RESUMEN
Since the term proteomics was coined by Marc Wilkins in 1994, there has been an explosion in the number of articles reporting the use of the proteomics technique. As the layers of biological organization and their regulation increase, the complexity of living beings increases. Thus, we go from the genome to tissues, cells, cellular compartments, and phenotypes and the complexity of the tools used to study this complexity also increases. Unlike the genome study, in the case of the proteome, we have a more complex panorama. We have a spatial and temporal proteome. Proteomics helps to answer complex biological questions since proteins' function depends on their molecular structure, subcellular localization, and posttranslational modifications. In this protocol, we describe a methodology to extract proteins using different methods, separating proteins by electrophoresis in double-dimensional gels and analyzing the gels using specialized software that allows obtaining information on the number and abundance of the proteins from the gels.
Asunto(s)
Coffea , Proteínas de Plantas , Proteómica , Proteómica/métodos , Proteínas de Plantas/metabolismo , Proteínas de Plantas/análisis , Coffea/metabolismo , Coffea/química , Coffea/genética , Proteoma/análisis , Electroforesis en Gel Bidimensional/métodos , Programas InformáticosRESUMEN
Natural-based compounds with repellent activity arise nowadays with the possibility to replace commercial synthetic repellents wholly or partially, such as N,N-Diethyl-m-toluamide (DEET). It is due to DEET's demonstrated toxicity and cutaneous irritation for human beings. Besides, research recommends avoiding using it with kids and pregnant women. The search for a repellent product implies early stages of detailed research that resolve the modes of action against the target insect. Therefore the objective of the current study was to analyze neuronal electrophysiological signals and olfactory system protein expression when the Aedes aegypti mosquito with exposition to natural-based repellents. Adult females of Ae. aegypti of Rockefeller strain were exposed to specific concentrations of repellent compounds like geranyl acetate, α-bisabolol, nerolidol, and DEET. The neuronal effect was measured by electroantennography technique, and the effect of exposure to either DEET or a mixture of natural molecules on protein expression was determined with 2D-PAGE followed by MALDI-TOF-mass spectrometry (MS). This approach revealed that DEET affected proteins related to synapses and ATP production, whereas natural-based repellents increased transport, signaling, and detoxification proteins. The proteomic and electrophysiology experiments demonstrated that repellent exposure disrupts ionic channel activity and modifies neuronal synapse and energy production processes.
Asunto(s)
Aedes , Repelentes de Insectos , Embarazo , Adulto , Animales , Femenino , Humanos , Proteómica , DEET/farmacología , Repelentes de Insectos/farmacología , Electroforesis en Gel BidimensionalRESUMEN
Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.
Asunto(s)
Bothrops , Venenos de Crotálidos , Animales , Bothrops/metabolismo , Brasil , Electroforesis en Gel Bidimensional , Venenos de Crotálidos/metabolismo , Venenos de Serpiente/metabolismo , Electroforesis en Gel de Poliacrilamida , Metaloproteasas/metabolismo , Serina Proteasas/metabolismo , Lectinas Tipo C/metabolismoRESUMEN
Trypanosoma rangeli is a non-virulent hemoflagellate parasite infecting humans, wild and domestic mammals in Central and Latin America. The share of genotypic, phenotypic, and biological similarities with the virulent, human-infective T. cruzi and T. brucei, allows comparative studies on mechanisms of pathogenesis. In this study, investigation of the T. rangeli Arginine Kinase (TrAK) revealed two highly similar copies of the AK gene in this taxon, and a distinct expression profile and activity between replicative and infective forms. Although TrAK expression seems stable during epimastigotes growth, the enzymatic activity increases during the exponential growth phase and decreases from the stationary phase onwards. No differences were observed in activity or expression levels of TrAK during in vitro differentiation from epimastigotes to infective forms, and no detectable AK expression was observed for blood trypomastigotes. Overexpression of TrAK by T. rangeli showed no effects on the in vitro growth pattern, differentiation to infective forms, or infectivity to mice and triatomines. Although differences in TrAK expression and activity were observed among T. rangeli strains from distinct genetic lineages, our results indicate an up-regulation during parasite replication and putative post-translational myristoylation of this enzyme. We conclude that up-regulation of TrAK activity in epimastigotes appears to improve proliferation fitness, while reduced TrAK expression in blood trypomastigotes may be related to short-term and subpatent parasitemia in mammalian hosts.
