| 1. |
- Raine, Amanda, 1969-
(författare)
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Ribosome Associated Factors Recruited for Protein Export and Folding
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein folding and export to the membrane are crucial events in the cell. Both processes may be initiated already at the ribosome, assisted by factors that bind to the polypeptide as it emerges from the ribosome. The signal recognition particle (SRP) scans the ribosome for nascent peptides destined for membrane insertion and targets these ribosomes to the site for translocation in the membrane. Trigger factor (TF) is a folding chaperone that interacts with nascent chains to promote their correct folding, prevent misfolding and aggregation. In this thesis, we first investigated membrane targeting and insertion of two heterologous membrane proteins in E. coli by using in vitro translation, membrane targeting and cross-linking. We found that these proteins are dependent on SRP for targeting and that they initially interact with translocon components in the same way as native nascent membrane proteins. Moreover we have characterised the SRP and TF interactions with the ribosome both with cross-linking experiments and with quantitative binding experiments. Both SRP and TF bind to ribosomal L23 close to the nascent peptide exit site where they are strategically placed for binding to the nascent polypeptide. Quantitative analysis of TF and SRP binding determined their respective KD values for binding to non translating ribosomes and reveals that they bind simultaneously to the ribosome, thus having separate binding sites on L23. Finally, binding studies on ribosome nascent chain adds clues as to how TF functions as a chaperone.
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| 2. |
- Raine, Amanda, et al.
(författare)
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Trigger factor binding to ribosomes with nascent peptide chains of varying lengths and sequences
- 2006
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:38, s. 28033-28038
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Tidskriftsartikel (refereegranskat)abstract
- Trigger factor (TF) is the first protein-folding chaperone to interact with a nascent peptide chain as it emerges from the ribosome. Here, we have used a spin down assay to estimate the affinities for the binding of TF to ribosome nascent chain complexes (RNCs) with peptides of varying lengths and sequences. An in vitro system for protein synthesis assembled from purified Escherichia coli components was used to produce RNCs stalled on truncated mRNAs. The affinity of TF to RNCs exposing RNA polymerase sequences increased with the length of the nascent peptides. TF bound to RNA polymerase RNCs with significantly higher affinity than to inner membrane protein leader peptidase and bacterioopsin RNCs. The latter two RNCs are substrates for signal recognition particle, suggesting complementary affinities of TF and signal recognition particle to nascent peptides targeted for cytoplasm and membrane.
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| 3. |
- Riihimaki, Miia, et al.
(författare)
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Epithelial expression of mRNA and protein for IL-6, IL-10 and TNF-alpha in endobronchial biopsies in horses with recurrent airway obstruction
- 2008
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Ingår i: BMC Veterinary Research. - : Springer Science and Business Media LLC. - 1746-6148. ; 4, s. 8-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The aim of this study was to evaluate the contribution of bronchial epithelium to airway inflammation, with focus on mRNA and protein expression of cytokines of innate immunity IL-6, IL-10 and TNF-alpha, in horses with Recurrent Airway Obstruction (RAO) during exacerbation and in remission. Results: Despite marked clinical and physiologic alterations between exacerbation and after remission in the RAO horses no differences were detected in either cytokine mRNA or protein levels. Moreover, the expression of investigated cytokines in RAO horses on pasture did not differ from controls. In comparing real-time PCR analysis to results of immunohistochemistry only IL-10 mRNA and protein levels in RAO horses on pasture were significantly correlated (r(s) = 0.893, p = 0.007). Curiously, in controls examined on pasture the TNF-alpha protein level was positively correlated to IL-10 mRNA expression (r(s) = 0.967, p = 0.007) and negatively correlated to IL-6 mRNA expression (r(s) = -0.971, p = 0.001). Conclusion: Given the complementary relationship of assessing cytokines directly by immunohistochemistry, or indirectly by PCR to mRNA, the lack of significant changes in either mRNA or protein levels of IL-6, IL-10 or TNF-alpha mRNA in RAO horses in exacerbation suggests that these particular cytokines in bronchial tissue may not play a substantive role in the active inflammation of this disease. To support this contention further studies examining time dependency of expression of IL-6, IL-10 or TNF-alpha are needed, as is expansion of the range of cytokines to include other key regulators of airway inflammation.
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| 4. |
- Riihimäki, M., et al.
(författare)
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Clinical alterations and mRNA levels of IL-4 and IL-5 in bronchoalveolar cells of horses with transient pulmonary eosinophilia
- 2008
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Ingår i: Research in Veterinary Science. - : Elsevier BV. - 0034-5288 .- 1532-2661. ; 85:1, s. 52-55
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to assess clinical signs and altered pulmonary cell expression of cytokines related to eosinophil kinetics in horses with pulmonary eosinophilia. Pulmonary eosinophilia was detected by bronchoalveolar lavage (BAL) in a group of standardbreds in training. Horses had detailed clinical examination, bronchoscopy, endobronchial biopsy and BAL on three occasions at approximately 6 month intervals. During the second sampling period BAL eosinophils were significantly elevated (p > ; 0.010), with five horses having from 5% to 37% eosinophils in BAL. Neither detailed clinical examination parameters nor gene expression of IL-4 and IL-5 mRNA (real-time-PCR) were associated with BAL eosinophilia. Pulmonary eosinophilia abated without treatment apart from deworming. It appears that pronounced lung eosinophilia in horses can be transient, abate without specific treatment, and in this instance, lack correlation to upregulation of expression of either IL-4 or IL-5.
