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  • 201.
    Eiríksdóttir, Emelía
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Konate, Karidia
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Divita, Gilles
    Deshayes, Sébastien
    Secondary Structure of Cell-Penetrating Peptides Controls Membrane Interaction and Insertion2010Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1798, nr 6, s. 1119-1128Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.

  • 202.
    Ek, Caroline
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
    Garbaras, Andrius
    Yu, Zhenyang
    Oskarsson, Hanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och botanik.
    Eriksson Wiklund, Ann-Kristin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
    Kumblad, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och botanik.
    Gorokhova, Elena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
    Increase in stable isotope ratios driven by metabolic alterations in amphipods exposed to the beta-blocker propranolol2019Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, nr 5, artikel-id e0211304Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Anthropogenic pressures, such as contaminant exposure, may affect stable isotope ratios in biota. These changes are driven by alterations in the nutrient allocation and metabolic pathways induced by specific stressors. In a controlled microcosm study with the amphipod Gammarus spp., we studied effects of the beta-blocker propranolol on stable isotope signatures (delta N-15 and delta C-13), elemental composition (%C and %N), and growth (protein content and body size) as well as biomarkers of oxidative status (antioxidant capacity, ORAC; lipid peroxidation, TBARS) and neurological activity (acetylcholinesterase, AChE). Based on the known effects of propranolol exposure on cellular functions, i.e., its mode of action (MOA), we expected to observe a lower scope for growth, accompanied by a decrease in protein deposition, oxidative processes and AChE inhibition, with a resulting increase in the isotopic signatures. The observed responses in growth, biochemical and elemental variables supported most of these predictions. In particular, an increase in %N was observed in the propranolol exposures, whereas both protein allocation and body size declined. Moreover, both ORAC and TBARS levels decreased with increasing propranolol concentration, with the decrease being more pronounced for TBARS, which indicates the prevalence of the antioxidative processes. These changes resulted in a significant increase of the delta N-15 and delta C-13 values in the propranolol-exposed animals compared to the control. These findings suggest that MOA of beta-blockers may be used to predict sublethal effects in non-target species, including inhibited AChE activity, improved oxidative balance, and elevated stable isotope ratios. The latter also indicates that metabolism-driven responses to environmental contaminants can alter stable isotope signatures, which should be taken into account when interpreting trophic interactions in the food webs.

  • 203.
    Ekdahl, Ylva
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    A-to-I RNA editing: Function and consequences during brain development2013Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The aim of my thesis has been to study how A-to-I RNA editing of miRNAs is regulated during brain development and the biological function of these editing events.

    Using high throughput RNA sequencing, we performed an unbiased search for edited, mature miRNAs in total mouse brain tissue from three developmental stages. We searched for known and novel editing sites within short RNA sequences approximately resembling the length of mature miRNAs.

    We can conclude that the gradual increase in editing efficiency seen for most selectively edited sites in transcripts encoding neurotransmission proteins, also applies to miRNAs during development of the mammalian brain. The most striking editing events all occur in the crucial seed sequence, essential for target recognition. These results indicate that A-to-I editing is utilized to diversify target recognition by the miRNAs during development.

    Furthermore, our data suggests that specific transcripts, targeted by either non-edited or edited miRNAs, are regulated in a manner that is consistent with the developmental shifts in editing frequencies. One example of this is the developmentally regulated editing of miR-381, targeting the Pum2 transcript in the brain. Pum2 is a translational repressor that regulates many mRNAs shown to be important for neurological functions, including memory formation and learning.

    We have further analyzed what determines a substrate to be edited by the ADAR enzymes, specifically in the context of the mammalian GABAA receptor. We found that long stem loop structures located close to exon sequences function as inducers of exonic editing.

    Taken together, my research demonstrate the power of combining, RNA-Seq, bioinformatics and specific experimental verifications in order to shed light on the impact of A-to-I editing on the process of RNA interference. Furthermore, we have expanded the knowledge of RNA structure requirements for ADAR editing to occur. 

  • 204.
    Ekdahl, Ylva
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Behm, Mikaela
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Öhman, Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Complex post-transcriptional regulation of Pumilio 2 fine-tunes the neuronManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Highly polarized cells, such as differentiated

    neurons, requires a sophisticated network of

    regulatory events to control gene expression

    in response to different environmental as

    well as developmental conditions. In this

    study we show how different RNA processing

    events can work in concert to regulate gene

    expression of Pumillio 2 (Pum2), a

    translational repressor important for

    neuronal homeostasis as well as memory and

    learning. We have previously shown that

    miRNAs, encoded within the miR379-410

    cluster, which regulate the Pum2 expression

    in turn are regulated by A-to-I editing. Here,

    we identify an alternative splicing event

    within the Pum2 3’UTR that facilitates the

    escape of targeting by many of these

    miRNAs. We propose that splicing and

    editing are two RNA processing events that

    can work in concert to fine-tune the

    expression of Pum2 and have different

    effects depending on the neuronal

    subcellular localization of the transcript.

