RESUMO
TcPR-10, a member of the pathogenesis-related protein 10 family, was identified in EST library of interactions between Theobroma cacao and Moniliophthora perniciosa. TcPR-10 has been shown to have antifungal and ribonuclease activities in vitro. This study aimed to identify proteins that are differentially expressed in M. perniciosa in response to TcPR-10 through a proteomic analysis. The fungal hyphae were subjected to one of four treatments: control treatment or 30-, 60- or 120-min treatment with the TcPR-10 protein. Two-dimensional maps revealed 191 differentially expressed proteins, 55 of which were identified by mass spectrometry. The proteins identified in all treatments were divided into the following classes: cell metabolism, stress response, zinc binding, phosphorylation mechanism, transport, autophagy, DNA repair, and oxidoreductases. The predominant class was stress-response proteins (29%), such as heat shock proteins; these proteins exhibited the highest expression levels relative to the control treatment and are known to trigger defense mechanisms against cytotoxic drugs as well as TcPR-10. Oxidoreductases (25%) were overexpressed in the control and in 30-min treatments but exhibited reduced expression at 120 min. These proteins are involved in the repair of damage caused by oxidative stress due to the contact with TcPR- 10. Consistent with the antifungal activity of TcPR-10, several proteins identified were related to detoxification, autophagy or were involved in mechanisms for maintaining fungal homeostasis, such as ergosterol biosynthesis. These results show that the sensitivity of the fungus to TcPR-10 involves several biochemical routes, clarifying the possible modes of action of this antifungal protein.
Assuntos
Basidiomycota/efeitos dos fármacos , Basidiomycota/metabolismo , Cacau/química , Proteínas Fúngicas/metabolismo , Proteínas de Plantas/farmacologia , Proteoma , Proteômica , Basidiomycota/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteômica/métodos , Estresse Fisiológico , Fatores de TempoRESUMO
Moniliophthora perniciosa (Stahel) Aime and Phillips-Mora is a hemibiotrophic basidiomycete (Agaricales, Tricholomataceae) that causes witches' broom disease in cocoa (Theobroma cacao L.). This pathogen carries a stable integrated invertron-type linear plasmid in its mitochondrial genome that encodes viral-like DNA and RNA polymerases related to fungal senescence and longevity. After culturing the fungus and obtaining its various stages of development in triplicate, we carried out total RNA extraction and subsequent complementary DNA synthesis. To analyze DNA and RNA polymerase expression levels, we performed real-time reverse transcriptase polymerase chain reaction for various fungal phases of development. Our results showed that DNA and RNA polymerase gene expression in the primordium phase of M. perniciosa is related to a potential defense mechanism against T. cacao oxidative attack.
Assuntos
Agaricales/genética , Cacau/microbiologia , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Mitocôndrias/genética , Estresse Oxidativo/genética , Agaricales/crescimento & desenvolvimento , Agaricales/isolamento & purificação , Agaricales/metabolismo , Cacau/metabolismo , DNA Polimerase Dirigida por DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Genoma Mitocondrial , Mitocôndrias/metabolismo , Doenças das Plantas/microbiologia , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cacao (Theobroma cacao) is one of the most important tropical crops; however, production is threatened by numerous pathogens, including the hemibiotrophic fungus Moniliophthora perniciosa, which causes witches' broom disease. To understand the mechanisms that lead to the development of this disease in cacao, we focused our attention on cacao transcription factors (TFs), which act as master regulators of cellular processes and are important for the fine-tuning of plant defense responses. We developed a macroarray with 88 TF cDNA from previously obtained cacao-M. perniciosa interaction libraries. Seventy-two TFs were found differentially expressed between the susceptible (Catongo) and resistant (TSH1188) genotypes and/or during the disease time course--from 24 h to 30 days after infection. Most of the differentially expressed TFs belonged to the bZIP, MYB and WRKY families and presented opposite expression patterns in susceptible and resistant cacao-M. perniciosa interactions (i.e., up-regulated in Catongo and down-regulated in TSH1188). The results of the macroarray were confirmed for bZIP and WRKY TFs by real-time PCR. These differentially expressed TFs are good candidates for subsequent functional analysis as well as for plant engineering. Some of these TFs could also be localized on the cacao reference map related to witches' broom resistance, facilitating the breeding and selection of resistant cacao trees.
