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  • 1.
    Bolognesi, B
    et al.
    University of Cambridge, Cambridge, UK.
    Jahn, T R
    University of Cambridge, Cambridge, UK.
    Brorsson, Ann-Christin
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik.
    Luheshi, L M
    University of Cambridge, Cambridge, UK.
    Yerbury, J J
    University of Wollongong, Wollongong, NSW, Australia .
    Crowther, D C
    University of Cambridge, Cambridge, UK.
    Dobson, C M
    University of Cambridge, Cambridge, UK.
    The N-terminus of amyloid-beta plays a crucial role in its aggregation and toxicity2010Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, nr Suppl. 1, s. 79-80Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    The aggregation of Amyloid Beta (Aß) peptide into insolubleamyloid fibrils that deposit in the brain is one of the primarypathogenic events in Alzheimer’s disease. We have previouslyshown, using a Drosophila model of Aß toxicity, that the N terminus of the Aß peptide, despite being unstructured in themature Aß fibril, nonetheless affects Aß induced neurodegeneration in vivo. In order to understand the contribution of the N terminusof Aß to its aggregation behaviour, we have investigated anumber of rationally designed N-terminal mutants in vitro. We find that single amino acid mutations in this region affect significantlythe kinetics of Aß aggregation in vitro as measured by arange of spectroscopic techniques. Furthermore, we observe striking differences in the morphology of the aggregated speciesformed by these different Aß mutants when imaged with TEM or  AFM  and  also  in the ß-sheet  content  of their  mature  fibrils. Interestingly, mutants with an increased net charge or lower hydrophobicity tend  to show slower aggregation  kinetics, and  to form more ordered  aggregates  whereas mutations that  reduce net charge   or   increase   hydrophobicity   favour   faster   aggregation kinetics   and   poorly   structured  aggregates.   In   addition,    the exposed  hydrophobicity of aggregates  formed  in the early stages of aggregation  is correlated  to their toxicity.  These findings demonstrate  not  only that  the N-terminus of the Aß peptide  plays a crucial  role  in its aggregation  and  toxicity  but  also  suggest that this  region  of Aß  may  modulate  in vivo toxicity  by altering  the conformations of aggregates that  it forms.

  • 2.
    Bresell, Anders
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
    Weinander, Rolf
    Department of Medicine, Division of Rheumatology Unit, Karolinska Institutet, Stockholm.
    Wiklund, Ronney
    Department of Plant Biology & Forestry Genetics, Swedish Agricultural University, Uppsala.
    Eriksson, Jan
    Department of Plant Biology & Forestry Genetics, Swedish Agricultural University, Uppsala.
    Jansson, Christer
    Department of Plant Biology & Forestry Genetics, Swedish Agricultural University, Uppsala.
    Persson, Bengt
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
    Jakobsson, Per-Johan
    Department of Medicine, Division of Rheumatology Unit, Karolinska Institutet, Stockholm.
    Morgenstern, Ralf
    Institute of Environmental Medicine Karolinska Institutet, Stockholm.
    Lundqvist, Gerd
    Institute of Environmental Medicine Karolinska Institutet, Stockholm.
    Raza, Haider
    Institute of Environmental Medicine Karolinska Institutet, Stockholm.
    Shimoji, Miyuki
    Institute of Environmental Medicine Karolinska Institutet, Stockholm.
    Sun, Tie-Hua
    Institute of Environmental Medicine Karolinska Institutet, Stockholm.
    Balk, Lennart
    Stockholm Marine Research Centre, University of Stockholm.
    Bioinformatic and enzymatic characterization of the MAPEG superfamily2005Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 272, nr 7, s. 1688-1703Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belonging to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calculations of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal glutathione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4), 5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2/FLAP/LTC4 synthase families. The insect members are most similar to MGST1/prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, showing an early origin for MAPEG family differentiation. Thus, the evolutionary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the subfamilies. We have further investigated and functionally characterized representative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity analysis demonstrated that all proteins catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 µmol·min−1·mg−1 in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N-terminal amino acid sequence that was at least 50% pure, with a specific activity towards 1-chloro-2,4-dinitrobenzene of 11 µmol·min−1·mg−1. Yeast microsomes expressing the Arabidopsis enzyme showed an activity of 0.02 µmol·min−1·mg−1, whereas the Drosophila enzyme expressed in E. coli was highly active at 3.6 µmol·min−1·mg−1. The purified pike enzyme is the most active MGST described so far with a specific activity of 285 µmol·min−1·mg−1. Drosophila and pike enzymes also displayed glutathione peroxidase activity towards cumene hydroperoxide (0.4 and 2.2 µmol·min−1·mg−1, respectively). Glutathione transferase activity can thus be regarded as a common denominator for a majority of MAPEG members throughout the kingdoms of life whereas glutathione peroxidase activity occurs in representatives from the MGST1, 2 and 3 and PGES subfamilies.

  • 3.
    Carlsson, Jonas
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
    Soussi, Thierry
    Department of Oncology-Pathology, Cancer Center Karolinska (CCK), Karolinska Institutet, Stockholm, Sweden.
    Persson, Bengt
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
    Investigation and prediction of the severity of p53 mutants using parameters from structural calculations2009Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, nr 15, s. 4142-4155Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A method has been developed to predict the effects of mutations in the p53 cancer suppressor gene. The new method uses novel parameters combined with previously established parameters. The most important parameter is the stability measure of the mutated structure calculated using molecular modelling. For each mutant, a severity score is reported, which can be used for classification into deleterious and nondeleterious. Both structural features and sequence properties are taken into account. The method has a prediction accuracy of 77% on all mutants and 88% on breast cancer mutations affecting WAF1 promoter binding. When compared with earlier methods, using the same dataset, our method clearly performs better. As a result of the severity score calculated for every mutant, valuable knowledge can be gained regarding p53, a protein that is believed to be involved in over 50% of all human cancers.

