Estudi de la diversitat de picoflagel·lats heterotròfics a la zona de Blanes mitjançant tècniques moleculars i de cultiu

S’ha estudiat la diversitat de picoflagel—·lats heterotròfics, d’aigües del nord-oest del mar Mediterrani, mitjançant DGGE , biblioteques de clons i enriquiments. S’ha trobat que la diversitat és alta (índex Shannon 2,227), com en d’altres estudis, però que disminueix en afegir matèria orgànica (extracte de llevat o d’arròs), amb índex de Shannon de fins a 0,731. Per una entrada d’aigua continental rica en nutrients, s’han desenvolupat millor les crisofícies en detriment d’altres organismes habituals al medi marí, com els MAST.

Estudo morfológico comparativo de ovos e ninfas de Triatoma maculata (Erichson, 1848) e Triatoma pseudomaculata Corrêa & Espínola, 1964 (Hemiptera, Reduviidae, Triatominae)

Utilizando a microscopia ótica (MO) e eletrônica de varredura (MEV) procurou-se fornecer dados a taxionomia através da estrutura dos ovos e morfologia das ninfas, destes vetores da doença de Chagas. Os ovos em MO apresentam a superfície exocorial do opérculo e do corpo dividida em áreas poligonais com ornamentação própria; em T. maculata o exocório do corpo tem áreas indefinidas. Em MEV o exocório dos opérculos apresenta áreas poligonais de superfície estofada com pequena sulcos irregulares e perfurações distribuídas aleatoriamente nas duas espécies. O exocório do corpo apresenta: em T. maculata áreas acolchoadas com perfurações mais numerosas nos bordos, visualizando-se a borda corial, goteira espermática, aerópilas e micrópilas; em T. pseudomaculata as áreas são planas com numerosas perfurações. Nas ninfas o sulco estridulatório e o rostro apresentam diferenças significativas. O sulco estridulatórios em MO possibilitou diferenciar ninfas de 1º, 2º e 3º estádios, os 4º e 5º estádios apresentam-se semelhantes. Em MEV a diferenciação é acentuada. O rostro em MO apresenta pilosidade característica a partir do 3º estádio. T. maculata apresenta pêlos curtos e esparsos no 1º e 2º artículos e longos e numerosos no 3º, em T. pseudo-maculata semelhantes, porém mais curtos no 3º artículo.

Estudo pareado da cardiopatia chagásica no Rio Grande do Sul, Brasil

São estudados 666 pares de indivíduos, sendo 344 masculinos e 322 femininos, com sorologia positiva/negativa para doença de Chagas, obtidos em inquérito sorológico-eletrocardiográfico entre populações rurais não selecionadas de 17 municípios do sul Rio Grande do Sul, Brasil. Na análise dos eletrocardiogramas foram consideradas só as alterações sugestivas de cardiopatia chagásica: bloqueio átrio-ventricular de 1º, e 2º e 3º, BCRD isolado ou associado ao HBAE, HBAE isolado, extrasístoles ventriculares freqüentes e/ou polifocais e/ou bigeminadas e trigeminadas; alterações de ST e T; zonas eletricamente inativas. Com este critério apresentaram eletrocardiogramas alterados 201 pessoas soropositivas (30,2%) e 66 soronegativas (9,9%). O gradiente de 20,3%, sendo 21,5% nos homens e 18,9% nas mulheres, mostrou-se significativo ao nível de p < 0,001.

Tripomastigota muito largo como padrão morfológico de cepas do Trypanosoma cruzi do extremo Sul do Brasil?

Foi estudado, em camundongos, o padrão morfológico-evolutivo de tripomastigotas sanguíneos de cinco cepas silvestres de Trypanosoma cruzi de Santa Catarina. Os resultados mostraram predomínio da forma muito larga ( > 70%) ao longo de toda a fase aguda de infecção em quatro cepas de média virulência. Com uma cepa de elevada virulência, a forma delgada foi prevalente no período inicial de infecção seguido de maior ocorrência da muito larga. Tendo em vista que a maioria das cepas de T. cruzi que possuem predomínio da forma muito larga foram isoladas no sul do Brasil (Rio Grande do Sul e Santa Catarina) e que existem evidências de diferentes comportamentos biológicos entre estes parasitas sangüíneos, os autores sugerem a investigação de possíveis correlações dos padrões morfológicos com os aspectos epidemiológicos e clínicos da doença de Chagas.