Asunto(s)
Arginina Quinasa/metabolismo , Procesamiento Proteico-Postraduccional , Trypanosoma cruzi/enzimología , Trypanosoma rangeli/enzimología , Secuencia de Aminoácidos , Animales , Arginina Quinasa/biosíntesis , Arginina Quinasa/clasificación , Arginina Quinasa/genética , Western Blotting , ADN Protozoario/aislamiento & purificación , Electroforesis en Gel Bidimensional , Femenino , Flagelos/enzimología , Técnica del Anticuerpo Fluorescente Indirecta , Ratones , Ratones Endogámicos BALB C , Filogenia , Alineación de Secuencia , Trypanosoma cruzi/clasificación , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidad , Trypanosoma rangeli/clasificación , Trypanosoma rangeli/genética , Trypanosoma rangeli/patogenicidad , Regulación hacia Arriba , VirulenciaRESUMEN
Two-dimensional gel electrophoresis (2D-GE) is an indispensable technique for the study of proteomes of biological systems, providing an assessment of changes in protein abundance under various experimental conditions. However, due to the complexity of 2D-GE gels, there is no systematic, automatic, and reproducible protocol for image analysis and specific implementations are required for each context. In addition, practically all available solutions are commercial, which implies high cost and little flexibility to modulate the parameters of the algorithms. Using the bacterial strain, Pseudomonas aeruginosaAG1 as a model, we obtained images from 2D-GE of periplasmic protein profiles when the strain was exposed to multiple conditions, including antibiotics. Then, we proceeded to implement and evaluate an image analysis protocol with open-source software, CellProfiler. First, a preprocessing step included a bUnwarpJ-Image pipeline for aligning 2D-GE images. Then, using CellProfiler, we standardized two pipelines for spots identification. Total spots recognition was achieved using segmentation by intensity, whose performance was evaluated when compared with a reference protocol. In a second pipeline with the same program, differential identification of spots was addressed when comparing pairs of protein profiles. Due to the characteristics of the programs used, our workflow can automatically analyze a large number of images and it is parallelizable, which is an advantage with respect to other implementations. Finally, we compared six experimental conditions of bacterial strain in the presence or absence of antibiotics, determining protein profiles relationships by applying clustering algorithms PCA (Principal Components Analysis) and HC (Hierarchical Clustering).
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Proteómica/métodos , Pseudomonas aeruginosa/citología , Humanos , Proteómica/instrumentaciónRESUMEN
Angiostrongylus cantonensis is the main causative agent of eosinophilic meningoencephalitis (EoM) in humans. Molecular diagnostic methods are essential since the identification of larvae in cerebrospinal fluid (CSF) is extremely rare. To date, the detection of a 31 kDa antigen by Western blotting has been the primary immunodiagnostic method for EoM caused by A. cantonensis. However, cross-reactivity with other parasites has been observed. Therefore, we conducted a comparative analysis using sera from individuals with angiostrongyliasis. We also characterized proteins isolated from different cellular sources of A. cantonensis, Toxocara canis, Schistosoma mansoni, and Strongyloides stercoralis with mass spectrometry. A total of 115 cross-reactive proteins were identified. Three of these proteins, heat shock protein, an intermediate filament protein, and galectin 1, represent potential markers for cross-reactivity. In addition, synthetic peptides were generated from previously identified diagnostic targets and tested against sera from individuals infected with several other parasites. As a result, two other markers of cross-reactivity were identified: peptide #4 derived from the 14-3-3 protein and peptide #12 derived from the Lec-5 protein. In contrast, 34 proteins were exclusively present in the Angiostrongylus extracts and represent promising diagnostic molecules for specific identification of A. cantonensis infection. In particular, cytochrome oxidase subunit I is of great interest as a possible immunodiagnostic target for angiostrongyliasis.
