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1.
Sci Rep ; 9(1): 12019, 2019 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-31427667

RESUMEN

Humic substances have been widely used as plant growth promoters to improve the yield of agricultural crops. However, the mechanisms underlying this effect remain unclear. Root soluble protein profiles in plants 11 days after planting and cultivated with and without humic acids (HA, 50 mg CL-1), were analyzed using the label-free quantitative proteomic approach. Cultivation of maize with HA resulted in higher fresh weight of roots than in untreated plants (control). Plants treated with HA showed increased number, diameter and length of roots. In the proteomics analysis, differences were detected in the following categories: energy metabolism, cytoskeleton, cellular transport, conformation and degradation of proteins, and DNA replication. Thirty-four proteins were significantly more abundant in the seedlings treated with HA, whereas only nine proteins were abundant in the control. The effects on root architecture, such as the induction of lateral roots and biomass increase were accompanied by changes in the energy metabolism-associated proteins. The results show that the main effect of HA is protective, mainly associated with increased expression of the 2-cys peroxidase, putative VHS/GAT, and glutathione proteins. Indeed, these proteins had the highest fold-difference. Overall, these results improve our understanding of the molecular mechanisms of HA-promoted plant growth.

2.
Parasit Vectors ; 11(1): 264, 2018 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-29690895

RESUMEN

Dengue virus (DENV), an arbovirus transmitted by mosquitoes, has become a major threat to American human life, reaching approximately 23 million cases from 1980 to 2017. Brazil is among the countries most affected by this terrible viral disease, with 13.6 million cases. DENV has four different serotypes, DENV1-4, which show a broad clinical spectrum. Dengue creates a staggering epidemiological and economic burden for endemic countries. Without a specific therapy and with a commercial vaccine that presents some problems relative to its full effectiveness, initiatives to improve vector control strategies, early disease diagnostics and the development of vaccines and antiviral drugs are priorities. In this study, we present the probable origins of dengue in America and the trajectories of its spread. Overall, dengue diagnostics are costly, making the monitoring of dengue epidemiology more difficult and affecting physicians' therapeutic decisions regarding dengue patients, especially in developing countries. This review also highlights some recent and important findings regarding dengue in Brazil and the Americas. We also summarize the existing DENV polymerase chain reaction (PCR) diagnostic tests to provide an improved reference since these tests are useful and accurate at discriminating DENV from other flaviviruses that co-circulate in the Americas. Additionally, these DENV PCR assays ensure virus serotyping, enabling epidemiologic monitoring.


Asunto(s)
Virus del Dengue/aislamiento & purificación , Dengue/diagnóstico , Dengue/epidemiología , Américas/epidemiología , Dengue/historia , Dengue/patología , Historia del Siglo XIX , Historia del Siglo XX , Historia del Siglo XXI , Humanos
3.
J Proteomics ; 151: 43-52, 2017 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-27478070

RESUMEN

BACKGROUND: Oral leukoplakia is the most common potentially malignant disorder in the oral cavity and can precede carcinoma. This study aimed to identify possible oral leukoplakia salivary biomarkers. METHODS: Unstimulated saliva was collected from participants and protein concentration was determined. Proteins were then precipitated with cold acetone and separated using 2DE over a pH range of 3-10. Spot demarcation and matching were performed and protein identification was done through MS analysis. Oral leukoplakia tissues were submitted to immunohistochemistry analysis for keratin 10 (CK10). A complementary analysis of oral leukoplakias that were not included previously was performed in addition. RESULTS: 226±10 spots were identified in oral leukoplakia 2DE gels, and 262±12 spots were identified in volunteers. Twenty-two spots were highly abundant in oral leukoplakias or not detected in the control group, such as apolipoprotein A1, alpha amylase, cystatins, keratin 10, and lysozyme precursor. All were identified. All oral leukoplakia cases were immunopositive for CK10, mainly in the superficial epithelial layers. CONCLUSIONS: The 2DE salivary protein profiles of individuals with and without oral leukoplakia were observably different. CK10 appears to be an interesting protein and should be further studied in oral carcinogenesis. SIGNIFICANCE: MS-based proteomics enables large-scale analysis of proteins. Proteomics can provide detailed descriptions of proteomes of cells and tissues, including body fluids, and appears as a powerful tool to study human disorders. Saliva is readily accessible through non invasive collection and can mirror diverse disease states. Saliva from both diseased and healthy subjects can be analyzed through 2DE and differences between groups could be found. Routine immunohistochemistry analysis confirmed one of these findings, with CK10 being positive tissues from individuals with oral leukoplakia. Therefore, the present study allows insights into development of an important potential oral cancer precursor, named oral leukoplakia. However, the results can be extrapolated and tested in other precancer states, such as proliferative verrucous leukoplakia, patients at risk of oral cancer due to lifestyle behavior and/or cancer history in the family or even those who are under surveillance after a treated primary oral cancer.