    This enables a differential gene expression in

    different compartments of the cell such as

    cell body and synaptic buds.

  • 205.
    Ekdahl, Ylva
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Farahani, Hossein Shahrabi
    Behm, Mikaela
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Lagergren, Jens
    Öhman, Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    A-to-I editing of microRNAs in the mammalian brain increases during development2012Ingår i: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 22, nr 8, s. 1477-1487Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Adenosine-to-inosine (A-to-I) RNA editing targets double-stranded RNA stem-loop structures in the mammalian brain. It has previously been shown that miRNAs are substrates for A-to-I editing. For the first time, we show that for several definitions of edited miRNA, the level of editing increases with development, thereby indicating a regulatory role for editing during brain maturation. We use high-throughput RNA sequencing to determine editing levels in mature miRNA, from the mouse transcriptome, and compare these with the levels of editing in pri-miRNA. We show that increased editing during development gradually changes the proportions of the two miR-376a isoforms, which previously have been shown to have different targets. Several other miRNAs that also are edited in the seed sequence show an increased level of editing through development. By comparing editing of pri-miRNA with editing and expression of the corresponding mature miRNA, we also show an editing-induced developmental regulation of miRNA expression. Taken together, our results imply that RNA editing influences the miRNA repertoire during brain maturation.

  • 206.
    EL Andaloussi, Samir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lehto, Taavi
    Laboratory of Molecular Biotechnology, Institute of Technology, Tartu University, Tartu, Estonia.
    Lundin, Per
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Application of PepFect peptides for the delivery of splice-correcting oligonucleotides2011Ingår i: Cell-penetrating peptides: Methods and Protocols, New York: Humana Press, 2011, s. 361-373Kapitel i bok, del av antologi (Övrigt vetenskapligt)
    Abstract [en]

    One oligonucleotide-based approach that appear very promising for the treatment of different genetic disorders are based on so-called splice-correcting oligonucleotides (SCOs) that are exploited to manipulate splicing patterns. In order to increase the bioavailability, cell-penetrating peptides (CPPs) have readily been covalently conjugated to SCOs to facilitate cellular internalization. While being a successful strategy for the delivery of uncharged oligonucleotides (ONs), it is extremely difficult to generate covalent conjugates between commonly used negatively charged ON analogs and cationic CPPs. Furthermore, high concentrations of ONs in the micromolar range are often needed to obtain biological responses, most likely as a result of endosomal entrapment of material. Therefore, exploring other vectorization methods using CPPs with endosomolytic properties are highly desired. A method of using stearyl modified CPP (i.e., TP10) analogs, named PepFect3 and PepFect4, are being described for the transfection of antisense SCOs using a simple one-step co-incubation procedure. These peptides form complexes with SCOs and efficiently promote cellular uptake by facilitating endosomal escape. This chapter describes the methods of how to form and characterize these nanoparticles and the cellular assay used to address the delivery.

  • 207.
    EL Andaloussi, Samir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Said Hassane, Fatouma
    Université Montpellier, Motpellier, France.
    Boisguerin, Prisca
    Université Montpellier, Motpellier, France.
    Sillard, Rannar
    University of Tartu, Institute of Technology, Tartu, Estonia.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lebleu, Bernard
    Université Montpellier, Motpellier, France.
    Cell-penetrating peptides-based strategies for the delivery of splice redirecting antisense oligonucleotides2011Ingår i: Therapeutic Oligonucleotides: Methods and Protocols / [ed] John Goodchild, New York: Humana Press, 2011, s. 75-89Kapitel i bok, del av antologi (Övrigt vetenskapligt)
    Abstract [en]

    Progress in our understanding of the molecular pathogenesis of human malignancies has provided therapeutic targets amenable to oligonucleotide (ON)-based strategies. Antisense ON-mediated splicing regulation in particular offers promising prospects since the majority of human genes undergo alternative splicing and since splicing defects have been found in many diseases. However, their implementation has been hampered so far by the poor bioavailability of nucleic acids-based drugs. Cell-penetrating peptides (CPPs) now appear as promising non-viral delivery vector for non-permeant biomolecules. We describe here new CPPs allowing the delivery of splice redirecting steric-block ON using either chemical conjugation or non-covalent complexation. We also describe a convenient and robust splice redirecting assay which allows the quantitative assessment of ON nuclear delivery.