  • 4.
    Cedersund, Gunnar
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Conclusions via unique predictions obtained despite unidentifiability - new definitions and a general method2012Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, nr 18, s. 3513-3527Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    It is often predicted that model-based data analysis will revolutionize biology, just as it has physics and engineering. A widely used tool within such analysis is hypothesis testing, which focuses on model rejections. However, the fact that a systems biology model is non-rejected is often a relatively weak statement, as such models usually are highly over-parametrized with respect to the available data, and both parameters and predictions may therefore be arbitrarily uncertain. For this reason, we formally define and analyse the concept of a core prediction. A core prediction is a uniquely identified property that must be fulfilled if the given model structure is to explain the data, even if the individual parameters are non-uniquely identified. It is shown that such a prediction is as strong a conclusion as a rejection. Furthermore, a new method for core prediction analysis is introduced, which is beneficial for the uncertainty of specific model properties, as the method only characterizes the space of acceptable parameters in the relevant directions. This avoids the curse of dimensionality associated with the generic characterizations used by previously proposed methods. Analysis on examples shows that the new method is comparable to profile likelihood with regard to practical identifiability, and thus generalizes profile likelihood to the more general problem of observability. If used, the concepts and methods presented herein make it possible to distinguish between a conclusion and a mere suggestion, which hopefully will contribute to a more justified confidence in systems biology analyses.

  • 5.
    Cedersund, Gunnar
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Roll, Jacob
    Linköpings universitet, Institutionen för systemteknik, Reglerteknik. Linköpings universitet, Tekniska högskolan.
    Systems biology: Model Based Evaluation and Comparison of Potential Explanations for Given Biological Data2009Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, nr 4, s. 903-922Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Systems biology and its usage of mathematical modeling to analyse biological data is rapidly becoming an established approach to biology. A crucial advantage of this approach is that more information can be extracted from observations of intricate dynamics, which allows nontrivial complex explanations to be evaluated and compared. In this minireview we explain this process, and review some of the most central available analysis tools. The focus is on the evaluation and comparison of given explanations for a given set of experimental data and prior knowledge. Three types of methods are discussed: (a) for evaluation of whether a given model is sufficiently able to describe the given data to be nonrejectable; (b) for evaluation of whether a slightly superior model is significantly better; and (c) for a general evaluation and comparison of the biologically interesting features in a model. The most central methods are reviewed, both in terms of underlying assumptions, including references to more advanced literature for the theoretically oriented reader, and in terms of practical guidelines and examples, for the practically oriented reader. Many of the methods are based upon analysis tools from statistics and engineering, and we emphasize that the systems biology focus on acceptable explanations puts these methods in a nonstandard setting. We highlight some associated future improvements that will be essential for future developments of model based data analysis in biology.

  • 6.
    Danielsson, Anna
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Öst, Anita
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Lystedt, Erika
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Kjölhede, Preben
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Obstetrik och gynekologi. Linköpings universitet, Hälsouniversitetet.
    Gustavsson, Johanna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Nyström, Fredrik H.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Strålfors, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Insulin resistance in human adipocytes occurs downstream of IRS1 after surgical cell isolation but at the level of phosphorylation of IRS1 in type 2 diabetes2005Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 272, nr 1, s. 141-151Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Insulin resistance is a cardinal feature of type 2 diabetes and also a consequence of trauma such as surgery. Directly after surgery and cell isolation, adipocytes were insulin resistant, but this was reversed after overnight incubation in 10% CO2 at 37 °C. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)1 was insulin sensitive, but protein kinase B (PKB) and downstream metabolic effects exhibited insulin resistance that was reversed by overnight incubation. MAP-kinases ERK1/2 and p38 were strongly phosphorylated after surgery, but was dephosphorylated during reversal of insulin resistance. Phosphorylation of MAP-kinase was not caused by collagenase treatment during cell isolation and was present also in tissue pieces that were not subjected to cell isolation procedures. The insulin resistance directly after surgery and cell isolation was different from insulin resistance of type 2 diabetes; adipocytes from patients with type 2 diabetes remained insulin resistant after overnight incubation. IRS1, PKB, and downstream metabolic effects, but not insulin-stimulated tyrosine phosphorylation of insulin receptor, exhibited insulin resistance. These findings suggest a new approach in the study of surgery-induced insulin resistance and indicate that human adipocytes should recover after surgical procedures for analysis of insulin signalling. Moreover, we pinpoint the signalling dysregulation in type 2 diabetes to be the insulin-stimulated phosphorylation of IRS1 in human adipocytes.

  • 7.
    Danø, Sune
    et al.
    Copenhagen University.
    Madsen, Mads F
    Copenhagen University.
    Schmidt, Henning
    Chalmers Technical University.
    Cedersund, Gunnar
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Reduction of a biochemical model with preservation of its basic dynamic properties.2006Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, nr 21, s. 4862-4877Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The complexity of full-scale metabolic models is a major obstacle for their effective use in computational systems biology. The aim of model reduction is to circumvent this problem by eliminating parts of a model that are unimportant for the properties of interest. The choice of reduction method is influenced both by the type of model complexity and by the objective of the reduction; therefore, no single method is superior in all cases. In this study we present a comparative study of two different methods applied to a 20D model of yeast glycolytic oscillations. Our objective is to obtain biochemically meaningful reduced models, which reproduce the dynamic properties of the 20D model. The first method uses lumping and subsequent constrained parameter optimization. The second method is a novel approach that eliminates variables not essential for the dynamics. The applications of the two methods result in models of eight (lumping), six (elimination) and three (lumping followed by elimination) dimensions. All models have similar dynamic properties and pin-point the same interactions as being crucial for generation of the oscillations. The advantage of the novel method is that it is algorithmic, and does not require input in the form of biochemical knowledge. The lumping approach, however, is better at preserving biochemical properties, as we show through extensive analyses of the models.

