Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae)

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.

Polymerase chain reaction and restriction fragment length polymorphism of cytocrome oxidase subunit I used for differentiation of Brazilian Biomphalaria species intermediate host of Schistosoma mansoni

The intermediate hosts of Schistosoma mansoni, in Brazil, Biomphalaria glabrata, B. tenagophila and B. straminea, were identified by restriction fragment length polymorphism analysis of the mitochondrial gene cytochrome oxidase I (COI). We performed digestions with two enzymes (AluI and RsaI), previously selected, based on sequences available in Genbank. The profiles obtained with RsaI showed to be the most informative once they were polymorphic patterns, corroborating with much morphological data. In addition, we performed COI digestion of B. straminea snails from Uruguay and Argentina

Identification of Biomphalaria havanensis and Biomphalaria obstructa populations from Cuba using polymerase chain reaction and restriction fragment length polymorphism of the ribosomal RNA intergenic spacer

In Cuba, several Biomphalaria species have been reported such as B. orbignyi, B. schrammi, B. helophila, B. havanensis and B. peregrina; only the latter three are considered as potential hosts of Schistosoma mansoni. The specific identification of Biomphalaria species is based on anatomical and morphological characters of genital organs and shells. The correct identification of these snails is complicated by the high variation in these characters, similarity among species and in some cases by the small size of the snails. In this paper, we reported the classical morphological identification, the use of PCR and RFLP analysis of the internal transcribed spacer region of the ribosomal RNA genes for molecular identification of seven snail populations from different localities in Cuba. Using morphological and molecular analysis, we showed that among the studied Cuban Biomphalaria populations only B. havanensis and B. obstructa species were found

Genetic variability in Brazilian populations of Biomphalaria straminea complex detected by simple sequence repeat anchored polymerase chain reaction amplification

Biomphalaria glabrata, B. tenagophila and B. straminea are intermediate hosts of Schistosoma mansoni, in Brazil. The latter is of epidemiological importance in the northwest of Brazil and, due to morphological similarities, has been grouped with B. intermedia and B. kuhniana in a complex named B. straminea. In the current work, we have standardized the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) technique, using the primers (CA)8RY and K7, to study the genetic variability of these species. The similarity level was calculated using the Dice coefficient and genetic distance using the Nei and Li coefficient. The trees were obtained by the UPGMA and neighbor-joining methods. We have observed that the most related individuals belong to the same species and locality and that individuals from different localities, but of the same species, present clear heterogeneity. The trees generated using both methods showed similar topologies. The SSR-PCR technique was shown to be very efficient in intrapopulational and intraspecific studies of the B. straminea complex snails

Frequency of the CCR5delta32 allele in Brazilians: a study in colorectal cancer and in HTLV-I infection

A observaçäo de que indivíduos homozigotos para uma deleçäo de 32 pares de base no gene que codifica para o receptor 5 de cc-quimiocinas apresentam um menor risco de contrair a infecçäo por HIV-1 levou à investigaçäo da freqüência deste polimorfismo em várias populaçöes mundiais. É importante investigar se o CCR5delta32 é um fator a ser considerado na epidemiologia do HIV em populaçöes individuais. Com estes pressupostos em mente nós estabelecemos a freqüência do CCR5delta32 em uma grande amostra (907 indivíduos näo-relacionados) da populaçäo urbana do sudeste brasileiro, estratificada da seguinte maneira: 322 indivíduos sadios, 354 pacientes com câncer colorretal e 229 doadores de sangue. Os três grupos apresentaram essencialmente a mesma freqüência alélica de CCR5delta32 e a comparaçäo par-a-par näo revelou diferenças significativas. Assim, os nossos resultados podem ser agrupados para fornecer uma estimativa confiável de 0,053 ñ 0,005 da freqüência alélica de CCR5delta32. Os doadores de sangue compreendiam 50 indivíduos soronegativos para HTLV-I, 115 indivíduos assintomáticos por ELISA mas com resultados indeterminados em Western blot, 49 indivíduos soropositivos para HTLV-I mas assintomáticos e 15 indivíduos soropositivos para HTLV-I sintomáticos com mielopatia. Foi observado um sugestivo gradiente decrescente da freqüência alélica de CCR5delta32 nestas categorias. Entretanto, quando aplicamos o teste exato de Fisher, näo emergiram diferenças significativas. Para uma melhor avaliaçäo da influência do alelo CCR5delta32 na probabilidade de infectar-se com HTLV-I ou de desenvolver doença clínica seräo necessários estudos com um maior número de doadores de sangue.

