RESUMEN
Fusarium head blight (FHB) is one of the most destructive diseases of wheat and other cereals worldwide. During infection, the Fusarium fungi produce mycotoxins that represent a high risk to human and animal health. Developing small-molecule inhibitors to specifically reduce mycotoxin levels would be highly beneficial since current treatments unspecifically target the Fusarium pathogen. Culmorin possesses a well-known important synergistically virulence role among mycotoxins, and longiborneol synthase appears to be a key enzyme for its synthesis, thus making longiborneol synthase a particularly interesting target. This study aims to discover potent and less toxic agrochemicals against FHB. These compounds would hamper culmorin synthesis by inhibiting longiborneol synthase. In order to select starting molecules for further investigation, we have conducted a structure-based virtual screening investigation. A longiborneol synthase structural model is first built using homology modeling, followed by molecular dynamics simulations that provided the required input for a protein-ligand ensemble docking procedure. From this strategy, the three most interesting compounds (hits) were selected among the 25 top-ranked docked compounds from a library of 15,000 drug-like compounds. These putative inhibitors of longiborneol synthase provide a sound starting point for further studies involving molecular modeling coupled to biochemical experiments. This process could eventually lead to the development of novel approaches to reduce mycotoxin contamination in harvested grain.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/enzimología , Ensayos Analíticos de Alto Rendimiento/métodos , Ligasas/metabolismo , Sesquiterpenos/metabolismo , Agroquímicos/química , Agroquímicos/metabolismo , Agroquímicos/farmacología , Secuencia de Aminoácidos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/química , Proteínas Fúngicas/clasificación , Fusarium/metabolismo , Fusarium/patogenicidad , Ligasas/antagonistas & inhibidores , Ligasas/genética , Modelos Moleculares , Estructura Molecular , Filogenia , Enfermedades de las Plantas/microbiología , Unión Proteica , Dominios Proteicos , Homología de Secuencia de AminoácidoRESUMEN
The brown marmorated stink bug, Halyomorpha halys, is a devastating invasive species in the USA. Similar to other insects, olfaction plays an important role in its survival and reproduction. As odorant-binding proteins (OBPs) are involved in the initial semiochemical recognition steps, we used RNA-Sequencing (RNA-Seq) to identify OBPs in its antennae, and studied their expression pattern in different body parts under semiochemical stimulation by either aggregation or alarm pheromone or food odorants. Thirty full-length putative HhalOBPs were identified, corresponding to 22 'classic' OBPs and eight 'Plus-C' OBPs. The similarity amongst them ranged from 4.95-70.92%, and with another 325 hemipteran OBPs similarity ranged from 1.94-91.51%, the highest levels being with other stink bug OBPs. Phylogenetic analysis confirmed the monophyly of seven groups of stink bug and other hemipteran OBPs. All 30 HhalOBPs were expressed and about 2/3 were expressed primarily in antennae. The expression of 21 HhalOBPs was higher in the antennae under alarm pheromone stimulus, indicating that multiple OBPs may be responding to this pheromone. Two were highest in antennae under aggregation pheromone stimulus. These findings should provide a basis for understanding the physiological functions of HhalOBPs and the chemosensory perception of this pest, which may help to uncover new control targets for behavioural interference.
Asunto(s)
Heterópteros/fisiología , Proteínas de Insectos/genética , Percepción Olfatoria , Receptores Odorantes/genética , Transcriptoma , Secuencia de Aminoácidos , Animales , Antenas de Artrópodos/metabolismo , ADN Complementario/genética , ADN Complementario/metabolismo , Femenino , Heterópteros/genética , Control de Insectos , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Masculino , Filogenia , Receptores Odorantes/química , Receptores Odorantes/metabolismo , Alineación de SecuenciaRESUMEN
Erythrinus erythrinus, a Neotropical fish species of the Erythrinidae family, has a wide distribution in South America. Previous cytogenetic analysis showed that this species presents extensive karyotype diversity, with 4 karyomorphs (A-D) described herein. This study investigated the karyotypic structure of 2 new populations of E. erythrinus from the Brazilian Pantanal region, in order to improve the knowledge of the chromosomal diversity in this species. Both populations showed typical characteristics of karyomorph A, with 2n=54 chromosomes (6m+2st+46a), without differentiation between males and females. In addition, identical supernumerary B chromosomes, appearing as double-minute chromosomes, were also found in both populations. These findings suggest the presence of mitotic instability in view of their high intra- and inter-individual numerical variation. The presence of these chromosomes is likely a basal characteristic for this group, since the same kind of Bs also occurs in some other populations and karyomorphs of E. erythrinus. As such, they are important markers of biodiversity found in this nominal species, which probably corresponds to a species complex.
