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1.
J Proteome Res ; 23(2): 633-643, 2024 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-38183416

RESUMO

One of the main challenges in compiling the complete collection of protein antigens from pathogens for the selection of vaccine candidates or intervention targets is to acquire a broad enough representation of them to be recognized by the highly diversified immunoglobulin repertoire in human populations. Dried serum spot sampling (DSS) retains a large repertoire of circulating immunoglobulins from each individual that can be representative of a population, according to the sample size. In this work, shotgun proteomics of an infectious pathogen based on DSS sampling coupled with IgM immunoprecipitation, liquid chromatography-mass spectrometry (LC-MS/MS), and bioinformatic analyses was combined to characterize the circulating IgM antigenome. Serum samples from a malaria endemic region at different clinical statuses were studied to optimize IgM binding efficiency and antibody leaching by varying serum/immunomagnetic bead ratios and elution conditions. The method was validated using Plasmodium falciparum extracts identifying 110 of its IgM-reactive antigens while minimizing the presence of human proteins and antibodies. Furthermore, the IgM antigen recognition profile differentiated between malaria-infected and noninfected individuals at the time of sampling. We conclude that a shotgun proteomics approach offers advantages in providing a high-throughput, reliable, and clean way to identify IgM-recognized antigens from trace amounts of serum. The mass spectrometry raw data and metadata have been deposited with ProteomeXchange via MassIVE with the PXD identifier PXD043800.


Assuntos
Doenças Transmissíveis , Malária , Humanos , Espectrometria de Massa com Cromatografia Líquida , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Imunoglobulina M
2.
Int J Mol Sci ; 24(3)2023 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-36769239

RESUMO

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


Assuntos
Hemocromatose , Sobrecarga de Ferro , Humanos , Masculino , Camundongos , Feminino , Animais , Hemocromatose/genética , Aldeídos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peroxidação de Lipídeos , Proteína da Hemocromatose/genética , Proteína da Hemocromatose/metabolismo
3.
Biochim Biophys Acta Mol Basis Dis ; 1863(12): 3049-3059, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28965885

RESUMO

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.


Assuntos
Suplementos Nutricionais , Ferro/administração & dosagem , Malária/dietoterapia , Malária/imunologia , Baço/efeitos dos fármacos , Baço/imunologia , Animais , Antígenos CD4/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Heme Oxigenase-1/metabolismo , Imunidade Inata/efeitos dos fármacos , Ferro/sangue , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Malária/parasitologia , Malária/prevenção & controle , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo/efeitos dos fármacos , Plasmodium yoelii/efeitos dos fármacos , Plasmodium yoelii/imunologia , RNA Mensageiro/metabolismo , Superóxido Dismutase-1/metabolismo
4.
Biochim Biophys Acta ; 1832(12): 2009-18, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23872112

RESUMO

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.


Assuntos
Encéfalo/patologia , Modelos Animais de Doenças , Glutationa Peroxidase/metabolismo , Heme Oxigenase-1/metabolismo , Malária Cerebral/patologia , Estresse Oxidativo , Animais , Antioxidantes/metabolismo , Western Blotting , Encéfalo/metabolismo , Catalase/metabolismo , Glutationa/metabolismo , Malária Cerebral/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oxirredução , Carbonilação Proteica , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Fatores de Transcrição/metabolismo
5.
Antimicrob Agents Chemother ; 57(12): 5878-88, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24041883

RESUMO

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.