Detection of microsatellite instability but not truncating APC mutations in gastric adenocarcinomas in Brazilian patients

Um papel crucial para o gene da polipose cólica adenomatosa (APC) na carcinogênese colo-retal já está estabelecido, mas sua participaçäo nos tumores gástricos permanece controversa. Mutaçöes no APC foram detectadas com uma freqüência relativamente alta em tumores gástricos em pacientes japoneses, mas tais mutaçöes foram relatadas como sendo muito raras em britânicos e em pacientes da regiäo norte-central da Itália. Nós relatamos aqui a análise de 40 adenocarcinomas gástricos esporádicos primários e 35 adenocarcinomas cólicos esporádicos primários (de pacientes residentes em Säo Paulo, Brasil), quanto a mutaçöes no gene APC entre os códons 686 e 1693, usando o teste da proteína truncada (PTT). Embora 19 mutaçöes que levem a formaçäo de uma proteína truncada tenham sido detectadas em 35 adenocarcinomas do colo (54,2 por cento), näo se encontrou nenhuma nos adenocarcinomas gástricos. Como um controle interno, as amostras tumorais foram também avaliadas quanto à presença de alteraçöes de microssatélites, que também säo características comuns a ambos os tipos de tumores. Encontrou-se instabilidade de microssatélites em uma amostra de tumor cólico e em 7 de tumores gástricos. Este fato sugere que, no que diz respeito a mutaçöes do APC, os adenocarcinomas gástricos de pacientes brasileiros säo semelhantes aos que ocorrem na Europa, e confirmam a diferença fundamental que existe tanto entre os carcinomas gástricos que ocorrem em diferentes regiöes geográficas como entre a etiologia molecular de adenocarcinomas gástricos e colo-retais que ocorrem em Säo Paulo, Brasil.

Further studies on the molecular systematics of Biomphalaria snails from Brazil

The polymerase chain reaction and restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rRNA gene, using the enzyme DdeI were used for the molecular identification of ten species and one subspecies of Brazilian Biomphalaria. Emphasis is given to the analysis of B. oligoza, B. schrammi and B. amazonica. The RFLP profiles obtained using this enzyme were highly distinctive for the majority of the species and exhibited low levels of intraspecific polymorphism among specimens from different regions of Brazil. However, B. peregrina and B. oligoza presented very similar profiles that complicated their identification at the molecular level and suggested a very close genetic similarity between the two species. Others enzymes including HaeIII, HpaII, AluI and MnlI were tested for their ability to differentiate these species. For B. amazonica three variant profiles produced with DdeI were observed. The study demonstrated that the ITS contains useful genetic markers for the identification of these snails.

Nova variante do gene do prionio em uma familia psicotica / New variant of the prion protein gene in a psychotic family

As doencas de prionio sao caracterizadas pelo acumulo no cerebro da PrP-sc, uma proteina prionica infectante e protease resistente. A Prp-sc difere da PrP-c, de funcao desconhecida, apenas em termos conformacionais. As doencas humanas de prionio conhecidas sao Kuru, Gertsman Straussler (GSS), Insonia Familiar Fatal(IFF) e doencas de Creutzfeldt-Jakob (CJD)...

