RESUMO
A validated, multigene-based method using real-time quantitative PCR (qPCR) and the Razor Ex BioDetection system was developed for detection of Phymatotrichopsis omnivora. This soilborne fungus causes Phymatotrichopsis root rot of cotton, alfalfa, and other dicot crops in the southwestern United States and northern Mexico, leading to significant crop losses and limiting the range of crops that can be grown in soils where the fungus is established. It is on multiple lists of regulated organisms. Because P. omnivora is difficult to isolate, accurate and sensitive culture-independent diagnostic tools are needed to confirm infections by this fungus. Specific PCR primers and probes were designed based on P. omnivora nucleotide sequences of the genes encoding rRNA internal transcribed spacers, beta-tubulin, and the second-largest subunit of RNA polymerase II (RPB2). PCR products were cloned and sequenced to confirm their identity. All primer sets allowed early detection of P. omnivora in infected but asymptomatic plants. A modified rapid DNA purification method, which facilitates a quick (â¼30-min) on-site assay capability for P. omnivora detection, was developed. Combined use of three target genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a multigene-based, field-deployable, rapid, and reliable identification method for a fungal plant pathogen and should serve as a model for the development of field-deployable assays of other phytopathogens.
Assuntos
Ascomicetos/isolamento & purificação , Doenças das Plantas/prevenção & controle , Reação em Cadeia da Polimerase em Tempo Real/métodos , Microbiologia do Solo , Ascomicetos/genética , Primers do DNA/genética , DNA Fúngico/genética , Proteínas Fúngicas/genética , Gossypium , Medicago sativa , México , Doenças das Plantas/microbiologia , Sensibilidade e Especificidade , Sudoeste dos Estados Unidos , Fatores de TempoRESUMO
OBJECTIVES: To determine whether demographic, clinical and immunological features may predict the outcome in juvenile SSc (JSSc). METHODS: Clinical and laboratory characteristics of patients with JSSc collected from paediatric rheumatology centres worldwide were analysed. First, univariate tests identified those features significantly related with fatal outcome, and then multivariate logistic regression analysis was applied to determine the predictors of mortality. RESULTS: One hundred and thirty-four patients from 40 centres were eligible for the analysis. Sixteen patients died and a rapidly fatal course was observed in most of them: 4/16 died within 1 yr after diagnosis and 10/16 within 5 yrs. At the moment of diagnosis, patients with poor outcome showed a significantly higher frequency of internal organ involvement, particularly cardiac, respiratory and gastrointestinal systems. No significant difference emerged for entity of skin, vascular and musculo-skeletal involvement, nor for auto-antibodies profile and laboratory tests. Multivariate analysis showed the following factors to be significant predictors of mortality: fibrosis on chest X-rays [odds ratio (OR) 11.2], raised creatinine levels (OR 22.7) and pericarditis (OR 41.3), while a short disease duration at diagnosis conferred protection (OR 0.3). CONCLUSIONS: All patients with JSSc and fatal outcome were affected by the diffuse form of the disease, and most of them showed a very rapid progression and early signs of internal organ involvement. This suggests that, in children, SSc may have two possible courses: a rapid development of internal organ failure leading to severe disability and eventually to death, or a slow course of the disease with lower mortality.
Assuntos
Escleroderma Sistêmico/mortalidade , Adolescente , Distribuição de Qui-Quadrado , Criança , Europa (Continente) , Seguimentos , Humanos , Análise Multivariada , América do Norte , Pericardite/complicações , Pericardite/mortalidade , Prognóstico , Fibrose Pulmonar/complicações , Fibrose Pulmonar/mortalidade , Estudos Retrospectivos , Escleroderma Sistêmico/complicações , América do Sul , SobrevidaRESUMO
Manufacturing process of medical grade silicon rubber trileaflet valves for VADs could propitiate important leaflet thickness variations which could result in partial opening of the valve and affect its hydrodynamic performance. The leaflets of a total of 10 valves were measured to assess its thickness variability. Two experiments were performed to asses the impact of the leaflets thickness variation under hypothetical situations. The first experiment was divided into three hypothetical cases. In each case either none, one or two leaflets of different valves were mechanically blocked, resembling possible critical working circumstances. The second experiment was intended to know how the variation on the leaflets thickness affects the hydrodynamic performance of the valves. The results demonstrated a significant variation on the leaflets thickness was found. As for the first experiment, a small variation on the hydrodynamic performance was found above 4 L/min flow rates and a slightly higher energy loss was found in one of the cases. As for the second experiment, the results showed that the variation of the leaflet thickness does not affect the general hydrodynamic performance of the valves. No relationship between the thickness variability and the hydrostatic performance of the valves was found.