  • 8.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    The dynamic amyloid landscape2010Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, nr Suppl. 1, s. 14-14Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    n/a

  • 9.
    Hedlund, Joel
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
    Cantoni, Roberto
    Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, Sweden.
    Baltscheffsky, Margareta
    Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, Sweden.
    Baltscheffsky, Herrick
    Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, Sweden.
    Persson, Bengt
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik.
    Analysis of ancient sequence motifs in the H+ -PPase family2006Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, s. 5183-5193Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The unique family of membrane-bound proton-pumping inorganic pyrophosphatases, involving pyrophosphate as the alternative to ATP, was investigated by characterizing 166 members of the UniProtKB ⁄ Swiss-Prot + UniProtKB ⁄TrEMBL databases and available completed genomes, using sequence comparisons and a hidden Markov model based upon a conserved 57-residue region in the loop between transmembrane segments 5 and 6. The hidden Markov model was also used to search the approximately one million sequences recently reported from a large-scale sequencing project of organisms in the Sargasso Sea, resulting in additional 164 partial pyrophosphatase sequences. The strongly conserved 57-residue region was found to contain two nonapeptidyl sequences, mainly consisting of the four ‘very early’ proteinaceous amino acid residues Gly, Ala, Val and Asp, compatible with an ancient origin of the inorganic pyrophosphatases. The nonapeptide patterns have charged amino acid residues at positions 1, 5 and 9, are apparent binding sites for the substrate and parts of the active site, and were shown to be so specific for these enzymes that they can be used for functional assignments of unannotated genomes.

  • 10.
    Jullesson, David
    et al.
    Linköpings universitet, Institutionen för medicinsk teknik. Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Johansson, Rikard
    Linköpings universitet, Institutionen för medicinsk teknik. Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Rohini Rajan, Meenu
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Strålfors, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Cedersund, Gunnar
    Linköpings universitet, Institutionen för medicinsk teknik. Linköpings universitet, Tekniska fakulteten. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Dominant negative inhibition data should be analyzed using mathematical modeling - re-interpreting data from insulin signaling.2015Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, nr 4, s. 788-802Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    As our ability to measure the complexity of intracellular networks has evolved, it has become increasingly clear that we need new methods for data analysis: methods involving mathematical modeling. Nevertheless, it is still uncontroversial to publish and interpret experimental results without a model-based proof that the reasoning is correct. In the present study, we argue that this attitude probably needs to change in the future. We illustrate this need for modeling by considering the common experimental technique of using dominant-negative constructs. More specifically, we consider published time-series and dose-response data which previously have been used to argue that the protein S6 kinase does not phosphorylate insulin receptor substrate-1 at a specific serine residue. Using a presented general approach to interpret such data, we now demonstrate that the given dominant-negative data are not conclusive (i.e. that in the absence of other proofs, S6 kinase still may be the kinase). Using simulations with uncertainty analysis and analytical solutions, we show that an alternative explanation is centered around depletion of substrate, which can be tested experimentally. This analysis thus illustrates both the necessity and the benefits of using mathematical modeling to fully understand the implications of biological data, even for a small system and relatively simple data.

  • 11.
    Kallberg, Yvonne
    et al.
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik.
    Persson, Bengt
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik.
    Prediction of coenzyme specificity in dehydrogenases/reductases2006Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, s. 1177-1184Artikel i tidskrift (Refereegranskat)
  • 12.
    Kurz, Tino
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och vård, Farmakologi.
    Gustafsson, Bertil
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan, Patologi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk patologi och klinisk genetik.
    Brunk, Ulf
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för medicin och vård, Farmakologi.
    Intralysosomal iron chelation protects against oxidative stress-induced cellular damage2006Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, nr 13, s. 3106-3117Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Oxidant-induced cell damage may be initiated by peroxidative injury to lysosomal membranes, catalyzed by intralysosomal low mass iron that appears to comprise a major part of cellular redox-active iron. Resulting relocation of lytic enzymes and low mass iron would result in secondary harm to various cellular constituents. In an effort to further clarify this still controversial issue, we tested the protective effects of two potent iron chelators - the hydrophilic desferrioxamine (dfo) and the lipophilic salicylaldehyde isonicotinoyl hydrazone (sih), using cultured lysosome-rich macrophage-like J774 cells as targets. dfo slowly enters cells via endocytosis, while the lipophilic sih rapidly distributes throughout the cell. Following dfo treatment, long-term survival of cells cannot be investigated because dfo by itself, by remaining inside the lysosomal compartment, induces apoptosis that probably is due to iron starvation, while sih has no lasting toxic effects if the exposure time is limited. Following preincubation with 1 mm dfo for 3 h or 10 μm sih for a few minutes, both agents provided strong protection against an ensuing ∼LD50 oxidant challenge by preventing lysosomal rupture, ensuing loss of mitochondrial membrane potential, and apoptotic/necrotic cell death. It appears that once significant lysosomal rupture has occurred, the cell is irreversibly committed to death. The results lend strength to the concept that lysosomal membranes, normally exposed to redox-active iron in high concentrations, are initial targets of oxidant damage and support the idea that chelators selectively targeted to the lysosomal compartment may have therapeutic utility in diminishing oxidant-mediated cell injury. © 2006 The Authors.