Genetic variability and identification of the intermediate snail hosts of Schistosoma mansoni

Studies based on shell or reproductive organ morphology and genetic considerations suggest extensive intraspecific variation in Biomphalaria snails. The high variability at the morphological and genetic levels, as well as the small size of some specimens and similarities between species complicate the correct identification of these snails. Here we review our work using methods based on polymerase chain reaction (PCR) amplification for analysis of genetic variation and identification of Biomphalaria snails from Brazil, Argentina, Uruguay and Paraguay. Arbitrarily primed-PCR revealed that the genome of B. glabrata exhibits a remarkable degree of intraspecific polymorphism. Low stringency-PCR using primers for 18S rRNA permited the identification of B. glabrata, B. tenagophila and B. occidentalis. The study of individuals obtained from geographically distinct populations exhibits significant intraspecific DNA polymorphism, however specimens from the same species, exhibit some species specific LSPs. We also showed that PCR-restriction fragment of length polymorphism of the internal transcribed spacer region of Biomphlaria rDNA, using Ddel permits the differentiation of the three intermediate hosts of Schistosoma mansoni. The molecular biological techniques used in our studies are very useful for the generation of new knowledge concerning the systematics and population genetics of Biomphalaria snails.

Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae) determined by a polymerase chain reaction-restriction fragment length polymorphism

The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath: deferens vans and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment length polymorphism technique was used for molecular identification of these molluscs. This techniques is based on the amplification of the internal transcribed spacer regions ITS1 and ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mn I, Hae III, RSA I, Hpa II and AluI. The restriction patterns obtained with Dde I presented the best profile for separation of the four species of Biomphalaria. The profiles obtained will all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns.

A genética molecular de sarcomas de partes moles e sua aplicação à clínica / The molecular genetics of soft tissue sarcomas and its clinical application

Sarcomas de partes moles, como outros tumores humanos, são causados por mutações em genes supressores de tumores e oncogenes. Entre eles alguns, como p53 por exemplo, são freqüentemente detectados na forma mutatada em sarcomas e outros tumores humanos. De outro lado, há várias mutações altamente específicas para determinados sarcomas que podem servir como marcadores diagnósticos de alto valor. Exemplos de mutações de potencial diagnóstico incluem a translocação entre PAX3 e FKHR em rabdomiossarcoma alveolar, a translocação entre EWS e FLI-1 em tumores neuroectodérmicos periféricos primitivos e EWS e ATF-1 e sarcomas de células claras. Todas essas translocações podem ser detectadas por metodologias moleculares e que podem constituir um componente importante de repertório diagnóstico futuro.

Frequencia da mutacao delta-mtDNA 4977 e acumulo de placas neuriticas e emaranhados neurofibrilares no giro para-hipocampal humano, no envelhecimento normal e na doenca de Alzheimer / Delta mtDNA 4977 mutational frequence and neuritic plaques and neurofibrillary tangles in human parahippocampal gyrus, in normal aging and Alzheimer's disease

A presenca da mutacao-delecao mtDNA no giro para-hipocampal humano foi investigada em 95 pacientes autopsiados de tres series de origens geograficas distintas, Alemanha, Brasil e Japao, incluindo 70 pacientes sem doencas neuropsiquiatricas e 25 pacientes portadores da doenca de Alzheimer. Somente a serie alema, caracterizada por maiores proporcoes de neuronios medios e grandes, e alta incidencia de placas neuriticas e emaranhados neurofibrilares no giro para-hipocampal, apresentou a delta-mtDNA em niveis detectaveis pela reacao de cadeia da polimerase (PCR). As series brasileira e japonesa, caracterizadas por menores proporcoes de neuronios medios e grandes e baixa incidencia de placas e emaranhados, nao apresentaram niveis detectaveis da alfa-mtDNA. A frequencia f da alfa-mtDNA foi tres vezes menor no grupo de pacientes portadores da doenca de Alzheimer (f=0,12) que no grupo controle (f=0,37) (p=0,03)...