Asunto(s)
Angiostrongylus cantonensis/inmunología , Antígenos Helmínticos/inmunología , Proteínas del Helminto/inmunología , Meningoencefalitis/diagnóstico , Meningoencefalitis/parasitología , Infecciones por Strongylida/diagnóstico , Secuencia de Aminoácidos , Angiostrongylus cantonensis/química , Animales , Antígenos Helmínticos/sangre , Antígenos Helmínticos/química , Antígenos Helmínticos/aislamiento & purificación , Western Blotting , Secuencia Conservada , Reacciones Cruzadas , Electroforesis , Electroforesis en Gel Bidimensional , Proteínas del Helminto/química , Proteínas del Helminto/aislamiento & purificación , Humanos , Inmunoensayo , Pruebas Inmunológicas , Espectrometría de Masas , Meningoencefalitis/inmunología , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitologíaRESUMEN
BACKGROUND: Two-dimensional gel electrophoresis (2-DGE) is a commonly used tool for proteomic analysis. This gel-based technique separates proteins in a sample according to their isoelectric point and molecular weight. 2-DGE images often present anomalies due to the acquisition process, such as: diffuse and overlapping spots, and background noise. This study proposes a joint pre-processing framework that combines the capabilities of nonlinear filtering, background correction and image normalization techniques for pre-processing 2-DGE images. Among the most important, joint nonlinear diffusion filtering, adaptive piecewise histogram equalization and multilevel thresholding were evaluated using both synthetic data and real 2-DGE images. RESULTS: An improvement of up to 46% in spot detection efficiency was achieved for synthetic data using the proposed framework compared to implementing a single technique of either normalization, background correction or filtering. Additionally, the proposed framework increased the detection of low abundance spots by 20% for synthetic data compared to a normalization technique, and increased the background estimation by 67% compared to a background correction technique. In terms of real data, the joint pre-processing framework reduced the false positives up to 93%. CONCLUSIONS: The proposed joint pre-processing framework outperforms results achieved with a single approach. The best structure was obtained with the ordered combination of adaptive piecewise histogram equalization for image normalization, geometric nonlinear diffusion (GNDF) for filtering, and multilevel thresholding for background correction.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Bases de Datos de Proteínas , Humanos , Procesamiento de Imagen Asistido por Computador , Proteínas/análisis , Proteómica/métodos , Relación Señal-RuidoRESUMEN
This chapter describes the application of two-dimensional gel electrophoresis (2DGE) combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the analysis of rat eye lens proteins. The main purpose was to identify proteins that may serve as potential biomarkers in age-related cataract formation. This includes the family of proteins known as the crystallins. Structural proteins and enzymes involved antioxidant activities. In addition, we also analyzed lenses from other species to illustrate the potential of using this technique in clinical and preclinical biomarker studies.
Asunto(s)
Envejecimiento/metabolismo , Biomarcadores/metabolismo , Catarata/metabolismo , Electroforesis en Gel Bidimensional/métodos , Proteínas del Ojo/metabolismo , Cristalino/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Perros , Humanos , Persona de Mediana Edad , RatasRESUMEN
Calf diarrhea causes substantial economic losses in the cattle industry worldwide. Bovine rotavirus A (RVA) is the main viral agent that leads to enteric infection and diarrhea outbreaks in calves throughout the world. The aim of this retrospective (2006-2015) study was to determine the frequency of RVA detection in diarrheic fecal samples from beef and dairy calves from the three main cattle-producing regions of Brazil. Diarrheic fecal samples (n=1,498) of 124 beef and 56 dairy cattle herds from the Midwest, South, and Southeast geographical regions of Brazil were evaluated using the silver-stained polyacrylamide gel electrophoresis (ss-PAGE) technique. RVA double stranded-RNA was identified by the ss-PAGE technique in 410 (27.4%) fecal samples. The frequency of positive samples found in beef calves (31.9%; 328/1,027) was higher than the frequency found in diarrheic fecal samples from dairy calves (17.4%; 82/471). RVA infection was identified in calves from the three Brazilian geographical regions analyzed. However, the frequency of positive diarrheic calves in the Midwest region (39.4%), predominantly beef calves, was higher than in the South (19.4%) and Southeast (17.6%) regions. The temporal distribution of RVA-infected calves evaluated by two five-year periods (2006-2010, 24.5%; 2011-2015, 28.8%) demonstrated a very similar frequency of RVA in both periods. Considering the wide regional and temporal scope of this study, it can be concluded that RVA remains an important etiology of neonatal diarrhea in calves of Brazilian cattle herds.