Analysis of Factor VIII polymorphic markers as a means for carrier detection in Brazilian families with haemophilia A

Haemophilia A is an X-linked, recessively inherited bleeding disorder of varying severity, which results from the deficiency of procoagulant factor VIII f(8). Linkage diagnosis using polymorphic markers in the f8 gene is widely used to detect carriers. The objective of this study was to verify the informativeness of three polymorphic markers in the Brazilian population, to evaluate the usefulness of such markers in carrier detection procedures. Sixty-three unrelated healthy volunteers and 10 haemophilic families were studied. Two microsatellite repeats and one HindIII RFLP markers were used. Carrier and non-carrier status could be determined in 80% of females investigated. Intron 13 markers presented the highest heterozygosity rate (79%) followed by intron 22 (68%) and intron 19 (57%). When all three markers were used together, linkage analysis informativeness increased significantly. We conclude that these markers are suitable for carrier detection in the Brazilian population and we recommend their use in combination to maximize diagnostic efficiency.

X-chromosome genetic variation in São Paulo State (Brazil) population

As data on X-chromosomal short tandem repeats (X-STRs) for the Brazilian population are scarse, the aim of this study was to determine the allele frequencies of five X-STRs (DXS6854, DXS7424, DXS101, DXS6808 and DXS7132) in the São Paulo State, Brazil. No deviations from the Hardy-Weinberg equilibrium were observed, with the exception of DXS101. The forensic efficiency parameters demonstrated that DXS101 was the most informative marker. Population comparisons revealed that the X-STR profile sampled in the state of São Paulo was more similar to European and African populations than Asiatic populations reported in this work.

Experimental investigation of smouldering in biomass

The smouldering process of wood logs was studied experimentally in a laboratory facility and in prescribed forest bums. The main goal was to check the parameters that initiate and control the stability of the smouldering process. To do so, sample temperatures at five different locations and concentrations of CO, CO2 and O-2 were measured and discussed. By varying the temperature and air supply of the flow tunnel apparatus, different rates of smoulder propagation were identified. In prescribed bums, the main characteristics of the self-sustained smouldering combustion front in logs of different sizes and species are reported. The average smouldering speed in the field is about one order of magnitude lower than that reported for different materials in laboratory experiments. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Log smoldering after an Amazonian deforestation fire

The characteristics of log smoldering after an Amazonian deforestation fire are described. The experiment was carried out in 2001 at the Caiabi farm, near the city of Alta Floresta, state of Mato Grosso, Brazil, as part of a set of tests that have been performed in the same area since 1997. A 200 x 200m(2) test area was slashed in the beginning of June and burned on 20 August. The area contained 507 logs with diameter at breast height (DBH) higher than 10 cm, per hectare. In the day following the main burn 59 logs were found to remain smoldering, a number that corresponds to 2.9% of the total in the area. We chose 11 of the 59 logs to have their smoldering process monitored. Their diameter, moisture content and CHN dry biomass composition after the plot burn and before smoldering were determined. Other parameters such as temperature distribution while smoldering, porosity, density and mass volatilized during thermogravimetric test were also determined. Average smoldering speeds were in the range from 0.8 to 1.5 cm h(-1) for logs that smoldered without transition to the flaming regime. The average speed increased to 2.1 cm h(-1) for those logs that oscillated between smoldering and flaming. The speeds were lower overnight as compared to values determined during daytime for the same log. Higher log moisture contents were found to produce decreased speeds. Micro-porous biomass was not observed in the set of the 11 selected logs. Smoldering was observed to occur at substantial intensity in crossing of logs, with no longitudinal propagation. (C) 2003 Elsevier Ltd. All rights reserved.

