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A Zaha

Publications and source records attributed to A Zaha.

At least 19 recordsLinked to original sources
PubMed2006

Detection of genetic polymorphism among and within Echinococcus granulosus strains by heteroduplex analysis of a microsatellite from the U1 snRNA genes.

Polymerase chain reaction of a pentanucleotide microsatellite in the U1 snRNA gene complex generated a multiple band pattern due to the priming of paralogous sequences. Denaturation and slow renaturation of polymerase chain reaction products allow the formation of heteroduplex DNA that can be detected by its differential mobility in polyacrylamide gel electrophoresis. Heteroduplex analysis was used to determine if the U1 snRNA microsatellite could be a useful genetic marker in Echinococcus granulosus. A U1 snRNA microsatellite fragment from E. granulosus was isolated and characterized by Southern blot and sequencing. Four E. granulosus strains were analyzed: sheep, Tasmanian sheep, cattle, and camel strains. The former two showed polymorphism and shared three of the six patterns found for sheep strain. The cattle strain displayed two patterns, and the camel strain was monomorphic. The electrophoretic profiles were used for statistical analysis in order to determine genetic distance and the relationship among strains. Heteroduplex analysis can be helpful in genotyping E. granulosus strains and is useful in detecting polymorphism within strains.

Animals↗
PubMed2006

Recent transmission of tuberculosis involving retired patients.

The reported incidence of tuberculosis (TB) in three different regions of Rio Grande do Sul State in Brazil varies considerably. We used IS6110-RFLP and spoligotyping methods to genotype Mycobacterium tuberculosis isolates obtained from 268 patients between 1998 and 2000 in order to assess the levels of recent transmission of TB in the three regions. The degree of clustering of the strain types did not differ among the three regions; neither did other characteristics such as demographic features, underlying medical conditions, or the proportion of resistant TB. As reported previously, male patients were at greater risk of developing TB and our data suggest that part of this may be related to the higher rates of recent transmission among them (P<0.05). In addition, we found that retired patients were almost 3 times more likely to be infected with cluster-pattern strains than patients reporting any other occupation (P<0.05), and more than 3 times more likely than non-retired patients in the same age group (P<0.05) to be infected with cluster-pattern strains. We conclude that recent transmission is not a major factor contributing to the differences in TB incidence in the three regions of Rio Grande do Sul. The reason for the suggested high proportion of recent transmission TB cases among the retired people needs further studies.

Adolescent↗
PubMed2005

Searching for antigen B genes and their adaptive sites in distinct strains and species of the helminth Echinococcus.

Twenty-seven PCR-derived antigen B (AgB) nucleotide sequences from four Echinococcus species (Echinococcus granulosus, Echinococcus multilocularis, Echinococcus oligarthrus and Echinococcus vogeli) were aligned with 78 already published sequences, to generate a maximum likelihood phylogeny of the AgB multigene family. The phylogenetic analysis confirms that the family is constituted by four groups of genes present in each one of the four species (AgB1, AgB2, AgB3 and AgB4), and suggests that it originated by ancient duplication events preceding speciation within the genus. AgB5 sequences, which had been formerly suggested to correspond to a putatively new AgB subunit, cluster with AgB3. Likelihood tests suggest that AgB gene evolution may have been driven by heterogeneous selection pressures acting on particular AgB1, AgB3 and AgB4 codons. No selection is detected in AgB2. We discuss implications of our findings in terms of AgB biology and its use as a diagnostic tool.

Adaptation, Physiological↗
PubMed2005

Early post-larval development of the endoparasitic platyhelminth Mesocestoides corti: trypsin provokes reversible tegumental damage leading to serum-induced cell proliferation and growth.

Mesocestoides corti is a suitable in vitro model for studying the development of human endoparasitic platyhelminthes. Treatment with trypsin, supplemented with fetal bovine serum (FBS), induces M. corti development from larvae (tetrathyridia) to segmented adult worm; however, the role of this protease and of FBS in post-larval development induction remains unknown. To characterize the participation of trypsin enzymatic activity and of FBS in the induction of tetrathyridia growth and development, both stimuli were added to the larvae either together or sequentially. Additionally, specific inhibition of trypsin activity was also monitored. Finally, the effect of the enzyme on the parasite tegument as well as the proliferative activity and location of proliferating cells after induction of tetrathyridia development were also studied. We conclude that trypsin-induced tetrathyridia development to adult worm is FBS-dependent and that the effect of serum factors is dependent upon a previous trypsin-induced reversible damage to the larva tegument. In dividing and non-dividing tetrathyridia, proliferative activity of cells is mainly located within the apical massif in the anterior region and nerve cords of larvae, respectively. In tetrathyridia stimulated to develop to adult worms, an intense proliferative activity is evident along the nerve cords. Our results suggest that in natural infections the tetrathyridia tegument is temporally made permeable to growth factors by proteolytic enzyme activity in the intestine juice of the definitive host, thus leading to development to adult worms.

