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  • 1.
    Aasa, Jenny
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Motwani, Hitesh V.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Törnqvist, Margareta
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Quantification of the mutagenic potency and repair of glycidol-induced DNA lesions2016In: Mutation research. Genetic toxicology and environmental mutagenesis, ISSN 1383-5718, E-ISSN 1879-3592, Vol. 805, 38-45 p.Article in journal (Refereed)
    Abstract [en]

    Glycidol (Gly) is an electrophilic low-molecular weight epoxide that is classified by IARC as probably carcinogenic to humans. Humans might be exposed to Gly from food, e.g. refined vegetable oils, where Gly has been found as a food process contaminant. It is therefore important to investigate and quantify the genotoxicity of Gly as a primary step towards cancer risk assessment of the human exposure. Here, quantification of the mutagenic potency expressed per dose (AUC: area under the concentration time curve) of Gly has been performed in Chinese hamster ovary (CHO) cells, using the HPRT assay. The dose of Gly was estimated in the cell exposure medium by trapping Gly with a strong nucleophile, cob(I)alamin, to form stable cobalamin adducts for analysis by LC-MS/MS. Gly was stable in the exposure medium during the time for cell treatment, and thus the dose in vitro is the initial concentration x cell treatment time. Gly induced mutations in the hprt-gene at ante of 0.08 +/- 0:01 mutations/10(5) cells/mMh. Through comparison with the effect of ionizing radiation in the same system a relative mutagenic potency of 9.5 rad-eq./mMh was obtained, which could be used for comparison of genotoxicity of chemicals and between test systems and also in procedures for quantitative cancer risk assessment. Gly was shown to induce strand breaks, that were repaired by base excision repair. Furthermore, Gly-induced lesions, present during replication, were found to delay the replication fork elongation. From experiments with repair deficient cells, homologous recombination repair and the ERCC1-XPF complex were indicated to be recruited to support in the repair of the damage related to the stalled replication elongation. The type of DNA damage responsible for the mutagenic effect of Gly could not be concluded from the present study.

  • 2.
    Abdel Rehim, Abbi
    et al.
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Abdel Rehim, Mohamed
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Screening and determination of drugs in human saliva utilizing microextraction by packed sorbent and liquid chromatography-tandem mass spectrometry2013In: BMC Biomedical chromotography, ISSN 0269-3879, E-ISSN 1099-0801, Vol. 27, no 9, 1188-1191 p.Article in journal (Refereed)
    Abstract [en]

    This study presents a new method for collecting and handling saliva samples using an automated analytical microsyringe and microextraction by packed syringe (MEPS). The screening and determination of lidocaine in human saliva samples utilizing MEPS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were carried out. An exact volume of saliva could be collected. The MEPS C-8-cartridge could be used for 50 extractions before it was discarded. The extraction recovery was about 60%. The pharmacokinetic curve of lidocaine in saliva using MEPS-LC-MS/MS is reported.

  • 3.
    Abdel-Rehim, Abbi
    et al.
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Abdel-Rehim, Mohamed
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Evaluation of microextraction by packed sorbent and micro-liquid chromatography-tandem mass spectrometry as a green approach in bioanalysis2013In: BMC Biomedical chromotography, ISSN 0269-3879, E-ISSN 1099-0801, Vol. 27, no 10, 1225-1233 p.Article in journal (Refereed)
    Abstract [en]

    In this study the use of micro-liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was investigated in routine bioanalysis application for separation and quantification of pro-drug AZD6319 (developed for aldezheimer treatment). Microextraction by packed sorbent (MEPS) was used as sample clean-up method. The focus of this study was put on the evaluation of the usability of smaller column diameters such as 1.0 and 0.3mm instead of 2.1mm in bioanalysis application to reduce solvent consumption and sample volumes. Solvent consumption was reduced by 80% when a 1.0mm column was used compared with 2.1mm column. Robustness of the micro-columns in terms of accuracy and precision was investigated. The application of LC-MS/MS for the quantitative analysis of AZD6319 in plasma samples showed good selectivity, accuracy and precision. The coefficients of determination (R-2) were >0.998 for all runs using plasma samples on the studied micro-columns. The inter-day accuracy values for quality control samples ranged from 99 to 103% and from 96 to 105% for 0.3x50mm and 1.0x50mm columns, respectively. The inter-day precision values ranged from 4.0 to 9.0% and from 4.0 to 8.0% for 0.3x50 and 1.0x50mm columns, respectively. In addition the sensitivity was increased by three times using a 1.0mm column compared with 2.1mm. Furthermore, robustness of the micro-columns from different manufacturers was investigated.

  • 4.
    Abedi-Valugerdi, Manuchehr
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mercury and silver induce B cell activation and anti-nucleolar autoantibody production in outbred mouse stocks: are environmental factors more important than the susceptibility genes in connection with autoimmunity?2009In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 155, no 1, 117-124 p.Article in journal (Refereed)
    Abstract [en]

    Environmental and predisposing genetic factors are known to play a crucial role in the development of systemic autoimmune diseases. With respect to the role of environmental factors, it is not known how and to what extent they contribute to the initiation and exacerbation of systemic autoimmunity. In the present study, I considered this issue and asked if environmental factors can induce autoimmunity in the absence of specific susceptible genes. The development of genetically controlled mercury- and silver-induced B cell activation and anti-nucleolar autoantibodies (ANolA) production in genetically heterozygous outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mouse stocks were analysed. Four weeks of treatment with both mercury and silver induced a strong B cell activation characterized by increased numbers of splenic antibody-secreting cells of at least one or more immunoglobulin (Ig) isotype(s) in all treated stocks. The three stocks also exhibited a marked increase in the serum IgE levels in response to mercury, but not silver. More importantly, in response to mercury a large numbers of ICR (88%), NMRI (96%) and Black Swiss (100%) mice produced different levels of IgG1 and IgG2a ANolA (a characteristic which is linked strictly to the H-2 genes). Similarly, but at lower magnitudes, treatment with silver also induced the production of IgG1 and IgG2a ANolA in 60% of ICR, 75% of NMRI and 100% of Black Swiss mice. Thus, the findings of this study suggest that long-term exposure to certain environmental factors can activate the immune system to produce autoimmunity per se, without requiring specific susceptible genes.

  • 5.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abrahams, Jan Pieter
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jarvet, Juri
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. National Institute of Chemical Physics and Biophysics, Estonia.
    Luo, Jinghui
    Tiiman, Ann
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wärmländer, Sebastian K. T. S.
    The hairpin conformation of the amyloid beta peptide is an important structural motif along the aggregation pathway2014In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 19, no 4-5, 623-634 p.Article, review/survey (Refereed)
    Abstract [en]

    The amyloid beta (A beta) peptides are 39-42 residue-long peptides found in the senile plaques in the brains of Alzheimer's disease (AD) patients. These peptides self-aggregate in aqueous solution, going from soluble and mainly unstructured monomers to insoluble ordered fibrils. The aggregation process(es) are strongly influenced by environmental conditions. Several lines of evidence indicate that the neurotoxic species are the intermediate oligomeric states appearing along the aggregation pathways. This minireview summarizes recent findings, mainly based on solution and solid-state NMR experiments and electron microscopy, which investigate the molecular structures and characteristics of the A beta peptides at different stages along the aggregation pathways. We conclude that a hairpin-like conformation constitutes a common motif for the A beta peptides in most of the described structures. There are certain variations in different hairpin conformations, for example regarding H-bonding partners, which could be one reason for the molecular heterogeneity observed in the aggregated systems. Interacting hairpins are the building blocks of the insoluble fibrils, again with variations in how hairpins are organized in the cross-section of the fibril, perpendicular to the fibril axis. The secondary structure propensities can be seen already in peptide monomers in solution. Unfortunately, detailed structural information about the intermediate oligomeric states is presently not available. In the review, special attention is given to metal ion interactions, particularly the binding constants and ligand structures of A beta complexes with Cu(II) and Zn(II), since these ions affect the aggregation process(es) and are considered to be involved in the molecular mechanisms underlying AD pathology.

  • 6.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bolognesi, Benedetta
    Dobson, Christopher M.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lendel, Christofer
    Hydrophobicity and conformational change as mechanistic determinants for nonspecific modulators of amyloid β self-assembly2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 1, 126-137 p.Article in journal (Refereed)
    Abstract [en]

    The link between many neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid β-peptide (Aβ) associated with Alzheimer's disease. We show that lacmoid binds Aβ(1-40) in a surfactant-like manner and counteracts the formation of all types of Aβ(1-40) and Aβ(1-42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide.