Asunto(s)
Leucoplasia Bucal/química , Proteómica/métodos , Saliva/química , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor , Estudios de Casos y Controles , Electroforesis en Gel Bidimensional , Humanos , Queratina-10/análisis , Queratina-10/aislamiento & purificación , Persona de Mediana Edad , Lesiones Precancerosas/diagnóstico , Proteoma/análisis
4.
Urol Oncol ; 34(1): 5.e11-25, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26420021

RESUMEN

OBJECTIVE: The aim of this study was to evaluate the differentially secreted protein profile in the urine from patients with clear cell renal cell carcinoma (ccRCC) using mass spectrometry-based methods. Urine composition can reflect kidney physiology and can be used to detect markers for renal diseases. Moreover, characterization of the secretome is likely to assist in the investigation of new drugs for biological targets and diagnose the ccRCC at an early stage. METHODS AND MATERIALS: Urine samples from patients were divided according to Fuhrman degree (FI-IV), which was associated with the cellular differentiation as good prognosis (GP) and poor prognosis (PP). Healthy individuals were used as the control group (CG). We used both qualitative and quantitative mass spectrometry-based analyses that involved the following approaches: 1-dimensional gel electrophoresis combined with liquid chromatography mass spectrometry in tandem (1DE LC-MS/MS), in-solution digestion combined with label-free 1-dimensional LC-MS(E) (1D LC-MS(E)), and bidimensional gel electrophoresis combined with matrix-assisted laser desorption/ionization time of flight in tandem (2DE MALDI-TOF/TOF) or combined with LC-MS/MS. RESULTS: All the strategies allowed the identification of 354 proteins from the CG, GP, and PP groups. Qualitative experiments using 1DE LC-MS/MS analysis detected different protein profiles, and 224 proteins were identified in all groups. The label-free MS(E) quantitative analysis identified 113 proteins and generated novel information on secreted protein profiles, including 49 up-secreted proteins in the urine from patients with ccRCC and 40 down-secreted proteins related to the CG. Proteins such as kininogen-1, uromodulin, apolipoprotein D, polyubiquitin, and CD59 glycoprotein were down secreted according to the groups CG>GP>PP. In contrast, apolipoprotein A, fibrinogen, and haptoglobin were up secreted in patient groups. The same expression profile observed for kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin was corroborated by 2DE LC-MS/MS or 2DE MALDI-TOF/TOF analyses. These 2 strategies also showed 13 differentially secreted proteins among the 3 groups. CONCLUSIONS: The proteins kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin presented similar quantitative protein profiles according to MS(E) and 2DE approaches. The latter proteins were up secreted and the former ones were down-regulated. The strategies used proved to be valuable in identifying proteins that were differentially secreted in urine from patients with RCC.