Asunto(s)
Characiformes/genética , Cromosomas , Evolución Molecular , Animales , Evolución Biológica , Brasil , Characiformes/clasificación , Femenino , Geografía , Cariotipo , Cariotipificación , Masculino , Metafase , Mitosis , Modelos GenéticosRESUMEN
A subtractive cDNA library from cotton buds was constructed to prospect for differentially expressed genes related to early bud development. A library was constructed and 768 cDNA sequences were obtained, comprising 168 clusters, with 126 contigs and 42 singlets. Both the Gossypium as well as Arabidopsis databases were utilized for the in silico analysis, since some genes identified in cotton have not yet been studied for functionality, although they have homology with genes from other species. The transcriptome revealed a large number of transcripts, some of them with unknown function, and others related to pollen development, pollen tubes, ovules, and fibers at different stages. The most populated contig was identified as fiber from 0-10 days after anthesis, with 12 reads. The success and novelty rates generated from the library were 67 and 51%, respectively. The information obtained here will provide a framework for research on functional cotton genomics.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Gossypium/crecimiento & desarrollo , Gossypium/genética , Fibra de Algodón , ADN Complementario/genética , Biblioteca de Genes , Transcriptoma/genéticaRESUMEN
AIMS: To evaluate the activity of cry1Ia gene against cotton pests, Spodoptera frugiperda and Anthonomus grandis. METHODS AND RESULTS: Had isolated and characterized a toxin gene from the Bacillus thuringiensis S1451 strain which have been previously shown to be toxic to S. frugiperda and A. grandis. The toxin gene (cry1Ia) was amplified by PCR, sequenced, and cloned into the genome of a baculovirus. The Cry1Ia protein was expressed in baculovirus infected insect cells, producing protein inclusions in infected cells. The Cry1Ia protein has used in bioassays against to S. frugiperda and A. grandis. CONCLUSIONS: Bioassays using the purified recombinant protein showed high toxicity to S. frugiperda and A. grandis larvae. Molecular modelling of the Cry1Ia protein translated from the DNA sequence obtained in this work, showed that this protein possibly posses a similar structure to the Cry3A protein. Ultrastructural analysis of midgut cells from A. grandis incubated with the Cry1Ia toxin, showed loss of microvilli integrity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the cry1Ia is a good candidate for the construction of transgenic plants resistant to these important cotton pests.
Asunto(s)
Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Insecticidas/toxicidad , Spodoptera/efectos de los fármacos , Gorgojos/efectos de los fármacos , Animales , Bacillus thuringiensis , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Baculoviridae/genética , Secuencia de Bases , Bioensayo , Clonación Molecular , Expresión Génica , Gossypium/microbiología , Mucosa Intestinal/metabolismo , Intestinos/ultraestructura , Larva/virología , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Proteínas Recombinantes/toxicidad , Spodoptera/virología , Transfección/métodos , Gorgojos/virologíaRESUMEN
The human fungal pathogen Paracoccidioides brasiliensis is an ascomycete that displays a temperature-dependent dimorphic transition, appearing as a mycelium at 22 degrees C and as a yeast at 37 degrees C, this latter being the virulent form. We report on the in silico search made of the P. brasiliensis transcriptome-expressed sequence tag database for components of signaling pathways previously known to be involved in morphogenesis and virulence in other species of fungi, including Saccharomyces cerevisiae, Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Using this approach, it was possible to identify several protein cascades in P. brasiliensis, such as i) mitogen-activated protein kinase signaling for cell integrity, cell wall construction, pheromone/mating, and osmo-regulation, ii) the cAMP/PKA system, which regulates fungal development and virulence, iii) the Ras protein, which allows cross-talking between cascades, iv) calcium-calmodulin-calcineurin, which controls cell survival under oxidative stress, high temperature, and membrane/cell wall perturbation, and v) the target of rapamycin pathway, controlling cell growth and proliferation. The ways in which P. brasiliensis responds to the environment and modulates the expression of genes required for its survival and virulence can be inferred through comparison with other fungi for which this type of data is already available.