(AU)
A diarreia neonatal ocasiona perdas econômicas importantes na pecuária bovina em todo o mundo. Rotavírus A (RVA) é o principal agente etiológico viral de infecções entéricas e surtos de diarreia em bezerros de rebanhos de corte e leite. O objetivo deste estudo retrospectivo (2006-2015) foi determinar a frequência de detecção de RVA em amostras de fezes diarreicas de bezerros de corte e leite das três principais regiões produtoras de bovinos do Brasil. Amostras de fezes diarreicas (n=1.498) de 124 rebanhos bovinos de corte e 56 rebanhos bovinos de leite das regiões Centro-Oeste, Sul e Sudeste do Brasil foram avaliadas utilizando a técnica de eletroforese em gel de poliacrilamida (EGPA). O genoma segmentado de RVA foi identificado pela técnica de EGPA em 410 (27,4%) amostras de fezes. A frequência de amostras positivas encontrada em bezerros de rebanhos de corte (31,9%; 328/1.027) foi maior que a frequência identificada em amostras de fezes diarreicas de bezerros de rebanhos leiteiros (17,4%; 82/471). A infecção por RVA foi identificada em bezerros das três regiões geográficas brasileiras analisadas. No entanto, a frequência de bezerros com diarreia positivos para RVA na região Centro-Oeste (39,4%), predominantemente de bezerros de rebanhos de corte, foi maior que nas regiões Sul (19,4%) e Sudeste (17,6%). A distribuição temporal dos bezerros infectados com RVA avaliados por dois períodos de cinco anos (2006-2010, 24,5%; 2011-2015, 28,8%) demonstrou uma frequência muito semelhante em ambos os períodos. Considerando a amplitude regional e temporal deste estudo, pode-se concluir que RVA continua sendo uma importante etiologia de diarreia neonatal em bezerros de rebanhos bovinos brasileiros.(AU)
Asunto(s)
Animales , Bovinos , Infecciones por Rotavirus/veterinaria , Infecciones por Rotavirus/epidemiología , Rotavirus/patogenicidad , Enfermedades Gastrointestinales/etiología , Electroforesis en Gel Bidimensional/veterinariaRESUMEN
Calf diarrhea causes substantial economic losses in the cattle industry worldwide. Bovine rotavirus A (RVA) is the main viral agent that leads to enteric infection and diarrhea outbreaks in calves throughout the world. The aim of this retrospective (2006-2015) study was to determine the frequency of RVA detection in diarrheic fecal samples from beef and dairy calves from the three main cattle-producing regions of Brazil. Diarrheic fecal samples (n=1,498) of 124 beef and 56 dairy cattle herds from the Midwest, South, and Southeast geographical regions of Brazil were evaluated using the silver-stained polyacrylamide gel electrophoresis (ss-PAGE) technique. RVA double stranded-RNA was identified by the ss-PAGE technique in 410 (27.4%) fecal samples. The frequency of positive samples found in beef calves (31.9%; 328/1,027) was higher than the frequency found in diarrheic fecal samples from dairy calves (17.4%; 82/471). RVA infection was identified in calves from the three Brazilian geographical regions analyzed. However, the frequency of positive diarrheic calves in the Midwest region (39.4%), predominantly beef calves, was higher than in the South (19.4%) and Southeast (17.6%) regions. The temporal distribution of RVA-infected calves evaluated by two five-year periods (2006-2010, 24.5%; 2011-2015, 28.8%) demonstrated a very similar frequency of RVA in both periods. Considering the wide regional and temporal scope of this study, it can be concluded that RVA remains an important etiology of neonatal diarrhea in calves of Brazilian cattle herds.(AU)