  • 208. El-Gebali, Sara
    et al.
    Mistry, Jaina
    Bateman, Alex
    Eddy, Sean R.
    Luciani, Aurelien
    Potter, Simon C.
    Qureshi, Matloob
    Richardson, Lorna J.
    Salazar, Gustavo A.
    Smart, Alfredo
    Sonnhammer, Erik L. L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Hirsh, Layla
    Paladin, Lisanna
    Piovesan, Damiano
    Tosatto, Silvio C. E.
    Finn, Robert D.
    The Pfam protein families database in 20192019Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, nr D1, s. D427-D432Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The last few years have witnessed significant changes in Pfam (https://pfam.xfam.org). The number of families has grown substantially to a total of 17,929 in release 32.0. New additions have been coupled with efforts to improve existing families, including refinement of domain boundaries, their classification into Pfam clans, as well as their functional annotation. We recently began to collaborate with the RepeatsDB resource to improve the definition of tandem repeat families within Pfam. We carried out a significant comparison to the structural classification database, namely the Evolutionary Classification of Protein Domains (ECOD) that led to the creation of 825 new families based on their set of uncharacterized families(EUFs). Furthermore, we also connected Pfam entries to the Sequence Ontology (SO) through mapping of the Pfam type definitions to SO terms. Since Pfam has many community contributors, we recently enabled the linking between authorship of all Pfam entries with the corresponding authors' ORCID identifiers. This effectively permits authors to claim credit for their Pfam curation and link them to their ORCID record.

  • 209.
    Eliasson, Maria
    Stockholms universitet.
    Xenobiotic-metabolizing enzymes and enzymes involved in the metabolism of reactive oxygen species in the porcine ovary: subcellular distributions and levels of expression during follicular maturation and in the non-pregnant and pregnant corpus luteum1998Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz(a)anthracene (DMBA) is metabolized by both porcine ovarian microsomes and mitochondria, with the highest activity being seen in the corpus luteum. Microsomal DMBA monooxygenase activity is inhibited by inhibitors of the CYP450 system and by 17-bestradiol, thus indicating involvement of CYP1B1. Mitochondrial DMBA monooxygenase activity is located in the inner membrane/matrix and is not affected by inhibitors of the CYP450-system, by 17-bestradiol or by inhibitors of the respiratory chain. Mass spectral analysis of metabolites produced by microsomal and mitochondrial fractions showed both common and specific metabolites for each fraction. Both fractions produce reactive intermediates with the capacity to bind covalently to protein, and, in the case of mitochondria, to mitochondrial DNA. The metabolism of DMBA by both porcine ovarian microsomes and mitochondria seems to involve pathways other than the CYP450 system, and the involvement of reactive oxygen species is suggested.

    The level of expression of certain enzymes indirectly or directly involved in cellular defenses against metabolites of xenobiotics and reactive oxygen species (i. e, catalase, sulphotransferase, DT-diaphorase, glutathione peroxidase and glutathione reductase) are elevated in the pregnant corpus luteum compared to the non-pregnant corpus luteum.

    GST activity toward CDNB is higher in non-pregnant corpora lutea than in both follicles and pregnant corpora lutea. Western blotting revealed the presence of the GST subunits A1/2,A3, A4, A5, M1/2, M2 and P1 in the cytosol from follicles of different levels of maturation and in corpora lutea. In general, the highest levels of expression of these subunits are observed in corpora lutea. GSTP1 is the only subunit expressed at higher levels in follicles.

  • 210. El-Sayed, R.
    et al.
    Waraky, A.
    Ezzat, Kariem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Albabtain, R.
    Eigammal, K.
    Shityakov, S.
    Muhammed, M.
    Hassan, M.
    Degradation of pristine and oxidized single wall carbon nanotubes by CYP3A42019Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 515, nr 3, s. 487-492Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Carbon nanotubes (CNTs) are a class of carbon based nanomaterials which have attracted substantial attention in recent years as they exhibit outstanding physical, mechanical and optical properties. In the last decade many studies have emerged of the underlying mechanisms behind CNT toxicity including malignant transformation, the formation of granulomas, inflammatory responses, oxidative stress, DNA damage and mutation. In the present investigation, we studied the biodegradation of single-walled carbon nanotubes (SWCNTs) by Cytochrome P450 enzymes (CYP3A4) through using Raman spectroscopy. CYP3A4 is known isozyme accountable for metabolizing various endogenous and exogenous xenobiotics. CYP3A4 is expressed dominantly in the liver and other organs including the lungs. Our results suggest that CYP3A4 has a higher affinity for p-SWNTs compared to c-SWNTs. HEK293 cellular viability was not compromised when incubated with SWNT. However, CYP3A4 transfected HEK293 cell line showed no digestion of cSWNTs after incubation for 96 h. Cellular uptake of c-SWNTs was observed by electron microscopy and localization of c-SWNTs was confirmed in endosomal vesicles and in the cytoplasm. This is the first study CYP3A4 degrading both p-SWNTs and c-SWNTs in an in vitro setup. Interestingly, our results show that CYP3A4 is more proficient in degrading p-SWNTs than c-SWNTs. We also employed computational modeling and docking assessments to develop a further understanding of the molecular interaction mechanism.