Assuntos
Compostos de Anilina/farmacologia , Antimaláricos/farmacologia , Antineoplásicos/farmacologia , Butanos/farmacologia , Colina Quinase/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Compostos de Piridínio/farmacologia , Compostos de Quinolínio/farmacologia , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Cloroquina/farmacologia , Colina/química , Colina/metabolismo , Colina Quinase/química , Colina Quinase/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Escherichia coli/genética , Humanos , Cinética , Testes de Sensibilidade Parasitária , Fosforilação/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Trofozoítos/efeitos dos fármacos , Trofozoítos/enzimologia , Trofozoítos/crescimento & desenvolvimento
6.
Trop Parasitol ; 12(2): 78-86, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36643988

RESUMO

Background: Plants are an important option in the treatment of malaria, especially in endemic regions, and are a less expensive and more accessible alternative with a lower risk of toxicity. Colombia has a great diversity of plants, and evaluation of natural extracts could result in the discovery of new compounds for the development of antimalarial drugs. The purpose of this work was to evaluate the in vitro antiplasmodial activity and the cytotoxicity of plant extracts from the Colombian North Coast against Plasmodium falciparum. Materials and Methods: The antiplasmodial activity of 12 plant species from the Colombian North Coast that are used in traditional medicine was evaluated through in vitro cultures of P. falciparum, and the cytotoxicity of extracts of these species to human cells was determined. Plant extracts with high antiplasmodial activity were subjected to preliminary phytochemical screening. Results: Extracts from five plants had promising antiplasmodial activity. Specifically, Bursera simaruba (Burseraceae) (bark), Guazuma ulmifolia Lam. (Malvaceae) (whole plant), Murraya exotica L. (Rutaceae) (leaves), Hippomane mancinella L. (Euphorbiaceae) (seeds), and Capparis odoratissima Jacq. (Capparaceae) (leaves). Extracts presented 50% inhibitory concentration values between 1 and 9 µg/ml. Compared to no extract, these active plant extracts did not show cytotoxic effects on mononuclear cells or hemolytic activity in healthy human erythrocytes. Conclusions: The results obtained from this in vitro study of antiplasmodial activity suggest that active plant extracts from the Colombian North Coast are promising for future bioassay-guided fractionation to allow the isolation of active compounds and to elucidate their mechanism of action against Plasmodium spp.

7.
Front Cell Infect Microbiol ; 12: 934321, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36118030

RESUMO

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


Assuntos
Malária Falciparum , Malária , Plasmodium , Anticorpos Antiprotozoários , Biomarcadores , Criança , Humanos , Imunoglobulina A , Imunoglobulina G , Imunoglobulina M , Malária Falciparum/diagnóstico , Plasmodium falciparum , Proteoma
8.
J Proteome Res ; 10(4): 1719-27, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21235272

RESUMO

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


Assuntos
Química Encefálica , Eletroforese em Gel Bidimensional/métodos , Proteínas do Tecido Nervoso/química , Carbonilação Proteica , Proteômica/métodos , Animais , Camundongos , Estrutura Molecular , Estresse Oxidativo , Fenil-Hidrazinas/química , Espécies Reativas de Oxigênio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
9.
J Clin Microbiol ; 49(12): 4401-4, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21976762

RESUMO

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


Assuntos
Linfoma de Células B/complicações , Malária/diagnóstico , Traço Falciforme/complicações , Antineoplásicos/administração & dosagem , Guiné Equatorial , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/cirurgia , Malária/patologia , Masculino , Pessoa de Meia-Idade , Parasitemia/diagnóstico , Parasitemia/parasitologia , Plasmodium/isolamento & purificação , Espanha , Baço/imunologia , Esplenectomia , Transplante de Células-Tronco , Transplante Autólogo
10.
Malar J ; 10: 82, 2011 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-21477369

RESUMO

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.


Assuntos
Antimaláricos/farmacologia , Atovaquona/farmacologia , Cloroquina/farmacologia , Eritrócitos/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Triterpenos/farmacologia , Malária Falciparum/sangue , Malária Falciparum/prevenção & controle , Parasitemia/sangue , Parasitemia/prevenção & controle , Plasmodium falciparum/crescimento & desenvolvimento
11.
Malar J ; 10: 103, 2011 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-21518429

RESUMO

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.