Influência da temperatura na biologia de triatomíneos: IX. Rhodnius nasutus Stal, 1859 (Hemiptera, Reduviidae)

Estudou-se a influência da temperatura na biologia de Rhodnius nasutus Stal, 1859 (Hemiptera, Reduviidae), tendo em vista a sua criação em grande escala, em laboratório, para serem utilizados no xenodiagnóstico e fornecer informações que subsidien as ações de controle. Os experimentos realizaram-se em duas câmaras climatizadas, a 25 ± 0,5ºC e 3º ± 1ºC, com umidade relativa de 70 ± 5% e fotoperíodo de 12 horas. Apresentam-se os dados relativos ao ciclo evolutivo, pré-postura, fecundidade, fertilidade, sobrevivência ao jejum e quantidade de sangue ingerido. A duração média do ciclo evolutivo foi de 145,7 dias a 25ºC e de 114,2 dias, a 30ºC. A quantidade de sangue ingerido para completar o ciclo evolutivo foi de 304,3mg e 350,3 mg, a 25º e 30ºC, respectivamente. A sobrevivência ao jejum absoluto foi significativamente maior a 25º do que a 30ºC, ao nível de 1%.

Genetic and genomic analysis modeling of germline c-MYC overexpression and cancer susceptibility

Background: Germline genetic variation is associated with the differential expression of many human genes. The phenotypic effects of this type of variation may be important when considering susceptibility to common genetic diseases. Three regions at 8q24 have recently been identified to independently confer risk of prostate cancer. Variation at 8q24 has also recently been associated with risk of breast and colorectal cancer. However, none of the risk variants map at or relatively close to known genes, with c-MYC mapping a few hundred kilobases distally. Results: This study identifies cis-regulators of germline c-MYC expression in immortalized lymphocytes of HapMap individuals. Quantitative analysis of c-MYC expression in normal prostate tissues suggests an association between overexpression and variants in Region 1 of prostate cancer risk. Somatic c-MYC overexpression correlates with prostate cancer progression and more aggressive tumor forms, which was also a pathological variable associated with Region 1. Expression profiling analysis and modeling of transcriptional regulatory networks predicts a functional association between MYC and the prostate tumor suppressor KLF6. Analysis of MYC/Myc-driven cell transformation and tumorigenesis substantiates a model in which MYC overexpression promotes transformation by down-regulating KLF6. In this model, a feedback loop through E-cadherin down-regulation causes further transactivation of c-MYC.Conclusion: This study proposes that variation at putative 8q24 cis-regulator(s) of transcription can significantly alter germline c-MYC expression levels and, thus, contribute to prostate cancer susceptibility by down-regulating the prostate tumor suppressor KLF6 gene.

Comparative analysis of cancer genes in the human and chimpanzee genomes

Background: Cancer is a major medical problem in modern societies. However, the incidence of this disease in non-human primates is very low. To study whether genetic differences between human and chimpanzee could contribute to their distinct cancer susceptibility, we have examined in the chimpanzee genome the orthologous genes of a set of 333 human cancer genes. Results: This analysis has revealed that all examined human cancer genes are present in chimpanzee, contain intact open reading frames and show a high degree of conservation between both species. However, detailed analysis of this set of genes has shown some differences in genes of special relevance for human cancer. Thus, the chimpanzee gene encoding p53 contains a Pro residue at codon 72, while this codon is polymorphic in humans and can code for Arg or Pro, generating isoforms with different ability to induce apoptosis or interact with p73. Moreover, sequencing of the BRCA1 gene has shown an 8 Kb deletion in the chimpanzee sequence that prematurely truncates the co-regulated NBR2 gene. Conclusion: These data suggest that small differences in cancer genes, as those found in tumor suppressor genes, might influence the differences in cancer susceptibility between human and chimpanzee. Nevertheless, further analysis will be required to determine the exact contribution of the genetic changes identified in this study to the different cancer incidence in non-human primates.