A diarreia neonatal ocasiona perdas econômicas importantes na pecuária bovina em todo o mundo. Rotavírus A (RVA) é o principal agente etiológico viral de infecções entéricas e surtos de diarreia em bezerros de rebanhos de corte e leite. O objetivo deste estudo retrospectivo (2006-2015) foi determinar a frequência de detecção de RVA em amostras de fezes diarreicas de bezerros de corte e leite das três principais regiões produtoras de bovinos do Brasil. Amostras de fezes diarreicas (n=1.498) de 124 rebanhos bovinos de corte e 56 rebanhos bovinos de leite das regiões Centro-Oeste, Sul e Sudeste do Brasil foram avaliadas utilizando a técnica de eletroforese em gel de poliacrilamida (EGPA). O genoma segmentado de RVA foi identificado pela técnica de EGPA em 410 (27,4%) amostras de fezes. A frequência de amostras positivas encontrada em bezerros de rebanhos de corte (31,9%; 328/1.027) foi maior que a frequência identificada em amostras de fezes diarreicas de bezerros de rebanhos leiteiros (17,4%; 82/471). A infecção por RVA foi identificada em bezerros das três regiões geográficas brasileiras analisadas. No entanto, a frequência de bezerros com diarreia positivos para RVA na região Centro-Oeste (39,4%), predominantemente de bezerros de rebanhos de corte, foi maior que nas regiões Sul (19,4%) e Sudeste (17,6%). A distribuição temporal dos bezerros infectados com RVA avaliados por dois períodos de cinco anos (2006-2010, 24,5%; 2011-2015, 28,8%) demonstrou uma frequência muito semelhante em ambos os períodos. Considerando a amplitude regional e temporal deste estudo, pode-se concluir que RVA continua sendo uma importante etiologia de diarreia neonatal em bezerros de rebanhos bovinos brasileiros.(AU)
Asunto(s)
Animales , Bovinos , Infecciones por Rotavirus/veterinaria , Infecciones por Rotavirus/epidemiología , Rotavirus/patogenicidad , Enfermedades Gastrointestinales/etiología , Electroforesis en Gel Bidimensional/veterinariaRESUMEN
The actinobacterium Streptomyces sp. MC1 has previously shown the capacity to resist and remove Cr(VI) from liquid culture media. The aim of this work is to analyze the differential expression pattern of intracellular proteins when Streptomyces sp. MC1 is exposed to Cr(VI) in order to explain the molecular mechanisms of resistance that this microorganism possesses. For this purpose, 2D-PAGE and shotgun proteomic analyses (2D-nanoUPLC-ESI-MS/MS) were applied. The presence of Cr(VI) induced the expression of proteins involved in molecular biosynthesis and energy generation, chaperones with a key role in the repair of misfolded proteins and stress response, transcription proteins, proteins of importance in the DNA supercoiling, repair and replication, and dehydrogenases involved in oxidation-reduction processes. These dehydrogenases can be associated with the reduction of Cr(VI) to Cr(III). The results of this study show that proteins from the groups mentioned before are important to face the stress caused by the Cr(VI) presence and help the microorganism to counteract the toxicity of the metal. The use of two proteomic approaches resulted in a larger number of peptides identified, which is also transduced in a significant number of protein ID. This decreased the potential complexity of the sample because of the protein dynamic range, as well as increased the recovery of peptides from the gel after digestion.
Asunto(s)
Cromo/farmacología , Proteómica/métodos , Streptomyces/efectos de los fármacos , Streptomyces/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biología Computacional , Electroforesis en Gel Bidimensional , Espectrometría de Masas en TándemRESUMEN
The high concentrations of mercury found in Amazon have been intensively studied by the scientific community in the last decades. These mercurial species bind preferentially to proteins. Therefore, this work proposal sought to obtain the fractionation, identification and study of mercury - bound proteins present in samples of muscular and hepatic tissue from fish collected in the reservoir of the Jirau Hydroelectric Power Plant - on the Madeira River. Two-dimensional electrophoresis (2D-PAGE) for protein fractionation, graphite furnace atomic absorption spectrometry (GFAAS) for the quantification of mercury and Mass Spectrometry (ESI-MS/MS) were used for the identification of proteins. Concluding the work with analysis of graphics from the Blast2go program. Two mercury - bound proteins were identified as triosephosphate isomerase A and Protein FAM45A. The data generated by the bioinformatics programs confirm the tendency of these proteins to be linked to mercury and elucity the possibles existing physiological and cellular interactions.