Assuntos
Antimaláricos/administração & dosagem , Malária/tratamento farmacológico , Malária/imunologia , Plasmodium yoelii/efeitos dos fármacos , Plasmodium yoelii/imunologia , Triterpenos/administração & dosagem , Animais , Anticorpos Antiprotozoários/sangue , Western Blotting , Ensaio de Imunoadsorção Enzimática , Eritrócitos/parasitologia , Feminino , Injeções Intraperitoneais , Malária/mortalidade , Malária/prevenção & controle , Camundongos , Camundongos Endogâmicos ICR , Microscopia , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Plasmodium yoelii/crescimento & desenvolvimento , Análise de Sobrevida
12.
J Proteome Res ; 9(12): 6392-404, 2010 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-20932057

RESUMO

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.


Assuntos
Proteínas de Peixes/análise , Gadiformes/metabolismo , Proteômica/métodos , Animais , Oceano Atlântico , Encéfalo/metabolismo , Análise por Conglomerados , Eletroforese em Gel Bidimensional , Europa (Continente) , Proteínas de Peixes/classificação , Gadiformes/crescimento & desenvolvimento , Geografia , Fígado/metabolismo , Espectrometria de Massas , Mar Mediterrâneo , Análise Multivariada , Dinâmica Populacional , Análise de Componente Principal , Isoformas de Proteínas/análise , Isoformas de Proteínas/classificação
13.
J Proteome Res ; 9(11): 5770-81, 2010 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-20818828

RESUMO

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 ß-spectrin under physiological conditions.


Assuntos
Aldeídos/metabolismo , Aminoácidos/análise , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Aminoácidos/metabolismo , Animais , Biomarcadores/análise , Bovinos , Membrana Eritrocítica/química , Histidina/metabolismo , Humanos , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Estresse Oxidativo , Soroalbumina Bovina/análise , Espectrina
14.
J Infect Dis ; 200(8): 1279-88, 2009 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-19758097

RESUMO

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.


Assuntos
Regulação da Expressão Gênica/fisiologia , Malária Cerebral/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/parasitologia , Encéfalo/patologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium berghei , Análise Serial de Proteínas , RNA Mensageiro/metabolismo
15.
Sci Rep ; 10(1): 9398, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523082

RESUMO

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-stage Plasmodium falciparum rapidly 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 for P. falciparum antigens and to identify epitopes associated with exposure and clinical disease. Sera from malaria cases identified five prevalent antigens recognized by all analyzed patients' IgGs. Epitope mapping of them, using adult and children sera samples from an endemic malaria region in Ghana segregated into patients with positive or negative subclinical detection of P. falciparum, revealed binding specificity for two 20-mer immunodominant antigenic regions within the START-related lipid transfer protein and the protein disulfide isomerase PDI8. These 20-mer epitopes 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 humoral response against START and PDI8 antigens may be triggered at submicroscopic parasitemia levels in children and may eventually be used to differentially diagnose subclinical malaria in children.


Assuntos
Epitopos/imunologia , Imunoglobulina G/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Adolescente , Adulto , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Criança , Mapeamento de Epitopos/métodos , Feminino , Gana , Humanos , Malária Falciparum/parasitologia , Masculino , Parasitemia/imunologia , Parasitemia/parasitologia , Proteínas de Protozoários/imunologia , Adulto Jovem
16.
Malar J ; 8: 279, 2009 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-19961586

RESUMO

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.


Assuntos
Cloroquina/farmacologia , Eritrócitos/parasitologia , Hemoglobinas/química , Parasitemia/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Benzotiazóis , Separação Celular , Citofotometria/métodos , DNA de Protozoário/efeitos dos fármacos , Diaminas , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Corantes Fluorescentes , Humanos , Concentração Inibidora 50 , Limite de Detecção , Malária Falciparum/diagnóstico , Compostos Orgânicos , Parasitemia/genética , Testes de Sensibilidade Parasitária/métodos , Plasmodium falciparum/genética , Quinolinas , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos
17.
Am J Hematol ; 84(2): 79-86, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19097174