  • 7.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kaspersen, Jørn Døvling
    Nielsen, Søren Bang
    Jensen, Grethe Vestergaard
    Christiansen, Gunna
    Pedersen, Jan Skov
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Otzen, Daniel E.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Formation of dynamic soluble surfactant-induced amyloid β peptide aggregation intermediates2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 32, 23518-23528 p.Article in journal (Refereed)
    Abstract [en]

    Intermediate amyloidogenic states along the amyloid β peptide (Aβ) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aβ aggregation, we here investigate surfactant-induced Aβ aggregation. This process leads to co-aggregates featuring a β-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aβ in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via β-structure to the fully formed α-helical state at high surfactant concentration. The β-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aβ co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (k(ex) ∼1100 s(-1)) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with β-structure promoting substances, such as surfactants, Aβ is kinetically driven toward an aggregation-prone state.

  • 8.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lendel, Christofer
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Transient small molecule interactions kinetically modulate amyloid beta peptide self-assembly2012In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 586, no 22, 3991-3995 p.Article in journal (Refereed)
    Abstract [en]

    Small organic molecules, like Congo red and lacmoid, have been shown to modulate the self-assembly of the amyloid beta peptide (A beta). Here, we show that A beta forms NMR invisible non-toxic co-aggregates together with lacmoid as well as Congo red. We find that the interaction involves two distinct kinetic processes and at every given time point only a small fraction of A beta is in the co-aggregate. These weak transient interactions kinetically redirect the aggregation prone A beta from self-assembling into amyloid fibrils. These findings suggest that even such weak binders might be effective as therapeutics against pathogenic protein aggregation.

  • 9.
    Abreu-Vieira, Gustavo
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. National Institute of Diabetes and Digestive and Kidney Diseases, NIH, USA.
    Xiao, Cuiying
    Gavrilova, Oksana
    Reitman, Marc L.
    Integration of body temperature into the analysis of energy expenditure in the mouse2015In: Molecular Metabolism, ISSN 2212-8778, Vol. 4, no 6, 461-470 p.Article in journal (Refereed)
    Abstract [en]

    Objectives: We quantified the effect of environmental temperature on mouse energy homeostasis and body temperature.Methods: The effect of environmental temperature (4e33 C) on body temperature, energy expenditure, physical activity, and food intake invarious mice (chow diet, high-fat diet, Brs3-/y, lipodystrophic) was measured using continuous monitoring.Results: Body temperature depended most on circadian phase and physical activity, but also on environmental temperature. The amounts ofenergy expenditure due to basal metabolic rate (calculated via a novel method), thermic effect of food, physical activity, and cold-inducedthermogenesis were determined as a function of environmental temperature. The measured resting defended body temperature matchedthat calculated from the energy expenditure using Fourier’s law of heat conduction. Mice defended a higher body temperature during physicalactivity. The cost of the warmer body temperature during the active phase is 4e16% of total daily energy expenditure. Parameters measured indiet-induced obese and Brs3-/y mice were similar to controls. The high post-mortem heat conductance demonstrates that most insulation in miceis via physiological mechanisms.Conclusions: At 22 C, cold-induced thermogenesis isw120% of basal metabolic rate. The higher body temperature during physical activity isdue to a higher set point, not simply increased heat generation during exercise. Most insulation in mice is via physiological mechanisms, with littlefrom fur or fat. Our analysis suggests that the definition of the upper limit of the thermoneutral zone should be re-considered. Measuring bodytemperature informs interpretation of energy expenditure data and improves the predictiveness and utility of the mouse to model human energyhomeostasis.

  • 10. Adase, Christopher A.
    et al.
    Draheim, Roger R.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Goethe University .
    Rueda, Garrett
    Desai, Raj
    Manson, Michael D.
    Residues at the Cytoplasmic End of Transmembrane Helix 2 Determine the Signal Output of the Tar(Ec) Chemoreceptor2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 16, 2729-2738 p.Article in journal (Refereed)
    Abstract [en]

    Baseline signal output and communication between the periplasmic and cytoplasmic domains of the Escherichia colt aspartate chemoreceptor Tar(Ec) are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). In particular, the cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in wild type Tar(Ec) is important for determining the CheA kinase-stimulating activity of the receptor and its ability to respond to chemoeffector-induced stimuli. Here, we have studied the effect on Tar(Ec) function of the six residue sequence at positions 207-212 Moving various combinations of aromatic residues among these positions generates substantial changes M receptor activity. Trp has the largest effect on function, both in maintaining normal activity and in altering activity when it is moved. Tyr has a weaker effect, and Phe has the weakest; however, all three aromatic residues can alter signal output when they are placed in novel positions. We also find that Gly-211 plays an important role in receptor function, perhaps because of the flexibility it introduces into the TM2-HAMP domain connector. The conservation of this Gly residue in the high-abundance chemoreceptors of E. coli and Salmonella enterica suggests that it may be important for the nuanced, bidirectional transmembrane signaling that occurs in these proteins.

  • 11.
    Adlerz, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Holback, Sofia
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Multhaup, Gerd
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    IGF-1-induced Processing of the Amyloid Precursor Protein Family Is Mediated by Different Signaling Pathways2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 14, 10203-10209 p.Article in journal (Refereed)
    Abstract [en]

    The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid beta-peptide (Abeta) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of Abeta. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of Abeta in the brain. In the present study, we show that IGF-1 increases alpha-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1.

  • 12. Ahn, Young O.
    et al.
    Mahinthichaichan, Paween
    Lee, Hyun Ju
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ouyang, Hanlin
    Kaluka, Daniel
    Yeh, Syun-Ru
    Arjona, Davinia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rousseau, Denis L.
    Tajkhorshid, Emad
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B.
    Conformational coupling between the active site and residues within the K-C-channel of the Vibrio cholerae cbb(3)-type (C-family) oxygen reductase2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 42, E4419-E4428 p.Article in journal (Refereed)
    Abstract [en]

    The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K-C-channel is a conserved glutamate in subunit III. However, the majority of the K-C-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K-C-channel, was found to depend on the conformation of Y241(Vc), located in subunit I at the interface with subunit III. Mutations of Y241(Vc) (to A/F/H/S) in the Vibrio cholerae cbb(3) eliminate catalytic activity, but also cause perturbations that propagate over a 28-angstrom distance to the active site heme b(3). The data suggest a linkage between residues lining the KC-channel and the active site of the enzyme, possibly mediated by transmembrane helix alpha 7, which contains both Y241(Vc) and the active site crosslinked Y255(Vc), as well as two Cu-B histidine ligands. Other mutations of residues within or near helix alpha 7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.

  • 13. Ali, Raja Hashim
    et al.
    Muhammad, Sayyed Auwn
    Khan, Mehmood Alam
    Arvestad, Lars
    Stockholm University, Faculty of Science, Numerical Analysis and Computer Science (NADA). Stockholm University, Science for Life Laboratory (SciLifeLab). Swedish e-Science Research Center, Sweden .
    Quantitative synteny scoring improves homology inference and partitioning of gene families2013In: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 14, no Suppl,15, S12- p.Article in journal (Refereed)
    Abstract [en]

    Background

    Clustering sequences into families has long been an important step in characterization of genes and proteins. There are many algorithms developed for this purpose, most of which are based on either direct similarity between gene pairs or some sort of network structure, where weights on edges of constructed graphs are based on similarity. However, conserved synteny is an important signal that can help distinguish homology and it has not been utilized to its fullest potential.

    Results

    Here, we present GenFamClust, a pipeline that combines the network properties of sequence similarity and synteny to assess homology relationship and merge known homologs into groups of gene families. GenFamClust identifies homologs in a more informed and accurate manner as compared to similarity based approaches. We tested our method against the Neighborhood Correlation method on two diverse datasets consisting of fully sequenced genomes of eukaryotes and synthetic data.

    Conclusions

    The results obtained from both datasets confirm that synteny helps determine homology and GenFamClust improves on Neighborhood Correlation method. The accuracy as well as the definition of synteny scores is the most valuable contribution of GenFamClust.