  • 211.
    Elvers, Ingegerd
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Hagenkort, Anna
    Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Johansson, Fredrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Djureinovic, Tatjana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Lagerqvist, Anne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Schultz, Niklas
    Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Stoimenov, Ivaylo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Erixon, Klaus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Helleday, Thomas
    Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    CHK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation2012Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 17, s. 8440-8448Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine-inhibiting ATM and ATR amongst other kinases-is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase eta (Pol eta), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.

  • 212.
    Elvers, Ingegerd
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Johansson, Fredrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Djureinovic, Tatjana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Lagerqvist, Anne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Stoimenov, Ivaylo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Schultz, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Erixon, Klaus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Helleday, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    UVC–stalled replication forks readily collapse into DNA double-strand breaksin the absence of DNA polymerase η and independently of Mus81 in humancellsManuskript (preprint) (Övrigt vetenskapligt)
  • 213.
    Elväng, Annelie
    Stockholms universitet.
    Quantitative monitoring of microorganisms in environmental samples using biomarkers1998Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The use of microorganisms in environmental biotechnology is an area of growing interest. Microorganisms have been used for diverse applications, such as biological fertilizers, biological pesticides and bioremediation of toxic chemicals. In many cases, the bacteria of interest have been genetically alterred to improve certain traits. Before large scale environmental release of genetically modified microorganisms is attempted, careful risk assessment of the fate and survival of the inoculum and potential effects on the ecosystem must be evaluated. In order to monitor the inocula in complex environmental samples among a large population of indigenous microorganisms, the methods for detection of the microbe must be very specific and sensitive. To achieve any reliable estimations of fate, survival and efficacy of the inocula the methods for detection also need to be quantitative.

    Bacteria of interest can be tagged with specific biomarkers of eukaryotic origin, such as the firefly luciferase gene, luc, or the Aequoria victoria green fluorescent protein gene, gfp. These biomarkers confer novel genotypes (the marker gene itself) and phenotypes (bioluminescence or fluorescence, respectively) that are absent in the natural microbial flora. For example, PCR amplification, with primers targeting the marker gene, can be used for sensitive detection of biomarked bacteria. Alternatively, the bioluminescent reaction of the firefly luciferase enzyme can be used as: 1) an in vivo indicator of metabolic activity of the cell since luciferase activity is dependent on cellular energy reserves, or 2) an in vitro assay (supplemented with ATP and substrate). The in vitro assay of luciferase activity is independent of the energy status of the cell. The in vivo fluorescence of the green fluorescent protein is also independent of cellular energy reserves or co-factors.

    Methods were developed for specific detection of bacteria with luc or gfp biomarkers in soil and sediment samples. Complementary methods were used for quantitative monitoring of bacteria based on: DNA sequence, cellular activity, cellular protein content and total numbers of cells. All methods developed were independent of traditional cultivation techniques.

    Different quantitative approaches were developed and applied for monitoring of specific bacteria with environmental relevance. For example, luc-tagged cyanobacteria were quantitated in Baltic Sea sediment by competitive PCR amplification of the luc biomarker. Luciferase activity, expressed by the luc biomarker, was quantitated by luminometry. In addition, Arthrobacter chlorophenolicus A6 cells were tagged with gfp or luc biomarkers and quantitated during degradation of high concentrations of 4-chlorophenol in soil. As the cells bioremediated the soil, the metabolic activity of the Arthrobacter population was monitored as bioluminescence by luminometry and the total number of fluorescent Arthrobacter cells were enumerated by flow cytometry. In conclusion, these quantitative approaches, based on biomarkers, were demonstrated to be useful for accurate monitoring of specific cells in environmental samples.

  • 214.
    Emanuelsson, Olof
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Large-scale prediction of protein subcellular localization: sequence-based methods and applications2002Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 215.
    Enquist, Karl
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Fransson, Mawritz
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Boekel, Carolina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bengtsson, Inger
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Geiger, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lang, Lisa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pettersson, Aron
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Johansson, Sofia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane-integration characteristics of two ABC transporters, CFTR and P-glycoprotein2009Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 387, nr 5, s. 1153-1164Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To what extent do corresponding transmembrane helices in related integral membrane proteins have different membrane-insertion characteristics? Here, we compare, side-by-side, the membrane insertion characteristics of the 12 transmembrane helices in the adenosine triphosphate-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and the cystic fibrosis transmembrane conductance regulator (CFTR). Our results show that 10 of the 12 CFTR transmembrane segments can insert independently into the ER membrane. In contrast, only three of the P-gp transmembrane segments are independently stable in the membrane, while the majority depend on the presence of neighboring loops and/or transmembrane segments for efficient insertion. Membrane-insertion characteristics can thus vary widely between related proteins.

  • 216.
    Ensterö, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    The multi-faceted RNA molecule: Characterization and Function in the regulation of Gene Expression2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In this thesis I have studied the RNA molecule and its function and characteristics in the regulation of gene expression. I have focused on two events that are important for the regulation of the transcriptome: Translational regulation through micro RNAs; and RNA editing through adenosine deaminations.