Assuntos
Coração Auxiliar , Materiais Biocompatíveis , Engenharia Biomédica , Coração Auxiliar/estatística & dados numéricos , Humanos , Pressão Hidrostática , Técnicas In Vitro , Teste de Materiais , Desenho de Prótese , Elastômeros de SiliconeRESUMO
Penile reflexes (PRs) were monitored in chronic spinal cord-transected rats by identifying them visually, and at the same time they were recorded as the electromyographic activity of bulbospongiosus muscles. Intraperitoneal injection of the agonist muscarine (10 microg) produced a facilitation of PRs. A decrease in the latency, an increase in the number of clusters and often an increase in the duration of cups were found after muscarine. In addition, 66% (six out of nine) of the animals ejaculated after muscarine. These results suggest that cholinergic receptor stimulation may be involved in erectile and ejaculatory mechanisms mediated by the spinal cord.
Assuntos
Ejaculação/efeitos dos fármacos , Disfunção Erétil/tratamento farmacológico , Muscarina/farmacologia , Agonistas Muscarínicos/farmacologia , Ereção Peniana/efeitos dos fármacos , Traumatismos da Medula Espinal/complicações , Animais , Ejaculação/fisiologia , Eletromiografia , Disfunção Erétil/etiologia , Disfunção Erétil/fisiopatologia , Masculino , Ereção Peniana/fisiologia , Ratos , Ratos Wistar , Receptores Muscarínicos/fisiologia , Reflexo/efeitos dos fármacos , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Chromium is a highly toxic non-essential metal for microorganisms and plants. Due to its widespread industrial use, chromium (Cr) has become a serious pollutant in diverse environmental settings. The hexavalent form of the metal, Cr(VI), is considered a more toxic species than the relatively innocuous and less mobile Cr(III) form. The presence of Cr in the environment has selected microbial and plant variants able to tolerate high levels of Cr compounds. The diverse Cr-resistance mechanisms displayed by microorganisms, and probably by plants, include biosorption, diminished accumulation, precipitation, reduction of Cr(VI) to Cr(III), and chromate efflux. Some of these systems have been proposed as potential biotechnological tools for the bioremediation of Cr pollution. In this review we summarize the interactions of bacteria, algae, fungi and plants with Cr and its compounds.
Assuntos
Cromo/farmacologia , Poluentes Ambientais/toxicidade , Sequência de Aminoácidos , Bactérias/efeitos dos fármacos , Biodegradação Ambiental , Cromo/análise , Cromo/química , Cromo/farmacocinética , Cromo/toxicidade , Microbiologia Ambiental , Poluentes Ambientais/análise , Eucariotos/efeitos dos fármacos , Fungos/efeitos dos fármacos , Dados de Sequência Molecular , Plantas/efeitos dos fármacosRESUMO
The gram-negative bacterium Myxobacter sp. AL-1 produces chitosanase-cellulase activity that is maximally excreted during the stationary phase of growth. Carboxymethylcellulase zymogram analysis revealed that the enzymatic activity was correlated with two bands of 32 and 35 kDa. Ion-exchange-chromatography-enriched preparations of the 32-kDa enzyme were capable of degrading the cellulose fluorescent derivatives 4-methylumbelliferyl-beta-D-cellobioside and 4-methylumbelliferyl-beta-D-cellotrioside. These enzymatic preparations also showed a greater capacity at 70 degrees C than at 42 degrees C to degrade chitosan oligomers of a minimum size of six units. Conversely, the beta-1,4 glucanolytic activity was more efficient at attacking carboxymethylcellulose and methylumbelliferyl-cellotrioside at 42 degrees C than at 70 degrees C. The 32-kDa enzyme was purified more than 800-fold to apparent homogeneity by a combination of ion-exchange and molecular-exclusion chromatography. Amino-terminal sequencing indicated that mature chitosanase-cellulase shares more than 70% identity with endocellulases produced by strains DLG, PAP115, and 168 of the gram-positive microorganism Bacillus subtilis.
Assuntos
Celulase/metabolismo , Glicosídeo Hidrolases/metabolismo , Myxococcales/enzimologia , Myxococcales/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Carboximetilcelulose Sódica/metabolismo , Celobiose/análogos & derivados , Celobiose/metabolismo , Celulase/química , Celulase/isolamento & purificação , Celulose/metabolismo , Quitina/análogos & derivados , Quitina/metabolismo , Quitosana , Cromatografia por Troca Iônica , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Himecromona/análogos & derivados , Himecromona/metabolismo , Dados de Sequência Molecular , Myxococcales/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Trissacarídeos/metabolismoRESUMO
EsigmaG-dependent transcription of the splAB operon in the forespore at stage III of Bacillus subtilis sporulation initiates from two promoters, P1 preceding splA (major) and P3 preceding splB (minor). To explore the possible role of splA in controlling splB-encoded spore photoproduct lyase expression, we measured beta-galactosidase from splB-lacZ fusions integrated at the SPbeta prophage locus which contained point mutations or deletions which either inactivated or physically removed P1 and/or splA. Paradoxically, inactivation of P1 by point mutation or its removal by deletion from upstream resulted in elevated beta-galactosidase expression of the resulting splB-lacZ fusion, as did an in-frame deletion of splA which left P1 and P3 intact;however, expression of all fusions remained sporulation specific and EsigmaG dependent.
