| 1. |
- Eriksson, Martin, 1970, et al.
(author)
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Community-Level Analysis of psbA Gene Sequences and Irgarol Tolerance in Marine Periphyton
- 2009
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In: Applied and Environmental Microbiology. - Washington, D.C. : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 75:4, s. 897-906
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Journal article (peer-reviewed)abstract
- This study analyzes psbA gene sequences, predicted D1 protein sequences, species relative abundance, and pollution-induced community tolerance in marine periphyton communities exposed to the antifouling compound Irgarol 1051. The mechanism of action of Irgarol is the inhibition of photosynthetic electron transport at photosystem II by binding to the D1 protein. The metagenome of the communities was used to produce clone libraries containing fragments of the psbA gene encoding the D1 protein. Community tolerance was quantified with a short-term test for the inhibition of photosynthesis. The communities were established in a continuous flow of natural seawater through microcosms with or without added Irgarol. The selection pressure from Irgarol resulted in an altered species composition and an inducted community tolerance to Irgarol. Moreover, there was a very high diversity in the psbA gene sequences in the periphyton, and the composition of psbA and D1 fragments within the communities was dramatically altered by increased Irgarol exposure. Even though tolerance to this type of compound in land plants often depends on a single amino acid substitution (Ser(264)-> Gly) in the D1 protein, this was not the case for marine periphyton species. Instead, the tolerance mechanism likely involves increased degradation of D1. When we compared sequences from low and high Irgarol exposure, differences in nonconserved amino acids were found only in the so-called PEST region of D1, which is involved in regulating its degradation. Our results suggest that environmental contamination with Irgarol has led to selection for high-turnover D1 proteins in marine periphyton communities at the west coast of Sweden.
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| 2. |
- Teneberg, Susanne, et al.
(author)
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Lactotetraosylceramide, a novel glycosphingolipid receptor for Helicobacter pylori, present in human gastric epithelium
- 2002
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In: Journal of Biological Chemistry. - Bethesda : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 277:22, s. 19709-19719
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Journal article (peer-reviewed)abstract
- The binding of Helicobacter pylori to glycosphingolipids was examined by binding of 35S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. In addition to previously reported binding specificities, a selective binding to a non-acid tetraglycosylceramide of human meconium was found. This H. pylori binding glycosphingolipid was isolated and, on the basis of mass spectrometry, proton NMR spectroscopy, and degradation studies, were identified as Galβ3GlcNAcβ3-Galβ4Glcβ1Cer (lactotetraosylceramide). When using non-acid glycosphingolipid preparations from human gastric epithelial cells, an identical binding of H. pylori to the tetraglycosylceramide interval was obtained in one of seven samples. Evidence for the presence of lactotetraosylceramide in the binding-active interval was obtained by proton NMR spectroscopy of intact glycosphingolipids and by gas chromatography-electron ionization mass spectrometry of permethylated tetrasaccharides obtained by ceramide glycanase hydrolysis. The lactotetraosylceramide binding property was detected in 65 of 74 H. pylori isolates (88%) Binding of H. pylori to lactotetraosylceramide on thin-layer chromatograms was inhibited by preincubation with lactotetraose but not with lactose. Removal of the terminal galactose of lactotetraosylceramide by galactosidase hydrolysis abolished the binding as did hydrazinolysis of the acetamido group of the N-acetylglucosamine. Therefore, Galβ3GlcNAc is an essential part of the binding epitope.
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| 3. |
- Kielland, Anders, et al.