Assuntos
Agaricales/fisiologia , Cacau/genética , Cacau/microbiologia , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Genes de Plantas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismoRESUMO
Toll-like receptors have been implicated in the recognition of various pathogens, including bacteria, viruses, protozoa and fungi. However, no information is available about Toll-like receptor 4 (TLR4) participation in Sporothrix schenckii recognition and the consequent triggering of the immune response to this fungal pathogen. Following activation of TLRs by ligands of microbial origin, several responses are provoked, including reactions in immune cells that may lead them to produce signalling factors that trigger inflammation. The present study was designed to elucidate the role of TLR4 during the host response to S. schenckii. TLR4-deficient (C3H/HeJ) and control mice (C3H/HePas) were infected with S. schenckii yeast cells and immune response was assessed over 10 weeks by assaying production of pro-inflammatory mediator (nitric oxide and tumour necrosis factor-alpha) and anti-inflammatory cytokine (interleukin-10) by peritoneal macrophages and their correlation with apoptosis in peritoneal exudate cells. We found that both pro-inflammatory and anti-inflammatory mediators are reduced in TLR4-deficient mice, suggesting the involvement of this receptor in the recognition of this infectious agent. Translocation into the nucleus of nuclear transcription factor, nuclear factor-kappaB, was also evaluated and showed higher levels in TLR-4 normal mice, consistent with the results found for cytokine production. We are showing here, for the first time, the involvement of TLR4 in S. schenckii recognition. Taken together, our results demonstrate that the activation of peritoneal macrophages in response to S. schenckii lipid extracts has different responses in these two mouse strains which differ in TLR4 expression, suggesting an important role for TLR4 in governing the functions of macrophages in this fungal infection.
Assuntos
Antígenos de Fungos/imunologia , Lipídeos/imunologia , Macrófagos Peritoneais/imunologia , Sporothrix/imunologia , Esporotricose/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Apoptose/imunologia , Tolerância Imunológica/imunologia , Mediadores da Inflamação/metabolismo , Interleucina-10/biossíntese , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Necrose/imunologia , Espécies Reativas de Nitrogênio/biossíntese , Fator de Transcrição RelA/metabolismo , Translocação Genética , Fator de Necrose Tumoral alfa/biossínteseRESUMO
In order to increase the efficiency of cacao tree resistance to witches' broom disease, which is caused by Moniliophthora perniciosa (Tricholomataceae), we looked for molecular markers that could help in the selection of resistant cacao genotypes. Among the different markers useful for developing marker-assisted selection, single nucleotide polymorphisms (SNPs) constitute the most common type of sequence difference between alleles and can be easily detected by in silico analysis from expressed sequence tag libraries. We report the first detection and analysis of SNPs from cacao-M. perniciosa interaction expressed sequence tags, using bioinformatics. Selection based on analysis of these SNPs should be useful for developing cacao varieties resistant to this devastating disease.
Assuntos
Cacau/genética , Etiquetas de Sequências Expressas , Doenças das Plantas/genética , Polimorfismo de Nucleotídeo Único/genética , Biblioteca Gênica , Fases de Leitura Aberta/genética , Biossíntese de Proteínas/genéticaRESUMO
We sought to determine whether hyposialorrhea is an early manifestation of Parkinson disease (PD). We measured basal and citric acid stimulated secretion of whole saliva in 20 patients with early stage (Hoehn-Yahr I-II) PD who had motor symptoms for less than 1 year and were on no medication and 11 age matched controls. Compared to controls, PD patients had significant reduction of both basal (0.0964+/-0.08 vs 0.293+/-0.112 ml/min, p<0.001) and reflex (0.263+/-0.213 vs 0.537+/-0.313 ml/min, p<0.001) salivary secretion. Our findings confirm that hyposialorrhea is an early autonomic manifestation of PD.
