Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Direct detection of infectious bursal disease virus from clinical samples by in situ reverse transcriptase-linked polymerase chain reaction

The presence of the very virulent (vv) Brazilian strain of infectious bursal disease virus (IBDV) was determined in the bursa of Fabricius, thymus and liver of 2-week-old broilers from a flock with a higher than expected mortality. For this purpose, a direct in situ reverse transcriptase (RT)-linked polymerase chain reaction (PCR) method was developed using specific primers for vvIBDV. Unlabelled forward and reverse biotinylated oligonucleotides were used for RT-PCR in a one-step method and the respective products were revealed by a direct enzymatic reaction. The results were compared with those obtained by standard RT-PCR using general primers for IBDV and virus isolation. The virus isolation, RT-PCR and in situ RT-PCR revealed positive results on the bursa of Fabricius in 86%, 80% and 100%, respectively. The in situ RT-PCR detected vvIBDV in all tested thymus and liver samples, whereas the standard RT-PCR detected virus in 80% and 90% of the samples, respectively. After three consecutive passages on chicken embryonated eggs, IBDV was isolated from 64% of the thymus samples and 30% of the liver samples. In the present study, no classical or antigenic variants of IBDV were detected. The developed in situ RT-PCR assay was able to detect the very virulent strain of IBDV with a higher sensitivity than the conventional RT-PCR and virus isolation.

Phenylalanine ammonia-lyase activity in soybean roots extract measured by reverse-phase high performance liquid chromatography

The high performance liquid chromatography (HPLC) technique was applied to measure phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity in soybean (Glycine max L. Merril cv. BR16) roots. t-Cinnamate, the catalytic product of the PAL reaction was quantified at 275 nm by isocratic elution with methanol:water through an ODS(M) column. Comparative experiments were carried out with 1.0 mM ferulic acid, an inducer of PAL activity. The results suggest that liquid chromatography is a rapid and sensitive method to analyze PAL activity in non-purified extract.

Leishmania amazonensis: Partial purification and study of the biochemical properties of the telomerase reverse transcriptase activity from promastigote-stage

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determination of toxigenic capacity by reverse transcription polymerase chain reaction in coagulase-negative staphylococci and Staphylococcus aureus isolated from newborns in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phase diagrams of a CTAB/organic solvent/buffer system applied to extraction of enzymes by reverse micelles

A partial pseudo-ternary phase diagram has been studied for the cethyltrimethylammonium bromide/isooctane:hexanol:butanol/potassium phosphate buffer system, where the two-phase diagram consisting of the reverse micelle phase (L-2) in equilibrium with the solvent is indicated. Based on these diagrams two-phase systems of reverse micelles were prepared with different compositions of the compounds and used for extraction and recovery of two enzymes, and the percentage of enzyme recovery yield monitored. The enzymes glucose-6-phosphate dehydrogenase (G6PD) and xylose redutase (XR) obtained from Candida guilliermondii yeast were used in the extraction procedures. The recovery yield data indicate that micelles having different composition give selective extraction of enzymes. The method can thus be used to optimize enzyme extraction processes. (c) 2007 Elsevier B.V. All rights reserved.

Effect of Ag additions on the reverse martensitic transformation in the Cu-10 mass% Al alloy

The effect of Ag additions on the reverse martensitic transformation in the Cu-10 mass% Al alloy was studied using differential thermal analysis (DTA), optical (OM) and scanning electron microscopies (SEM) and X-ray diffractometry. The results indicated that Ag additions to the Cu-10 mass% Al alloy shift the equilibrium concentration to higher Al contents, allow to obtain both beta(1)' and beta' martensitic phases in equilibrium and that Ag precipitation is a process associated with the perlitic phase formation.

