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  • 1.
    Aronsson, Henrik
    et al.
    Department of Plant Physiology, Göteborg University, Göteborg, Sweden.
    Sundqvist, Christer
    Department of Plant Physiology, Göteborg University, Göteborg, Sweden.
    Timko, Michael P.
    Department of Biology, University of Virginia, Charlottesville, United States.
    Dahlin, Clas
    Halmstad University, School of Business, Engineering and Science, Biological and Environmental Systems (BLESS).
    Characterisation of the assembly pathway of the pea NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR), with emphasis on the role of its substrate, Pchlide2001In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 111, no 2, p. 239-244Article in journal (Refereed)
    Abstract [en]

    The homologous import and membrane association of a key enzyme for chlorophyll biosynthesis, the NADPH:protochlorophyllide (Pchlide) oxidoreductase (PAR, EC 1.6.99.1) into pea chloroplasts was investigated in vitro. The co-factor, NADPH, decreased binding of the precursor protein (pPOR) to the envelope membranes in the presence of ATP. The decrease of the binding reaction with NADPH was not observed with the precursor of the small subunit of Rubisco (pSS). To investigate possible substrate-dependency for the import reaction, internal Pchlide concentrations in the plastids were raised by either an addition of ÎŽ-aminolevulinic acid to isolated plastids or etiolation of the seedlings prior to plastid isolation. Increased amounts of plastid-bound Pchlide gave no observable differences in POR import. The capacity of POR and 11 different POR mutants, carrying charged-to-alanine scanning substitutions, to form a catalytically active POR-Pchlide-NADPH complex and to associate with the thylakoid membranes in a protease-resistant way were tested. Wild-type POR, as well as the mutants with charge substitutions in the N-terminal region of the protein, exhibited higher catalytic activity than the POR mutants carrying substitutions in the C-terminal region. Formation of a catalytically active complex did not, however, increase the association efficiency onto the thylakoids. We can, therefore, postulate that the import of pea POR into pea chloroplasts was not substrate-dependent, nor did formation of catalytically active complexes stimulate or inhibit the membrane association reaction of POR.

  • 2.
    Farkas, Daniel
    et al.
    Institutionen för kemi, Göteborgs Universitet, Department of Chemistry, University of Gothenburg.
    Franzén, Lars-Gunnar
    Halmstad University, School of Business and Engineering (SET), Biological and Environmental Systems (BLESS), Plant Cell Biology: Energy transduction in plant cells.
    Hansson, Örjan
    Institutionen för kemi, Göteborgs Universitet, Department of Chemistry, University of Gothenburg.
    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 folding2011In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 78, no 2, p. 156-166Article in journal (Refereed)
    Abstract [en]

    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.

  • 3.
    Funes, Soledad
    et al.
    Institut für Physiologische Chemie, Ludwig-Maximilians-Universität München, Germany.
    Franzén, Lars-Gunnar
    Halmstad University, School of Business and Engineering (SET), Biological and Environmental Systems (BLESS), Plant Cell Biology: Energy transduction in plant cells.
    González-Halphen, Diego
    Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, D.F., México.
    Chlamydomonas reinhardtii: the model of choice to study mitochondria from unicellular photosynthetic organisms.2007In: Methods in Molecular Biology, ISSN 1064-3745, Vol. 372, p. 137-149Article in journal (Other academic)
    Abstract [en]

    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.

  • 4.
    Jakobs, Kristin
    Halmstad University, School of Business and Engineering (SET). Halmstad University, School of Business and Engineering (SET), Biological and Environmental Systems (BLESS), Biomechanics and Biomedicine.
    ­Hur påverkar dietärt nitrat muskelfunktionen och återhämtningen vid styrketräning?: En pilotstudie i samarbete med Karolinska Institutet och Åstrands laboratoriet.2011Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Research on human physiology and how it is working is updated daily. In the world of sports they are testing new as old, natural as unnatural preparations and different training methods continuously in order to optimize athletic performance. A substance that´s been research on, up till today is nitric oxide and its influence in the body. From being interpreted as a harmful substance in the body, it went to possibly help heart disease patients, and also optimize the physic in sport performance. Nitric oxide is formed in the body naturally by oxygen, but it can also be formed without oxygen through the ingestion of nitrates found in many vegetables. Studies on nitrate in the sport field have concentrated on the effect on endurance sports and the effect has been shown to increase the efficiency and the blood flow to the muscles. Later on they also found that nitrate supplementation seems to give a lower Vo2max together with an increased time to exhaustion. These findings are really interesting because normally a reduction in Vo2max leads us to a decrease in workability. All these studies give an idea on how nitrate works aerobic, that is with oxygen. The research has not yet an explanation on how nitrate affect anaerobic work and maximum performance that occurs in weight training.