(author)
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In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012
- 2009
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In: Free Radical Biology & Medicine. - Philadelphia, PA : Elsevier. - 0891-5849 .- 1873-4596. ; 47:6, s. 760-766
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Journal article (peer-reviewed)abstract
- Production of reactive oxygen and nitrogen species (ROS/RNS) is an important part of the inflammatory response, but prolonged elevated levels of ROS/RNS as under chronic inflammation can contribute to the development of disease. Monitoring ROS/RNS in living animals is challenging due to the rapid turnover of ROS/RNS and the limited sensitivity and specificity of ROS/RNS probes. We have explored the use of the chemiluminescent probe L-012 for noninvasive imaging of ROS/RNS production during inflammation in living mice. Various inflammatory conditions were induced, and L-012-dependent luminescence was recorded with an ultrasensitive CCD camera. Strong luminescent signals were observed from different regions of the body corresponding to inflammation. The signal was reduced by administration of the SOD mimetic tempol, the NADPH oxidase inhibitor apocynin, and the inhibitor of nitric oxide synthesis L-NAME, signifying the requirement for the presence of ROS/RNS. Additionally, the L-012 signal was abolished in mice with a mutation in the Ncf1 gene, encoding a protein in the NADPH oxidase complex 2, which generates ROS/RNS during inflammation. In conclusion, L-012 is well distributed in the mouse body and mediates a strong ROS/RNS-dependent luminescent signal in vivo and is useful for monitoring the development and regulation of inflammation in living organisms. © 2009 Elsevier Inc. All rights reserved.
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| 4. |
- Rögnvaldsson, Thorsteinn, et al.
(author)
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Improving automatic peptide mass fingerprint protein identification by combining many peak sets
- 2004
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In: Journal of chromatography. B. - : Elsevier. - 1570-0232 .- 1873-376X. ; 807:2, s. 209-215
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Journal article (peer-reviewed)abstract
- An automated peak picking strategy is presented where several peak sets with different signal-to-noise levels are combined to form a more reliable statement on the protein identity. The strategy is compared against both manual peak picking and industry standard automated peak picking on a set of mass spectra obtained after tryptic in gel digestion of 2D-gel samples from human fetal fibroblasts. The set of spectra contain samples ranging from strong to weak spectra, and the proposed multiple-scale method is shown to be much better on weak spectra than the industry standard method and a human operator, and equal in performance to these on strong and medium strong spectra. It is also demonstrated that peak sets selected by a human operator display a considerable variability and that it is impossible to speak of a single “true” peak set for a given spectrum. The described multiple-scale strategy both avoids time-consuming parameter tuning and exceeds the human operator in protein identification efficiency. The strategy therefore promises reliable automated user-independent protein identification using peptide mass fingerprints.
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| 5. |
- Aronsson, H., et al.
(author)
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Characterization of the plastid import reaction of the pea NADPH-protochlorophyllide oxidoreductase (POR)
- 1998
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In: The Chloroplast. - New York : Springer-Verlag. - 9780792355779 - 0792355776 ; , s. 167-170
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Conference paper (peer-reviewed)abstract
- NADPH: protochlorophyllide (POR) is a vital enzyme in the biosynthesis of chlorophyl where it catalyzes the reduction of Pchlide into Chlide in a light-dependent manner. POR is nucleus-encoded and imported into the plastids where it is found at the inner membranes. Together with its substrate and the co-factor NADPH it forms a ternary complex which is needed for catalytical activity. The anomaly of a decreasing POR level during active chlorophyll synthesis was cleared with the discovery of two different POR proteins, POR-A and POR-B, in barley and Arabidopsis thaliana. During greening, POR-A is negatively regulated by light both at transcriptional and proteolytical levels. In addition, the import of POR-A, but not POR-B, has been suggestedto require Pchlide in order to be translocated into the plastid. In this respect, POR-A differs from other known nucleus-encoded plastid proteins, and as it appears, this requirements represents a novel and exclusive import characteristic. In pea, only one POR gene has been found indicating that the situation for the regulation of POR import and accumulation is far from clear. We here present a characterization of the import conditions of the pea POR, including the potentional role of Pchlide inthe translocation step.
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| 6. |
- Farkas, Daniel, et al.
(author)
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Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding
- 2011
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In: Protein Expression and Purification. - San Diego : Academic Press. - 1046-5928 .- 1096-0279. ; 78:2, s. 156-166
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Journal article (peer-reviewed)abstract
- The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c6 in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1-79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His6 tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 Å-resolution crystal structure of photosystem I Ref. [5] and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50-65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and 15N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.
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| 7. |
- Funes, Soledad, et al.
(author)
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Chlamydomonas reinhardtii : the model of choice to study mitochondria from unicellular photosynthetic organisms.