  • 14.
    Alikhani, Nyosha
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ankarcrona, Maria
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mitochondria and Alzheimer's disease: amyloid-beta peptide uptake and degradation by the presequence protease, hPreP2009In: Journal of Bioenergetics and Biomembranes, ISSN 0145-479X, E-ISSN 1573-6881, Vol. 41, no 5, 447-451 p.Article in journal (Refereed)
    Abstract [en]

    Several lines of evidence suggest mitochondrial dysfunction as a possible underlying mechanism of Alzheimer's disease (AD). Accumulation of the amyloid-beta peptide (Abeta), a neurotoxic peptide implicated in the pathogenesis of AD, has been detected in brain mitochondria of AD patients and AD transgenic mouse models. In vitro evidence suggests that the Abeta causes mitochondrial dysfunction e.g. oxidative stress, mitochondrial fragmentation and decreased activity of cytochrome c oxidase and TCA cycle enzymes. Here we review the link between mitochondrial dysfunctions and AD. In particular we focus on the mechanism for Abeta uptake by mitochondria and on the recently identified Abeta degrading protease in human brain mitochondria.

  • 15.
    Alikhani, Nyosha
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berglund, Anna-Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Engmann, Tanja
    Pavlov, Pavel
    Langer, Thomas
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Matrix localized AtPreP complements intermembrane space located homologue, MOP112, in Saccaromyces cerevisiaeManuscript (preprint) (Other academic)
  • 16.
    Alikhani, Nyosha
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berglund, Anna-Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Engmann, Tanja
    Spånning, Erika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Voegtle, F. -Nora
    Pavlov, Pavel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Meisinger, Chris
    Langer, Thomas
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Targeting Capacity and Conservation of PreP Homologues Localization in Mitochondria of Different Species2011In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 410, no 3, 400-410 p.Article in journal (Refereed)
    Abstract [en]

    Mitochondrial presequences and other unstructured peptides are degraded inside mitochondria by presequence proteases (PrePs) identified in Arabidopsis thaliana (AtPreP), humans (hPreP), and yeast (Cym1/Mop112). The presequences of A. thaliana and human PreP are predicted to consist of 85 and 29 amino acids, respectively, whereas the Saccharomyces cerevisiae Cym1/Mop112 presequence contains only 7 residues. These differences may explain the reported targeting of homologous proteins to different mitochondrial subcompartments. Here we have investigated the targeting capacity of the PreP homologues' presequences. We have produced fusion constructs containing N-terminal portions of AtPreP(1-125), hPreP(1-69), and Cym1(1-40) coupled to green fluorescent protein (GFP) and studied their import into isolated plant, mammalian, and yeast mitochondria, followed by mitochondrial subfractionation. Whereas the AtPreP presequence has the capacity to target GFP into the mitochondrial matrix of all three species, the hPreP presequence only targets GFP to the matrix of mammalian and yeast mitochondria. The Cym1/Mop112 presequence has an overall much weaker targeting capacity and only ensures mitochondrial sorting in its host species yeast. Revisiting the submitochondrial localization of Cym1 revealed that endogenous Cym1/Mop112 is localized to the matrix space, as has been previously reported for the plant and human homologues. Moreover, complementation studies in yeast show that native AtPreP restores the growth phenotype of yeast cells lacking Cym1, demonstrating functional conservation.

  • 17.
    Alikhani, Nyosha
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Guo, Lan
    Pinho, Catarina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Yan, Shi Du
    Decreased proteolytic activity of the PreP peptidasome in Alzheimer disease brain mitochondria and transgenic modelsManuscript (preprint) (Other academic)
  • 18.
    Alikhani, Nyosha
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Guo, Lan
    Yan, Shiqiang
    Du, Heng
    Pinho, Catarina Moreira
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chen, John Xi
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Yan, Shirley ShiDu
    Decreased proteolytic activity of the mitochondrial amyloid-β degrading enzyme, PreP peptidasome, in Alzheimer's disease brain mitochondria2011In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 27, no 1, 75-87 p.Article in journal (Refereed)
    Abstract [en]

    Accumulation of amyloid-β peptide (Aβ), the neurotoxic peptide implicated in the pathogenesis of Alzheimer's disease (AD), has been shown in brain mitochondria of AD patients and of AD transgenic mouse models. The presence of Aβ in mitochondria leads to free radical generation and neuronal stress. Recently, we identified the presequence protease, PreP, localized in the mitochondrial matrix in mammalian mitochondria as the novel mitochondrial Aβ-degrading enzyme. In the present study, we examined PreP activity in the mitochondrial matrix of the human brain's temporal lobe, an area of the brain highly susceptible to Aβ accumulation and reactive oxygen species (ROS) production. We found significantly lower hPreP activity in AD brains compared with non-AD age-matched controls. By contrast, in the cerebellum, a brain region typically spared from Aβ accumulation, there was no significant difference in hPreP activity when comparing AD samples to non-AD controls. We also found significantly reduced PreP activity in the mitochondrial matrix of AD transgenic mouse brains (Tg mAβPP and Tg mAβPP/ABAD) when compared to non-transgenic aged-matched mice. Furthermore, mitochondrial fractions isolated from AD brains and Tg mAβPP mice had higher levels of 4-hydroxynonenal, an oxidative product, as compared with those from non-AD and nonTg mice. Accordingly, activity of cytochrome c oxidase was significantly reduced in the AD mitochondria. These findings suggest that decreased PreP proteolytic activity, possibly due to enhanced ROS production, contributes to Aβ accumulation in mitochondria leading to the mitochondrial toxicity and neuronal death that is exacerbated in AD. Clearance of mitochondrial Aβ by PreP may thus be of importance in the pathology of AD.

  • 19. Allendorf, Fred W.
    et al.
    Berry, Oliver
    Ryman, Nils
    Stockholm University, Faculty of Science, Department of Zoology.
    So long to genetic diversity, and thanks for all the fish2014In: Molecular Ecology, ISSN 0962-1083, E-ISSN 1365-294X, Vol. 23, no 1, 23-25 p.Article in journal (Refereed)
    Abstract [en]

    The world faces a global fishing crisis. Wild marine fisheries comprise nearly 15% of all animal protein in the human diet, but, according to the U.N. Food and Agriculture Organization, nearly 60% of all commercially important marine fish stocks are overexploited, recovering, or depleted (FAO 2012; Fig. 1). Some authors have suggested that the large population sizes of harvested marine fish make even collapsed populations resistant to the loss of genetic variation by genetic drift (e. g. Beverton 1990). In contrast, others have argued that the loss of alleles because of overfishing may actually be more dramatic in large populations than in small ones (Ryman et al. 1995). In this issue, Pinsky & Palumbi (2014) report that overfished populations have approximately 2% lower heterozygosity and 12% lower allelic richness than populations that are not overfished. They also performed simulations which suggest that their estimates likely underestimate the actual loss of rare alleles by a factor of three or four. This important paper shows that the harvesting of marine fish can have genetic effects that threaten the long-term sustainability of this valuable resource.

  • 20.
    Alling, Vanja
    et al.
    Stockholm University, Faculty of Science, Department of Applied Environmental Science (ITM).
    Humborg, Christoph
    Stockholm University, Faculty of Science, Department of Applied Environmental Science (ITM).
    Mörth, Carl-Magnus
    Stockholm University, Faculty of Science, Department of Geological Sciences.
    Rahm, Lars
    Polehne, Falk
    Tracing terrestrial organic matter by delta34S and delta13C signatures in a subarctic estuary2008In: Limnology and Oceanography, ISSN 0024-3590, E-ISSN 1939-5590, Vol. 53, no 6, 2594-2602 p.Article in journal (Refereed)
    Abstract [en]

    A key issue to understanding the transformations of terrestrial organic carbon in the ocean is to disentangle the latter from marine-produced organic matter. We applied a multiple stable isotope approach using 34S and 13C isotope signatures from estuarine dissolved organic matter (DOM), enabling us to constrain the contribution of terrestrial-derived DOM in an estuarine gradient of the northern Baltic Sea. The stable isotope signatures for dissolved organic sulfur (34SDOS) have twice the range between terrestrial and marine end members compared to the stable isotope signatures for dissolved organic carbon (13CDOC); hence, the share of terrestrial DOM in the total estuarine DOM can be calculated more precisely. DOM samples from the water column were collected using ultrafiltration on board the German RV Maria S Merian during a winter cruise, in the Bothnian Bay, Bothnian Sea, and Baltic proper. We calculated the terrestrial fraction of the estuarine DOC (DOCter) from both 13CDOC and 34SDOS signatures and applying fixed C: S ratios for riverine and marine end members to convert S isotope signatures into DOC concentrations. The 34SDOS signature of the riverine end member was +7.02‰, and the mean signatures from Bothnian Bay, Bothnian Sea, and Baltic proper were +10.27, +12.51, and +13.67‰, respectively, showing an increasing marine signal southwards (34SDOS marine end member 5 18.1‰). These signatures indicate that 87‰, 75‰, and 67‰, respectively, of the water column DOC is of terrestrial origin (DOCter) in these basins. Comparing the fractions of DOCter in each basin—that are still based on few winter values only—with the annual river input of DOC, it appears that the turnover time for DOCter in the Gulf of Bothnia is much shorter than the hydraulic turnover time, suggesting that high-latitude estuaries might be efficient sinks for DOCter.