Reverse martensitic transformation in the Cu-10 wt% Al-6 wt%Ag alloy

The reverse martensitic transformation in the Cu-10 wt%Al-6 wt%Ag alloy was studied by classical differential thermal analysis (IDTA), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX) and X-ray diffractometry (XRD). The results indicated that the presence of Ag in the Cu-10%Al alloy is responsible for the separation of the competitive reactions that occur during the reverse martensitic transformation and is also associated to an increase in the disordering degree at high temperatures, when compared with alloys without Ag addition. (c) 2005 Springer Science + Business Media, Inc.

Activation energy for the reverse eutectoid reaction in hypo-eutectoid Cu-Al alloys

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of Ag additions on the activation energy for the reverse eutectoid reaction in Cu-Al alloys

The eutectoid transformation may be defined as a solid-state diffusion-controlled decomposition process of a high-temperature phase into a two-phase lamellar aggregate behind a migrating boundary on cooling below the eutectoid temperature. In substitutional solid solutions, the eutectoid reaction involves diffusion of the solute atoms either through the matrix or along the boundaries or ledges. The effect of Ag on the non-isothermal kinetics of the reverse eutectoid reaction in the Cu-9 mass%Al, Cu-10 mass%Al, and Cu-11 mass%Al alloys were studied using differential scanning calorimetry (DSC), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The activation energy for this reaction was obtained using the Kissinger and Ozawa methods. The results indicated that Ag additions to Cu-Al alloys interfere on the reverse eutectoid reaction, increasing the activation energy values for the Cu-9 mass%Al and Cu-10 mass%Al alloys and decreasing these values for the Cu-11 mass%Al alloy for additions up to 6 mass%Ag. The changes in the activation energy were attributed to changes in the reaction solute and in Ag solubility due to the increase in Al content.

Mutagenicity of Flavonoids Assayed by Bacterial Reverse Mutation (Ames) Test

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Quantitative determination of anti-fungal and insecticide amides in adult plants, plantlets and callus from Piper tuberculatum by reverse-phase high-performance liquid chromatography

A rapid, sensitive and reliable reverse-phase HPLC method was used for the quantitative determination of the anti-fungal and insecticide amides, dihydropiplartine (1), piplartine (2), Delta(alpha,beta)-dihydropiperine (3) and pellitorine (4) in plants in natura, in plantlets in vitro and ex vitro, and in callus of Piper tuberculatum. Well-resolved peaks were obtained with good detection response and linearity in the range of 15.0-3000 mug/mL. The plants in natura contained compounds 1-4, the plantlets ex vitro and in vitro accumulated compounds 1-2 and 1-4, respectively, while only amide 4 was found in callus. Copyright (C) 2003 John Wiley Sons, Ltd.

Quantitative determination of maitenin and 22 beta-hydroxymaitenin in callus of Maytenus aquifolium (Celastraceae) by reverse phase high performance liquid chromatography

Explants of Maytenus aquifolium were induced to form callus and, subsequently, suspension cultures. The isolation of natural products from callus led to the identification of the cytotoxic triterpene quinonemethides, maitenin (1) and 22 beta-hydroxymaitenin (2), A rapid, sensitive and reliable reversed-phase high-performance liquid chromatography method was developed using a Cls column and isocratic elution for the determination of 1 and 2, the elaborated method gave well-resolved peaks for these compounds with good detection response and linearity in the range of 0.08-72.0 mu g. The quantification of 1 and 2 was performed by an external standard method. (C) 1998 John Wiley & Sons, Ltd.

Low triioothyronine (T-3) or reverse triioothyronine (rT(3)) syndrome modifies gene expression in rats with congestive heart failure

Heart failure (NF) is frequently associated with euthyroid sicksyndrome (low T-3 and elevated rT(3)). We investigated if altered thyroid hormone in HF could affect expression of the TH receptor (TR alpha 1), and alpha and beta myosin heavy chains (alpha-MHC beta-MHC). HF was provoked in rats by aortic stenosis. We showed that rT(3) generated front liver and kidney deiodination significantly increased and T-3 decreased in HE; there was significantly higher TR alpha 1 expression, no alpha-MHC expression, but beta-MHC expression. Changes in TR alpha 1 could be compensating for low T-3 from HF.