Asunto(s)
Biomarcadores de Tumor/orina , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/metabolismo , Proteoma/análisis , Proteómica/métodos , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/orina , Estudios de Casos y Controles , Cromatografía Liquida/métodos , Electroforesis en Gel Bidimensional/métodos , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Renales/patología , Neoplasias Renales/orina , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , Adulto Joven
5.
Funct Integr Genomics ; 15(2): 197-210, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25403594

RESUMEN

The genome of Xanthomonas citri subsp. Citri strain 306 pathotype A (Xac) was completely sequenced more than 10 years; to date, few studies involving functional genomics Xac and its host compatible have been developed, specially related to adaptive events that allow the survival of Xac within the plant. Proteomic analysis of Xac showed that the processes of chemotactic signal transduction and phosphate metabolism are key adaptive strategies during the interaction of a pathogenic bacterium with its plant host. The results also indicate the importance of a group of proteins that may not be directly related to the classical virulence factors, but that are likely fundamental to the success of the initial stages of the infection, such as methyl-accepting chemotaxis protein (Mcp) and phosphate specific transport (Pst). Furthermore, the analysis of the mutant of the gene pstB which codifies to an ABC phosphate transporter subunit revealed a complete absence of citrus canker symptoms when inoculated in compatible hosts. We also conducted an in silico analysis which established the possible network of genes regulated by two-component systems PhoPQ and PhoBR (related to phosphate metabolism), and possible transcriptional factor binding site (TFBS) motifs of regulatory proteins PhoB and PhoP, detaching high degree of conservation of PhoB TFBS in 84 genes of Xac genome. This is the first time that chemotaxis signal transduction and phosphate metabolism were therefore indicated to be fundamental to the process of colonization of plant tissue during the induction of disease associated with Xanthomonas genus bacteria.


Asunto(s)
Quimiotaxis , Citrus/microbiología , Fosfatos/metabolismo , Enfermedades de las Plantas/microbiología , Transducción de Señal , Xanthomonas/metabolismo , Adaptación Biológica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Flagelos/fisiología , Mutación , Regulón , Factores de Transcripción/metabolismo , Xanthomonas/genética , Xanthomonas/patogenicidad , Xanthomonas/fisiología
6.
J Proteomics ; 73(7): 1334-41, 2010 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-20215060

RESUMEN

Chronic periodontal disease is a chronic inflammatory process affecting tooth supporting tissues in the presence of pathogenic bacterial biofilm. There is some evidence for changes in the protein composition of whole saliva from chronic periodontitis patients, but there have been no studies using a proteomic approach. Hence, the aim of this study was to compare the protein profiles of unstimulated whole saliva from patients with periodontitis and healthy subjects by two complementary approaches (2D-gel electrophoresis and liquid chromatography). Protein spots of interest were analyzed by MALDI-TOF-TOF, and the data was complemented by an ESI-Q-TOF experiment. The analyses revealed that subjects with periodontal disease have increased amounts of blood proteins (serum albumin and hemoglobin) and immunoglobulin, and they have a lower abundance of cystatin compared to the control group. A higher number of protein spots were observed in the periodontitis group, of which most were identified as alpha-amylase. This higher number of alpha-amylase variants seems to be caused by hydrolysis by cysteine proteases under such inflammatory conditions. This approach gives novel insights into alterations of salivary protein in presence of periodontal inflammation and may contribute to the improvement of periodontal diagnosis.


Asunto(s)
Biomarcadores/análisis , Periodontitis/diagnóstico , Periodontitis/metabolismo , Proteoma/análisis , Saliva/química , Adulto , Enfermedad Crónica , Femenino , Humanos , Masculino , Persona de Mediana Edad
7.
Toxicon ; 55(7): 1222-35, 2010 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-20060013