Structural and functional properties of genes involved in human cancer

Background: One of the main goals of cancer genetics is to identify the causative elements at the molecular level leading to cancer.Results: We have conducted an analysis of a set of genes known to be involved in cancer in order to unveil their unique features that can assist towards the identification of new candidate cancer genes. Conclusion: We have detected key patterns in this group of genes in terms of the molecular function or the biological process in which they are involved as well as sequence properties. Based on these features we have developed an accurate Bayesian classification model with which human genes have been scored for their likelihood of involvement in cancer.

Análise das relações taxonômicas e sistemáticas entre espécies de triatomíneos (Hemiptera, Reduviidae) de colônias mantidas pelo Serviço Especial de Saúde de Araraquara, inferida de seqüências do 16S rDNA mitocondrial

Foram analisadas seqüências de nucleotídeos do gene 16S do rDNA mitocondrial em 14 populações de triatomíneos mantidos em colônias no insetário SESA de Araraquara- SP, comparando-as com seqüências do mesmo gene disponíveis no GenBank. Os fragmentos variaram de 311 a 317 pb com baixa variação intra-específica entre as distâncias genéticas (0% a 0,6%), exceto para os relacionamentos entre espécimes de Triatoma sordida (1%) e espécimes de T. brasiliensis (1,3%) atribuídos a populações geográficas diferentes. A parafilia de Rhodniini e do gênero Panstrongylus foi evidenciada pelas analises, confirmando resultados anteriores entre estes e os estreitos relacionamentos de R. prolixus com R. robustus e de T. infestans e T. platensis. O relacionamento entre T. maculata e T. pseudomaculata não foi solucionado, uma vez que, esses táxons apareceram tanto em monofilia quanto em parafilia: T. pseudomaculata (SESA) está agrupado com T. maculata (seqüência do GenBank) e associados a T . brasiliensis (SESA), enquanto T. maculata (SESA) aparece agrupado com T. pseudomaculata do SESA e do GenBank. Os resultados evidenciam a utilidade do gene 16S como marcador de espécies de triatomíneos e sua importância em questões de sistemática e taxonomia. Há necessidade de novos estudos envolvendo outros marcadores associados a caracteres sistemáticos clássicos de morfologia, ecologia e comportamento para decisões sistemáticas adequadas uma vez, que teriam impacto não apenas sistemático mas, para as estratégias de controle.

Efeito de extratos de plantas do Cerrado em Dipetalogaster maxima (Uhler) (Hemiptera, Reduviidae)

A transmissão da doença de Chagas ocorre, principalmente, por meio de fezes de hemípteros hematófagos (Triatominae), os quais ingerem o Trypanosoma cruzi ao se alimentarem do sangue de pessoas ou outros mamíferos infectados. Para o controle dos triatomíneos, os piretróides são os principais inseticidas utilizados. Entretanto, algumas populações de insetos demonstraram resistência a determinados piretróides, indicando a necessidade do desenvolvimento de novos inseticidas eficazes no controle desses vetores. Assim, foi avaliada a atividade inseticida de 83 extratos vegetais, pertencentes a 35 espécies diferentes, em ninfas do primeiro estádio de Dipetalogaster maxima (Uhler, 1894) (Hemiptera: Reduviidae), triatomíneo encontrado no México. Para o teste tópico, foram aplicados 50 ìg de cada extrato nos tergitos abdominais de dez ninfas, em duplicata. Como controles, foram utilizados insetos tratados com etanol, acetona ou sem nenhum tipo de tratamento. Os triatomíneos foram observados durante 28 dias. Nenhum extrato apresentou atividade inseticida significativa, entretanto, o extrato hexânico do fruto e o etanólico da casca do caule de Simarouba versicolor (Simaroubaceae) inibiram a taxa de ecdise em D. maxima (40% e 25%, respectivamente). Sugere-se que estes extratos sejam quimicamente investigados e monitorados por ensaios biológicos a fim de determinar os componentes, para que estes possam ser utilizados como modelos moleculares ou como compostos biorracionais nos programas de controle de insetos.