  • 13.
    Lindgren, Mikael
    et al.
    Norwegian University Science and Technology.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Amyloid oligomers: spectroscopic characterization of amyloidogenic protein states2010Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, nr 6, s. 1380-1388Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    It is assumed that protein fibrils manifested in amyloidosis result from an aggregation reaction involving small misfolded protein sequences being in an oligomeric or prefibrillar state. This review covers recent optical spectroscopic studies of amyloid protein misfolding, oligomerization and amyloid fibril growth. Although amyloid fibrils have been studied using established protein-characterization techniques throughout the years, their oligomeric precursor states require sensitive detection in real-time. Here, fluorescent staining is commonly performed using thioflavin T and other small fluorescent molecules such as 4-(dicyanovinyl)- julolidine and 1-amino-8-naphtalene sulphonate that have high affinity to hydrophobic patches. Thus, populated oligomeric intermediates and related prefibrillar structures have been reported for several human amyloidogenic systems, including amyloid-beta protein, prion protein, transthyretin, alpha-synuclein, apolipoprotein C-II and insulin. To obtain information on the progression of the intermediate states, these were monitored in terms of fluorescence parameters, such as anisotropy, and quantum efficiency changes upon protein binding. Recently, new antibody stains have allowed precise monitoring of the oligomer size and distributions using multicolor labelling and single molecule detection. Moreover, a pentameric thiophene derivative (p-FTAA) was reported to indicate early precursors during A-beta(1-40) fibrillation, and was also demonstrated in real-time visualization of cerebral protein aggregates in transgenic AD mouse models by multiphoton microscopy. Conclusively, molecular probes and optical spectroscopy are now entering a phase enabling the in vivo interrogation of the role of oligomers in amyloidosis. Such techniques used in parallel with in vitro experiments, of increasing detail, will probably couple structure to pathogenesis in the near future.

  • 14.
    Nyman, Elin
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Fagerholm, Siri
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Jullesson, David
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Strålfors, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Cedersund, Gunnar
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Mechanistic explanations for counter-intuitive phosphorylation dynamics of the insulin receptor and insulin receptor substrate-1 in response to insulin in murine adipocytes2012Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, nr 6, s. 987-999Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Insulin signaling through insulin receptor (IR) and insulin receptor substrate-1 (IRS1) is important for insulin control of target cells. We have previously demonstrated a rapid and simultaneous overshoot behavior in the phosphorylation dynamics of IR and IRS1 in human adipocytes. Herein, we demonstrate that in murine adipocytes a similar overshoot behavior is not simultaneous for IR and IRS1. The peak of IRS1 phosphorylation, which is a direct consequence of the phosphorylation and the activation of IR, occurs earlier than the peak of IR phosphorylation. We used a conclusive modeling framework to unravel the mechanisms behind this counter-intuitive order of phosphorylation. Through a number of rejections, we demonstrate that two fundamentally different mechanisms may create the reversed order of peaks: (i) two pools of phosphorylated IR, where a large pool of internalized IR peaks late, but phosphorylation of IRS1 is governed by a small plasma membrane-localized pool of IR with an early peak, or (ii) inhibition of the IR-catalyzed phosphorylation of IRS1 by negative feedback. Although (i) may explain the reversed order, this two-pool hypothesis alone requires extensive internalization of IR, which is not supported by experimental data. However, with the additional assumption of limiting concentrations of IRS1, (i) can explain all data. Also, (ii) can explain all available data. Our findings illustrate how modeling can potentiate reasoning, to help draw nontrivial conclusions regarding competing mechanisms in signaling networks. Our work also reveals new differences between human and murine insulin signaling.

  • 15.
    Nyman, Elin
    et al.
    Linköpings universitet, Institutionen för medicinsk teknik. Linköpings universitet, Tekniska fakulteten. CVMD iMED DMPK AstraZeneca R&D, Mölndal, Sweden.
    Lindgren, Isa
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biologi. Linköpings universitet, Tekniska högskolan.
    Lövfors, William
    Linköpings universitet, Institutionen för medicinsk teknik. Linköpings universitet, Tekniska högskolan.
    Lundengård, Karin
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för radiologiska vetenskaper. Linköpings universitet, Hälsouniversitetet.
    Cervin, Ida
    Linköpings universitet, Institutionen för medicinsk teknik. Linköpings universitet, Tekniska högskolan.
    Arbring, Theresia
    Linköpings universitet, Institutionen för teknik och naturvetenskap, Fysik och elektroteknik. Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för medicinsk teknik.
    Altimitas, Jordi
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biologi. Linköpings universitet, Tekniska högskolan.
    Cedersund, Gunnar
    Linköpings universitet, Institutionen för medicinsk teknik. Linköpings universitet, Tekniska fakulteten. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Mathematical modeling improves EC50 estimations from classical dose–response curves2015Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, nr 5, s. 951-962Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The beta-adrenergic response is impaired in failing hearts. When studying beta-adrenergic function in vitro, the half-maximal effective concentration (EC50) is an important measure of ligand response. We previously measured the in vitro contraction force response of chicken heart tissue to increasing concentrations of adrenaline, and observed a decreasing response at high concentrations. The classical interpretation of such data is to assume a maximal response before the decrease, and to fit a sigmoid curve to the remaining data to determine EC50. Instead, we have applied a mathematical modeling approach to interpret the full dose–response curvein a new way. The developed model predicts a non-steady-state caused by a short resting time between increased concentrations of agonist, which affect the dose–response characterization. Therefore, an improved estimate of EC50 may be calculated using steady-state simulations of the model. The model-based estimation of EC50 is further refined using additional time resolved data to decrease the uncertainty of the prediction. The resulting model-based EC50 (180–525 nM) is higher than the classically interpreted EC50 (46–191 nM). Mathematical modeling thus makes it possible to reinterpret previously obtained datasets, and to make accurate estimates of EC50 even when steady-state measurements are not experimentally feasible.