Animals↗
PubMed2005

High polymorphism in genes encoding antigen B from human infecting strains of Echinococcus granulosus.

Echinococcus granulosus antigen B (AgB) is encoded by a gene family and is involved in the evasion of the host immune response. E. granulosus exists as a number of strains (G1-G10) that differ in biological characteristics. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and transcription profile of AgB in 5 E. granulosus strains. Twenty-four genomic sequences were isolated and clustered in 3 groups related to 2 of the 5 reported AgB genes. AgB4 genes were present in almost all strains, whereas AgB2 were present as functional genes exclusively in G1/G2 cluster, and as non-functional genes in G5 and the G6/G7 cluster, suggesting inter-strain variation. The AgB transcription patterns, analysed by RT-PCR, showed that AgB2 and AgB4 genes were transcribed in G1, while only the AgB4 gene was transcribed in G7 strain. Cysts from the same strain or cluster shared more genomic and cDNA variants than cysts from different strain or cluster. The level of nucleotide and deduced amino acid sequence variation observed is higher than that reported so far for coding genes of other helminths. Neutrality was rejected for AgB2 genes. These data show the genetic polymorphism of antigen-coding genes among genetically characterized strains of E. granulosus.

Amino Acid Sequence↗
PubMed2004

Contingent, non-neutral evolution in a multicellular parasite: natural selection and gene conversion in the Echinococcus granulosus antigen B gene family.

Recent studies have demonstrated that the Echinococcus granulosus antigen B (AgB) interferes with the intermediate hosts' immune response and is encoded by a multigene family. The number of members within the family is still uncertain, but there are several evidences of a large genetic variability. The E. granulosus AgB genomic sequences available in nucleotide databases can be grouped into four clades, corresponding to genes EgAgB1, EgAgB2, EgAgB3 and EgAgB4. In the present study, we use PCR amplifications followed by cloning and sequencing to evaluate the genetic variability for AgB isoforms. Two pairs of primers were independently used for PCR amplification. Both PCR reactions from each of three isolated protoscolex (larvae) were cloned in a plasmid vector and the plasmid inserts of 30 colonies from each cloning experiment were sequenced. Using phylogenetic tools, the 113 EgAgB clones are classified as follows: 25 are related to EgAgB1, 24 to EgAgB2, 9 to EgAgB3 and 39 to EgAgB4. The remaining 16 clones form a separate cluster, which we name EgAgB5, more closely related to EgAgB3 than to any of the other genes. Within each gene group, a number of variant sequences occur, which differ from one another by one or few nucleotides. One EgAgB3 clone has a premature stop codon (pseudogene) and an EgAgB2 clone lacks the region corresponding to the intron. The overall variation cannot be explained by differences among the asexual protoscoleces, or by experimental artifacts. Using Echinococcuss AgB genes from other species/strains as outgroups, neutrality is rejected for EgAgB2, and balancing selection is detected for EgAgB5, which also seems to be involved in gene conversion. We suggest that EgAgB1-EgAgB5 represent a family of contingency genes, that is, genes that are variably expressed, so that some but not others are expressed in each individual parasite. Contingency genes are common in parasitic protozoa and other microparasites, but the EgAgB family is the first set identified in a multicellular parasite.

Alleles↗
PubMed2004

Detection of Mycobacterium tuberculosis by a polymerase chain reaction colorimetric dot-blot assay.

SETTING: A public health laboratory in a tuberculosis-endemic region in Brazil. OBJECTIVE: To evaluate the accuracy of a combined polymerase chain reaction (PCR) colorimetric dot-blot protocol for Mycobacterium tuberculosis detection in clinical samples in a public health laboratory. DESIGN: Eighty clinical samples (13 cerebrospinal fluid, 31 induced sputum, 17 expectorated sputum, eight bronchoalveolar lavage and 11 pleural fluid) were assayed with the developed protocol. The accuracy of polymerase chain reaction (PCR) dot-blot methodology was compared to PCR agarose gel electrophoresis (PCR-AG) using as a gold standard the bacteriological result (culture and biochemical identification) combined with clinical follow-up. One internal region of the IS6110 repetitive element of the M. tuberculosis complex was selected for amplification and the amplified product transferred to nylon membranes to be detected by biotinylated DNA probe. RESULTS: Overall sensitivity and specificity obtained were respectively 90% and 97% for PCR-AG and 95% and 97% for the PCR dot-blot. Among the 56 respiratory specimens, the sensitivity and specificity results for PCR-AG were respectively 88% and 95%, and for PCR dot-blot they were 94% and 95%. Among the 24 non-respiratory specimens the sensitivity and specificity results were respectively 83% and 100% for PCR-AG, and 100% and 100% for the PCR dot-blot protocol. CONCLUSION: The results demonstrated that the PCR dot-blot assay may be helpful in the diagnosis of tuberculosis, and feasible even in resource-poor countries.

