A Comparative Study of the Outer Membrane Proteome from an Atypical and a Typical Enteropathogenic Escherichia coli

This study compared the proteomic profile of outer membrane proteins (OMPs) from one strain of atypical enteropathogenic Escherichia coli (aEPEC) and one of typical EPEC (tEPEC). The OMPs fractions were obtained using sarcosine extraction, and analyzed by one- and two-dimensional gel electrophoresis (1DE and 2DE, respectively). The 1DE OMPs analysis of typical and atypical EPEC evidenced similar patterns; however, the 2DE OMP profile from the aEPEC revealed more protein spots in the 40- to 70-kDa region. 2DE image analysis identified 159 protein spots in both strains whereas 53 protein spots were observed only in tEPEC and 128 were observed only in aEPEC. Remarkably, 41.5% of aEPEC spots showed higher levels of expression compared to tEPEC, some of which with two, others four or even five times more. Twenty-four selected spots were identified using MALDI-TOF mass spectrometry and they corresponded to proteins involved in cell structure and metabolism, as well as in gene regulation. Some of these proteins showed similarity with proteins identified in other E. coli pathotypes. Besides, the differential expression of some proteins in aEPEC may suggest that it could be related to their features that ascertain the adaptation to distinct environments and the worldwide spread distribution of these pathogens

Comparative expression analysis in mature gonads, liver and brain of turbot (Scophthalmus maximus) by cDNA-AFLPS

Turbot is one of the most important farmed fish in Europe. This species exhibits a considerable sexual dimorphism in growth and sexual maturity that makes the all-female production recommended for turbot farming. Our knowledge about the genetic basis of sex determination and the molecular regulation of gonad differentiation in this species is still limited. Our goal was to identify and compare gene expression and functions between testes and ovaries in adults in order to ascertain the relationship between the genes that could be involved in the gonad differentiation or related to the sex determination system. The identification of differentially expressed sex related genes is an initial step towards understanding the molecular mechanisms of gonad differentiation. For this, we carried out a transcriptome analysis based on cDNA-AFLP technique which allowed us to obtain an initial frame on sex-specific gene expression that will facilitate further analysis especially along the critical gonad differentiating period. With the aim of widening the study on sex-biased gene expression we reproduced the same experiments in two somatic tissues: liver and brain. We have selected the liver because it is the most analyzed one regarding sexual dimorphic gene expression and due to its importance in steroid hormones metabolism and the brain because the functional relationship between brain and gonad is documented. We found slight but important differences between sexes which deserve further investigationThis research work was financially supported by the Xunta of Galicia (07MMA004200PR) to A. Viñas. X. Taboada was supported by a fellowship from the European Social Fund and Consellería de Educación e Ordenación Universitaria- Xunta de Galicia (Spain)S

Unravelling the mechanisms of resistance to imatinib mesylate in chronic myeloid leukemia: a proteomic approach