  • 16.
    Ruiz-Pavon, L
    et al.
    Linnaeus University, School of Natural Sciences, Kalmar, Sweden,.
    Karlsson, Patrik
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär genetik. Linköpings universitet, Tekniska högskolan.
    Carlsson, Jonas
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
    Samyn, D
    Linnaeus University, School of Natural Sciences, Kalmar, Sweden,.
    Persson, Bengt
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
    Persson, B L
    Linnaeus University, School of Natural Sciences, Kalmar, Sweden,.
    Spetea Wiklund, Cornelia
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär genetik. Linköpings universitet, Tekniska högskolan.
    Modeling and Mutational analysis of Anion transporter 1 protein of Arabidopsis thaliana2010Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, nr Suppl. 1, s. 231-231Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    The  thylakoid   anion  transporter 1  (ANTR1)   from  Arabidopsisthaliana,  has been characterized as a Na-dependent Pi transporter when expressed in E. coli (1), but  no data  is yet available  for the protein  structure  and  amino  acids involved in transport of Pi. In this  study  a  three-dimensional structural  model  of  ANTR1  was constructed in silico using the crystal structure  of glycerol-3- phosphate/phosphate antiporter from E. coli as a template.  Based on Multiple  Sequence Alignments (MSAs) with other plant  ANT- Rs  and  mammalian   SLC17  homologues,   five  highly  conserved amino  acids involved in Pi transport have been identified,  namely Arg-120, Ser-124 and Arg-201 inside the putative translocation pathway,  Arg-228  and  Asp-382  exposed  at  the  cytoplasmic  sur- face of the protein.  The activity of the protein  as a Na-dependent Pi transporter in the wild type and mutants  was analyzed  by het- erologous  expression  and  uptake   of  radioactive   Pi  into  E.  coli cells. Substitution of the three Arg (120, 201 and 228) for Glu residues  and  of Asp-382 for  an  Asn residue  resulted  in an  inac- tive ANTR1  transporter. All other  mutants  had sufficient activity to  allow  measurement   of  kinetic  parameters, attesting   that  the mutated  proteins  were functional.  Based on  our  results,  we pro- pose that Arg-201 is a critical residue for substrate  binding and translocation, whereas Ser-124 may function  as periplasmic  gate- way for  Na+   ions.  Residue  Arg-120  plays  an  important role  in Pi  binding  and  associated   conformational  changes,  and  finally that Arg-228 and Asp-382 only weakly participate  in interactions allowing conformational changes to occur at the cytoplasmic  sur-face of the transporter.

  • 17.
    Sandin, Linnea
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Bergkvist, Liza
    Linköpings universitet, Tekniska fakulteten. Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi.
    Nath, Sangeeta
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Kielkopf, Claudia
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Janefjord, Camilla
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten.
    Helmfors, Linda
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Zetterberg, Henrik
    Clinical Neurochemistry Laboratory, Department of Neuroscience and Physiology, Sahlgrenska University Hospital, Mölndal, Sweden / UCL Institute of Neurology, London, UK.
    Blennow, Kaj
    Clinical Neurochemistry Laboratory, Department of Neuroscience and Physiology, Sahlgrenska University Hospital, Mölndal, Sweden.
    Li, Hongyun
    Illawarra Health and Medical Research Institute, University of Wollongong, Australia.
    Nilsberth, Camilla
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Närsjukvården i centrala Östergötland, Medicinska och geriatriska akutkliniken.
    Garner, Brett
    Illawarra Health and Medical Research Institute, University of Wollongong, Australia / School of Biological Sciences, University of Wollongong, Australia.
    Brorsson, Ann-Christin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Kågedal, Katarina
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Beneficial effects of increased lysozyme levels in Alzheimer’s disease modelled in Drosophila melanogaster2016Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, nr 19, s. 3508-3522Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimer’s disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome-wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aβ1-42 or AβPP together with BACE1 in the eyes, the rough eye phenotype indicative of toxicity was completely rescued by coexpression of lysozyme. In Drosophila flies bearing the Aβ1-42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aβ1-42 in the Drosophila eye was discovered. We propose that the increased levels of lysozyme, seen in mouse models of AD and in human AD cases, were triggered by Aβ1-42 and caused a beneficial effect by binding of lysozyme to toxic species of Aβ1-42, which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD.

  • 18.
    Sarg, Bettina
    et al.
    Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Austria.
    Gréen, Anna
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Söderkvist, Peter
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi.
    Helliger, Wilfried
    Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Austria.
    Rundquist, Ingemar
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi.
    Lindner, Herbert H.
    Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Austria.
    Characterization of sequence variations in human histone H1.2 and H1.4 subtypes2005Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 272, nr 14, s. 3673 -3683Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In humans, eight types of histone H1 exist (H1.1–H1.5, H1°, H1t and H1oo), all consisting of a highly conserved globular domain and less conserved N- and C-terminal tails. Although the precise functions of these isoforms are not yet understood, and H1 subtypes have been found to be dispensable for mammalian development, it is now clear that specific functions may be assigned to certain individual H1 subtypes. Moreover, microsequence variations within the isoforms, such as polymorphisms or mutations, may have biological significance because of the high degree of sequence conservation of these proteins. This study used a hydrophilic interaction liquid chromatographic method to detect sequence variants within the subtypes. Two deviations from wild-type H1 sequences were found. In K562 erythroleukemic cells, alanine at position 17 in H1.2 was replaced by valine, and, in Raji B lymphoblastoid cells, lysine at position 173 in H1.4 was replaced by arginine. We confirmed these findings by DNA sequencing of the corresponding gene segments. In K562 cells, a homozygous GCC→GTC shift was found at codon 18, giving rise to H1.2 Ala17Val because the initial methionine is removed in H1 histones. Raji cells showed a heterozygous AAA→AGA codon change at position 174 in H1.4, corresponding to the Lys173Arg substitution. The allele frequency of these sequence variants in a normal Swedish population was found to be 6.8% for the H1.2 GCC→GTC shift, indicating that this is a relatively frequent polymorphism. The AAA→AGA codon change in H1.4 was detected only in Raji cells and was not present in a normal population or in six other cell lines derived from individuals suffering from Burkitt's lymphoma. The significance of these sequence variants is unclear, but increasing evidence indicates that minor sequence variations in linker histones may change their binding characteristics, influence chromatin remodeling, and specifically affect important cellular functions.