  • 21.
    Alling, Vanja
    et al.
    Stockholm University, Faculty of Science, Department of Applied Environmental Science (ITM).
    Sanchez-Garcia, Laura
    Stockholm University, Faculty of Science, Department of Applied Environmental Science (ITM).
    Porcelli, Don
    Pugach, Sveta
    Vonk, Jorien E.
    van Dongen, Bart
    Mörth, Carl-Magnus
    Stockholm University, Faculty of Science, Department of Geological Sciences. Stockholm University, Stockholm Resilience Centre.
    Anderson, Leif G.
    Sokolov, Alexander
    Stockholm University, Stockholm Resilience Centre.
    Andersson, Per
    Humborg, Christoph
    Stockholm University, Faculty of Science, Department of Applied Environmental Science (ITM). Stockholm University, Stockholm Resilience Centre.
    Semiletov, Igor
    Gustafsson, Örjan
    Stockholm University, Faculty of Science, Department of Applied Environmental Science (ITM).
    Non-conservative behavior of dissolved organic carbon across the Laptev and East Siberian Seas2010In: Global Biogeochemical Cycles, ISSN 0886-6236, E-ISSN 1944-9224, Vol. 24, GB4033- p.Article in journal (Refereed)
    Abstract [en]

    Climate change is expected to have a strong effect on the Eastern Siberian Arctic Shelf (ESAS) region, which includes 40% of the Arctic shelves and comprises the Laptev and East Siberian seas. The largest organic carbon pool, the dissolved organic carbon (DOC), may change significantly due to changes in both riverine inputs and transformation rates; however, the present DOC inventories and transformation patterns are poorly understood. Using samples from the International Siberian Shelf Study 2008, this study examines for the first time DOC removal in Arctic shelf waters with residence times that range from months to years. Removals of up to 10%–20% were found in the Lena River estuary, consistent with earlier studies in this area, where surface waters were shown to have a residence time of approximately 2 months. In contrast, the DOC concentrations showed a strong nonconservative pattern in areas with freshwater residence times of several years. The average losses of DOC were estimated to be 30%–50% during mixing along the shelf, corresponding to a first-order removal rate constant of 0.3 yr−1. These data provide the first observational evidence for losses of DOC in the Arctic shelf seas, and the calculated DOC deficit reflects DOC losses that are higher than recent model estimates for the region. Overall, a large proportion of riverine DOC is removed from the surface waters across the Arctic shelves. Such significant losses must be included in models of the carbon cycle for the Arctic Ocean, especially since the breakdown of terrestrial DOC to CO2 in Arctic shelf seas may constitute a positive feedback mechanism for Arctic climate warming. These data also provide a baseline for considering the effects of future changes in carbon fluxes, as the vast northern carbon-rich permafrost areas draining into the Arctic are affected by global warming.

  • 22. Al-Minawi, Ali Z.
    et al.
    Lee, Yin-Fai
    Håkansson, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lundin, Cecilia
    Saleh-Gohari, Nasrollah
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bryant, Helen E.
    Meuth, Mark
    Hinz, John M.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The ERCC1/XPF endonuclease is required for completion of homologous recombination at DNA replication forks stalled by inter-strand cross-links2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 19, 6400-6413 p.Article in journal (Refereed)
    Abstract [en]

    Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1-XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival.

  • 23.
    Al-Minawi, Ali Z
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Saleh-Gohari, Nasrollah
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The ERCC1/XPF endonuclease is required for efficient single-strand annealing and gene conversion in mammalian cells2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 1, 1-9 p.Article in journal (Refereed)
    Abstract [en]

    The mammalian ERCC1-XPF endonuclease has a suggested role in the repair of DNA double-strand breaks (DSB) by single-strand annealing (SSA). Here, we investigated the role of ERCC1 in homologous recombination in mammalian cells, and confirm a role of ERCC1 in SSA. Interestingly, we also report an unexpected role for ERCC1 in gene conversion. This provides support that gene conversion in mammalian somatic cells is carried out through synthesis-dependent strand annealing, rather than through a double Holliday Junction mechanism. Moreover, we find low frequencies of SSA and gene conversion in G1-arrested cells, suggesting that SSA is not a frequent DSB repair pathway in G1-arrested mammalian cells, even in the presence of perfect repeats. Furthermore, we find that SSA is not influenced by inhibition of CDK2 (using Roscovitine), ATM (using Caffeine and KU55933), Chk1 (using CEP-3891) or DNA-PK (using NU7026).

  • 24.
    Amelina, Hanna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proteomics in biomarker research: Insights into the effects of aging and environment on biological systems2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteomics is the global analysis of proteins that covers a broad range of technologies aimed at determining the identity and quantity of proteins expressed in the cell, their three-dimensional structure and interaction partners. In contrast to genome, proteome reflects more accurately on the dynamic state of the cell, tissue, or an organism. Therefore much is expected from proteomics to yield better disease markers for early diagnosis and therapy monitoring, as well as biomarkers that would indicate environmental exposure or provide prediction of biological age.

    In this thesis, I have developed and applied robust and sensitive subproteomic approaches to study the effect of aging as well as and environmental pollution using different animal models.

    In the first part, a high-throughput proteomic method based on liquid chromatography coupled to 2-dimensional gel electrophoresis (LC/2-DE) was developed. The usefulness of this method has been demonstrated by applying it to the assessment of marine pollution in a field experiment.

    Next, I have utilized this subproteomic approach to study the effect of aging in mouse kidney of both genders. As a result, a protein expression signature of aging kidney was obtained, revealing gender-dependent alterations in proteome profiles of aging mouse kidney.

    In order to further reduce the dynamic range of protein expression and increase the sensitivity of proteomic analysis, I have applied a shotgun mass spectrometry-based proteomic approach using isobaric tags for relative and absolute quantification (iTRAQ) coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to study age-related differences in peroxisome-enriched fractions from mouse liver. Only eight proteins showed statistically significant difference in expression (p<0.05) with moderate folds. This study indicates that age-depended changes in the liver proteome are minimal, suggesting that its proteome is efficiently maintained until certain age.

    Finally, in the context of aging studies and the role of peroxisomes in aging, I have tested the utility of cell-penetrating peptides (CPPs) as agents for protein delivery into acatalasemic peroxisomes using yeast as a model. The results obtained suggest that CPPs may be suitable for the delivery of antioxidants to peroxisomes and in future could provide a tool for the protein therapy of age-related diseases.

  • 25.
    Amelina, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Apraiz, Itxaso
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sun, Wei
    Cristobal, Susana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proteomics-based method for the assessment of marine pollution using liquid chromatography coupled with two-dimensional electrophoresis2007In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 6, 2094-2104 p.Article in journal (Refereed)
    Abstract [en]

    Using a proteomic approach, we have developed a new method for the assessment of marine pollution that generates highly reproducible protein expression patterns and it is simple and scalable. The protocol is based on applying liquid chromatography (LC) coupled with two-dimensional electrophoresis (2-DE) to analyze changes in the protein expression pattern after exposure to marine pollution. The digestive gland of the sentinel “blue mussel” (Mytilus edulis) was batch-processed through a simple cell fractionation followed by ion-exchange chromatography and 2-DE. The selection of ligands, elution method, and small volume design was carefully considered to define a protocol that could be mainly robotized. A pilot study with samples collected from different Gothenburg harbor areas indicated that the clean area could be distinguished from the polluted ones based on a protein expression pattern (PES) composed of 13 proteins. Principal component analysis (PCA) and hierarchical clustering confirmed that the PES was sufficient to discriminate polluted and unpolluted areas and to provide a spatial gradient from the polluted source. Several proteins from the PES were identified by electrospray ionization tandem mass spectrometry (ESI−MS/MS), and they are involved in β-oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding. In the near future, this methodology could show potential advantages to assess marine pollution and could become a stable platform to elucidate ecotoxicological questions.