Experimental measurements of the NOx and CO concentrations operating in oscillatory and non-oscillatory burning conditions

The effects of combustion driven acoustic oscillations in carbon monoxide and nitrogen oxides emission rates of a combustor operated with liquefied petroleum gas (LPG) were investigated. Because the fuel does not contain nitrogen, tests were also conducted with ammonia injected in the fuel, in order to study the formation of fuel NOx. The main conclusions were: (a) the pulsating combustion process is more efficient than the non-pulsating one and (b) the pulsating combustion process generates higher rates of NOx, with and without ammonia injection, as shown by CO and NO concentrations as function of the O-2 concentration. An increase in the LPG flow rate, keeping constant the air to fuel ratio, increased the acoustic pressure amplitude and the frequency of oscillation. The injection of ammonia had no influence on either pressure amplitude or frequency. (c) 2005 Elsevier Ltd. All rights reserved.

Satellyptus: analysis and database of microsatellites from ESTs of Eucalyptus

The main goal of our research was to search for SSRs in the Eucalyptus EST FORESTs database (using a software for mining SSR-motifs). With this objective, we created a database for cataloging Eucalyptus EST-derived SSRs, and developed a bioinformatics tool, named Satellyptus, for finding and analyzing microsatellites in the Eucalyptus EST database. The search for microsatellites in the FORESTs database containing 71,115 Eucalyptus EST sequences (52.09 Mb) revealed 20,530 SSRs in 15,621 ESTs. The SSR abundance detected on the Eucalyptus ESTs database (29% or one microsatellite every four sequences) is considered very high for plants. Amongst the categories of SSR motifs, the dimeric (37%) and trimeric ones (33%) predominated. The AG/CT motif was the most frequent (35.15%) followed by the trimeric CCG/CGG (12.81%). From a random sample of 1,217 sequences, 343 microsatellites in 265 SSR-containing sequences were identified. Approximately 48% of these ESTs containing microsatellites were homologous to proteins with known biological function. Most of the microsatellites detected in Eucalyptus ESTs were positioned at either the 5 or 3 end. Our next priority involves the design of flanking primers for codominant SSR loci, which could lead to the development of a set of microsatellite-based markers suitable for marker-assisted Eucalyptus breeding programs.

Genotyping of AR and PSA polymorphisms in a patient with Klinefelter syndrome, non-Hodgkin lymphoma, and adenocarcinoma of the prostate

It has been hypothesized that the AR (androgen receptor) gene binds the two PSA (prostate-specific antigen) alleles with differing affinities and may differentially influence prostate cancer risk. In this article, we report a case of adenocarcinoma of the prostate in a 56-year-old man with Klinefelter syndrome (47,XXY) and non-Hodgkin lymphoma, as well as the AR and PSA genotype. AR and PSA gene polymorphisms were analyzed by polymerase chain reaction-based methods using DNA from peripheral white blood cells and the prostate cancer. We determined the methylation status of the AR gene on the X chromosome. The patient presents with the AG genotype for the ARE-I (androgen response element) region of the PSA gene. We detect the presence of two short AR alleles with 19 and 11CAG repeats each. Unmethylated alleles were demonstrated for both. The shorter allele was inactive in more than 60% of total DNA in both control blood and prostate cancer cells. The presence of short AR alleles and the G allele of the PSA gene may contribute to the development of prostate cancer in a 47,XXY patient. (C) 2004 Elsevier B.V. All rights reserved.

Biopatologia do Helicobacter pylori

A infecção pelo Helicobacter pylori (H. pylori) induz inflamação persistente na mucosa gástrica com diferentes lesões orgânicas em humanos, tais como gastrite crônica, úlcera péptica e câncer gástrico. Os fatores determinantes desses diferentes resultados incluem a intensidade e a distribuição da inflamação induzida pelo H. pylori na mucosa gástrica. Evidências recentes demonstram que cepas do H. pylori apresentam diversidade genotípica, cujos produtos acionam o processo inflamatório por meio de mediadores e citocinas, que podem levar a diferentes graus de resposta inflamatória do hospedeiro, resultando em diferentes destinos patológicos. Cepas H. pylori com a ilha de patogenicidade cag induzem resposta inflamatória mais grave, através da ativação da transcrição de genes, aumentando o risco para desenvolvimento de úlcera péptica e câncer gástrico. O estresse oxidativo e nitrosativo induzido pela inflamação desempenha importante papel na carcinogênese gástrica como mediador da formação ou ativação de cancerígenos, danos no DNA, bem como de alterações da proliferação celular e da apoptose.