  • 19.
    Uludag, Yildiz
    et al.
    Cranfield Health, Cranfield University, Silsoe, UK.
    Piletsky, Sergey A.
    Cranfield Health, Cranfield University, Silsoe, UK .
    Turner, Anthony P. F.
    Cranfield University, UK.
    Cooper, Matthew A.
    Akubio Ltd, Cambridge, UK.
    Piezoelectric sensors based on molecular imprinted polymers for detection of low molecular mass analytes2007Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, nr 21, s. 5471-5480Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Biomimetic recognition elements employed for the detection of analytes are commonly based on proteinaceous affibodies, immunoglobulins, single-chain and single-domain antibody fragments or aptamers. The alternative supra-molecular approach using a molecularly imprinted polymer now has proven utility in numerous applications ranging from liquid chromatography to bioassays. Despite inherent advantages compared with biochemical/biological recognition (which include robustness, storage endurance and lower costs) there are few contributions that describe quantitative analytical applications of molecularly imprinted polymers for relevant small molecular mass compounds in real-world samples. There is, however, significant literature describing the use of low-power, portable piezoelectric transducers to detect analytes in environmental monitoring and other application areas. Here we review the combination of molecularly imprinted polymers as recognition elements with piezoelectric biosensors for quantitative detection of small molecules. Analytes are classified by type and sample matrix presentation and various molecularly imprinted polymer synthetic fabrication strategies are also reviewed.

  • 20.
    Vainonen, Julia P.
    et al.
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Sakuragi, Yumiko
    Department of Plant Biology, Faculty of Life Sciences University of Copenhagen, Denmark.
    Stael, Simon
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Tikkanen, Mikko
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Allahverdiyeva, Yagut
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Paakkarinen, Virpi
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Aro, Eveliina
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Suorsa, Marjaana
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Scheller, Henrik V.
    Department of Plant Biology, Faculty of Life Sciences University of Copenhagen, Denmark.
    Vener, Alexander
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi.
    Aro, Eva-Mari
    Department of Biology, Plant Physiology and Molecular Biology University of Turku, Finland.
    Light regulation of CaS, a novel phosphoprotein in the thylakoid membrane of Arabidopsis thaliana2008Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, nr 8, s. 1767-1777Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Exposure of Arabidopsis thaliana plants to high levels of light revealed specific phosphorylation of a 40 kDa protein in photosynthetic thylakoid membranes. The protein was identified by MS as extracellular calcium-sensing receptor (CaS), previously reported to be located in the plasma membrane. By confocal laser scanning microscopy and subcellular fractionation, it was demonstrated that CaS localizes to the chloroplasts and is enriched in stroma thylakoids. The phosphorylation level of CaS responded strongly to light intensity. The light-dependent thylakoid protein kinase STN8 is required for CaS phosphorylation. The phosphorylation site was mapped to the stroma-exposed Thr380, located in a motif for interaction with 14-3-3 proteins and proteins with forkhead-associated domains, which suggests the involvement of CaS in stress responses and signaling pathways. The knockout Arabidopsis lines revealed a significant role for CaS in plant growth and development. © 2008 The Authors.

  • 21.
    Viitanen, Lenita
    et al.
    Department of Biochemistry and Pharmacy ° bo Akademi University, Turku, Finland.
    Nylund, Matts
    Department of Biochemistry and Pharmacy ° bo Akademi University, Turku, Finland.
    Eklund, D. Magnus
    Department of Plant Biology and Forest Genetics Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Alm, Christina
    Department of Plant Biology and Forest Genetics Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Eriksson, Ann-Katrin
    Department of Plant Biology and Forest Genetics Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Tuuf, Jessica
    Department of Biochemistry and Pharmacy A ° bo Akademi University, Turku, Finland.
    Salminen, Tiina A.
    Department of Biochemistry and Pharmacy ° bo Akademi University, Turku, Finland.
    Mattjus, Peter
    Department of Biochemistry and Pharmacy ° bo Akademi University, Turku, Finland.
    Edqvist, Johan
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Characterization of SCP-2 from Euphorbia lagascae reveals that a single Leu/Met exchange enhances sterol transfer activity2006Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, s. 5641-5655Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sterol carrier protein-2 (SCP-2) is a small intracellular basic protein domain implicated in peroxisomal beta-oxidation. We extend our knowledge of plant SCP-2 by characterizing SCP-2 from Euphorbia lagascae. This protein consists of 122 amino acids including a PTS1 peroxisomal targeting signal. It has a molecular mass of 13.6 kDa and a pI of 9.5. It shares 67% identity and 84% similarity with SCP-2 from Arabidopsis thaliana. Proteomic analysis revealed that E. lagascae SCP-2 accumulates in the endosperm during seed germination. It showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC). The transfer of BODIPY-PC was almost completely inhibited after addition of phosphatidylinositol, palmitic acid, stearoyl-CoA and vernolic acid, whereas sterols only had a very marginal inhibitory effect. We used protein modelling and site-directed mutagenesis to investigate why the BODIPY-PC transfer mediated by E. lagascae SCP-2 is not sensitive to sterols, whereas the transfer mediated by A. thaliana SCP-2 shows sterol sensitivity. Protein modelling suggested that the ligand-binding cavity of A. thaliana SCP-2 has four methionines (Met12, 14, 15 and 100), which are replaced by leucines (Leu11, 13, 14 and 99) in E. lagascae SCP-2. Changing Leu99 to Met99 was sufficient to convert E. lagascae SCP-2 into a sterol-sensitive BODIPY-PC-transfer protein, and correspondingly, changing Met100 to Leu100 abolished the sterol sensitivity of A. thaliana SCP-2.