    The purpose of this study was to investigate how nitrate affects muscle function and endurance in strength training. In a randomized, double-blind, crossover trial, eight men (age 19-26, 23 (±2, 3)) consumed nitrate or a placebo (0.1 mmol/kg bodyweight/day) for three days. During the fourth day the test persons were tested in four different strength tests to see how they performed. Lactate and glucose concentrations were measured to see how the laktacid system was influenced.

    The study gave no support that dietary nitrate affects weight training. The results from the occasion with nitrate respectively placebo remained essentially unchanged.

    It was concluded that an intake of nitrate not will give any significant effects on the model of strength training. The main reason for this may be that nitrate provides the greatest impact on long-term work-duration and mainly during aerobic work. In this case the main use is mostly stored energy in the body, and the energy systems in which oxygen is required will probably not be of major importance.

  • 5.
    Rögnvaldsson, Thorsteinn
    et al.
    Halmstad University, School of Information Science, Computer and Electrical Engineering (IDE), Halmstad Embedded and Intelligent Systems Research (EIS).
    Häkkinen, Jari
    Department of Theoretical Physics, Lund University, Sölvegatan 14A, SE-223 62 Lund, Sweden.
    Lindberg, Claes
    Molecular Sciences, AstraZeneca RandD Lund, SE-221 87 Lund, Sweden.
    Marko-Varga, György
    Molecular Sciences, AstraZeneca RandD Lund, SE-221 87 Lund, Sweden.
    Potthast, Frank
    Funct. Genomics Center Zürich, Winterthurerstr. 190 Y32 H52, CH-8057 Zürich, Switzerland.
    Samuelsson, Jim
    Genedata GmbH, Lena-Christ-Str. 50, D-82152 Martinsried, Germany.
    Improving automatic peptide mass fingerprint protein identification by combining many peak sets2004In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 807, no 2, p. 209-215Article in journal (Refereed)
    Abstract [en]

    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.

  • 6.
    Rögnvaldsson, Thorsteinn
    et al.
    Halmstad University, School of Information Technology, Halmstad Embedded and Intelligent Systems Research (EIS), Intelligent systems (IS-lab).
    You, Liwen
    Lund University, Department of Theoretical Physics, Lund, Sweden.
    Why neural networks should not be used for HIV-1 protease cleavage site prediction2004In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 20, no 11, p. 1702-1709Article in journal (Refereed)
    Abstract [en]

    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.

  • 7.
    Teneberg, Susanne
    et al.
    Halmstad University, School of Information Technology. Institute of Medical Biochemistry, Göteborg University, P. O. Box 440, SE 405 30 Göteborg, Sweden.
    Leonardsson, I.
    Department of Clinical Microbiology and Immunology, Örebro Medical Centre Hospital, SE 701 85 Örebro, Sweden.
    Karlsson, H.
    Department of General Surgery, Örebro Medical Centre Hospital, SE 701 85 Örebro, Sweden.
    Jovall, P.-A.
    Department of Medical Microbiology, Dermatology, and Infection, University of Lund, SE 223 62 Lund, Sweden.
    Ångström, J.
    Institute of Medical Biochemistry, Göteborg University, P. O. Box 440, SE 405 30 Göteborg, Sweden.
    Danielsson, D.
    Institute of Medical Biochemistry, Göteborg University, P. O. Box 440, SE 405 30 Göteborg, Sweden.
    Näslund, I.
    Institute of Medical Biochemistry, Göteborg University, P. O. Box 440, SE 405 30 Göteborg, Sweden.
    Ljungh, A.
    Institute of Medical Biochemistry, Göteborg University, P. O. Box 440, SE 405 30 Göteborg, Sweden.
    Wadström, T.
    Institute of Medical Biochemistry, Göteborg University, P. O. Box 440, SE 405 30 Göteborg, Sweden.
    Karlsson, K.-A
    Institute of Medical Biochemistry, Göteborg University, P. O. Box 440, SE 405 30 Göteborg, Sweden.
    Lactotetraosylceramide, a novel glycosphingolipid receptor for Helicobacter pylori, present in human gastric epithelium2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 22, p. 19709-19719Article in journal (Refereed)
    Abstract [en]

    The binding of Helicobacterpylori 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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