  • 26.
    Amelina, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cristobal, Susana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proteomic study on gender differences in aging kidney of mice2009In: Proteome Science, ISSN 1477-5956, E-ISSN 1477-5956, Vol. 7, no 16Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: This study aims to analyze sex differences in mice aging kidney. We applied a proteomic technique based on subfractionation, and liquid chromatography coupled with 2-DE. Samples from male and female CD1-Swiss outbred mice from 28 weeks, 52 weeks, and 76 weeks were analysed by 2-DE, and selected proteins were identified by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS).

    RESULTS: This proteomic analysis detected age-related changes in protein expression in 55 protein-spots, corresponding to 22 spots in males and 33 spots in females. We found a protein expression signature (PES) of aging composed by 8 spots, common for both genders. The identified proteins indicated increases in oxidative and proteolytic proteins and decreases in glycolytic proteins, and antioxidant enzymes.

    CONCLUSION: Our results provide insights into the gender differences associated to the decline of kidney function in aging. Thus, we show that proteomics can provide valuable information on age-related changes in expression levels of proteins and related modifications. This pilot study is still far from providing candidates for aging-biomarkers. However, we suggest that the analysis of these proteins could suggest mechanisms of cellular aging in kidney, and improve the kidney selection for transplantation.

  • 27.
    Amelina, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjodin, Marcus O. D.
    Bergquist, Jonas
    Cristobal, Susana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Quantitative subproteomic analysis of age-related changes in mouse liver peroxisomes by iTRAQ LC-MS/MS2011In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 879, no 30, 3393-3400 p.Article in journal (Refereed)
    Abstract [en]

    Aging is a complex multifactorial phenomenon, which is believed to result from the accumulation of cellular damage to biological macromolecules. Peroxisomes recently emerged as another important source of reactive oxygen species (ROS) production in addition to mitochondria. However, the role of these organelles in the process of aging is still not clear. The aim of this study was to characterize the changes in protein expression profiles of young (10 weeks old) versus old (18 months old) mouse liver peroxisome-enriched fractions. We have applied shotgun proteomic approach based on liquid chromatography and tandem mass spectrometry (LC-MS/MS) combined with iTRAQ (isobaric tags for relative and absolute quantitation) labeling that allows comparative quantitative multiplex analysis. Our analysis led to identification and quantification of 150 proteins, 8 out of which were differentially expressed between two age groups at a statistically significant level (p < 0.05), with folds ranging from 1.2 to 4.1. These proteins involved in peroxisornal beta-oxidation, detoxification of xenobiotics and production of ROS. Noteworthy, differences in liver proteome have been observed between as well as within different age groups. In conclusion, our subproteomic quantitative study suggests that mouse liver proteome is sufficiently maintained until certain age.

  • 28.
    Andersson, August
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Magnetic resonance investigations of lipid motion in isotropic bicelles2005In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 21, no 7, 7702-7709 p.Article in journal (Refereed)
    Abstract [en]

    The dynamics of DMPC in different isotropic bicelles have been investigated by NMR and EPR methods. The local dynamics were obtained by interpretation of 13C NMR relaxation measurements of DMPC in the bicelles, and these results were compared to EPR spectra of spin-labeled lipids. The overall size of the bicelles was investigated by PFG NMR translational diffusion measurements. The dynamics and relative sizes were compared among three different bicelles: [DMPC]/[DHPC] = 0.25, [DMPC]/[DHPC] = 0.5, and [DMPC]/[CHAPS] = 0.5. The local motion is found to depend much more strongly on the choice of the detergent, rather than the overall size of the bicelle. The results provide an explanation for differences in apparent dynamics for different peptides, which are bound to bicelles. This in turn determines under what conditions reasonable NMR spectra can be observed. A model is presented in which extensive local motion, in conjunction with the overall size, affects the spectral properties. An analytical expression for the size dependence of the bicelles, relating the radius of the bilayer region with physical properties of the detergent and the lipid, is also presented.

  • 29.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berthold, Catrine L.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A dynamic c terminal segment in the mycobacterium tuberculosis mn/fe r2lox protein can adopt a helical structure with possible functional consequences2012In: Chemistry and Biodiversity, ISSN 1612-1872, E-ISSN 1612-1880, Vol. 9, no 9, 1981-1988 p.Article in journal (Refereed)
    Abstract [en]

    Mycobacterium tuberculosis R2-like ligand-binding oxidase (MtR2lox) belongs to a recently discovered group of proteins that are homologous to the ribonucleotide reductase R2 proteins. MtR2lox carries a heterodinuclear Mn/Fe cofactor and, unlike R2 proteins, a large ligand-binding cavity. A unique tyrosine-valine cross link is also found in the vicinity of the active site. To date, all known structures of R2 and R2lox proteins show a disordered C-terminal segment. Here, we present two new crystal forms of MtR2lox, revealing an ordered helical C-terminal. The ability of alternating between an ordered and disordered state agrees well with bioinformatic analysis of the protein sequence. Interestingly, ordering of the C-terminal helix shields a large positively charged patch on the protein surface, potentially used for interaction with other cellular components. We hypothesize that the dynamic C-terminal segment may be involved in control of protein function in vivo.

  • 30.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundgren, Camilla A. K.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Magnusdottir, Auour
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ge, Changrong
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Molina, Daniel Martinez
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The Mycobacterium tuberculosis Very-Long-Chain Fatty Acyl-CoA Synthetase: Structural Basis for Housing Lipid Substrates Longer than the Enzyme2012In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 20, no 6, 1062-1070 p.Article in journal (Refereed)
    Abstract [en]

    The Mycobacterium tuberculosis acid-induced operon MymA encodes the fatty acyl-CoA synthetase FadD13 and is essential for virulence and intracellular growth of the pathogen. Fatty acyl-CoA synthetases activate lipids before entering into the metabolic pathways and are also involved in transmembrane lipid transport. Unlike soluble fatty acyl-CoA synthetases, but like the mammalian integral-membrane very-long-chain acyl-CoA synthetases, FadD13 accepts lipid substrates up to the maximum length tested (C-26). Here, we show that FadD13 is a peripheral membrane protein. The structure and mutational studies reveal an arginine- and aromatic-rich surface patch as the site for membrane interaction. The protein accommodates a hydrophobic tunnel that extends from the active site toward the positive patch and is sealed by an arginine-rich lid-loop at the protein surface. Based on this and previous data, we propose a structural basis for accommodation of lipid substrates longer than the enzyme and transmembrane lipid transport by vectorial CoA-esterification.

  • 31.
    Andersson, Charlotta Selina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural studies of R2 and R2–like proteins with a heterodinuclear Mn/Fe cofactor and enzymes involved in Mycobacterium tuberculosis lipid metabolism2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Tuberculosis is a notorious disease responsible for the deaths of 1.4 million people worldwide. A third of the world's population is infected with Mycobacterium tuberculosis, the bacterium causing the disease. The increase of multi drug-resistant strains worsens the situation, and the World Health Organization has declared tuberculosis to be a global emergency. The bacterium envelopes itself with a unique set of very long-chain lipids that play an important role in virulence and drug resistance. Therefore enzymes involved in lipid metabolism are putative drug targets. 

    To allow entry into different metabolic pathways and transmembrane transport, fatty acids have to be activated. This is done primarily by fatty acyl-CoA synthetases (ACSs). We identified an ACS possibly involved in the bacterium’s virulence and solved its structure. Structural interpretation combined with previously reported data gives us insights into the details of its function. This enzyme is known to harbor lipid substrates longer than the enzyme itself, and we now propose how this peripheral membrane protein accommodates its substrates. 

    Some of the most chemically challenging oxidations are performed by dinuclear metalloproteins belonging to the ferritin-like superfamily. We show that the ferritin-like protein, R2lox, from M. tuberculosis contains a new type of heterodinuclear Mn/Fe cofactor. This protein cofactor is capable of performing potent 2-electron oxidations as demonstrated by a novel tyrosine-valine crosslink observed in the protein. 

    Recently a new subclass of ribonucleotide reductase (RNR) R2 proteins, was identified in the intracellular pathogen Chlamydia trachomatis containing the same type of Mn/Fe cofactor mentioned above. The RNR R2 proteins use their metal site to generate a stable radical, essential for the reduction of ribonucleotides to their deoxy forms, the building blocks of DNA. With this work, we were able to characterize the architecture of this metal cofactor.