Blotting, Southern↗
PubMed2003

A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease.

Several recombinant clones expressing antigens from Echinococcus granulosus were isolated previously from a parasite cDNA library using cystic hydatid disease (CHD) patients' sera or rabbit hyperimmune antiserum against a lipoproteic fraction from bovine cyst fluid. Six of these antigens were expressed in Escherichia coli and the purified recombinant proteins were tested in enzyme-linked immunosorbent assay (ELISA) for specific IgG with a panel of sera from patients with surgically confirmed (n = 58) or immunologically diagnosed (n = 71) CHD. Sera from clinically normal individuals (n = 203) and sera from individuals with other helminthic infections (n = 65) were assayed for the assessment of specificity. A cut-off value was determined by receiver-operating-characteristic plots for each antigen. A recombinant antigen B subunit (AgB8/2) presented the highest sensitivity (93.1%), considering the group of sera from patients with CHD surgically confirmed, and specificity (99.5%) and is proposed as the basis for an immunodiagnostic test. The other recombinant antigens tested presented sensitivities between 58.6% and 89.7%, and three of them were considered of complementary value. In subclass-specific ELISA, different IgG isotypes showed dominance in the response for each of the recombinant antigens. There was a clear predominance of IgG4 response for all antigens tested, indicating that this would be the subclass of choice to be assessed for these recombinant proteins.

Animals↗
PubMed2003

Direct-test PCR for detection of meningococcal DNA and its serogroup characterization: standardization and adaptation for use in a public health laboratory.

A direct PCR test (DT-PCR) was established to detect Neisseria meningitidis DNA in clinical samples from patients with suspected bacterial meningitis. Specific primers for the 16S rDNA of N. meningitidis were designed to amplify a 600 bp DNA fragment. One hundred and ninety-three clinical samples were analysed, corresponding to 114 samples from patients diagnosed as positive and 79 as negative for infection by N. meningitidis using conventional methods (culture, latex agglutination and counterimmunoelectrophoresis). These samples were submitted to PCR by two different clinical sample preparation approaches (with and without DNA extraction and purification) and submitted to different PCR protocols to improve the results. In agarose gel detection, the sensitivity value for DT-PCR was 88.5 % and, using dot-blot DNA detection, the sensitivity increased to 96.4 %. The detection limit for meningococcus in cerebrospinal fluid was 2x10(2) c.f.u. ml(-1). Serogroup prediction was done using a multiplex PCR protocol and the sensitivity was 83 % for agarose gel DNA detection and 96.4 % using dot-blot DNA detection.

Cerebrospinal Fluid↗
PubMed2003

Isolation and characterization of microsatellites from the tapeworm Echinococcus granulosus.

The Echinococcus granulosus genome was searched for microsatellites using 8 different repeated oligonucleotides as probes (GT15, CT15, AT15, CG15, CAT10, CAA10, CGG10 and CATA10). Southern blot experiments revealed that DNA regions containing GT, CAA, CATA and CT repeats are the most frequent in the E. granulosus genome. AT and CG probes showed no hybridization signal. Two loci containing CA/GT (Egmsca1 and Egmsca2) and 1 locus containing GA/CT (Egmsga1) repeats were cloned and sequenced. The locus Egmsca1 was analysed in 73 isolates from Brazil and Argentina whose strains were previously characterized. Brazilian isolates from cattle strain and Argentinean isolates from camel strain were monomorphic and shared the allele (CA)7. Argentinean isolates of sheep and Tasmanian sheep strains shared 2 alleles [(CA)8 and (CA)10] with Brazilian isolates of sheep strain. The allele (CA)11 was found only in Brazilian isolates of sheep strain at a low frequency. The Brazilian and the Argentinean sheep strain populations were tested for the Hardy-Weinberg equilibrium, and only the former was in agreement with the expectations. No polymorphism was found among individual protoscoleces from a single hydatid cyst, validating the utilization of pooled protoscoleces from 1 cyst, grouped as an isolate, in population studies. This work describes for the first time the isolation and characterization of microsatellites from E. granulosus.

Animals↗
PubMed2001

Echinococcus granulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein.