Imatinib mesylate is a potent inhibitor of the Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. The aim of this study was to obtain further insights into the Bcr-Abl activity-independent mechanisms underlying imatinib resistance, in chronic myeloid leukemia. The imatinib-resistant KCL22R and sensitive KCL22S cells were used as experimental model. None of the already described resistance mechanisms has been detected so far in KCL22R cells; therefore additional mechanisms independent of Bcr-Abl kinase activity could be envisaged. Moreover, KCL22S cells exhibited typical features of the quiescent hematopoietic Ph+ stem cells, thereby representing a good experimental model to investigate imatinib resistance. To this aim differentially expressed proteins between KCL22S and KCL22R cells were characterized using a proteomic approach: two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Tandem Mass Spectrometry. 51 proteins were identified: 27 over-expressed and 24 under-expressed in KCL22R cells versus KCL22S cells. Bioinformatic analysis with GeneSpring and Ingenuity Pathway Analysis (IPA) softwares showed that several of these proteins were involved in the modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-kB and Ras-MAPK signaling. Since the Erk pathway has been shown to influence chemotherapeutic drug resistance of hematopoietic cells, the level of activation of Erk in KCL22R and KCL22S cells was investigated. This analysis demonstrated that continuous activation of Erk occurred in KCL22R cells as compared to sensitive cells. Interestingly, examination of the most statistically significant protein network showed that several differentially expressed proteins, between KCL22R and KCL22S cells, were directly or indirectly connected with Erk. In particular, among them, this study focused on two SH2-containing, non-receptor protein tyrosine phosphatases: Shp1 (PTPN6) and Shp2 (PTPN11). It has been shown that Shp2 positively regulates the Ras-Erk pathway and is activated by phosphorylation. This study demonstrated that the level of phosphorylation and hence of activation of Shp2 in KCL22R cells was higher than in KCL22S cells. In addition the knock-down of Shp2 expression, in combination with imatinib treatment, significantly reduced the activation of Erk 1/2 in KCL22R cells and produced a reversion of the KCL22R phenotype, suggesting that Shp2 plays a role in the Bcr-Abl activity-independent mechanisms of imatinib resistance. Interestingly this study also demonstrated that Shp1, that was found down-regulated in KCL22R cells, interacted with Shp2 and that Shp1 played a negative role in the Shp2 activation in KCL22S cells. Moreover Annexin A1 and Hsp70, belonging to the same protein network, were found down-regulated in KCL22R cells. They could also play a role in imatinib resistance by the direct or indirect interaction with Shp2. Taken together these results suggest that a reduced Shp1 expression in KCL22R cells could contribute to a continuous Shp2 activation, sustaining a Bcr-Abl activity-independent pathway of proliferation and survival to imatinib treatment. These two proteins could be used as putative biomarkers to evaluate the efficacy of imatinib treatment and to develop new combinatorial therapeutic approaches

Produção bacteriana em ambientes estuarinos: condições de incubação e factores de conversão

Mestrado em MicrobiologiaO bacterioplâncton heterotrófico desempenha um papel muito importante no ciclo biogeoquímico oceânico do carbono. A produtividade de biomassa bacteriana (PBB) converte a matéria orgância dissolvida (MOD) em matéria orgânica particulada (MOP), que fica disponível para os niveis tróficos superiores da cadeia alimentar. A PBB é determinada através da taxa de incorporação da leucina. Existem dois grandes problemas com este método: a utilização de factores de conversão para uma medição correcta da PBB e as condições de incubação sob as quais a leucina é incorporada. De modo a dar resposta a estes problemas amostras de água de duas estações de colheita (zona marinha e zona salobra) do sistema estuarino da Ria de Aveiro foram incubadas sob duas condições principais (condições de campo e de laboratório) e foram determinados factores de conversão específicos (tanto empíricos como semi-teóricos) para ambas as zonas deste sistema estuarino. Relativamente aos ensaios das condições de incubação, descobriu-se que, em condições laboratoriais (luz PAR vs. escuro), a produtividade foi superior quando as amostras são incubadas no escuro e que, para as condições de campo (in situ luz vs. in situ escuro) não foi observado nenhum