    Micro RNAs (miRNAs) are ~22 nucleotides long RNA molecules that by semi complementarity bind to untranslated regions of a target messenger RNA (mRNA). The interaction manifests through an RNA/protein complex and act mainly by repressing translation of the target mRNA. I have shown that a pre-cursor miRNA molecule have significantly different information content of sequential composition of the two arms of the pre-cursor hairpin. I have also shown that sequential composition differs between species.

    Selective adenosine to inosine (A-to-I) RNA editing is a post-transcriptional process whereby highly specific adenosines in a (pre-)messenger transcript are deaminated to inosines. The deamination is carried out by the ADAR family of proteins and require a specific sequential and structural landscape for target recognition. Only a handful of messenger substrates have been found to be site selectively edited in mammals. Still, most of these editing events have an impact on neurotransmission in the brain.

    In order to find novel substrates for A-to-I editing, an experimental setup was made to extract RNA targets of the ADAR2 enzyme. In concert with this experimental approach, I have constructed a computational screen to predict specific positions prone to A-to-I editing.

    Further, I have analyzed editing in the mouse brain at four different developmental stages by 454 amplicon sequencing. With high resolution, I present data supporting a general developmental regulation of A-to-I editing. I also present data of coupled editing events on single RNA transcripts suggesting an A-to-I editing mechanism that involve ADAR dimers to act in concert. A different editing pattern is seen for the serotonin receptor 5-ht2c.

  • 217.
    Ericsson, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Prenyltransferases: branch-point enzymes in the biosynthesis of cholesterol, dolichol, ubiquinone and prenylated proteins1992Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 218. Eriksson, A M
    et al.
    Lundgren, B
    Andersson, K
    DePierre, J W
    Is the cytosolic catalase induced by peroxisome proliferators in mouse liver on its way to the peroxisomes?1992Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 308, nr 2Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Dietary treatment of male C57B1/6 mice with clofibrate, nafenopin or WY-14.643 resulted in a modest (at most 2-fold) increase in the total catalase activity in the whole homogenate and mitochondrial fraction prepared from the livers of these animals. On the other hand, the catalase activity recovered in the cytosolic fraction was increased 12- to 18-fold, i.e. 30-35% of the total catalase activity in the hepatic homogenate was present in the high-speed supernatant fraction after treatment with these peroxisome proliferators. A study of the time course of the changes in peroxisomal and cytosolic catalase activities demonstrated that the peroxisomal activity both increased upon initiation of exposure and decreased after termination of treatment several days after the increase and decrease, respectively, in the corresponding cytosolic activity. This finding suggests that the cytosolic catalase may be on its way to incorporation into peroxisomes.

  • 219. Eriksson, A M
    et al.
    Zetterqvist, M A
    Lundgren, B
    Andersson, K
    Beije, B
    DePierre, J W
    Studies on the intracellular distributions of soluble epoxide hydrolase and of catalase by digitonin-permeabilization of hepatocytes isolated from control and clofibrate-treated mice.1991Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 198, nr 2Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Digitonin permeabilization of hepatocytes from control and clofibrate-treated (0.5% by mass, 10 days) male C57bl/6 mice was used to study the intracellular distributions of soluble ('cytosolic') epoxide hydrolase and of catalase. The following conclusions were drawn. (1) About 60% of the total soluble epoxide hydrolase activity in control mouse hepatocytes is situated in the cytosol. (2) The rest is not mitochondrial, but probably peroxisomal. (3) Of the total catalase activity in control mouse hepatocytes, 5-10% is found in the cytosol. (4) Treatment of mice with clofibrate increases the total hepatocyte activity of soluble epoxide hydrolase 4-fold, but does not influence the relative distribution of this enzyme between cytosol and peroxisomes. (5) The total catalase activity is increased 3.5-fold by clofibrate treatment and 15-35% of this activity is shifted from the peroxisomes to the cytosol.

  • 220.
    Eriksson, AnnaCarin
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Functional and structural studies on the integrated mitochondrial processing peptidase/bc₁ complex from Spinacia oleracea1995Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 221.
    Eriksson, Hanna M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Intracellular vesicles induced by monotopic membrane protein in Escherichia coli2009Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The monotopic membrane protein alMGS, a glycosyltransferase catalyzing glucolipid synthesis in Acholeplasma laidlawii, was overexpressed in Escherichia coli. Optimization of basic growth parameters was performed, and a novel method for detergent and buffer screening using a small size-exclusion chromatography was developed. This resulted in a tremendous increase in protein yields, as well as the unexpected discovery that the protein induces intracellular vesicle formation in E. coli. This was confirmed by sucrose density separation and Cryo-TEM of membranes, and the properties of the vesicles were analyzed using SDS-PAGE, western blot and lipid composition analysis. It is concluded that both alMGS and alDGS, the next enzyme in glucolipid pathway, have the ability to make the membrane bend and eventually form vesicles. This is likely due to structural and electrostatic properties, such as the way the proteins penetrate the membrane interface and thereby expand one monolayer. The highly positively charged binding surfaces of the glycosyltransferases may bind negatively charged lipids, such as Phosphatidylglycerol (PG), in the membrane and withdraw it from the general pool of lipids. This would increase the overall lipid synthesis, since PG is a pace-keeper, and the local concentration of nonbilayer prone lipids, such as Phosphatidylethanolamine, can increase and also induce bending of the membrane. The formation of surplus membrane inside the E. coli cell was used to develop a generic method for overexpression of membrane proteins. A proof-of-principle experiment with a test set of twenty membrane proteins from E. coli resulted in elevated expression levels for about half of the set. Thus, we believe that this method will be a useful tool for overexpression of many membrane proteins. By engineering E. coli mutants with different lipid compositions, fine-tuning membrane properties for different proteins is also possible.