Asunto(s)
Peces , Hígado/química , Mercurio/análisis , Músculo Esquelético/química , Animales , Brasil , Electroforesis en Gel Bidimensional , Ríos/química , Espectrofotometría Atómica , Espectrometría de Masas en Tándem , Contaminantes Químicos del Agua/análisisRESUMEN
Obesity is a pandemic associated with a high incidence of cardiovascular disease; however, the mechanisms are not fully elucidated. Proteomics may provide a more in-depth understanding of the pathophysiological mechanisms and contribute to the identification of potential therapeutic targets. Thus, our study evaluated myocardial protein expression in healthy and obese rats, employing two proteomic approaches. Male Wistar rats were established in two groups (n = 13/group): control diet and Western diet fed for 41 weeks. Obesity was determined by the adipose index, and cardiac function was evaluated in vivo by echocardiogram and in vitro by isolated papillary muscle analysis. Proteomics was based on two-dimensional gel electrophoresis (2-DE) along with mass spectrometry identification, and shotgun proteomics with label-free quantification. The Western diet was efficient in triggering obesity and impaired contractile function in vitro; however, no cardiac dysfunction was observed in vivo. The combination of two proteomic approaches was able to increase the cardiac proteomic map and to identify 82 differentially expressed proteins involved in different biological processes, mainly metabolism. Furthermore, the data also indicated a cardiac alteration in fatty acids transport, antioxidant defence, cytoskeleton, and proteasome complex, which have not previously been associated with obesity. Thus, we define a robust alteration in the myocardial proteome of diet-induced obese rats, even before functional impairment could be detected in vivo by echocardiogram.
Asunto(s)
Enfermedades Cardiovasculares/patología , Miocardio/patología , Obesidad/metabolismo , Proteoma/análisis , Animales , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/metabolismo , Citoesqueleto/metabolismo , Dieta Occidental/efectos adversos , Modelos Animales de Enfermedad , Electroforesis en Gel Bidimensional , Ácidos Grasos/metabolismo , Humanos , Masculino , Espectrometría de Masas , Miocardio/metabolismo , Obesidad/etiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Ratas , Ratas WistarRESUMEN
Manganese (Mn) might stimulate the valve closure reflex in the freshwater bivalve Anodontites trapesialis, leading to metabolic suppression, whereas zinc (Zn) is not able to modify this behavior. To investigate this particular response, we exposed A. trapesialis specimens to Mn (0.5 mg L-1 ) and Zn (1.0 mg L-1 ) alone, and to their mixture, to measure further endpoints in different clam tissues: glycogen level in gills, and calcium (Ca2+ ), sodium (Na+ ), and chloride (Cl- ) concentrations in the hemolymph. Furthermore, we used cutting-edge technology, proteomics, to evaluate modifications in protein patterns under the 3 exposure tests. The main results highlighted that only Mn caused a clear drop in glycogen levels in gills, an increase in Ca2+ and Na+ , and a simultaneous decrease in Cl- concentration in the hemolymph. The proteomic analysis confirmed that Mn promoted more effects in A. trapesialis than the other tested conditions, because the number of proteins modulated was higher than the results obtained after exposure to Zn and the mixture. Moreover, 11 of the 12 modulated proteins were down-expressed. These results consolidate the hypothesis that Mn might suppress gill metabolic rate in A. trapesialis. Environ Toxicol Chem 2019;38:2480-2485. © 2019 SETAC.
Asunto(s)
Bivalvos/efectos de los fármacos , Bivalvos/metabolismo , Determinación de Punto Final , Branquias/metabolismo , Manganeso/toxicidad , Zinc/toxicidad , Animales , Electroforesis en Gel Bidimensional , Proteínas de Peces/metabolismo , Agua Dulce/química , Branquias/efectos de los fármacos , Glucógeno/metabolismo , Hemolinfa/efectos de los fármacos , Iones/metabolismo , Concentración Osmolar , Proteómica , Contaminantes Químicos del Agua/toxicidadRESUMEN
Neospora caninum is an obligate intracellular parasite related to cases of abortion and fertility impairment in cattle. The control of the parasite still lacks an effective protective strategy and the understanding of key mechanisms for host infection might be crucial for identification of specific targets. There are many proteins related to important mechanisms in the host cell infection cycle such as adhesion, invasion, proliferation and immune evasion. The surface proteins, especially SRS (Surface Antigen Glycoprotein - Related Sequences), have been demonstrated to have a pivotal role in the adhesion and invasion processes, making them potential anti-parasite targets. However, several predicted surface proteins were not described concerning their function and importance in the parasite life cycle. As such, a novel SRS protein, NcSRS57, was described. NcSRS57 antiserum was used to detect SRS proteins by immunofluorescence in parasites treated or not with phosphatidylinositol-specific phospholipase C (PI-PLC). The treatment with PI-PLC also allowed the identification of NcSRS29B and NcSRS29C, which were the most abundant SRS proteins in the soluble fraction. Our data indicated that SRS proteins in N. caninum shared a high level of sequence similarity and were susceptible to PI-PLC. In addition, the description of the SRS members, regarding abundance, function and immunogenicity will be useful in guiding specific methods to control the mechanism of adhesion and invasion mediated by these surface proteins.
