Browsing by Issue Date, starting with "2012-01-26"
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- Possible respiratory infection due to Aspergillus in workers from swineries and poultriesPublication . Sabino, Raquel; Mateus, V.; Viegas, Susana; Veríssimo, Cristina; Viegas, CarlaIntroduction: Aspergillus is among a growing list of allergens that can aggravate asthmatic responses. Significant pulmonary pathology is associated with Aspergillus-induced allergic and asthmatic lung disease. Environments with high levels of exposure to fungi are found in animal production facilities like swineries and poultry which farmers are at increased risk of occupational respiratory diseases. Purpose: Seven Portuguese poultries and seven swineries were analyzed in order to estimate the prevalence, amount and distribution of Aspergillus species, as well as to evaluate the existence of clinical symptoms associated with asthma and other allergy diseases in these highly contaminated settings. Methods: A total of 83 air samples (through impaction method), 66 surface samples (swab sampling) and 28 samples of different material covering their floor were analyzed. Results: The collected fungal isolates (699) were distributed by 40 different genus, Aspergillus presented a prevalence of 17.6% (average) and regarding poultries only, its prevalence showed a higher value (22.7%). The most frequently isolated Aspergillus species were A. versicolor, A. flavus and A. fumigatus. In poultries, A. flavus presented the highest level of airborne spores (>2000 ufc/m3) whereas in swineries was A. versicolor, with a prevalence four times higher than the other referred species. Eighty workers of these settings were analyzed, with ages ranging from 17 to 93 years old. Seven of these workers were medical diagnosed with asthma and four of them reported the first attack after the age of 40 years old, which possibly could be related with their occupational exposure. A high prevalence of respiratory symptoms in professionals without asthma was observed, namely wheezing associated with dyspnea (23.8%) and dyspnea after strenuous activities (12.3%), suggesting an under diagnosed respiratory problem. Moreover, 32.5% of all the inquired workers refer an improvement of their respiratory ability during the resting days and holiday. Conclusions: Considering the strong association between fungal sensitization and severity of asthma and that Aspergillus species are one of the most important fungal agents causing sensitization of the individual, this study contributes to the knowledge of Aspergillus prevalence and distribution in Portuguese poultries and swineries. These data, together with data regarding determinations of specific IgE levels and monitorization of mycotoxin exposure using biomarkers will also help to understand how and which Aspergillus species can affect workers of these settings.
- Translational regulation of the human erythropoietin transcript by an upstream open reading framePublication . Barbosa, Cristina; Romão, LuísaAmong the various cis-acting elements in mRNAs that participate in regulating protein synthesis are AUG codons within transcript leader sequences (uAUGs) and, in some cases, associated short upstream open reading frames (uORFs). Although about 15% of the human mRNAs present uORFs (mainly, those transcripts encoding growth factors or hormones), there is no general model for the mechanism by how and when an uORF downregulates the main ORF expression. Leaky scanning and reinitiation are mechanisms that allow translational control of the main ORF in a transcript bearing at least one uORF. In the leaky scanning mechanism ribosomes either ignore the uAUG codon and scan past it or recognize it, initiating translation. Short regulatory uORFs may also permit the small ribosomal subunit to stay mRNA-bound after termination and resume scanning for efficient reinitiation downstream at the main ORF. Even though, short uORFs may also downregulate expression of a main ORF by their special ability to mediate ribosome stalling at coding or termination codons, or by influencing the mRNA stability through the nonsense mediated mRNA decay (NMD) pathway. Erythropoietin (EPO) is a glycoprotein synthesized and released mainly from the kidney, with a key role in hematopoiesis. However, many other non-hematopoietic functions have recently been reported. Consequently, it might be used as a therapeutic target for the treatment of several human disorders. Understanding the molecular mechanisms of translational control of EPO mRNA may be valuable in the determination of these therapies. Knowing that human EPO transcript presents a 5’ leader with 181 nucleotides containing a 14-codon-uORF and given that it seems to be conserved among different species, which might indicate its role in translational regulation, we aimed to prove this hypothesis. To analyze the effect of this uORF, HepG2 and HEK293 cells were transfected with several constructs carrying the luciferase reporter gene with the intact or disrupted uORF. Luciferase activity was measured by luminometry and normalized to the corresponding mRNA levels to obtain translation efficiencies. The mRNA levels were quantified by RT-qPCR. Results show that the EPO uORF can decrease the main ORF translational efficiency in about 70%. In addition, our data support the conclusion that reinitiation, and in less extent leaky scanning, are responsible for the main ORF translation. Furthermore, this uORF does not block the translational machinery and does not trigger NMD. The interaction between the EPO uORF and its 3’ untranslated region (3’UTR) was also analyzed. Results show that the 3’UTR is able to increase the protein levels independently of the uORF.