  • 222.
    Eriksson, Hanna M.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Persson, Karina
    Umeå Universitet.
    Zhang, Shuguang
    Massachusetts Institute of Technology.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    High-yield expression and purification of a monotopic membrane glycosyltransferase2009Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 66, nr 2, s. 143-148Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Membrane proteins are essential to many cellular processes. However, the systematic study of membrane protein structure has been hindered by the difficulty in obtaining large quantities of these proteins. Protein overexpression using Escherichia coli is commonly used to produce large quantities of protein, but usually yields very little membrane protein. Furthermore, optimization of the expressing conditions, as well as the choice of detergent and other buffer components, is thought to be crucial for increasing the yield of stable and homogeneous protein. Herein we report high-yield expression and purification of a membrane-associated monotopic protein, the glycosyltransferase monoglucosyldiacylglycerol synthase (alMGS), in E. coli. Systematic optimization of protein expression was achieved through controlling a few basic expression parameters, including temperature and growth media, and the purifications were monitored using a fast and efficient size-exclusion chromatography (SEC) screening method. The latter method was shown to be a powerful tool for fast screening and for finding the optimal protein-stabilizing conditions. For alMGS it was found that the concentration of detergent was just as important as the type of detergent, and a low concentration of n-Dodecyl-β-D-maltoside (DDM) (~1× critical micelle concentration) was the best for keeping the protein stable and homogeneous. By using these simply methods to optimize the conditions for alMGS expression and purification, the final expression level increase by two orders of magnitude, reaching 170 mg of pure protein per litre culture.

  • 223.
    Eriksson, Hanna M.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wessman, Per
    Ge, Changrong
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Edwards, Katarina
    Wieslander, Ake
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Massive formation of intracellular membrane vesicles in Escherichia coli by a monotopic membrane-bound lipid glycosyltransferase2009Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, nr 49, s. 33904-33914Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of > or = 60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter approximately 100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.

  • 224.
    Eriksson, Jan
    Stockholms universitet.
    Regulation of psbA gene expression in the cyanobacterium Synechocystis 68032001Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 225.
    Eriksson, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gene therapy tools: oligonucleotides and peptides2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Genetic mutations can cause a wide range of diseases, e.g. cancer. Gene therapy has the potential to alleviate or even cure these diseases. One of the many gene therapies developed so far is RNA-cleaving deoxyribozymes, short DNA oligonucleotides that specifically bind to and cleave RNA. Since the development of these synthetic catalytic oligonucleotides, the main way of determining their cleavage kinetics has been through the use of a laborious and error prone gel assay to quantify substrate and product at different time-points. We have developed two new methods for this purpose. The first one includes a fluorescent intercalating dye, PicoGreen, which has an increased fluorescence upon binding double-stranded oligonucleotides; during the course of the reaction the fluorescence intensity will decrease as the RNA is cleaved and dissociates from the deoxyribozyme. A second method was developed based on the common denominator of all nucleases, each cleavage event exposes a single phosphate of the oligonucleotide phosphate backbone; the exposed phosphate can simultaneously be released by a phosphatase and directly quantified by a fluorescent phosphate sensor. This method allows for multiple turnover kinetics of diverse types of nucleases, including deoxyribozymes and protein nucleases.

    The main challenge of gene therapy is often the delivery into the cell. To bypass cellular defenses researchers have used a vast number of methods; one of these are cell-penetrating peptides which can be either covalently coupled to or non-covalently complexed with a cargo to deliver it into a cell. To further evolve cell-penetrating peptides and understand how they work we developed an assay to be able to quickly screen different conditions in a high-throughput manner. A luciferase up- and downregulation experiment was used together with a reduction of the experimental time by 1 day, upscaling from 24- to 96-well plates and the cost was reduced by 95% compared to commercially available assays. In the last paper we evaluated if cell-penetrating peptides could be used to improve the uptake of an LNA oligonucleotide mimic of GRN163L, a telomerase-inhibiting oligonucleotide. The combination of cell-penetrating peptides and our mimic oligonucleotide lead to an IC50 more than 20 times lower than that of GRN163L.