  • 32.
    Andersson, Jessica
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Activation, reaction mechanism and allosteric regulation of the anaerobic ribonucleotide reductase from bacteriophage T42000Doctoral thesis, monograph (Other academic)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyse the conversion of ribonucleotides to their corresponding deoxyribonucleotides in all organisms. The deoxyribonucleotides are the building blocks for DNA. Three different classes of RNR are found, class I, II and III. The class I RNRs operate under aerobic conditions, the class III RNRs operate under anaerobic conditions and the class II RNRs are indifferent to oxygen. All classes of RNR catalyse the reaction using a free radical mechanism. The free radical is generated to initiate the reaction mechanism but the generation differs between the classes.

    I have worked with the anaerobic class III RNR from bacteriophage T4 and the work presented in this thesis involves several different aspects of the enzyme. The class III RNR from phage T4 can be used as a model for other class III RNRs.

    From isotope labelling experiments, we show that a stable glycyl radical forms in the phage T4 class III RNR. I used site-directed mutagenesis to locate the glycyl radical to Gly580 in the NrdD protein of the T4 class III RNR. The glycyl radical is absolutely required for enzymatic activity.

    Also using protein engineering, I show for the first time, the importance of cysteines in radical generation and the reaction mechanism of the class III RNRs. Four cysteines in the C-terminal of T4 NrdD are responsible for the last step in the generation of the glycyl radical at Gly580. Two cysteines in the active site of T4 NrdD, Cys79 and Cys290 are required for the reaction mechanism of the enzyme. A third residue within the active site, Asn311 is most likely also important for catalytic activity. A reaction mechanism that is different from the class I and II RNRs has been proposed.

    The first crystal structure of a class III RNR, the class III RNR from phage T4 is presented. Structural relationships with the known class I RNR structure is discussed as well as similarities with another glycyl-radical enzyme.

    Finally, the allosteric regulation of the class III RNR from phage T4 was characterized. Almost all RNRs are allosterically regulated to keep the deoxynucleotide pools balanced in the cell. Similarities to other RNRs as well as a unique feature of the class III RNR from phage T4 is discussed.

  • 33.
    Andersson, Martin
    Stockholm University.
    Structural studies and design of Di-iron carboxylate proteins2000Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Di-iron carboxylate proteins perform a wide range of chemical reactions in the cell. These reactions often involve activation of dioxygen to generate highly oxidative species that can be used in catalysis. One member of the di-iron carboxylate family is the enzyme ribonucleotide reductase (RNR). This enzyme is essential for DNA synthesis since it is a key enzyme on the pathway for de-novo synthesis of deoxyribonucleotides, the building blocks of DNA. To perform its catalytic function RNR is dependent on a protein radical. In this thesis I have used x-ray crystallographic methods to investigate the mechanism of O2 activation and radical generation in the R2 subunit of RNR. These structural studies have lead to a proposal of a detailed mechanism, which could be common to most O2 activating di-iron carboxylate proteins. The alternative oxidase is a membrane protein that has been proposed to belong to the di-iron carboxylate family. This protein is a ubiquinol/oxygen oxidoreductase and can act as a terminal electron acceptor in the respiratory chain. I have used homology modelling to make a structural model of this enzyme, which provides new insights into its functions and its relationship to the other di-iron carboxylate proteins.

    Given the strong oxidative power of the di-iron carboxylate proteins they would be very useful as oxidants in various industrial applications. Another part of my project has been aimed towards the design of a di-iron carboxylate enzyme tailored for industrial and environmental applications using a small and stable di-iron carboxylate protein as a starting framework, namely the bacterioferritin protein. This work has lead to the synthesis of a gene library with many potential enzymatic activities.

  • 34.
    Andrys, Rudolf
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry. Charles University Prague, Czech Republic .
    Zurita, Javier
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Zguna, Nadezda
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Verschueren, Klaas
    De Borggraeve, Wim M.
    Ilag, Leopold L.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Improved detection of beta-N-methylamino-L-alanine using N-hydroxysuccinimide ester of N-butylnicotinic acid for the localization of BMAA in blue mussels (Mytilus edulis)2015In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 407, no 13, 3743-3750 p.Article in journal (Refereed)
    Abstract [en]

    beta-N-Methylamino-l-alanine (BMAA) is an important non-protein amino acid linked to neurodegenerative diseases, specifically amyotrophic lateral sclerosis (ALS). Because it can be transferred and bioaccumulated higher up the food chain, it poses significant public health concerns; thus, improved detection methods are of prime importance for the identification and management of these toxins. Here, we report the successful use of N-hydroxysuccinimide ester of N-butylnicotinic acid (C-4-NA-NHS) for the efficient separation of BMAA from its isomers and higher sensitivity in detecting BMAA compared to the current method of choice using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization. Implementation of this efficient method allowed localization of BMAA in the non-visceral tissues of blue mussels, suggesting that more efficient depuration may be required to remove this toxin prior to consumption. This is a crucial method in establishing the absence or presence of the neurotoxic amino acid BMAA in food, environmental or biomedical samples.

  • 35. Anko, Maja
    et al.
    Majhenc, Janja
    Kogej, Ksenija
    Sillard, Rannard
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Anderluh, Gregor
    Zorko, Matjaz
    Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane2012In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1818, no 3, 915-924 p.Article in journal (Refereed)
    Abstract [en]

    The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PR and PF6 observed in previous studies.

  • 36. Arbesu Valdivia, Alejandro
    et al.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Romero Batista, Yamilet
    Kumar, Saroj
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Characterization of recombinant antibodies for cancer therapy by infrared spectroscopy2013In: Biologicals (Print), ISSN 1045-1056, E-ISSN 1095-8320, Vol. 41, no 2, 104-110 p.Article in journal (Refereed)
    Abstract [en]

    Fourier transform infrared (FTIR) spectroscopy was used to study the structure of the recombinant antibodies 1E10, anti-CD20 and hR3, which are used as anti-cancer therapeutic drugs. We tested their sensitivity against different conditions and treatments such as pH, temperature, freeze-thaw cycles and drying, which are relevant for the practical usefulness of the drugs. All antibodies were stable against moderate temperature increases (up to 50 degrees C) and pH changes (range 5-9). 1E10 was sensitive to extreme pH values (pH 3 and 12), whereas hR3 was most sensitive to temperature (at and above 60 degrees C). We did not observe any significant changes upon freeze-thaw and drying treatments. The secondary structure content of all three antibodies was estimated to be similar to that of IgG with similar to 64% beta-sheet, 0% alpha-helix and similar to 36% other structure. (C) 2012 The International Alliance for Biological Standardization.

  • 37.
    Arefin, Md. Badrul
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University.
    Molecular characterization of the Drosophila responses towards nematodes2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A sophisticated evolutionary conserved innate immune system has evolved in insects to fight pathogens and to restrict damage in harmful (danger) situations including cancer. A significant amount of knowledge about different infection models in Drosophila has been generated in past decades, which revealed functional resemblances and implications for vertebrate systems. However, how Drosophila responds towards multicellular parasitic nematodes and in danger situations is still little understood. Therefore, the aim of the thesis was to characterize multiple aspects of the host defense in the two important contexts mentioned above.

    We analyzed the transcriptome profiles of nematode-infected Drosophila larvae with uninfected samples. For this we employed the entomopathogenic nematode Heterorhabditis bacteriophora with its symbiont Photorhabdus luminescence to infect Drosophila larvae. We found 642 genes were differentially regulated upon infection. Among them a significant portion belonged to immune categories. Further functional analysis identified a thioester containing protein TEP3, a recognition protein GNBP-like 3, the basement membrane component protein Glutactin and several other small peptides. Upon loss or reduced expression of these genes hosts showed mortality during nematode infections. This study uncovers a novel function for several of the genes in immunity.

    Furthermore, we investigated the cellular response towards nematodes. When we eliminated hemocytes genetically (referred to as hml-apo) in Drosophila, we found hml-apo larvae are resistant to nematodes. Subsequent characterization of hml-apo larvae showed massive lamellocyte differentiation (another blood cell type which is rare in naïve larvae), emergence of melanotic masses, up- and down-regulation of Toll and Imd signaling respectively suggesting a pro-inflammatory response. Moreover, a striking defective leg phenotype in adult escapers from pupal lethality was observed. We identified nitric oxide (NO) as a key regulator of these processes. We also showed that imaginal disc growth factors 3 (IDGF3): (a) protects hosts against nematodes, (b) is a clotting component and (c) negatively regulates Wnt and JAK/STAT signaling. To follow larval behavior in the presence or absence of nematodes we monitored Drosophila larval locomotion behaviors using FIMtrack (a recently devised automated method) to elucidate evasive strategies of hosts. Finally, we characterized host defenses in three Drosophila leukemia models with and without nematode infection. Taken together, these studies shed light on host responses in two crucial circumstances, nematode infections and danger situations.