- 2007
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In: Methods in Molecular Biology. - Clifton, NJ : Humana Press. - 1064-3745. - 9781588296672 - 9781597453653 ; 372, s. 137-149
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Journal article (other academic/artistic)abstract
- Chlamydomonas reinhardtii is a model organism to study photosynthesis, cellular division, flagellar biogenesis, and, more recently, mitochondrial function. It has distinct advantages in comparison to higher plants because it is unicellular, haploid, and amenable to tetrad analysis, and its three genomes are subject to specific transformation. It also has the possibility to grow either photoautotrophically or heterotrophically on acetate, making the assembly of the photosynthetic machinery not essential for cell viability. Methods developed allow the isolation of C. reinhardtii mitochondria free of thylakoid contaminants. We review the general procedures used for the biochemical characterization of mitochondria from this green alga.
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| 8. |
- Rögnvaldsson, Thorsteinn, 1963-, et al.
(author)
-
Why neural networks should not be used for HIV-1 protease cleavage site prediction
- 2004
-
In: Bioinformatics. - Oxford : Oxford University Press. - 1367-4803 .- 1367-4811. ; 20:11, s. 1702-1709
-
Journal article (peer-reviewed)abstract
- Several papers have been published where non-linear machine learning algorithms, e.g. artificial neural networks, support vector machines and decision trees, have been used to model the specificity of the HIV-1 protease and extract specificity rules. We show that the dataset used in these studies is linearly separable and that it is a misuse of nonlinear classifiers to apply them to this problem. The best solution on this dataset is achieved using a linear classifier like the simple perceptron or the linear support vector machine, and it is straightforward to extract rules from these linear models. We identify key residues in peptides that are efficiently cleaved by the HIV-1 protease and list the most prominent rules, relating them to experimental results for the HIV-1 protease. Motivation: Understanding HIV-1 protease specificity is important when designing HIV inhibitors and several different machine learning algorithms have been applied to the problem. However, little progress has been made in understanding the specificity because nonlinear and overly complex models have been used. Results: We show that the problem is much easier than what has previously been reported and that linear classifiers like the simple perceptron or linear support vector machines are at least as good predictors as nonlinear algorithms. We also show how sets of specificity rules can be generated from the resulting linear classifiers.
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| 9. |
- Levander, Fredrik, et al.
(author)
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Automated methods for improved protein identification by peptide mass fingerprinting
- 2004
-
In: Proteomics. - Weinheim : Wiley. - 1615-9861 .- 1615-9853. ; 4:9, s. 2594-2601
-
Journal article (peer-reviewed)abstract
- In order to maximize protein identification by peptide mass fingerprinting noise peaks must be removed from spectra and recalibration is often required. The preprocessing of the spectra before database searching is essential but is time-consuming. Nevertheless, the optimal database search parameters often vary over a batch of samples. For high-throughput protein identification, these factors should be set automatically, with no or little human intervention. In the present work automated batch filtering and recalibration using a statistical filter is described. The filter is combined with multiple data searches that are performed automatically. We show that, using several hundred protein digests, protein identification rates could be more than doubled, compared to standard database searching. Furthermore, automated large-scale in-gel digestion of proteins with endoproteinase LysC, and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis, followed by subsequent trypsin digestion and MALDI-TOF analysis were performed. Several proteins could be identified only after digestion with one of the enzymes, and some less significant protein identifications were confirmed after digestion with the other enzyme. The results indicate that identification of especially small and low-abundance proteins could be significantly improved after sequential digestions with two enzymes.
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| 10. |
- Guo, Xiaoyi, et al.
(author)
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Kernel Risk Sensitive Loss-based Echo State Networks for Predicting Therapeutic Peptides with Sparse Learning
- 2022
-
In: Proceedings - 2022 IEEE International Conference on Bioinformatics and Biomedicine, BIBM 2022. - Piscataway : IEEE. - 9781665468190 ; , s. 6-11
-
Conference paper (peer-reviewed)abstract
- The detection of therapeutic peptides is usually a biochemical experimental method, which is time-consuming and labor-intensive. Lots of computational biology methods had been proposed to solve the problem of therapeutic peptide prediction. However, the existing methods did not consider the processing of noisy samples. We propose a kernel risk-sensitive mean p-power error-based echo state network with sparse learning (KRP-ESN-SL). An efficient iterative optimization algorithm is used to train the model. The KRP-ESN-SL has better performance than other methods. © 2022 IEEE.
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