Assuntos
Doença de Parkinson/diagnóstico , Doença de Parkinson/fisiopatologia , Salivação/fisiologia , Taxa Secretória/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Ácido Cítrico/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reflexo Anormal/efeitos dos fármacos , Reflexo Anormal/fisiologia , Salivação/efeitos dos fármacos , Taxa Secretória/efeitos dos fármacosRESUMO
El blefaroespasmo es una distonía focal caracterizada por el cierre involuntario de los ojos debido a la contracción anormal de los músculos orbiculares de los párpados. Cuando el blefaroespasmo se asocia a la presencia de movimientos involuntarios oromandibulares se denomina síndrome de Meige. El objetivo de este estudio fue investigar la presencia de alteraciones deglutorias en pacientes con blefaroespasmo y síndrome de Meige. Se incluyeron 20 pacientes consecutivos que fueron estudiados mediante vídeo fluoroscopia con técnica de bario. Se investigaron las 4 etapas de la deglución. El 90% de los pacientes (18 casos) presentó trastornos en la deglución. Las alteraciones más comúnmente halladas fueron caída prematura del alimento, 15 casos (83%) y resíduos valeculares, 14 casos (78%). El 67% de anormalidades se observó en la tercera etapa de la deglución. El 89% de los pacientes (16) presentó más de un trastorno deglutorio. Se observó una correlación positiva y estadísticamente significativa entre el número de hallazgos patológicos y la edad de los pacientes y la duración de la enfermedad. De acuerdo a lo publicado, la prevalencia de desórdenes de la deglución en pacientes sanos de edad avanzada es del 44%. En nuestra serie alcanzó el 90%, lo cual sugiere que nuestros hallazgos podrían estar relacionados no sólo con la edad, sino también con la posibilidad de que el compromiso distónico en pacientes con blefaroespasmo sea mayor de lo que se aprecia clínicamente extendiéndose más allá de los músculos orofaciales.
Assuntos
Adulto , Pessoa de Meia-Idade , Idoso de 80 Anos ou mais , Humanos , Masculino , Feminino , Blefarospasmo/complicações , Transtornos de Deglutição/complicações , Síndrome de Meige/complicaçõesRESUMO
El blefaroespasmo es una distonía focal caracterizada por el cierre involuntario de los ojos debido a la contracción anormal de los músculos orbiculares de los párpados. Cuando el blefaroespasmo se asocia a la presencia de movimientos involuntarios oromandibulares se denomina síndrome de Meige. El objetivo de este estudio fue investigar la presencia de alteraciones deglutorias en pacientes con blefaroespasmo y síndrome de Meige. Se incluyeron 20 pacientes consecutivos que fueron estudiados mediante vídeo fluoroscopia con técnica de bario. Se investigaron las 4 etapas de la deglución. El 90% de los pacientes (18 casos) presentó trastornos en la deglución. Las alteraciones más comúnmente halladas fueron caída prematura del alimento, 15 casos (83%) y resíduos valeculares, 14 casos (78%). El 67% de anormalidades se observó en la tercera etapa de la deglución. El 89% de los pacientes (16) presentó más de un trastorno deglutorio. Se observó una correlación positiva y estadísticamente significativa entre el número de hallazgos patológicos y la edad de los pacientes y la duración de la enfermedad. De acuerdo a lo publicado, la prevalencia de desórdenes de la deglución en pacientes sanos de edad avanzada es del 44%. En nuestra serie alcanzó el 90%, lo cual sugiere que nuestros hallazgos podrían estar relacionados no sólo con la edad, sino también con la posibilidad de que el compromiso distónico en pacientes con blefaroespasmo sea mayor de lo que se aprecia clínicamente extendiéndose más allá de los músculos orofaciales. (AU)
Assuntos
Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Humanos , Masculino , Feminino , Transtornos de Deglutição/complicações , Blefarospasmo/complicações , Síndrome de Meige/complicaçõesRESUMO
The development of dyskinesias is a common side effect during chronic levodopa therapy in parkinsonian patients. Recent reports suggest that amantadine, a drug with well known antiparkinsonian activity, is effective in the treatment of this complication. In order to evaluate its usefulness we conducted an open label, prospective and longitudinal study in 26 patients with Parkinson's disease (PD) on chronic levodopa therapy who presented peaks of dose dyskinesias. After 3 weeks' treatment dyskinesia severity was reduced by 70% (p < 0.0001) on the I SAPD scale and by 68.8% (p < 0.0002) on the UPDRS IV subscale. Patients were later evaluated every 45 days showing persistent drug benefit during follow-up ranging from 2 to 11 months (mean 6.5 months). One third of our series presented unwanted effects which were only severe enough in 2 cases to discontinue treatment. In the others, side effects were transient or readily abated with amantadine dose reduction. Our findings support amantadine as a safe and useful drug to treat levodopa-induced dyskinesias which on occasion prove as disabling as PD itself. Treatment with amantadine should routinely be considered before indicating pallidotomy for levodopa-induced dyskinesias.