Comparative Analysis by Polymerase Chain Reaction Amplified Minicircles of Kinetoplast DNA of a Stable Strain of Trypanosoma cruzi from São Felipe, Bahia, its Clones and Subclones: Possibility of Predominance of a Principal Clone in this Area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicicles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment lenght polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%.This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.

An introduction to model-independent diffusion magnetic resonance imaging.

ABSTRACT: q-Space-based techniques such as diffusion spectrum imaging, q-ball imaging, and their variations have been used extensively in research for their desired capability to delineate complex neuronal architectures such as multiple fiber crossings in each of the image voxels. The purpose of this article was to provide an introduction to the q-space formalism and the principles of basic q-space techniques together with the discussion on the advantages as well as challenges in translating these techniques into the clinical environment. A review of the currently used q-space-based protocols in clinical research is also provided.

Magnetic resonance imaging determinants of intraindividual variability in the elderly: combined analysis of grey and white matter.

Elderly individuals display a rapid age-related increase in intraindividual variability (IIV) of their performances. This phenomenon could reflect subtle changes in frontal lobe integrity. However, structural studies in this field are still missing. To address this issue, we computed an IIV index for a simple reaction time (RT) task and performed magnetic resonance imaging (MRI) including voxel based morphometry (VBM) and the tract based spatial statistics (TBSS) analysis of diffusion tensor imaging (DTI) in 61 adults aged from 22 to 88 years. The age-related IIV increase was associated with decreased fractional anisotropy (FA) as well as increased radial (RD) and mean (MD) diffusion in the main white matter (WM) fiber tracts. In contrast, axial diffusion (AD) and grey matter (GM) densities did not show any significant correlation with IIV. In multivariate models, only FA has an age-independent effect on IIV. These results revealed that WM but not GM changes partly mediated the age-related increase of IIV. They also revealed that the association between WM and IIV could not be only attributed to the damage of frontal lobe circuits but concerned the majority of interhemispheric and intrahemispheric corticocortical connections.

Colony polymerase chain reaction of stably transfected Trypanosoma cruzi grown on solid medium

Tools for the genetic manipulation of Trypanosoma cruzi are largely unavailable, although several vectors for transfection of epimastigotes and expression of foreign or recombinant genes have been developed. We have previously constructed several plasmid vectors in which recombinant genes are expressed in T. cruzi using the rRNA promoter. In this report, we demonstrate that one of these vectors can simultaneously mediate expression of neomycin phosphotransferase and green fluorescent protein when used to stably transfect cultured epimastigotes. These stably transfected epimastigotes can be selected and cloned as unique colonies on solid medium. We describe a simple colony PCR approach to the screening of these T. cruzi colonies for relevant genes. Thus, the methodologies outlined herein provide important new tools for the genetic dissection of this important parasite.

Identification of planorbids from Venezuela by polymerase chain reaction amplification and restriction fragment length polymorphism of internal transcriber spacer of the RNA ribosomal gene

Snails of the genus Biomphalaria from Venezuela were subjected to morphological assessment as well as polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. Morphological identification was carried out by comparison of characters of the shell and the male and female reproductive apparatus. The PCR-RFLP involved amplification of the internal spacer region ITS1 and ITS2 of the RNA ribosomal gene and subsequent digestion of this fragment by the restriction enzymes DdeI, MnlI, HaeIII and MspI. The planorbids were compared with snails of the same species and others reported from Venezuela and present in Brazil, Cuba and Mexico. All the enzymes showed a specific profile for each species, that of DdeI being the clearest. The snails were identified as B. glabrata, B. prona and B. kuhniana.

Polymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1

Especial conditions were developed for the amplification of five DNA segments from US region of BHV-1 by polymerase chain reaction. In order to eliminate most nonspecific products it was found that addition of three cosolvents DMSO, glycerol and NP 40 was a simple method for increasing the specificity of amplification.

A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis

Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.

On Lundh's percolation diffusion

A collection of spherical obstacles in the unit ball in Euclidean space is said to be avoidable for Brownian motion if there is a positive probability that Brownian motion diffusing from some point in the ball will avoid all the obstacles and reach the boundary of the ball. The centres of the spherical obstacles are generated according to a Poisson point process while the radius of an obstacle is a deterministic function. If avoidable con gurations are generated with positive probability Lundh calls this percolation di usion. An integral condition for percolation di ffusion is derived in terms of the intensity of the point process and the function that determines the radii of the obstacles.

Characterization of Biomphalaria orbignyi, Biomphalaria peregrina and Biomphalaria oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene

The correct identification of Biomphalaria oligoza, B. orbignyi and B. peregrina species is difficult due to the morphological similarities among them. B. peregrina is widely distributed in South America and is considered a potential intermediate host of Schistosoma mansoni. We have reported the use of the polymerase chain reaction and restriction fragment length polymorphism analysis of the internal transcribed spacer region of the ribosomal DNA for the molecular identification of these snails. The snails were obtained from different localities of Argentina, Brazil and Uruguay. The restriction patterns obtained with MvaI enzyme presented the best profile to identify the three species. The profiles obtained with all enzymes were used to estimate genetic similarities among B. oligoza, B. peregrina and B. orbignyi. This is also the first report of B. orbignyi in Uruguay.

A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory.

Resistance of Biomphalaria occidentalis from Varzea das Flores dam, Minas Gerais, to Schistosoma mansoni infection detected by low stringency polymerase chain reaction

Biomphalaria occidentalis Paraense, 1981 from Varzea das Flores dam, MG, Brazil, was exposed to infection with Schistosoma mansoni. Individual infection was performed with 140 B. occidentalis and 100 B. glabrata snails using LE and SJ strains. Two groups of B. occidentalis were killed after seven day-miracidia exposure to detect S. mansoni DNA, through the low stringency polymerase chain reaction (LS-PCR), and were negative. The infection rates were 69.2% (LE strain) and 96.7% (SJ strain) for B. glabrata and 0% for B. occidentalis. LS-PCR enabled early resistance diagnosis.

Genetic markers from Biomphalaria tenagophila (Gastropoda: Pulmonata: Planorbidae) obtained by the double stringency polymerase chain reaction technique

Biomphalaria tenagophila, one of the intermediate hosts of the trematoda Schistosoma mansoni, is a simultaneous hermafrodite snail species. In order to analyse the genetic structure of these populations, we performed a double-stringency PCR technique to obtain genetic markers with microsatellites and arbitrary primers in a single reaction.