Array-CGH in patients with a clinical diagnosis of Kabuki syndrome: identification of two cases with terminal 2q37 deletion and no other recurrent rearrangement

Background: Kabuki syndrome (KS) is a multiple congenital anomaly syndrome characterized by specific facial features, mild to moderate mental retardation, postnatal growth delay, skeletal abnormalities, and unusual dermatoglyphic patterns with prominent fingertip pads. A 3.5 Mb duplication at 8p23.1-p22 was once reported as a specific alteration in KS but has not been confirmed in other patients. The molecular basis of KS remains unknown. Methods: We have studied 16 Spanish patients with a clinical diagnosis of KS or KS-like to search for genomic imbalances using genome-wide array technologies. All putative rearrangements were confirmed by FISH, microsatellite markers and/or MLPA assays, which also determined whether the imbalance was de novo or inherited. Results: No duplication at 8p23.1-p22 was observed in our patients. We detected complex rearrangements involving 2q in two patients with Kabuki-like features: 1) a de novo inverted duplication of 11 Mb with a 4.5 Mb terminal deletion, and 2) a de novo 7.2 Mb-terminal deletion in a patient with an additional de novo 0.5 Mb interstitial deletion in 16p. Additional copy number variations (CNV), either inherited or reported in normal controls, were identified and interpreted as polymorphic variants. No specific CNV was significantly increased in the KS group. Conclusion: Our results further confirmed that genomic duplications of 8p23 region are not a common cause of KS and failed to detect other recurrent rearrangement causing this disorder. The detection of two patients with 2q37 deletions suggests that there is a phenotypic overlap between the two conditions, and screening this region in the Kabuki-like patients should be considered.

Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene.

We report a Spanish family with autosomal-dominant non-neuropathic hereditary amyloidosis with a unique hepatic presentation and death from liver failure, usually by the sixth decade. The disease is caused by a previously unreported deletion/insertion mutation in exon 4 of the apolipoprotein AI (apoAI) gene encoding loss of residues 60-71 of normal mature apoAI and insertion at that position of two new residues, ValThr. Affected individuals are heterozygous for this mutation and have both normal apoAI and variant molecules bearing one extra positive charge, as predicted from the DNA sequence. The amyloid fibrils are composed exclusively of NH2-terminal fragments of the variant, ending mainly at positions corresponding to residues 83 and 92 in the mature wild-type sequence. Amyloid fibrils derived from the other three known amyloidogenic apoAI variants are also composed of similar NH2-terminal fragments. All known amyloidogenic apoAI variants carry one extra positive charge in this region, suggesting that it may be responsible for their enhanced amyloidogenicity. In addition to causing a new phenotype, this is the first deletion mutation to be described in association with hereditary amyloidosis and it significantly extends the value of the apoAI model for investigation of molecular mechanisms of amyloid fibrillogenesis.

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. Conclusions We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data.

Characterization of Arabidopsis FPS isozymes and FPS gene expression analysis provide insight into the biosynthesis of isoprenoid precursors in seeds

Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP) synthase (FPS), the prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Both enzymes were expressed in E. coli, purified and biochemically characterized. Despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 was found to be more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Homology modelling for FPS1S and FPS2 and analysis of the amino acid differences between the two enzymes revealed an increase in surface polarity and a greater capacity to form surface salt bridges of FPS2 compared to FPS1S. These factors most likely account for the enhanced thermostability of FPS2. Expression analysis of FPS::GUS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the Arabidopsis fps2 knockout mutant seed phenotypes demonstrated that under normal conditions FPS1S and FPS2 are functionally interchangeable. A putative role for FPS2 in maintaining seed germination capacity under adverse environmental conditions is discussed.

SSCP-polymorphism in intron 12 of the CFTR gene recognized by BclI

Source/Description: SSCP analysis of intron 12 of the CFTR gene from PCR products showed an extra band in several DNA samples. Sequencing of the additional fragment extra band revealed a T- A change in the position 1898 + 152 of CFTR (Fig. 1). The change is a polymorphism which can be identified by SSCP or by BclI digestion...