RESUMEN

A combination of anti-bothropic and anti-crotalic sera has been reported to be more effective in neutralizing the effects of Bothrops jararacussu venom than anti-bothropic serum alone. The role of proteins from B. jararacussu venom in the horse immune response was evaluated via the analysis of cross-reactivity with homologous and heterologous sera. Many of the proteins in B. jararacussu venom were identified via 2D gel electrophoresis. Western blots revealed that anti-jararacussu showed higher reactivity to l-aminoxidase (LAOs) and snake venom metalloproteinase, (SVMPs) and weaker reactivity towards Snake venom serine proteases (SVSPs), PLA(2), C-type lectin and cysteine-rich proteins. Anti-jararaca preferentially recognized LAOs, SVMPs and SVSPs. Both of these sera failed to recognize low-molecular weight proteins. Anti-crotalic serum clearly recognized LAOs, C-type lectin, SVSP, cysteine-rich proteins, SVMP and Asp49-PLA(2). The cross-reactivity with anti-PLA(2) revealed the immunoreactivity of these antibodies to proteins with molecular masses in a range that is poorly recognized by other studied anti-sera. Our results suggest that the contribution of anti-crotalic serum to the neutralization of B. jararacussu by may be due to its cross-reactivity with proteins such as C-type lectins, SVSPs, Asp49-PLA(2). These results also reinforce the importance of neutralizing the highly toxic proteins inclusive those with low immunogenicity in commercial antivenom production to obtain a highly protective serum against snake venoms.


Asunto(s)
Bothrops/genética , Bothrops/inmunología , Venenos de Serpiente/genética , Venenos de Serpiente/inmunología , Animales , Especificidad de Anticuerpos , Antivenenos/química , Antivenenos/inmunología , Reacciones Cruzadas , Venenos de Crotálidos/química , Venenos de Crotálidos/inmunología , Venenos de Crotálidos/aislamiento & purificación , Interpretación Estadística de Datos , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Inmunoquímica , Indicadores y Reactivos , Peso Molecular , Fosfolipasas A2/química , Hidrolisados de Proteína/química , Proteómica , Conejos , Venenos de Serpiente/enzimología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
8.
Proteomics ; 6(5): 1495-511, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16447160

RESUMEN

A proteomic analysis of a wild-type and of a phoB mutant showed that Vibrio cholerae expresses genes of two major regulons in response to phosphate starvation. The Pho regulon, expressed by the wild-type, allowed the cells to adapt to the new environment. Induction of the general stress regulon was mainly observed in the phoB mutant as a strategy to resist stress and survive. Some functions of the adaptative and survival responses play roles in the pathogenicity of the bacteria. Among the members of the Pho regulon, we found a porin described as an important factor for the intestinal colonisation. Other functions not obviously related to phosphate metabolism, expressed preferentially by the wild-type cells, have also been implicated in virulence. These findings might explain the lack of virulence of the phoB mutant. The Pho regulon picture of V. cholerae, however, will not be complete until minor members and membrane proteins are identified. Among the phosphate-starvation induced genes we have found 13 hypothetical ones and for some of them functions have been assigned. The majority of the genes identified here have not been described before, thus they could be used to expand the proteomic reference map of V. cholerae El Tor.


Asunto(s)
Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Fosfatos/metabolismo , Proteoma/análisis , Regulón , Vibrio cholerae O1/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Electroforesis en Gel Bidimensional , Datos de Secuencia Molecular , Operón , Vibrio cholerae O1/genética , Vibrio cholerae O1/patogenicidad
9.
Rapid Commun Mass Spectrom ; 19(12): 1703-8, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15912471