Evidentiation of paramyosin (Sm-97) as a modulating antigen on granulomatous hypersensitivity to Schistosoma mansoni eggs

A Schistosoma mansoni adult worm anionic fraction (PIII) has previously been shown to protect mice against challenge infection and to reduce pulmonary and hepatic granulomatous hypersensitivity. Serum from PIII-immunized rabbit was used to screen a gt11 cDNA library from S. mansoni adult worm in order to identify antigens capable of modulating granulomatous hypersensitivity. We obtained four clones with 400 (Sm-III.11), 900 (Sm-III.16), 1100 (Sm-III.10) and 1300 (Sm-III.12) bp of length. All clone-specific antibodies were able to recognize most of the PIII components. The sequence analysis showed that these clones presented high homology with S. mansoni paramyosin (Sm-97). These findings describe a new function to this antigen with an important role in modulation of granulomatous hypersensitivity to S. mansoni eggs.

Contrasting genomic variability between clones from field isolates and laboratory populations of Schistosoma mansoni

The extent of genomic variability of clones of Schistosoma mansoni obtained from field isolates was compared with that of strains that have been laboratory maintained. Analysis was undertaken using ramdomly amplified polymorphic DNAs (RAPDs) generated with three primers. Phenograms showing the similarity among the clones were constructed. The data showed that while the laboratory strain is highly homogeneous the clones derived from the field populations were highly variable with 43 per cent of RAPDs exhibiting polymorphisms among 23 clones. Clones isolated from the same infected individual were always more closely grouped than clones from different individuals. The data clearly demonstrated that earlier analysis of the genomic variability in S. mansoni have underestimated this phenomenon due to the failure to examine field isolates.

Specific identification of Biomphalaria tenagophila and Biomphalaria occidentalis populations by the low stringency polymerase chain reaction

Althrough Biomphalaria occidentalis and B. tenagophila are indisguishable on the basis of shell morphology and the majority of their genital organs, only the latter is susceptible to infection with Schistosoma mansoni. Thus, the identification of these species is fundamental to epidemiological studies of schistosomiasis. Here we describe a simple and rapid method for differentiating B. tenagophila from B. occidentalis based on low stringency polymere chain reaction and using a pair of primers specific for the amplification of the 18S rRNA gene. Analysis of the low stringency product profiles of populations of these snails from different geographical regions confirmed this approach as being applicable to the identification of B. tenagophila and B. occidentalis in cases where classical morphology is inconclusive.

A low stringency polymerase chain reaction approach to the identification of Biomphalaria glabrata and B. tenagophila, intermediate snail hosts of Schistosoma mansoni in Brazil

The low stringency polymerase chain reaction (LS-PCR) with a pair of specific primers for the amplification of the 18S rRNA gene was evaluted as a means of differentiating between the two Schistosoma mansoni intermediate host species in Brazil: Biomphalaria glabrata and B. tenagophila. Individual snails obtained from different states of Brazil were used and the amplification patterns obtained showed a high degree of gentic variability in these species. Nevertheless, 4 and 3 clearly defined specific diagnostic bands was observed in individuals from B. glabrata and B. tenagophila respectively. The detection of snail specific diagnostic bands suggests the possibility of reliable species differentiation at DNA level using LS-PCR.

Suscetibilidade aos agentes quimioterápicos de isolados de Schistosoma mansoni oriundos de pacientes tratados com oxamniquine e praziquantel e näo curados / Susceptibility to the chemotherapeutical compounds of Schistosoma mansoni from patients treated with oxamniquine and praziquantel and not cured ones

Ten inhabitants of Itaquara, Bahia, Brazil treated with oxamniquine and subsequently praziquantel were not cured. Schistosoma mansoni isolates derived from these patients were studied. Snails were infected with miracidia derived from the feces of these patients and the cercariae produced used to infect albino mice. The animals were then treated with a single oral dose of oxamniquine (25, 50 and 100mg/kg) or praziquantel (100, 200 and 400 mg/kg). The response to chemotherapy was significantly different in some of the isolates although it was not possible to characterize any of them as resistant. In addition, DNA analysis of the isolates by means of ®Random Amplified Polymorphic DNA® indicated a low degree of variability as compared with a laboratory strain, LE. Thus, it was not possible to characterize these organisms at a genetic level as a distinct strain.