Ca(2+) Mg(2+)-ATPase ,Rabbits ,medicine.drug - Abstract
The local anesthetics dibucaine and tetracaine inhibit the (Ca2+ + Mg2+)-ATPase from skeletal muscle sarcoplasmic reticulum [DeBoland, A. R., Jilka, R. L., & Martonosi, A. N. (1975) J. Biol. Chem. 250, 7501-7510; Suko, J., Winkler, F., Scharinger, B., & Hellmann, G. (1976) Biochim. Biophys. Acta 443, 571-586]. We have carried out differential scanning calorimetry and fluorescence measurements to study the interaction of these drugs with sarcoplasmic reticulum membranes and with purified (Ca2+ + Mg2+)-ATPase. The temperature range of denaturation of the (Ca2+ + Mg2+)-ATPase in the sarcoplasmic reticulum membrane, determined from our scanning calorimetry experiments, is ca. 45-55 degrees C and for the purified enzyme ca. 40-50 degrees C. Millimolar concentrations of dibucaine and tetracaine, and ethanol at concentrations higher than 1% v/v, lower a few degrees (degrees C) the denaturation temperature of the (Ca2+ + Mg2+)-ATPase. Other local anesthetics reported to have no effect on the ATPase activity, such as lidocaine and procaine, did not significantly alter the differential scanning calorimetry pattern of these membranes up to a concentration of 10 mM. The order parameter of the sarcoplasmic reticulum membranes, calculated from measurements of the polarization of the fluorescence of diphenylhexatriene, is not significantly altered at the local anesthetic concentrations that shift the denaturation temperature of the (Ca2+ + Mg2+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1989
43. Short-term modulation of lipogenesis by macronutrients in rainbow trout (Oncorhynchus mykiss) hepatocytes
- Author
-
Amalia Diez, José M. Bautista, Marcelino Álvarez, M Gallego, and Clemente J. Lopez-Bote
- Subjects
chemistry.chemical_classification ,Nutrition and Dietetics ,biology ,Linolenic acid ,Medicine (miscellaneous) ,Fatty acid ,Eicosapentaenoic acid ,Enzyme assay ,Biochemistry ,chemistry ,Docosahexaenoic acid ,Casein ,Lipogenesis ,biology.protein ,Polyunsaturated fatty acid - Abstract
Rainbow trout (Oncorhynchus mykiss) hepatocytes were cultured under simulated conditions of varying nutritional status to explore the short-term modulation by dietary substrates of the main lipogenic enzymes: glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACoAC) and fatty acid synthetase (FAS). Primary cultures were individually exposed to varying amounts of glucose, hydrolysed casein and long-chain polyunsaturated fatty acids (PUFA) for 12 h. A second set of experiments was designed to evaluate the effects of mixing different relative amounts of these macronutrients in the culture medium. Glucose concentrations of up to 20–25 mM SHOWED A STIMULATORY EFFECT ON G6PD, ME, ACL AND ACOAC ACTIVITY (Pin vivodietary conditions. It is felt that such an approach may serve to investigate the macronutrient regulation of other metabolic pathways.
44. Purification and properties of a high-affinity L-2-haloacid dehalogenase from Azotobacter sp. strain RC26
- Author
-
M.I. Prieto, José M. Bautista, Garrido Pertierra, Antonio Puyet, Marcelino Álvarez, and Amalia Diez
- Subjects
chemistry.chemical_classification ,Gel electrophoresis ,Azotobacter ,Strain (chemistry) ,biology ,Chemistry ,Stereochemistry ,Substrate (chemistry) ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Enzyme ,Biotransformation ,Biochemistry ,Dehalogenase ,Azotobacteraceae - Abstract
A monomeric 29 kDa protein showing dehalogenase activity on several halogenated carboxylic acids has been purified from Azotobacter sp. strain RC26. The purified enzyme is specific for the L isomer of optically active 2-haloacids leading to the inversion of the product configuration. The dehalogenase is active at temperatures ranging from 30 to 60°C and shows a relatively high affinity for the substrate. The combined thermal stability, high substrate affinity and resistance to enzyme inhibitors found for the RC26 dehalogenase may be relevant for its use as catalyst in biotransformation processes.