Use of crude, FML and rK39 antigens in ELISA to detect anti-Leishmania spp. antibodies in Felis catus

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

Polymorphisms and Haplotypes in the Interleukin-4 Gene are Associated With Chronic Periodontitis in a Brazilian Population

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

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE) x S(SM)(EK) x S(SM)(SSRs)). Clustering analyses showed a mean of 9 +/- 12.4 isolates per cluster (3.8 +/- 8 isolates/taxon) for MLEE, 6.2 +/- 4.9 isolates per cluster (4 +/- 4.5 isolates/taxon) for SSRs, and 4.1 +/- 2.3 isolates per cluster (2.6 +/- 2.3 isolates/taxon) for EK. A total of 45 (13%), 39(11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (Si) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships. (C) 2010 Elsevier B.V. All rights reserved.

Comparison Between the Polymerase Chain Reaction-Based Screening and the Southern Blot Methods for Identification of Fragile X Syndrome

The fragile X syndrome (FXS), the most common cause of hereditary mental retardation, is caused by expansions of CGG repeats in the FMR1 gene. The gold-standard method to diagnose FXS is the Southern blot (SB). Because SB is laborious and costly, some adaptations in the polymerase chain reaction (PCR) method have been utilized for FXS screening. A previous PCR-based screening method for FXS identification utilizing small amounts of DNA was reported as simple and efficient. The aim of this study was to reproduce the mentioned PCR-based screening method for identification of expanded alleles of the FMR1 gene in Brazilian individuals and to investigate the efficiency of this method in comparison with SB. Utilizing the enzyme Expand Long Template PCR System, 78 individuals were investigated by that PCR-based screening method for FXS identification. Conclusive results were obtained for 75 samples. Considering all the allelic forms of FXS (normal [NL], premutation [PM], and full-mutation [FM]), the comparison of the PCR-based screening method with SB demonstrated 100% of accuracy, sensitivity, and specificity. However, when the PM and the FM were analyzed separately from each other, but together with the NL allele, the accuracy, sensitivity, and specificity decreased (to 42.9%-97.4%). We concluded that the PCR-based screening method was reproducible and capable of identifying all different FXS alleles, but because the differentiation between the PM and the FM alleles was not accurate, SB is still the gold-standard method for the molecular diagnosis of FXS.

Characterization and transferability of microsatellite markers of the cultivated peanut (Arachis hypogaea)

Background: the genus Arachis includes Arachis hypogaea (cultivated peanut) and wild species that are used in peanut breeding or as forage. Molecular markers have been employed in several studies of this genus, but microsatellite markers have only been used in few investigations. Microsatellites are very informative and are useful to assess genetic variability, analyze mating systems and in genetic mapping. The objectives of this study were to develop A. hypogaea microsatellite loci and to evaluate the transferability of these markers to other Arachis species.Results: Thirteen loci were isolated and characterized using 16 accessions of A. hypogaea. The level of variation found in A. hypogaea using microsatellites was higher than with other markers. Cross-transferability of the markers was also high. Sequencing of the fragments amplified using the primer pair AhII from 17 wild Arachis species showed that almost all wild species had similar repeated sequence to the one observed in A. hypogaea. Sequence data suggested that there is no correlation between taxonomic relationship of a wild species to A. hypogaea and the number of repeats found in its microsatellite loci.Conclusion: These results show that microsatellite primer pairs from A. hypogaea have multiple uses. A higher level of variation among A. hypogaea accessions can be detected using microsatellite markers in comparison to other markers, such as RFLP, RAPD and AFLP. The microsatellite primers of A. hypogaea showed a very high rate of transferability to other species of the genus. These primer pairs provide important tools to evaluate the genetic variability and to assess the mating system in Arachis species.

Telomere biology of trypanosomatids: beginning to answer some questions

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organ ism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

5S rDNA characterization in twelve Sciaenidae fish species (Teleostei, Perciformes): depicting gene diversity and molecular markers

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