Assuntos
Bacillus subtilis/genética , Desoxirribodipirimidina Fotoliase/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Regiões Promotoras Genéticas , Proteínas , Bacillus subtilis/fisiologia , Deleção de Genes , Óperon Lac , Mutação , Esporos Bacterianos/fisiologiaRESUMO
Two copper-resistant (Copr) mutants, strains P1 and P3, were obtained from the dimorphic fungus Mucor rouxii. They were characterized as to their ability to take up copper in a growth medium supplemented with this metal ion. Detection of copper by linear sweep striping voltammetry in cell walls and in the cell wall-free fraction of disrupted cells revealed a higher content of the metal in both mutant fractions, as compared with those of the copper-sensitive (Cops) parental strain. Copper binding by M. rouxii growing cells was also studied through the use of a cytochemical method based on the compounds neocuproine (NCP) and sodium diethyldithiocarbamate (DTC). This method indicated that the P1 Copr strain accumulated more metal than the parental Cops strain, both on the cellular surface and in the intracellular milieu.
RESUMO
Tityus discrepans venom was fractionated by gel filtration on Sephadex G-50 column. The peptides in fraction II from Sephadex were further purified by high performance liquid chromatography, through a C4 reverse-phase column. Lethality of purified peptides was determined by injection into mice and crabs, and their effects were verified electrophysiologically on frog (Hyla crepitans) sartorius neuromuscular junction. Toxins having retention times between 39.6 and 40.7 min depolarized the muscle membrane and caused acetylcholine release at the endplate. The toxin eluted at 42.67 min increased the frequency of miniature endplate potentials without depolarizing muscle fibres. The four most active toxins were reduced, carboxymethylated and sequenced by automatic Edman degradation and named TdII-1 to II-4. Toxin gamma from Tityus serrulatus venom and the toxins from T. discrepans venom were found to be structurally distinct. TdII-1 to II-4 lack the pancreatic effects of T. serrulatus' toxin gamma; yet, the five toxins act on Na+ channels.
Assuntos
Venenos de Escorpião/isolamento & purificação , Acetilcolina/metabolismo , Sequência de Aminoácidos , Animais , Anuros , Braquiúros , Fracionamento Químico , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dextranos/química , Eletrofisiologia , Géis , Masculino , Camundongos , Dados de Sequência Molecular , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Oxirredução , Intoxicação/mortalidade , Venenos de Escorpião/química , Venenos de Escorpião/toxicidadeRESUMO
Bacterial spores are highly resistant to killing by UV radiation and exhibit unique DNA photochemistry. UV irradiation of spore DNA results in formation of spore photoproduct (SP), the thymine dimer 5-thyminyl-5,6-dihydrothymine. Repair of SP occurs during germination of Bacillus subtilis spores by two distinct routes, either by the general nucleotide excision repair (uvr) pathway or by a novel SP-specific monomerization reaction mediated by the enzyme SP lyase, which is encoded by the spl gene. Repair of SP occurs early in spore germination and is independent of de novo protein synthesis, suggesting that the SP repair enzymes are synthesized during sporulation and are packaged in the dormant spore. To test this hypothesis, the expression of a translational spl-lacZ fusion integrated at the spl locus was monitored during B. subtilis growth and sporulation. beta-Galactosidase expression from the spl-lacZ fusion was silent during vegetative growth and was not DNA damage inducible, but it was activated at morphological stage III of sporulation specifically in the forespore compartment, coincident with activation of expression of the stage III marker enzyme glucose dehydrogenase. Expression of the spl-lacZ fusion was shown to be dependent upon the sporulation-specific RNA polymerase containing the sigma-G factor (E sigma G), as spl-lacZ expression was abolished in a mutant harboring a deletion in the sigG gene and restored by expression of the sigG gene in trans. Primer extension analysis of spl mRNA revealed a major extension product initiating upstream from a small open reading frame of unknown function which precedes spl, and it revealed two other shorter minor extension products. All three extension products were present in higher quantities during sporulation and after sigG induction. The three putative transcripts are all preceded by sequences which share homology with the consensus sigma-G factor-type promoter sequence, but in vitro transcription by purified sigma-G RNA polymerase was detected only from the promoter corresponding to the major extension product. The open reading frame-spl operon therefore appears to be an additional member of the sigma-G regulon, which also includes as members the small, acid-soluble spore proteins which are in large part responsible for spore DNA photochemistry. Therefore, sporulating bacteria appear to coordinately regulate genes whose products not only alter spore DNA photochemistry but also repair the major spore-specific photoproduct during germination