RESUMO

The aim of this study was to evaluate the biological correlation and prognostic impact of Gata-1, Gata-2, EKLF, and c-MPL transcript level in a group of 41 acute myeloid leukemia (AML) patients. Gata-1 overexpression was related to advanced age and a low percentage of bone marrow blasts and was associated with the expression of CD34 antigen and lymphoid T markers. The negative impact of Gata-1 expression on the probability of achieving complete remission has been confirmed. Gata-2 overexpression was associated with a low percentage of blasts in BM and males. Expression of c-MPL was associated with CD34+ AML and M2 FAB AML subtype. A higher expression of EKLF was found in secondary AML versus primary AML. Nevertheless, patients expressing EKLF had a longer overall survival and event free survival than those patients that did not express EKLF. Our study has identified expression of EKLF as a factor with a favorable impact on prognosis in AML.


Assuntos
Fator de Transcrição GATA1/fisiologia , Fator de Transcrição GATA2/fisiologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/fisiologia , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/fisiologia , Receptores de Trombopoetina/fisiologia , Adolescente , Adulto , Idoso , Medula Óssea/patologia , Aberrações Cromossômicas , Intervalo Livre de Doença , Eritropoese/genética , Fator de Transcrição GATA1/análise , Fator de Transcrição GATA2/análise , Humanos , Fatores de Transcrição Kruppel-Like/análise , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/metabolismo , Segunda Neoplasia Primária/mortalidade , Segunda Neoplasia Primária/patologia , Prognóstico , Receptores de Trombopoetina/análise , Análise de Sobrevida , Adulto Jovem
18.
Ther Adv Infect Dis ; 6: 2049936119851464, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31205689

RESUMO

BACKGROUND: Recent advances in antiviral therapy show potential for a cure and/or control of most human infections caused by hepatitis viruses and retroviruses. However, medical success is largely dependent on the identification of the large number of people unaware of these infections, especially in developing countries. Dried blood spots (DBS) have been demonstrated to be a good tool for collecting, storing and transporting clinical specimens from rural areas and limited-resource settings to laboratory facilities, where viral infections can be more reliably diagnosed. METHODS: The seroprevalence and virological characterization of hepatitis B virus (HBV) and hepatitis C virus (HCV), as well as human retroviruses (HIV-1, HIV-2, human T-cell leukaemia virus type 1 [HTLV-1] and human T-cell leukaemia virus type 2 [HTLV-2]), were investigated in clinical specimens collected from DBS in Ghana. RESULTS: A total of 305 consecutive DBS were collected. A high prevalence of chronic HBV (8.5%) and occult hepatitis B (14.2%) was found, whereas rates were lower for HIV-1, HTLV-1 and HCV (3.2%, 1.3% and 0.6%, respectively). HIV-2 and HTLV-2 were absent. CRF02_AG was the predominant HIV-1 subtype, whereas genotype E was the most frequent HBV variant. CONCLUSIONS: DBS are helpful in the diagnosis and virological characterization of hepatitis and retrovirus infections in resource-limited settings. The high rate of hepatitis B in Ghana, either overt or occult, is noteworthy and confirms recent findings from other sub-Saharan countries. This should encourage close clinical follow up and antiviral treatment assessment in this population, as well as universal HBV vaccine campaigns.

19.
Free Radic Biol Med ; 44(12): 2034-42, 2008 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-18397762

RESUMO

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.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Eritrócitos/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Antígenos de Protozoários/metabolismo , Humanos , Oxirredução , Fenil-Hidrazinas/farmacologia , Plasmodium falciparum/metabolismo
20.
FASEB J ; 21(3): 851-65, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17244817

RESUMO

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.


Assuntos
Carboxipeptidases/química , Proteínas de Ligação ao GTP/genética , Peptídeo Hidrolases/classificação , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , Animais , Bactérias/enzimologia , Bactérias/genética , Sequência de Bases , Sítios de Ligação , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Proteínas de Ligação ao GTP/química , Camundongos , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , D-Ala-D-Ala Carboxipeptidase Tipo Serina/química
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