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| 5. |
- Riihimäki, Miia, et al.
(författare)
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Markers of respiratory inflammation in horses in relation to seasonal changes in air quality in a conventional racing stable
- 2008
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Ingår i: Canadian Journal of Veterinary Research-Revue Canadienne de Recherche Veterinaire. - 0830-9000. ; 72:5, s. 432-439
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Tidskriftsartikel (refereegranskat)abstract
- Airborne factors in a conventionally managed racing stable and markers of pulmonary inflammation in the stabled horses were investigated on 3 occasions at 6-month intervals, including 2 winter periods and the intervening summer period. The stable measurements included inside and outside ambient temperature and relative humidity, levels of total and respirable dust, endotoxin, and 1,3-beta-glucan. Horses (n = 12) were examined in detail clinically as well as by endoscopy, bronchoalveolar lavage (BAL) cytology, and real-time polymerase chain reaction (RT-PCR) analysis of BAL-cells for IL-6 and IL-10 mRNA. Indoor measurements showed low dust levels irrespective of season. Inhalable dust, as well as 1,3-beta-glucan, were higher during the winter stabling period, whereas endotoxin levels were higher during summer. Complete data from all sampling occasions to be used for further evaluation was obtained for only 8 of the horses. There was a trend for elevation of BAL neutrophils in the horses during winter stabling that coincided with a 3.7-fold increased expression of IL-6 mRNA in BAL cells (P = 0.014). Compared to summer sampling, IL-10 mRNA expression was significantly upregulated in only 1 of the winter sampling occasions, implicating influence on immune regulation by factor/s apart from seasonal differences. Our findings suggest up-regulation of innate immunity in the airways of stabled horses; in particular involving IL-6 in association with mild elevations in respirable dust, 1,3-beta-glucan, and/or cold ambient air. However, given that this study was observational, other unmeasured environmental factors associated with winter stabling need to be considered.
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| 6. |
- Riihimäki, M., et al.
(författare)
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Partial divergence of cytokine mRNA expression in bronchial tissues compared to bronchoalveolar lavage cells in horses with recurrent airway obstruction
- 2008
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Ingår i: Veterinary Immunology and Immunopathology. - : Elsevier BV. - 0165-2427 .- 1873-2534. ; 122:3-4, s. 256-264
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to investigate mRNA levels of cytokines in bronchial epithelium in horses with recurrent airway obstruction (RAO) during acute crisis and remission. Additionally, cytokine mRNA levels in endobronchial biopsies and bronchoalveolar lavage (BAL) cells were compared. Seven RAO horses were examined while in respiratory crisis following provocation and again while in remission after 2 months on pasture, during which time six healthy horses on pasture were also examined. Quantitative real-time PCR (RT-PCR) was used to assess mRNA expression for cytokines IL-5, IL-6, IL-8, IL- 10, IL- 17 and transforming growth factor beta 1 (TGF-beta 1) in endobronchial biopsies and bronchoalveolar lavage. Expression of IL-8 mRNA was significantly upregulated during crisis in both endobronchial biopsies and BAL cells (p = 0.036), while there was a similar trend for upregulation of IL-10 mRNA only in BAL cells that approached significance (p = 0.059). Moreover, during crisis the expression of IL-8 mRNA in BAL cells was positively correlated to relative IL-6 mRNA expression (r(s) = 0.971, p = 0.001) and bronchial epithelial expression of IL-10 and TGF-beta 1 mRNA were positively correlated (r(s) = 0.943,p = 0.005). In comparing the relationship of mRNA expression in BAL to biopsy in individual RAO horses, there was a positive correlation with IL-6 to IL-8 mRNA expression in BAL during respiratory crisis (r(s) = 0.971,p = 0.001) that also correlated positively with IL-8 expression in biopsies on pasture (r(s) = 0.986, p < 0.0001 for both). Regarding RAO horses at pasture versus controls neither the cytokine mRNA levels in endobronchial biopsy nor in BAL cells differed significantly. These results further support previous findings that IL-8 mRNA in both BAL cells and bronchial epithelium is upregulated in RAO horses during crisis. However, apart from IL-8, it appears that expression of other cytokines, including IL-5, IL6, IL-10, IL-17 and TGF-beta 1 in bronchial epithelium does not necessarily mirror cytokine expression in BAL cells in individual horses with RAO. Accordingly, examination of markers of inflammation in endobronchial tissue provides complementary but not necessarily identical information to that obtained in BAL cells. Given the potential for repeated sampling over time bronchial biopsy can serve as an invaluable additional tool for investigation of time-dependent changes in inflammatory process in this animal model of asthma.
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