  • 22.
    Wennerstrand, Patricia
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Mårtensson, Lars-Göran
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Dametto, P
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Hennig, Janosch
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Skoglund, Karin
    Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet.
    Peterson, Curt
    Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Onkologiska kliniken US.
    Different mechanisms behind low enzyme activity in vivo of two different variants of Thiopurine S-methyltransferase, TPMT2010Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, nr Suppl. 1, s. 257-258Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    In treatment of acute lymphoblastic leukemia and inflammatorybowel disease (IBD) thiopurines such as azathioprine and 6-mercaptopurineare used. All of these drugs are prodrugs and are, inthe cell, converted to 6-thioguanines (6-TGNs) and incorporatedinto DNA or inhibiting purine synthesis. A key enzyme for thisregulation is the cytosolic enzyme thiopurine S-methyltransferase(TPMT). This enzyme degrades azathioprine and 6-mercaptopurineto methylmercapto-purine and thereby reduces the bioavailabilityof the 6-TGNs incorporated into DNA. TPMT is apolymorphic enzyme with at least 29 different allelic variantsknown today and is one of the more classical examples of pharmacogeneticswhere the TPMT enzyme activity of the allelic variantsis directly correlated to the clinical dosages of the thiopurines, with a 10–15 fold dosage reduction for an allelic variantwith low TPMT enzyme activity. Even though TPMT is awell studied protein. Many studies have been performed in yeast‘‘suspensions’’ and not on pure protein solutions. It has beenspeculated and in a few cases shown that the reason for the lowactivity for most of the allelic variants is mainly due to the lowstability and/or tendency to aggregate. The mutations in thisstudy TPMT *2 (A80P) and TPMT * 5 (L49S) are both situatedat a distance far from the active site, however the enzyme activitiesare severely affected at 37°C. Preliminary results, using a repertoireof techniques such as CD, fluorescence and limitedproteolysis experiments suggest two different mechanisms for thelow enzyme activity at a temperature corresponding to in vivo conditions.

  • 23. West, Gun
    et al.
    Viitanen, Lenita
    Alm, Christina
    Mattjus, Peter
    Salminen, Tiina A.
    Edqvist, Johan
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär genetik.
    Identification of a glycosphingolipid transfer protein GLTP1 in Arabidopsis thaliana2008Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, nr 13, s. 3421-3437Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Arabidopsis thaliana At2g33470 encodes a glycolipid transfer protein (GLTP) that enhances the intervesicular trafficking of glycosphingolipids in vitro. GLTPs have previously been identified in animals and fungi but not in plants. Thus, At2g33470 is the first identified plant GLTP and we have designated it AtGTLP1. AtGLTP1 transferred BODIPY-glucosylceramide at a rate of 0.7 pmol·s-1, but BODIPY-galactosylceramide and BODIPY-lactosylceramide were transferred slowly, with rates below 0.1 pmol·s-1. AtGLTP1 did not transfer BODIPY-sphingomyelin, monogalactosyldiacylglycerol or digalactosyldiacylglycerol. The human GLTP transfers BODIPY-glucosylceramide, BODIPY-galactosylceramide and BODIPY-lactosylceramide with rates greater than 0.8 pmol·s-1. Structural models showed that the residues that are most critical for glycosphingolipid binding in human GLTP are conserved in AtGLTP1, but some of the sugar-binding residues are unique, and this provides an explanation for the distinctly different transfer preferences of AtGLTP1 and human GLTP. The AtGLTP1 variant Arg59Lys/Asn95Leu showed low BODIPY-glucosylceramide transfer activity, indicating that Arg59 and/or Asn95 are important for the specific binding of glucosylceramide to AtGLTP1. We also show that, in A. thaliana, AtGLTP1 together with At1g21360 and At3g21260 constitute a small gene family orthologous to the mammalian GLTPs. However, At1g21360 and At3g21260 did not transfer any of the tested lipids in vitro. © 2008 The Authors.