  • 226.
    Eriksson, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kinetic assays for RNA-cleaving deoxyribozymes and other nucleases2016Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In this thesis two different assays for real-time RNA-cleaving deoxyribozyme and general nuclease kinetics are presented. Previous publications on nuclease kinetic assays have been riddled with drawbacks of labeling, discontinuity, cost etc. To tackle some of the drawbacks two assays were developed; the first specifically for RNA-cleaving deoxyribozymes to allow real-time kinetic measurements independently of whether the deoxyribozyme has low or high levels of secondary structure and when cleaving a full length messenger RNA (mRNA) substrate; the second assay was developed as a means to measure kinetics of virtually any nuclease by utilizing the single ubiquitous phenomenon in nuclease cleavage, the exposure of a phosphate upon hydrolysis of the phosphate backbone.

    In Paper I the assay for RNA-cleaving deoxyribozyme kinetics is presented as a development of a previously published assay. The search for a fluorescent intercalating dye with more preferential properties than ethidium bromide resulted in PicoGreen. This dye allowed the assay to be used for deoxyribozymes with low and high levels of secondary structure as well as using full length mRNA substrates.

    Paper II presents the second assay of this thesis, an assay where phosphates exposed by nuclease cleavage are released from their products by phosphatases; the released inorganic phosphates are quantified in real-time by a biosensor. The assay allows for real-time kinetics without the use of labels (i.e. natural enzymes and substrates). Regardless of whether the nuclease was a protein, nucleic acid-based, an exo- or endonuclease, processive or single-target nuclease the assay suited them equally well.

  • 227.
    Eriksson, Olivia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Simplicity within Complexity: Understanding dynamics of cellular networks by model reduction2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Cellular networks composed of interactions between genes, proteins and metabolites, determines the behavioural repertoire of the cell. Recent developments in high-throughput experimental techniques and computational methods allow static descriptions of these networks on a genome scale. There are also several dynamical mathematical models characterizing small subnetworks of the cell such as a signaling cascade or cell division. These networks exhibit a considerable complexity, and mathematical analysis are therefore essential in order to uncover the underlying dynamical core driving the systems. A core description can reveal the relative functional contributions of the various molecular interactions and goes to the heart of what kind of computations biological circuits perform. Partially successful methodologies toward this end includes bifurcation analysis, which only considers a small number of dimensions, and large-scale computer simulations.

    In this thesis we explore a third route utilizing the inherent biological structure and dynamics of the network as a tool for model simplification. Using the well studied cell cycle, as a model system, we observe that the this network can be divided into dynamical modules displaying a switch-like behaviour. This allows a transformation into a piecewise linear system with delay, the subsequent use of tools from linear systems theory and finally a core dynamical description. Analytical expressions capturing important cell cycle features such as cell mass, as well as necessary constraints for cell cycle oscillations, are thereby retrieved. Finally we use the dynamical core together with large-scale simulations in order to study the balance between robustness and sensitivity.

    It appears that biological features such as switches, modularity and robustness provide a means to reformulate intractable mathematical problems into solvable ones, as biology appears to suggest a path of simplicity within the realm of mathematical complexity.

  • 228.
    Eriksson, Sylvia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Molecular properties of disordered plant dehydrins: Membrane interaction and function in stress2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Dehydrins are intrinsically disordered plant stress-proteins. Repetitively in their sequence are some highly conserved stretches of 7-17 residues, the so called K-, S-, Y- and lysine rich segments. This thesis aims to give insight into the possible role dehydrins have in the stressed plant cell with main focus on membrane interaction and protection. The work includes four recombinant dehydrins from the plant Arabidopsis thaliana: Cor47 (SK3), Lti29 (SK3), Lti30 (K6) and Rab18 (Y2SK2).

    Initially, we mimicked crowded cellular environment in vitro to verify that dehydrins are truly disordered proteins. Thereafter, the proposal that the compulsory K-segment determines membrane binding was tested. Experiments show that only Lti30 and Rab18 bind, whereas Cor47 and Lti29 does not. As Lti30 and Rab18 binds they assembles vesicles into clusters in vitro, a feature used to characterize the interaction. From this it was shown that membrane binding of Lti30 is electrostatic and determined by global as well as local charges. Protonation of histidine pairs flanking the K-segments works as an on/off-binding switch. By NMR studies it was shown that the K-segments form a dynamic α-helix upon binding, so called disorder-to-order behaviour. Also, dehydrins electrostatic interaction with lipids can be further tuned by posttranslational phosphorylation or coordination of calcium and zinc ions.

    Finally, specific binding of Rab18 to inositol lipids, mainly PI(4,5)P2, is reported. The interaction is mainly coordinated by two arginines neighboring one of the K-segments. In conclusion, the K-segments are indeed involved in the binding of dehydrins to membrane but only in combination with extensions (Lti30) or modified (Rab18). 