We report the isolation and characterization of an Echinococcus granulosus gene that codes for a protein with actin filament fragmenting and nucleating activities (EgAFFP). The genomic region corresponding to the EgAFFP gene presents a coding sequence of 1110 bp that is interrupted by eight introns. The EgAFFP deduced amino acid sequence is about 40% homologous to those of several members of the gelsolin family, such as Physarum polycephalum fragmin, Dictyostelium discoideum severin, and Lumbricus terrestris actin modulator. As do other proteins of the same family, EgAFFP presents three repeated domains, each one characterized by internal conserved amino acid motifs. Assays with fluorescence-labeled actin showed that the full-length recombinant EgAFFP effectively binds actin monomers in both a calcium-dependent and calcium-independent manner and also presents actin nucleating and severing activities.

Actins↗
PubMed2001

Comparative analysis of the 14-3-3 gene and its expression in Echinococcus granulosus and Echinococcus multilocularis metacestodes.

It was suggested that the unlimited proliferative capacity of the Echinococcus multilocularis metacestode may be related to overproduction of the 14-3-3 protein. As is known, the proliferative capacities of E. granulosus and E. multilocularis metacestodes are very different. By comparing the expression levels of the 14-3-3 gene between in vitro-obtained E. granulosus and E. multilocularis metacestodes, we were able to provide experimental evidence of the potential relation between 14-3-3 over-expression and tumour-like growth in E. multilocularis metacestodes. RT-PCR and Northern blot experiments indicated that 14-3-3 expression level is about 4-fold higher in the E. multilocularis metacestode. This differential expression was confirmed both by immunoblotting and immunocytochemistry experiments, which allowed detection of the protein in the cyst wall from E. multilocularis but not in the cyst wall from E. granulosus. The alignment of the Echinococcus 14-3-3 cDNA sequence with known 14-3-3 isoforms from other organisms, grouped the parasite sequence into the tumour growth-related isoforms. The known relation between over-expression of some 14-3-3 isoforms and tumour-related processes, together with the present results, suggest that the Echinococcus 14-3-3 protein could be one of the molecules responsible for the differences between E. granulosus and E. multilocularis metacestode growth behaviour.

14-3-3 Proteins↗
PubMed2000

Comparative analysis of two different subunits of antigen B from Echinococcus granulosus: gene sequences, expression in Escherichia coli and serological evaluation.

Two different Echinococcus granulosus antigen B subunits (AgB8/1 and AgB8/2) were characterized and the structure of the genes encoding these two proteins were compared. DNA sequences were expressed in Escherichia coli and the antigens' diagnostic value was then assessed. The genomic sequence of AgB8/1 has a 92 bp intron in the position corresponding to amino acid 16; the AgB8/2 genomic sequence presents a 68 bp intron in the position corresponding to amino acid 20. Both introns are located between the putative N-terminal hydrophobic sequence and the secreted peptide. A comparison between the AgB8/1 and AgB8/2 nucleotide sequences showed a 53.5% identity among exons and a 50% identity between introns. According to the molecular diversity analysis, the elapsed time since both genes shared a common ancestor would be around 4.2x10(7) years. When the native AgB and the two recombinant antigens (rAgB8/1 and rAgB8/2) were tested in an anti-IgG ELISA, the sensitivity of the native antigen B was 77.41% and its specificity was 81.9%, while rAgB8/1 showed 54.84% of sensitivity and 80.17% of specificity and rAg138/2 had an 83.87% sensitivity and a 98.28% specificity. Statistical analysis confirms that rAgB8/2 has a better performance than rAgB8/1 and native AgB in ELISA.

Amino Acid Sequence↗
PubMed2000

The 14-3-3 protein is secreted by the adult worm of Echinococcus granulosus.

The 14-3-3 protein, already described in the metacestode of Echinococcus multilocularis, has been characterized in the Echinococcus granulosus adult worm. Immunolocalization studies show the presence of the 14-3-3 protein in the periphery of testes and externally associated with the apical rostellum and adjacent worm tegument. The alcian blue staining in consecutive parasite sections gave similar reactivity patterns, suggesting that the 14-3-3 protein is produced and secreted by rostellar glands. Immunoblot analysis showed the presence of the 14-3-3 protein in somatic and excretory-secretory worm products with higher and smaller apparent molecular masses, respectively, than those detected in E. multilocularis or E. granulosus metacestode tissues. Conversely, the 14-3-3 protein was not detected in metacestode secretory products. Detection of anti-E. granulosus 14-3-3 reactivity in sera of experimentally infected dogs was achieved at early stages of infection. Specific antibody titres decreased during the course of infection. The possible origin and functions of the 14-3-3 protein produced by the adult worm are discussed.

14-3-3 Proteins↗
PubMed2000

Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line.

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines.

3T3 Cells↗