  • 24.
    Yin, Lan
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär genetik. Linköpings universitet, Tekniska högskolan.
    Karlsson, Patrik
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär genetik. Linköpings universitet, Tekniska högskolan.
    Spetea Wiklund, Cornelia
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär genetik. Linköpings universitet, Tekniska högskolan.
    Aro, E-M
    University of Turku, Turku, Finland.
    Schoefs, B
    Université de Bourgogne, Dijon, France.
    Chloroplast thylakoid transporters2010Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, nr Suppl. 1, s. 231-231Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    The aim of this study is to identify and functionally characterizesolute transporters from the chloroplast thylakoid membrane ofArabidopsis thaliana. As compared to chloroplast envelope transporters,much less information is available for transport processesacross the thylakoid membrane, which is mostly studied as thesite of light-driven photosynthetic reactions coupled to ATP synthesis.Although there are many reported examples of transportactivities, only a few thylakoid transporters have been identifiedat the gene level. Using bioinformatics analyses, we have predictedthe existence of approx. fifteen thylakoid transporters. Forexperimental validation, we have carried out immuno-localizationstudies used peptide-specific antibodies, functional analyses inheterologous system and validation using knockout mutants. Wehave recently identified one ATP/ADP carrier (Thuswaldneret al. JBC 2007) and one Na(+)-dependent phosphate transporter(Ruiz Pavon et al. JBC 2008). They are proposed to participatein the nucleotide metabolism in the thylakoid lumen(Spetea et al. PNAS 20004) as well as to balance the transthylakodproton electrochemical gradient storage. Based on phenotypicanalyses of knockout mutants, we will present novel dataabout the key physiological role of the two transporters duringthe high-light-induced repair of photosystem II complex in thethylakoid membrane. Subsequently, we will make a survey on theoutlook of thylakoid activities awaiting identification of responsibleproteins. Such knowledge is necessary to understand the thylakoidnetwork of transporters, and its role in photosynthesisand adaptation to environmental stress.

  • 25.
    Zako, T.
    et al.
    Bioengineering Laboratory, RIKEN Institute, Saitama, Japan.
    Kobayashi, T.
    Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.
    Sakono, M.
    Bioengineering Laboratory, RIKEN Institute, Saitama, Japan.
    Lindgren, M.
    Department of Physics, The Norwegian University of Science and Technology, Trondheim, Norway.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Maeda, M.
    Bioengineering Laboratory, RIKEN Institute, Saitama, Japan.
    Structure and cytotoxicity of novel insulin noodle-like filamentous amyloids2009Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, nr Suppl. s1, s. 173-173Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    Insulin is a small peptide hormone that is known to form proteinassembly called amyloid fibrils under acidic conditions. We havepreviously shown that filamentous (‘noodle’-like) insulin amyloidwhich was morphologically different from fibrous (‘needle’-like)insulin amyloid was formed in the presence of a reducing agent,tris (2-carboxyethyl) phosphine hydrochloride (TCEP). The CDspectra showed that both of insulin fibrils and filaments containa beta-sheet structure. Nevertheless, Thioflavin T (ThT) bindingproperty was very different between them, suggesting a differencein inner structure. In this study, we examined their cell toxicitiesusing two different cell lines with MTT assay, and also examineddifference in their inner structures using novel luminescent conjugatedpolyelectrolyte probes (LCPs)1–4. The cytotoxicity of theinsulin filaments against rat PC12 and human HEK293 cell linewas also extremely low while the fibrils were toxic, suggestingthat the insulin filaments were generally nontoxic. This findingsupports the idea that cell toxicity of amyloids correlates withtheir morphology. The fluorescence measurement in the presenceof Polythiophene acetic acid (PTAA)1–4, one of the conformationsensitive LCPs, showed that PTAA weakly bound to the insulinfilaments and that the insulin fibrils’ spectrum revealed a spectral red shift in comparison to the PTAA-filaments interaction. Thissuggests that the insulin ‘noodle’-like filaments are formed byloose assembly of insulin molecules.

    References:

    1. Nilsson et al., Adv Mat 2008; 20: 2639.

    2. Chem Bio Chem 2006; 7:1096.

    3. ACS Chem Biol 2007; 4: 553.

    4. Sigurdson et al., Nature Methods 2007; 4: 1023.

  • 26.
    Örtegren Kugelberg, Unn
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Yin, Lan
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Öst, Anita
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Karlsson, Helen
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Yrkes- och miljömedicin. Linköpings universitet, Hälsouniversitetet.
    Nyström, Fredrik
    Linköpings universitet, Institutionen för medicin och vård, Internmedicin. Linköpings universitet, Hälsouniversitetet.
    Strålfors, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Separation and characterization of caveolae subclasses in the plasma membrane of primary adipocytes: segregation of specific proteins and functions2006Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, nr 14, s. 3381-3392Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Caveolae are nearly ubiquitous plasma membrane domains that in adipocytes vary in size between 25 and 150 nm. They constitute sites of entry into the cell as well as platforms for cell signalling. We have previously reported that plasma membrane-associated caveolae that lack cell surface access can be identified by electron microscopy. We now report the identification, after density gradient ultracentrifugation, of a subclass of very high-density apparently closed caveolae that were not labelled by cell surface protein labelling of intact cells. These caveolae contained caveolin-1 and caveolin-2. Another class of high-density caveolae contained caveolin-1, caveolin-2 and specifically fatty acid transport protein-1, fatty acid transport protein-4, fatty acyl-CoA synthetase, hormone-sensitive lipase, perilipin, and insulin-regulated glucose transporter-4. This class of caveolae was specialized in fatty acid uptake and conversion to triacylglycerol. A third class of low-density caveolae contained the insulin receptor, class B scavenger receptor-1, and insulin-regulated glucose transporter-4. Small amounts of these proteins were also detected in the high-density caveolae. In response to insulin, the insulin receptor autophosphorylation and the amount of insulin-regulated glucose transporter-4 increased in these caveolae. The molar ratio of cholesterol to phospholipid in the three caveolae classes varied considerably, from 0.4 in very high-density caveolae to 0.9 in low-density caveolae. There was no correlation between the caveolar contents of caveolin and cholesterol. The low-density caveolae, with the highest cholesterol concentration, were particularly enriched with the cholesterol-rich lipoprotein receptor class B scavenger receptor-1, which mediated cholesteryl ester uptake from high-density lipoprotein and generation of free cholesterol in these caveolae, suggesting a specific role in cholesterol uptake/metabolism. These findings demonstrate a segregation of functions in caveolae subclasses.

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