padrão de variação. Nas experiências de DGGE encontrou-se, com excepção da água de superfície da zona marinha, que as várias condições de incubação não eram representativas das amostras originais. A PBB deve ser determinada nas condições de in situ luz mas, quando isto não é possível, e é necessário determinar a PBB em condições laboratoriais, deve-se realizar as incubações no escuro. Das experiências dos factores de conversão foi determinada uma diluição isotópica média e um factor de conversão semi-teórico médio de 5.07 e 7.51 Kg C mol-1 para a zona marinha e de 5.15 e 7.75 Kg C mol-1 para a zona salobra, respectivamente. Uma média de um factor de conversão empírico de 20.18 Kg C mol-1 foi obtido para a zona marinha e de 10.91 Kg C mol-1 para a zona salobra. Os resultados mostram que a PBB tem sido subestimada no sistema estuarino da Ria de Aveiro. As experiências de DGGE realizadas ao longo dos ensaios dos factores de conversão mostram que a consituição da comunidade bacteriana das amostras originais é diferente da comunidade nas amostras filtradas e diluídas utilizadas para a determinação dos factores de conversão empíricos. Também foi observado que a estrutura da comunidade bacteriana muda ao longo dos tempos de incubação e que esta é seleccionada quando se adiciona [3H]leucina às amostras. À luz das nossas descobertas concluímos que a PBB não tem sido correctamente determinada ao longo dos anos e que, para além de ser necessária a determinação de factores de conversão específicos para cada sistema, um novo problema surge quando a PBB é determinada com métodos radioactivos: a selecção da comunidade bacteriana, uma vez que a comunidade que incorpora a [3H]leucina é diferente, tornando-se óbvio que esta afecta grandemente a constituição da comunidade bacteriana.Heterotrophic bacterioplankton play a very important role in the ocean’s biogeochemical carbon cycle. Bacterial biomass production (BBP) converts dissolved organic matter (DOM) into particulate organic matter (POM), which becomes available to the higher trophic levels of the food web. BBP is assessed by leucine incorporation rates. Two major problems are found with this method: the incubation conditions under which the leucine is incorporated and the use of specific conversion factors to an accurate measurement of BBP. In order to give an answer to these problems, water samples from two sampling stations (marine and brackish zone) of the estuarine system Ria de Aveiro were incubated under two main conditions (field and laboratory conditions) and specific conversion factors (both empirical and semitheoretical) were determined in both zones of the estuarine system. Concerning the incubation conditions assays we found that, in laboratory conditions (PAR light vs. dark), BBP was superior when samples were incubated in the dark and that in the field conditions (in situ light vs. in situ dark) no pattern of variation was observed. In the DGGE experiments it was found that with the exception of surface water of marine zone, the several incubation conditions were not generally representative of the original samples. BBP must be determined in situ conditions, but when it is not possible, laboratory incubation for BPP determination must be done in dark. From the experiments of the conversion factors it was determined an average isotope dilution and an average semitheoretical conversion factor of 5.07 and 7.51 Kg C mol-1 for the marine station and of 5.15 and 7.75 Kg C mol-1 for the brackish water station, respectively. An average empirical conversion factor of 20.18 Kg C mol-1 was obtained for the marine zone and of 10.91 Kg C mol-1 was obtained for the brackish water station. The results show that BBP has been underestimated in the estuarine system of Ria de Aveiro. The DGGE experiments performed throughout the empirical conversion factor assays show that bacterial assemblages of original samples are different from the filtered and diluted samples used for empirical factor determination. It was also observed that bacterial community structure changes over the incubation periods and that this one is selected when [3H]leucine is added to the samples. In light of our findings we conclude that BBP has not been correctly measured over the years and that besides the necessity to determine the best incubation conditions and the specific conversion factors to each system, a new problem arises when BBP is determined with radioactive methods: the selection of bacterial community since community that uptakes the [3H]leucine, becoming obvious that this one affects greatly bacterial community assemblages