  • 38. Arestrom, Irene
    et al.
    Zuber, Bartek
    Bengtsson, Theresa
    Ahlborg, Niklas
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Immunology.
    Measurement of human latent transforming growth factor beta 1 using a latency associated protein reactive elisa2012In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 379, no 1-2, 23-29 p.Article in journal (Refereed)
    Abstract [en]

    Human Transforming Growth Factor (TGF)-beta 1, one of three TGF-beta isoforms, is a pleotropic cytokine critical for many physiological and immunological processes. TGF-beta 1 is secreted in a latent form, linked to Latency Associated Protein (LAP). Analysis of Latent TGF-beta 1 by TGF-ELISA requires dissociation of TGF-beta 1 from LAP, e.g. by acidification of samples. The ELISA then measures total TGF-beta 1, equivalent to dissociated Latent TGF-beta 1 plus any free TGF-beta 1 present prior to acidification. Evolutionary conservation of TGF-beta 1 across mammals also renders TGF-beta 1 ELISAs reactive with TGF-beta 1 in bovine serum often used in human cell cultures. To enable a direct analysis of Latent TGF-beta 1, monoclonal antibodies were made against LAP from human latent TGF-beta 1 and used to develop a LAP ELISA detecting Latent TGF-beta 1. The ELISA did not react with LAP from human Latent TGF-beta 2 or 3, respectively, nor with Latent TGF-beta in bovine serum. EDTA-containing plasma from healthy subjects (n = 20) was analyzed by conventional TGF-beta 1 ELISA and LAP ELISA. By TGF-beta 1 ELISA, total TGF-beta 1 were detected in all samples (median 133 pM, range 34-348 pM); low levels of free TGF-beta 1 found in 8/20 non-addified samples showed that >98.5% of the total TGF-beta 1 derived from Latent TGF-beta 1. Latent TGF-beta 1 found in non-acidified samples by LAP ELISA (median 154 pM, range 48-403 pM) was comparable in molar levels to, and correlated with, total TGF-beta 1 (r(s) 0.96, p<0.0001). A similar agreement between the total TGF-beta 1 and the LAP ELISA was found with citrate- and heparin-containing plasma. The LAP ELISA facilitates analysis of Latent TGF-beta 1 without sample acidification and is not compromised by the presence of bovine serum in human cell supernatants.

  • 39.
    Ariöz, Candan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Götzke, Hansjörg
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindholm, Ljubica
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, Jonny
    Edwards, Katarina
    Daley, Daniel O.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Heterologous overexpression of a monotopic glucosyltransferase (MGS) induces fatty acid remodeling in Escherichia coli membranes:  2014In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 7, 1862-1870 p.Article in journal (Refereed)
    Abstract [en]

    The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellular membranes when overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22 h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~ 62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE) was detected to be the most affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition.

  • 40.
    Ariöz, Candan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ye, Weihua
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    al Bakali, Amin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ge, Changrong
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liebau, Jobst
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Götzke, Hansjörg
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in Escherichia coli2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 33, 5533-5544 p.Article in journal (Refereed)
    Abstract [en]

    Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by P-31 NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a feed-forward stimulation of lipid synthesis in E. coil. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.

  • 41.
    Arukuusk, Piret
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Paernaste, Ly
    Margus, Helerin
    Eriksson, N. K. Jonas
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Vasconcelos, Luis
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Padari, Kaert
    Pooga, Margus
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Differential Endosomal Pathways for Radically Modified Peptide Vectors2013In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 10, 1721-1732 p.Article in journal (Refereed)
    Abstract [en]

    In the current work we characterize the uptake mechanism of two NickFect family members, NF51 and NF1, related to the biological activity of transfected plasmid DNA (pDNA). Both vectors condense pDNA into small negatively charged nanoparticles that transfect He La cells with equally high efficacy and the delivery is mediated by SCARA3 and SCARA.5 receptors. NF1 condenses DNA into less homogeneous and less stable nanoparticles than NF51. NF51/pDNA nanoparticles enter the cells via macropinocytosis, while NF1/pDNA complexes use clathrin- or caveolae-mediated endocytosis and macropinocytosis. Analysis of separated endosomal compartments uncovered lysomotropic properties of NF51 that was also proven by cotransfection with chloroquine. In summary we characterize how radical modifications in peptides, such as introducing a kink in the structure of NF51 or including extra negative charge by phospho-tyrosine substitution in NF1, resulted in equally high efficacy for gene delivery, although this efficacy is achieved by using differential transfection pathways.

  • 42. Arukuusk, Piret
    et al.
    Paernaste, Ly
    Oskolkov, Nikita
    Copolovici, Dana-Maria
    Margus, Helerin
    Padari, Kaert
    Moell, Kaidi
    Maslovskaja, Julia
    Tegova, Radi
    Kivi, Gaily
    Tover, Andres
    Pooga, Margus
    Ustav, Mart
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    New generation of efficient peptide-based vectors, NickFects, for the delivery of nucleic acids2013In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 5, 1365-1373 p.Article in journal (Refereed)
    Abstract [en]

    Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160 nm. Such nanoparticles have a negative surface charge (-11 to -18 mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine (TM) 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.

  • 43.
    Ashri, Nadia Y.
    et al.
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Daryanavard, Mosayeb
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Abdel-Rehim, Mohamed
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    micMicroextraction by packed sorbent and liquid chromatography-tandem mass spectrometry as a tool for quantification of peptides in plasma samples: determination of sensory neuron-specific receptors agonist BAM8-22 and antagonist BAM22-8 in plasma samples2013In: BMC Biomedical chromotography, ISSN 0269-3879, E-ISSN 1099-0801, Vol. 27, no 3, 396-403 p.Article in journal (Refereed)
    Abstract [en]

    Microextraction by packed sorbent (MEPS) is a miniaturized, solid-phase extraction (SPE) technique that works online with gas chromatography (GC) and liquid chromatography (LC). Not only is the automation process with MEPS advantageous, but the much smaller volumes of the samples, solvents and dead space in the system also provide other significant advantages such as the speed and the simplicity of the sample preparation process. In this study MEPS has been evaluated for quantification of sensory neuron-specific receptors agonist (BAM8-22). Owing to the instability of BAMs, the focus was on fast extraction and determination of the peptide online using LC-MS/MS. Sorbents such as C2, C8 and ENV+ (hydroxylated polystyrenedivinylbenzene copolymer) were investigated in the present study. MEPS-C8 gave the best results compared with C2 and ENV and it was used for the method validation. The calibration curve was obtained within the concentration range of 20.03045nmol/L in plasma. The regression correlation coefficients for plasma samples were 0.99 for all runs (n=6). The between-batch accuracy and precision for BAM8-22 ranged from 13 to 2.0% and from 4.0 to 14%, respectively. Additionally, the accuracy and precision for BAM22-8 ranged from 13 to 7.0% and from 3.0 to 12%, respectively. The present method was used for pharmacokinetic studies for BAMs in plasma samples.

  • 44.
    Asp, Patrik
    Stockholm University, Faculty of Science, Wenner-Gren Institute for Experimental Biology.
    Chromatin Remodeling by BRG1 and SNF2H: Biochemistry and Function2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition.

    We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing.

    By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.

  • 45. Atmeh, Ragheb F.
    et al.
    Kana'an, Belal M.
    Massad, Tariq T.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Isolation of High Density Lipoprotein Subclasses by Electrofiltration and Their Chemical Components2009In: Preparative Biochemistry & Biotechnology, ISSN 1082-6068, E-ISSN 1532-2297, Vol. 39, no 3, 248-265 p.Article in journal (Refereed)
    Abstract [en]

    The exact role of high density lipoprotein in atheroprotection is not well understood yet due to its complex nature; it comprises more than ten subclasses that vary in size, composition, and function. Isolation and characterization of these subclasses is an important step for further studies addressing their functions in health and disease. In this work, we present a novel method that is relatively simple and efficient for isolation of high density lipoprotein subclasses. The method depends on fractional filtration of the subclasses through a preformed gel membrane system under the effect of an electric field, where the stepwise isolation of the subclasses depends on differences in their rates of migration in polyacrylamide gel. Using this design, we were able to isolate seven high density lipoprotein subclasses with relative molecular masses of 42,000-50,000; 71,000; 103,000; 124,000; 150,000; 182,000; and 219,000. All the subclasses contained apolipoprotein A-I, phosphatidylcholine, sphingomyelin, free cholesterol, esterified cholesterol, and triacylglycerols. Some fractions of some samples contained the apolipoproteins A-II, C-I, C-II, C-III, and E. A subclass of molecular mass of 106,000 was identified and isolated from a healthy young subject that contained albumin and apoA-I with some free and esterified cholesterol, but with no triacylglycerols. This electrofiltration technique offers a novel tool for isolating pure native high density lipoprotein subclasses in a concentrated form that can be used directly for detailed studies of their physicochemical and physiological properties.