Asunto(s)
Humanos , Etiquetas de Secuencia Expresada , Paracoccidioides/fisiología , Proteínas Fúngicas/metabolismo , Transcripción Genética , Transducción de Señal/genética , Alineación de Secuencia , Feromonas/metabolismo , Hongos/citología , Hongos/metabolismo , Hongos/patogenicidad , Ósmosis/fisiología , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas ras/metabolismo , Transducción de Señal/fisiologíaRESUMEN
In order to discover genes expressed in leaves of Musa acuminata ssp. burmannicoides var. Calcutta 4 (AA), from plants submitted to temperature stress, we produced and characterized two full-length enriched cDNA libraries. Total RNA from plants subjected to temperatures ranging from 5 degrees C to 25 degrees C and from 25 degrees C to 45 degrees C was used to produce a COLD and a HOT cDNA library, respectively. We sequenced 1,440 clones from each library. Following quality analysis and vector trimming, we assembled 2,286 sequences from both libraries into 1,019 putative transcripts, consisting of 217 clusters and 802 singletons, which we denoted Musa acuminata assembled expressed sequence tagged (EST) sequences (MaAES). Of these MaAES, 22.87% showed no matches with existing sequences in public databases. A global analysis of the MaAES data set indicated that 10% of the sequenced cDNAs are present in both cDNA libraries, while 42% and 48% are present only in the COLD or in the HOT libraries, respectively. Annotation of the MaAES data set categorized them into 22 functional classes. Of the 2,286 high-quality sequences, 715 (31.28%) originated from full-length cDNA clones and resulted in a set of 149 genes.
Asunto(s)
Etiquetas de Secuencia Expresada , Genes de Plantas/genética , Musa/genética , Hojas de la Planta/genética , Temperatura , Secuencia de Bases , Cartilla de ADN , Biblioteca de Genes , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Since the Haemophilus influenzae genome sequence was completed in 1995, 172 other prokaryotic genomes have been completely sequenced, while 508 projects are underway. Besides pathogens, organisms important in several other fields, such as biotechnology and bioremediation, have also been sequenced. Institutions choose the organisms they wish to sequence according to the importance that these species represent to them, the availability of the microbes, and based on the similarity of a species of interest with others that have been sequenced previously. Improvements in sequencing techniques and in associated methodologies have been achieved; however, scientists need to continue working on the development of this field. In Brazil, a multicentered, centrally coordinated and research-focused network was adopted and successfully used for the sequencing of several important organisms. We analyzed the current status of microbial genomes, the trends for criteria used to choose new sequencing projects, the future of microbial sequencing, and the Brazilian genome network.
Asunto(s)
Genoma Arqueal , Genoma Bacteriano , Genómica/tendencias , BrasilRESUMEN
The conformational stability and the folding process of alpha, beta and Psi bovine trypsin at pH 3.0 followed by circular dichroism (CD) and size exclusion in HPLC have been analyzed as a function of urea concentration. The thermodynamic stability for a and b are deltaG = 15.91 +/- 0.28 kcal/mol, deltaG = 15.54 +/- 2.39 kcal/mol. respectively, and y trypsin is deltaG = 16.10 +/- 2.51 kcal/mol. The transition curves for alpha, beta and Psi forms suggest a molten globule state.
Asunto(s)
Tripsina/química , Animales , Bovinos , Cromatografía en Gel/métodos , Dicroismo Circular , Concentración de Iones de Hidrógeno , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Termodinámica , Tripsina/clasificación , Urea/químicaRESUMEN
Crotamine, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus is a strongly basic 42-amino acid polypeptide belonging to the small basic myotoxin family. As no tridimensional structure is available for this myotoxin subfamily, despite its important pharmacological interest, we propose in this paper a theoretical 3D model for crotamine. Starting from a homology modelling procedure, followed by intensive molecular dynamics (MD) simulations in water and complementary CD experiments, the designed 3D model is the first example of a tridimensional structure in this family of small basic myotoxins. Crotamine, therefore, belongs to a newly identified structural family presenting a common fold also found in beta-defensin and antopleurine-B. The proposed 3D model will be used for future calculations about crotamine aggregation and interaction with membranes.
Asunto(s)
Venenos de Crotálidos/química , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Gráficos por Computador , Venenos de Crotálidos/genética , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , TermodinámicaRESUMEN
The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was delta GH2O = 6.99 +/- 1.40 kcal/mol for guanidine hydrochloride and delta GH2O = 6.37 +/- 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 +/- 0.4 A to 26.0 +/- 0.3 A for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 +/- 0.3 A to 25.7 +/- 0.6 A for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.
Asunto(s)
Pliegue de Proteína , Tripsinógeno/metabolismo , Animales , Bovinos , Diuréticos Osmóticos/farmacología , Guanidina/farmacología , Parasimpaticomiméticos/farmacología , Desnaturalización Proteica , Urea/farmacologíaRESUMEN
The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was delta GH2O = 6.99 + ou - 1.40 kcal/mol for guanidine hydrochloride and delta GH2O = 6.37 + ou - 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 + ou - 0.4 angstron to 26.0 + ou - 0.3 angstron 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 + ou - 0.3 angstron to 25.7 + ou - 0.6 angstron for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.