- Toxinfecções alimentares Investigação epidemiológica laboratorial - dados do INSAPublication . Correia, Cristina BeloInfecções e intoxicações alimentares. Reporte dos dados do INSA, I.P. para a Autoridade Europeia de Segurança Alimentar.
- The same Bcl2 protein is produced by two human Bcl2 transcript isoforms that present different mechanisms of post-transcriptional regulation.Publication . Marques-Ramos, Ana; Teixeira, Alexandre; Lacerda, Rafaela; Romão, LuísaThe Bcl-2 family of proteins has an essential role in apoptosis since it comprises both anti- and pro-survival factors. Bcl-2 prevents apoptosis in response to stress conditions such as DNA damage and endoplasmatic reticulum stress. Deregulated expression of Bcl-2 is observed in a variety of tumours such as leukemia, linfoma, neuroblastoma and non-small-cell lung carcinoma. Additionally, increased cell survival due to elevated levels of Bcl-2 confers resistance to anticancer therapies. With such an important role in regulating programmed cell death, expression of Bcl-2 is highly regulated at multiple levels, both transcriptionally and posttranscriptionally. There are three human Bcl2 transcript isoforms, two of which encoding the same protein but differing in the 5´ untranslated region (UTR) length. The upstream promoter originates a 5´UTR longer-transcript which harbors an upstream open reading frame and an internal ribosome entry site (IRES) that regulate Bcl-2 protein production. IRES is an RNA structure present mainly in the 5’UTRs, that assists protein synthesis initiation without the requirement of the cap structure and some canonical translation initiation factors. Since the main feature of the IRES-mediated translation initiation is its cap-independence, it bypasses this regulation step and allows the cell to continue to produce some proteins, although the overall reduction of protein synthesis. The shorter Bcl-2 transcript isoform, encoding the same protein, lacks a 425-bp 5´ sequence. This shorter Bcl-2 5´UTR has not been analyzed for IRES activity until now. To test the shorter 5’UTR for IRES activity, we have transfected HEK293 cells with renilla/firefly luciferase dicistronic reporter plasmids where the firefly cistron is linked to the Bcl-2 shorter 5’UTR. We found no IRES activity in the Bcl-2 shorter 5´UTR, which was reflected by no firefly luciferase protein activity in luminescent assays. Accordingly, we conclude that in the Bcl-2 shorter mRNA isoform, the 425-bp missing sequence is essential for IRES activity. Since IRES activity has been assumed to be a backup mechanism to bypass stressful conditions in which overall protein synthesis is impaired and as these two transcription variants encode the same protein, it remains to be elucidated the biological relevance of both Bcl-2 mRNA isoforms having such different mechanisms of function.
- Translation initiation factors are involved in the mechanism of NMD resistance of mRNAs carrying a short open reading frame.Publication . Peixeiro, Isabel; Barbosa, Cristina; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The unified model for NMD, proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmatic poly(A)-binding protein 1 (PABPC1) and the NMD effector UPF1 for the termination complex. Consequently, PTCs located far, in a linear sense, from the poly(A) tail and associated PABPC1, in mRNAs containing residual downstream exon junction complexes (EJCs) are expected to elicit NMD. Nevertheless, it has been reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we focused on the mechanistic basis for this NMD resistance, exemplified by human β-globin transcripts carrying an AUG-proximal PTC. We demonstrate that the establishment of an efficient translation termination reaction at the AUG-proximal PTC is dependent on PABPC1 interaction with the initiation factor eIF4G and with the release factor eRF3 at the terminating ribosome. The PABPC1-eIF4G interaction allows the mRNA to acquire a closed-loop conformation, which, in concert with PABPC1-eRF3 interaction underlies linkage of translation initiation with efficient termination at the AUG-proximal PTC. Thus, these interactions contribute to an NMD-resistant PTC definition at an early phase of translation elongation. Furthermore, we provide strong evidence that the eukaryotic initiation factor 3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC through eIF3h and eIF3f subunits. In fact, our preliminary data suggest that eIF3h and eIF3f depletion in HeLa cells affect the association of the eIF3 complex with the ribosome and eIF4G, respectively. These data corroborate a role for PABPC1 on NMD evasion of AUG-proximal transcripts and provides further insights into the mechanistic details of PTC definition and translation initiation.