RESUMEN

Characterization of the peptide content in snake venoms can be an important tool for the investigation of new pharmacological lead compounds. For this purpose, single-step analysis of crude venoms has recently been demonstrated using mass spectrometry (MS) techniques. Reproducible profiles of ions in MS and MS/MS spectra may also be used to compare venoms from different species. In this work matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to obtain mass patterns of the major peptides (<8 kDa) found in pooled venoms from the genera Bothrops and Crotalus. Venoms from five different Bothrops species (B. jararaca, B. insularis, B. alternatus, B. jararacussu, and B. neuwiedi) and three Crotalus species (C. viridis, C. adamanteus and C. durissus terrificus) were analyzed. In agreement with other reports, venoms from Bothrops species contained a variety of peptides in the range m/z 1000-1500, and in some samples larger components (m/z 7000-8000) were detected. In the Crotalus species venoms were rich in peptides ranging from m/z 1000-1500 and 4000-5500. MS/MS experiments on the low molecular mass peptides (m/z 1000-1500) confirmed the presence of ten new bradykinin-potentiating peptides among venoms from genera Bothrops and Crotalus. In order to determine whether additional peptides could be identified after partial purification, B. jararaca venom was subjected to size-exclusion chromatography on Sephacryl S-200, and two distinct low molecular mass pools were analyzed further by MALDI-TOFMS. No additional peptides were detected from the pool with masses below 2000 Da but a substantial improvement with better resolution was observed for the pool with masses above 7000 Da, indicating that complex samples such as crude snake venoms can be analyzed for low molecular mass peptides using a single-step procedure.


Asunto(s)
Bothrops , Venenos de Crotálidos/química , Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Animales , Cromatografía en Gel/métodos , Resinas de Intercambio Iónico/química , Datos de Secuencia Molecular , Peso Molecular , Péptidos/aislamiento & purificación , Reproducibilidad de los Resultados , Análisis de Secuencia de Proteína
10.
Toxicon ; 46(1): 31-8, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15876444

RESUMEN

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.


Asunto(s)
Bradiquinina/metabolismo , Venenos de Crotálidos/química , Péptido Natriurético Tipo-C/análisis , Péptidos/análisis , Viperidae/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Isoformas de Proteínas , Alineación de Secuencia , Homología de Secuencia de Aminoácido
11.
Proteomics ; 4(5): 1491-504, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15188416

RESUMEN

A proteome reference map has been constructed for Vibrio cholerae El Tor, in the pI range of 4.0 to 7.0. The map is based on two-dimensional gels (2-D) and the identification, by peptide mass fingerprint, of proteins in 94 spots, corresponding to 80 abundant proteins. Two strains are compared, strain N16961 and a Latin American El Tor strain C3294. The consensus map contains 340 spots consistently seen with both strains grown in Luria-Bertani broth (LB) or minimal M9 medium. The results were obtained from nine gels run with 18 cm immobilized pH gradient strips and precast gels. The 2-D gels were anchored to real N16961 proteins identified by mass spectrometry. Various energy metabolism components and periplasmic ATP-binding cassette (ABC) transporter proteins were identified among the abundant proteins. Two isoforms of OmpU were found. Five operons are proposed and seven hypothetical proteins were experimentally confirmed. Comparisons are made with protein 2-D gels for a classical strain and to microarray analysis available for the N16961 El Tor strain. New results were obtained from the proteome analysis, indicating an abundance of periplasmic ABC transporter proteins not found in microarray studies.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteoma/normas , Vibrio cholerae/clasificación , Vibrio cholerae/metabolismo , Proteínas Bacterianas/química , Medios de Cultivo , Electroforesis en Gel Bidimensional , Focalización Isoeléctrica , Espectrometría de Masas , Peso Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Operón , Mapeo Peptídico , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estándares de Referencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vibrio cholerae/genética , Vibrio cholerae/crecimiento & desarrollo
12.
J. bras. med ; 71(4): 94-6, out. 1996. ilus
Artículo en Portugués | LILACS | ID: lil-186637

RESUMEN

Os autores apresentam um caso de colangiocarcinoma da junçäo dos ductos hepáticos principais (tumor de Klatskin), que fora tratado com drenagem biliar externa. Discutem os aspectos do diagnóstico e tratamento.


Asunto(s)
Humanos , Masculino , Adulto , Neoplasias del Sistema Biliar/diagnóstico , Colangiocarcinoma/diagnóstico , Neoplasias del Sistema Biliar/cirugía , Colangiocarcinoma/cirugía , Ictericia/diagnóstico , Ictericia/etiología
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