45. Parasitostatic effect of maslinic acid. II. Survival increase and immune protection in lethal Plasmodium yoelii-infected mice
- Author
-
Antonio Puyet, Carlos Moneriz, Patricia Marín-García, Amalia Diez, and José M. Bautista
- Subjects
lcsh:Arctic medicine. Tropical medicine ,Erythrocytes ,lcsh:RC955-962 ,Blotting, Western ,Antibodies, Protozoan ,Enzyme-Linked Immunosorbent Assay ,Parasitemia ,Biology ,Parasite load ,lcsh:Infectious and parasitic diseases ,Antimalarials ,Mice ,Immune system ,Antigen ,parasitic diseases ,medicine ,Animals ,lcsh:RC109-216 ,Mice, Inbred ICR ,Microscopy ,Research ,Plasmodium falciparum ,Plasmodium yoelii ,biology.organism_classification ,medicine.disease ,Acquired immune system ,Survival Analysis ,Triterpenes ,Malaria ,Infectious Diseases ,Immunology ,biology.protein ,Female ,Parasitology ,Antibody ,Injections, Intraperitoneal - Abstract
Background: The anti-malarial activity of maslinic acid (MA), a natural triterpene which has been previously shown to exert a parasitostatic action on Plasmodium falciparum cultures, was analysed in vivo by using the Plasmodium yoelii 17XL murine model. Methods: ICR mice were infected with P. yoelii and treated with a single dose of MA by a intraperitoneal injection of MA (40 mg kg -1 day -1 ) followed by identical dose administration for the following three days. Parasitaemia and accumulation of intraerythrocytic stages was monitored microscopically. To assess protective immunity, cured mice were challenged with the same dose of parasites 40 days after recovery from the primary infection and parasitaemia was further monitored for 30 days. Humoral response was tested by ELISA and visualization of specific anti-P. yoelii antibodies was performed by Western-blotting. Results: ICR mice treated with MA increased the survival rate from 20% to 80%, showing an arrest of parasite maturation from day 3 to 7 after infection and leading to synchronization of the intraerythrocytic cycle and accumulation of schizonts by day 6, proving that MA also behaves as a parasitostatic agent in vivo. Mice which survived the primary infection displayed lower rates of parasitic growth, showing a decline of parasitaemia after day 15, and complete clearance at day 20. These mice remained immunoprotected, showing not malaria symptoms or detectable parasitaemia after rechallenge with the same lethal strain. The analysis of specific antibodies against P. yoelii, present in mice which survived the infection, showed a significant increase in the number and intensity of immunoreactive proteins, suggesting that the protected mice may trigger a strong humoral response. Conclusion: The survival increase observed in MA-treated mice can be explained considering that the parasitostatic effect exerted by this compound during the first days of infection increases the chances to develop effective innate and/or acquired immune responses. MA may represent a new class of anti-malarial compounds which, as a consequence of its parasitostatic action, favours the development of more effective sterilizing immune responses. Background Despite the extensive research carried out to find new anti-malarial drugs or antigenic targets which can be eventually used in vaccine formulations, most of these efforts have been unsuccessful so far due to several factors, such as the emergence of resistant strains, the requirement of both low-toxicity and low-cost antimalarials, and the failure to develop a practical vaccine that prevents malaria. There is increasing evidence that the in vivo response to anti-malarial treatments is affected by factors other than the intrinsic susceptibility of Plasmodium species to the drugs. The parasite load, innate host resistance to the parasite [1] and naturally acquired immunity are known to play an important role in the infection progress and the outcome of the treatment with anti-malarials (reviewed by [2,3]). While most evidence on this subject was gathered from epidemiological surveys in
- Full Text
- View/download PDF
46. Parasitostatic effect of maslinic acid. I. Growth arrest of Plasmodium falciparum intraerythrocytic stages
- Author
-
José M. Bautista, Andrés García-Granados, Carlos Moneriz, Amalia Diez, Antonio Puyet, and Patricia Marín-García
- Subjects
lcsh:Arctic medicine. Tropical medicine ,Erythrocytes ,lcsh:RC955-962 ,Plasmodium falciparum ,Pharmacology ,Parasitemia ,Plasmodium ,Eimeria ,lcsh:Infectious and parasitic diseases ,Antimalarials ,chemistry.chemical_compound ,Neospora ,Maslinic acid ,Chloroquine ,parasitic diseases ,medicine ,lcsh:RC109-216 ,Malaria, Falciparum ,Atovaquone ,biology ,Research ,Antimicrobial ,biology.organism_classification ,Triterpenes ,In vitro ,Malaria ,Infectious Diseases ,chemistry ,Parasitology ,medicine.drug - Abstract
Background Natural products have played an important role as leads for the development of new drugs against malaria. Recent studies have shown that maslinic acid (MA), a natural triterpene obtained from olive pomace, which displays multiple biological and antimicrobial activities, also exerts inhibitory effects on the development of some Apicomplexan, including Eimeria, Toxoplasma and Neospora. To ascertain if MA displays anti-malarial activity, the main objective of this study was to asses the effect of MA on Plasmodium falciparum-infected erythrocytes in vitro. Methods Synchronized P. falciparum-infected erythrocyte cultures were incubated under different conditions with MA, and compared to chloroquine and atovaquone treated cultures. The effects on parasite growth were determined by monitoring the parasitaemia and the accumulation of the different infective stages visualized in thin blood smears. Results MA inhibits the growth of P. falciparum Dd2 and 3D7 strains in infected erythrocytes in, dose-dependent manner, leading to the accumulation of immature forms at IC50 concentrations, while higher doses produced non-viable parasite cells. MA-treated infected-erythrocyte cultures were compared to those treated with chloroquine or atovaquone, showing significant differences in the pattern of accumulation of parasitic stages. Transient MA treatment at different parasite stages showed that the compound targeted intra-erythrocytic processes from early-ring to schizont stage. These results indicate that MA has a parasitostatic effect, which does not inactivate permanently P. falciparum, as the removal of the compound allowed the infection to continue Conclusions MA displays anti-malarial activity at multiple intraerythrocytic stages of the parasite and, depending on the dose and incubation time, behaves as a plasmodial parasitostatic compound. This novel parasitostatic effect appears to be unrelated to previous mechanisms proposed for current anti-malarial drugs, and may be relevant to uncover new prospective plasmodial targets and opens novel possibilities of therapies associated to host immune response.
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.