  • 46.
    Attoff, Kristina
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. Stockholm University.
    Kertika, Dimitra
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lundqvist, Jessica
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Oredsson, Stina
    Lund University.
    Forsby, Anna
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5YManuscript (preprint) (Other academic)
  • 47. Azimzadeh, Omid
    et al.
    Scherthan, Harry
    Yentrapalli, Ramesh
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Barjaktarovic, Zarko
    Ueffing, Marius
    Conrad, Marcus
    Neff, Frauke
    Calzada-Wack, Julia
    Aubele, Michaela
    Buske, Christian
    Atkinson, Michael J.
    Hauck, Stefanie M.
    Tapio, Soile
    Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediatemitochondrial impairment after ionising radiation2012In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, no 8, 2384-2395 p.Article in journal (Refereed)
    Abstract [en]

    Qualitative proteome profiling of formalin-fixed, paraffin-embedded (FFPE) tissue is advancing the field of clinical proteomics. However, quantitative proteome analysis of FFPE tissue is hampered by the lack of an efficient labelling method. The usage of conventional protein labelling on FFPE tissue has turned out to be inefficient. Classical labelling targets lysine residues that are blocked by the formalin treatment. The aim of this study was to establish a quantitative proteomics analysis of FFPE tissue by combining the label-free approach with optimised protein extraction and separation conditions. As a model system we used FFPE heart tissue of control and exposed C57BL/6 mice after total body irradiation using a gamma ray dose of 3 gray. We identified 32 deregulated proteins (p <= 0.05) in irradiated hearts 24 h after the exposure. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation. In good agreement with our previous results using fresh-frozen tissue, the analysis indicated radiation-induced alterations in three main biological pathways: respiratory chain, lipid metabolism and pyruvate metabolism. The label-free approach enables the quantitative measurement of radiation-induced alterations in FFPE tissue and facilitates retrospective biomarker identification using clinical archives.

  • 48. Azimzadeh, Omid
    et al.
    Sievert, Wolfgang
    Sarioglu, Hakan
    Yentrapalli, ramesh
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Barjaktarovic, Zarko
    Sriharshan, Arundhathi
    Ueffing, Marius
    Janik, Dirk
    Aichler, Michaela
    Atkinson, Michael J
    Multhoff, Gabriele
    Tapio, Soile
    PPAR Alpha: A Novel Radiation Target in Locally Exposed Mus musculus Heart Revealed by Quantitative Proteomics2013In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, no 6, 2700-2714 p.Article in journal (Refereed)
    Abstract [en]

    Radiation exposure of the thorax is associated with a markedly increased risk of cardiac morbidity and mortality with a latency period of decades. Although many studies have confirmed the damaging effect of ionizing radiation on the myocardium and cardiac endothelial structure and function, the molecular mechanism behind this damage is not yet elucidated. Peroxisome proliferator-activated receptor alpha (PPAR alpha), a transcriptional regulator of lipid metabolism in heart tissue, has recently received great attention in the development of cardiovascular disease. The goal of this study was to investigate radiation-induced cardiac damage in general and the role of PPAR alpha in this process in particular. C57BL/6 mice received local heart irradiation with X-ray doses of 8 and 16 gray (Gy) at the age of 8 weeks. The mice were sacrificed 16 weeks later. Radiation-induced changes in the cardiac proteome were quantified using the Isotope Coded Protein Label (ICPL) method followed by mass spectrometry and software analysis. Significant alterations were observed in proteins involved in lipid metabolism and oxidative phosphorylation. Ionizing radiation markedly changed the phosphorylation and ubiquitination status of PPAR alpha. This was reflected as decreased expression of its target genes involved in energy metabolism and mitochondrial respiratory chain confirming the proteomics data. This study suggests that persistent alteration of cardiac metabolism due to impaired PPAR alpha activity contributes to the heart pathology after radiation.

  • 49. Bach, Dominik R.
    et al.
    Guitart-Masip, Marc
    Stockholm University, Faculty of Social Sciences, Aging Research Center (ARC), (together with KI).
    Packard, Pau A.
    Miro, Julia
    Falip, Merce
    Fuentemilla, Lluis
    Dolan, Raymond J.
    Human Hippocampus Arbitrates Approach-Avoidance Conflict2014In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 24, no 5, 541-547 p.Article in journal (Refereed)
    Abstract [en]

    Animal models of human anxiety often invoke a conflict between approach and avoidance [1, 2]. In these, a key behavioral assay comprises passive avoidance of potential threat and inhibition, both thought to be controlled by ventral hippocampus [2-6]. Efforts to translate these approaches to clinical contexts [7, 8] are hampered by the fact that it is not known whether humans manifest analogous approach-avoidance dispositions and, if so, whether they share a homologous neurobiological substrate [9]. Here, we developed a paradigm to investigate the role of human hippocampus in arbitrating an approach-avoidance conflict under varying levels of potential threat. Across four experiments, subjects showed analogous behavior by adapting both passive avoidance behavior and behavioral inhibition to threat level. Using functional magnetic resonance imaging (fMRI), we observe that threat level engages the anterior hippocampus, the human homolog of rodent ventral hippocampus [10]. Testing patients with selective hippocampal lesions, we demonstrate a causal role for the hippocampus with patients showing reduced passive avoidance behavior and inhibition across all threat levels. Our data provide the first human assay for approach-avoidance conflict akin to that of animal anxiety models. The findings bridge rodent and human research on passive avoidance and behavioral inhibition and furnish a framework for addressing the neuronal underpinnings of human anxiety disorders, where our data indicate a major role for the hippocampus.

  • 50.
    Baez, Sofia
    Stockholm University.
    Dopachrome and aminochrome, the oxidized metabolites of dopa and dopamine: studies on the molecular mechanisms underlying their reduction and conjugation with glutathione1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Dopamine, like other catecholamines can be oxidized to the corresponding ortho-quinone, which undergoes cyclization of its amino chain to cyclized dopamine ortho-quinone (aminochrome). Both o-quinones are toxic metabolites that may contribute to neurotoxic effects of these catecholamines. The oxidation of dopamine to o-quinone is believed to be one of the causes of the neurodegenerative process of Parkinson's disease.

    Although it is in generally accepted that free radicals are involved in the neurodegenerative process occurring in the dopaminergic neuron system in Parkinson's disease, the exact mechanism of neurodegeneration in vivo is still unknown. However, one possible source of free radicals in the dopaminergic neuron system in the central nerve system may involve the oxidation of dopamine to the corresponding o-quinone.

    L-Dopa is commonly used in the clinical management of Parkinson's disease. However, this treatment becomes gradually ineffective, due either to loss of efficacy or to appearance of side-effects, probably produced by the oxidative injury generated by the oxidation of dopa or of its metabolites.

    We report in this study that the oxidation of dopa and dopamine to the corresponding o-quinone and the followed cyclization, at physiological pH, is not itself responsible for the formation of reactive oxygen species. In addition, we show that the reduction of cyclized o-quinones of dopamine and the subsequent autoxidation is the step in which reactive species are formed.

    Formation of reactive oxygen species during one- or two-electron reduction of aminochrome and dopachrome has been studied in vitro by using NADPH-cytochrome P450 reductase and DT-diaphorase, respectively. The results suggest that DT-diaphorase, SOD, catalase (glutathione peroxidase) and sulfotransferase constitute the cellular defenses against formation of reactive oxygen species during reduction of aminochrome and dopachrome.

    We also studied the ability of human glutathione transferases to conjugate cyclized catechol o-quinones such as aminochrome and dopachrome and found that GSTs, in particularly GST M2-2 catalyzes GSH conjugation of dopachrome and aminochrome, preventing the formation of ROS and reactive catechol metabolites. The neuroprotective role of GST M2-2 suggested in the present study is depend on the presence of the enzyme in relevants regions of the human brain, regions where cell damage is observed in diseases such as schizoprenia and Parkinson's disease.

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