Asunto(s)
Antígenos de Protozoos/metabolismo , Antígenos de Superficie/metabolismo , Neospora/química , Fosfoinositido Fosfolipasa C/farmacología , Proteínas Protozoarias/metabolismo , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Chlorocebus aethiops , Clonación Molecular , ADN Protozoario/aislamiento & purificación , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Sueros Inmunes/inmunología , Sueros Inmunes/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Neospora/efectos de los fármacos , Neospora/genética , Neospora/inmunología , Fosfoinositido Fosfolipasa C/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Espectrometría de Masas en Tándem , Fosfolipasas de Tipo C/metabolismo , Fosfolipasas de Tipo C/farmacología , Células VeroRESUMEN
BACKGROUND: Angiostrongyliasis is caused by the nematode Angiostrongylus cantonensis and can lead to eosinophilic meningitis and meningoencephalitis in humans. The young adult worms play central pathogenic roles in the central nervous system (CNS); however, the underlying mechanism is unclear. Excretory-secretory products (ESPs) are good investigation targets for studying the relationship between a host and its parasite. OBJECTIVES: We aimed to profile, identify, and characterise the proteins in the ESPs of A. cantonensis young adults. METHODS: The ESPs of young adult worms were collected from culture medium after incubation ranging from 24 to 96 h. Proteomic and bioinformatics analyses were performed to characterise the ESPs. FINDINGS: A total of 51 spots were identified, and the highly expressed proteins included two protein disulphide isomerases, one calreticulin, and three uncharacterised proteins. Subsequently, approximately 254 proteins were identified in the ESPs of A. cantonensis young adults via liquid chromatography-mass spectrometry (LC-MS/MS) analysis, and these were further classified according to their characteristics and biological functions. Finally, we identified the immunoreactive proteins from a reference map of ESPs from A. cantonensis young adults. Approximately eight proteins were identified, including a protein disulphide isomerase, a putative aspartic protease, annexin, and five uncharacterised proteins. The study established and identified protein reference maps for the ESPs of A. cantonensis young adults. MAIN CONCLUSIONS: The identified proteins may be potential targets for the development of diagnostic or therapeutic agents for human angiostrongyliasis.
Asunto(s)
Angiostrongylus cantonensis/metabolismo , Proteínas del Helminto/análisis , Proteómica , Animales , Western Blotting , Cromatografía Liquida/métodos , Electroforesis en Gel Bidimensional , Proteínas del Helminto/metabolismo , Espectrometría de Masas/métodos , Valores de ReferenciaRESUMEN
Schistosoma mansoni venom allergen-like proteins (SmVALs) are part of a diverse protein superfamily partitioned into two groups (group 1 and group 2). Phylogenetic analyses of group 1 SmVALs revealed that members could be segregated into subclades (A-D); these subclades share similar gene expression patterns across the parasite lifecycle and immunological cross-reactivity. Furthermore, whole-mount in situ hybridization demonstrated that the phylogenetically, transcriptionally and immunologically-related SmVAL4, 10, 18 and 19 (subclade C) were all localized to the pre-acetabular glands of immature cercariae. Our results suggest that SmVAL group 1 phylogenetic relationships, stage-specific transcriptional profiles and tissue localization are predictive of immunological cross-reactivity.