Molecular mechanisms of disiccation tolerance in Fucus

Intertidal algae (brown, red and green) are three understudied and independent multicellular lineages possessing related intolerant and desiccation tolerant species, making them good models for desiccation tolerance research. Recent focus on distribution of Fucus vesiculosus under climate change led us to determine the upper thermal limits of this brown algae, using physiological indicators and gene expression responses to describe the induction and thermal characteristics of the heat-shock response in diverse populations. Ambient temperatures were poor predictors of the heat-stress experienced by intertidal algae, instead the microhabitats created by the algal canopy modulated the local thermal environment and influenced the stress response. Surprisingly, in the hottest microhabitat algae appeared to be protected from thermal stress by fast and intense desiccation. Proteomic research in brown algae has recently been facilitated by genomic resources, complete genome sequencing of model species and large-scale transcriptomic resources from Fucus species, and by technical advances in work on organisms with similar interfering compounds. We tested and optimized a protein extraction protocol suitable for intertidal Fucus algae and used it to investigate differential expression of proteins in response to desiccation, both by conventional 2DE and by DIGE. No significant changes of the protein profiles were detected after desiccation or rehydration, suggesting the importance of constitutive tolerance mechanisms, minimizing the metabolic cost of gene expression, while the desiccated state provides protection against heat stress. Studies of distinct field environments (desiccation-prone or –protected), of sequential emersion stress exposure and of laboratory desiccation under controlled conditions, all failed to identify robust protein expression changes attributable to desiccation tolerance. We characterized the first extractable proteome of F. vesiculosus by LC-MS/MS identification and annotation against brown algal protein databases, with considerable success despite limited functional annotation in brown algae proteins, and the presence of multiple proteins in some spots.Na zona entre marés existem diversas macroalgas (castanhas, verdes e vermelhas), pertencentes a três linhagens multicelulares independentes e pouco estudadas, contendo espécies tolerantes e espécies intolerantes à dessecação, o que faz delas bons modelas para o estudo da tolerância à dessecação. Recentemente, a atenção dada à distribuição de Fucus vesiculosus sob efeito das alterações climáticas levou-nos a querer determinar os limites subletais máximos de temperatura desta alga castanha, utilizando indicadores fisiológicos e alterações da expressão genética para descrever a temperatura de indução e o perfil da resposta ao choque térmico em diversas populações desta espécie. Vimos que conhecer a temperatura ambiente não é suficiente para antecipar o choque térmico sentido pela alga, ao mesmo tempo que os microhabitats formados pelo tapete de algas vão influenciar a temperatura local e afectar a resposta ao choque térmico. Surpreendentemente, no microhabitat mais quente as algas aparentavam estar protegidas do choque térmico pela dessecação rápida e intensa. Estudos de proteómica em algas castanhas foram facilitados recentemente graças a recursos genéticos, a sequenciação do genoma completo da espécie modelo, Ectocarpus siliculosus, numerosas sequências de transcriptos de Fucus, obtidas pelas novas técnicas de sequenciação e avanços técnicos no estudo de outros organismos com compostos secundários semelhantes que interferem com a qualidade e a quantidade das proteínas extraídas. Foi optimizado um protocolo de extracção de proteínas de algas do género Fucus, que foi utilizado para investigar a expressão diferencial de proteínas em resposta à dessecação tanto por 2DE convencional como por 2D-DIGE. Não foram detectadas alterações significativas nos perfis de proteínas na sequência da dessecação ou da rehidratação, o que sugere a importância de mecanismos constitutivos de tolerância, minimizando os custos metabólicos da expressão de novos genes, enquanto a dessecação protege do choque térmico. Estudos de campo, em locais de intensa dessecação ou protegidos, após exposição consecutiva ao stress de emersão, e após dessecação em condições controladas no laboratório, todos falharam na identificação de alterações robustas na expressão de proteínas envolvidas na tolerância à dessecação. Caracterizámos o primeiro proteoma extraível de F. vesiculosus, identificando as proteínas por LC-MS/MS e anotando utilizando as bases de dados de algas castanhas. Esta anotação foi bem-sucedida, apesar da fraca anotação funcional das proteínas de algas castanhas e da presença de múltiplas proteínas em alguns dos spots

Haemorrhagic Septicaemia

Livestock Production/Industries,

Study of two bipolar susceptibility genes: Slynar and IGF1

Linkage studies have implicated the 12q22-24 region in susceptibility to bipolar disorder. In this region alleles at the “Slynar” and Insulin Like Growth Factor 1 (IGF1) genes showed association with bipolar disorder. The Slynar gene is contained within a region of 278 kb on chromosome 12q24 and expresses the sequence AY070435 in the human brain. AY070435 has no known function. A Macaque brain expressed cDNA which is highly homologous to human AY070435 has been cloned and sequenced. To further characterise the human Slynar gene and expressed mRNA transcript studies were carried out to identify Slynar in the mouse and in human neuroblastoma cell lines. Exhaustive efforts were taken to find a mouse homologue but these proved negative. Slynar shared no homology, or partial homology with any other gene in the human genome. The other 12q24 bipolar susceptibility gene IGF1 is highly expressed in the human brain and a well known for its neuromodulatory functions. IGF1 protein has been shown to have an antidepressant and anxiolytic-like effect in the mouse brain. On a genome wide association study (GWAS) with the UCL case control sample, IGF1 was found to be associated to disease with 5 SNPs showing association within the gene. In order to further implicate IGF1 and find the aetiological base pair changes responsible for disease, IGF1 was sequenced. New three new non database SNPs, three previously characterised polymorphisms and a CA repeat were found and genotyped in an extended UCL sample of 1,000 cases and 1,000 controls. One of the novel SNPs and the CA repeat, both located in the promoter region, were associated with bipolar disorder. Haplotype analysis of the GWAS and new markers data confirmed association to bipolar disorder