Asunto(s)
Antígenos Helmínticos/inmunología , Proteínas del Helminto/inmunología , Filogenia , Schistosoma mansoni/química , Alérgenos/clasificación , Alérgenos/genética , Alérgenos/inmunología , Animales , Antígenos Helmínticos/clasificación , Antígenos Helmínticos/genética , Western Blotting , Reacciones Cruzadas , Vacunas contra el Dengue/inmunología , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica , Proteínas del Helminto/clasificación , Proteínas del Helminto/genética , Sueros Inmunes/inmunología , Espectrometría de Masas , Familia de Multigenes , Schistosoma mansoni/clasificación , Schistosoma mansoni/genética , Schistosoma mansoni/inmunología , Transcripción GenéticaRESUMEN
Metal contamination exerts environmental pressure on several lifeforms. Since metals are non-biodegradable and recalcitrant, they accumulate in living beings and spread through the food chain. Thus, many life forms are affected by environmental metal contamination, such as plants and microorganisms. In the case of microorganisms, scarce information is available on how metals affect them. As a highly resistant form of life, microorganisms can adapt to several environmental pressures through genetic modifications, changing their metabolism to overcome new conditions, and continuing to thrive in the same place. In this study, an Acinetobacter sp. strain was isolated from a copper mine, which presented very high resistance to copper, growing in copper concentrations of up to 7 mM. As a result of its metabolic response in the presence of 3 mM of copper, the expression of 35 proteins in total was altered. The proteins were identified to be associated with the glycolytic pathway, membrane transport, biosynthesis and two proteins directly involved in copper homeostasis (CopA and CopB).
Asunto(s)
Acinetobacter/metabolismo , Cobre/toxicidad , Proteómica , Acinetobacter/efectos de los fármacos , Acinetobacter/crecimiento & desarrollo , Acinetobacter/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Electroforesis en Gel Bidimensional , Amplificación de Genes , Genes Bacterianos , Pruebas de Sensibilidad Microbiana , Transducción de Señal/efectos de los fármacosRESUMEN
Von Willebrand disease (VWD) is a common cause of bleeding worldwide. Analysis of von Willebrand factor (VWF) multimer distribution (VWF:MD) is essential to properly classify and treat different types of VWD, and it is performed using a SDS agarose gel electrophoresis followed by Western blotting, a handmade technique that demands days to be completed and requires skillful execution. Aiming both to facilitate gel production and to shorten the preparation time, we developed an uncomplicated technique to provide agility in the analysis of VWF:MD, so that it can be easily accomplished in the routine practice of hemostasis laboratories. On that account, we used a commercial vertical mini-gel electrophoresis system for SDS-PAGE and a semi-dry transfer system, which allowed us to analyze VWF:MD of various samples in a period shorter than 12â¯h. This technique differentiated VWF:MD in human and animal plasmas under normal, congenital and acquired (experimental envenomation by Bothrops jararaca snake) conditions. This optimized method is cheap, rapid, reproducible, easy to be performed, and uses electrophoresis and Western blotting systems available in most laboratories. All these advantages encourage hemostasis professionals to use it in their routine practices. In order to facilitate the setup and accomplishment of the whole procedure step by step, videos were appended to the article.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Enfermedades de von Willebrand/diagnóstico , Factor de von Willebrand/metabolismo , Animales , Humanos , Masculino , Ratas , Ratas Wistar , Enfermedades de von Willebrand/patología , Factor de von Willebrand/análisisRESUMEN
Apicomplexan parasites have unconventional actins that play a central role in important cellular processes such as apicoplast replication, motility of dense granules, endocytic trafficking and force generation for motility and host cell invasion. In this study, we investigated the actin of the apicomplexan Neospora caninum - a parasite associated with infectious abortion and neonatal mortality in livestock. Neospora caninum actin was detected and identified in two bands by one-dimensional (1D) western blot and in nine spots by the 2D technique. The mass spectrometry data indicated that N. caninum has at least nine different actin isoforms, possibly caused by post-translational modifications. In addition, the C4 pan-actin antibody detected specifically actin in N. caninum cellular extract. Extracellular N. caninum tachyzoites were treated with toxins that act on actin, jasplakinolide and cytochalasin D. Both substances altered the peripheric cytoplasmic localization of actin on tachyzoites. Our findings add complexity to the study of the apicomplexan actin in cellular processes, since the multiple functions of this important protein might be regulated by mechanisms involving post-translational modifications.