  • 229.
    Eriksson, Sylvia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Harryson, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane binding of disordered plant dehydrins is tuned by phosphorylation and coordination of Ca2+ and Zn2+ ions.Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Dehydrins are intrinsically disordered proteins expressed under water- related stress in plants. As a clue to their function, some dehydrins are found to interact in an orderly manner with negatively-charged lipids, supporting the idea of a key role in safeguarding membrane integrity. We have earlier reported that this lipid interaction is modulated electrostatically. Of particular interest is the pronounced effect of local charge that shed light on how dehydrin function is regulated in vivo. In this study we test the generality of this proposition on four dehydrins from Arabidopsis thaliana representing different dehydrin subgroups. The results show that membrane interaction of dehydrins in their apo state is correlated to their protein net charge. Also, we explore further putative regulation mechanism by investigating the additive role of ion coordination and phosphorylation on membrane binding. The results show that coordination of Ca2+ and Zn2+ have markedly different effects. Coordination of Ca2+ augments mainly the membrane affinity of dehydrins that already bind lipids in their apo states (Lti30 and Rab18). Coordination of Zn2+, on the other hand, induces membrane binding and vesicle assembly of all tested proteins, also those that fail to bind membranes in the absence of metal ions (Cor47 or Lti29). Finally, we observe that the effect of Ca2+ is effectively enhanced by phosphorylation. The observations corroborate the idea of a sensitive and multifaceted regulatory mechanism of the dehydrin function in stressed plant cells but point also at a functional diversity. 

  • 230.
    Eriksson, Sylvia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Harryson, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The plant Rab18 dehydrin - a disordered stressed induced conditional peripheral membrane protein. Specific interaction with PI(4,5)P2Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The Arabidosis dehydrin Rab18 is expressed in response to drought and the phytohormone ABA. As a clue to a general dehydrin function, some dehydrins are found to interact in an orderly manner with negatively-charged lipids, supporting the idea of a key role in safeguarding membrane integrity. Interaction between dehydrins and membranes studied so far has been driven by general electrostatic attractions. Here, we report on specific binding of Rab18 with inositol lipids, especially PI(4,5)P2. This binding is not purely under the control of global protein electrostatics since Rab18 fails to bind to lipid vesicles with a high negative net charge (DOPC:DOPG, 3:1). Instead, Rab18 binds strongly to inositol lipids even at low negative vesicle net charge (i.e. DOPC:DOPI(4,5)P2, 98:2). Further, Rab18 show a high specificity to inositol lipids with a phosphate in the 5th position on the inositol ring i.e. PI(5)P and PI(4,5)P2 whereas a phosphate at 3rd position restrains Rab18 binding (i.e. PI(4,5)P2>PI(3,5)P2>PI(3,4)P2 and PI(5)P>> PI(3)P). Moreover, Rab18 specificity to inositol lipids is mainly augmented by the Arg in the protein sequence and when all six Arg are replaced by Lys is the binding of Rab18 to PI(4,5)P2 almost abolished. The two Arg preceding the first K-seg (Rab18Arg125-126) are key residues in binding PI(4,5)P2 and the mechanistic implication of these Arg is elucidated. We put forward the idea that Rab18 is a conditional peripheral membrane protein that sense and respond to lipid alterations at membrane surfaces during stress. Moreover, by binding to PI(4,5)P2 Rab18 could take part in the regulation of different cellular processes, showing a new role of dehydrins as regulators of plant stress. A possible role of Rab18 in the regulation of ABA induced stomata movements are presented. 

  • 231. Eriste, Elo
    et al.
    Kurrikoff, Kaido
    Suhorutsenko, Julia
    Osokolkov, Nikita
    Copolovici, Dana Maria
    Jones, Sarah
    Laakkonen, Pirjo
    Howl, John
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Peptide-Based Glioma-Targeted Drug Delivery Vector gHoPe22013Ingår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, nr 3, s. 305-313Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Gliomas are therapeutically challenging cancers with poor patient prognosis. New drug delivery strategies are needed to achieve a more efficient chemotherapy-based approach against brain tumors. The current paper demonstrates development of a tumor-targeted delivery vector that is based on a cell-penetrating peptide pVEC and a novel glioma-targeting peptide sequence gHo. The unique tumor-homing peptide gHo was identified using in vitro phage display technology. The novel delivery vector, which we designated as gHoPe2, was constructed by a covalent conjugation of pVEC, gHo, and a cargo; the latter could be either a labeling moiety (such as a fluorescent marker) or a cytostatic entity. Using a fluorescent marker, we demonstrate efficient uptake of the vector in glioma cells and selective labeling of glioma xenograft tumors in a mouse model. This is the first time that we know where in vitro phage display has yielded an efficient, in vivo working vector. We also demonstrate antitumor efficacy of the delivery vector gHoPe2 using a well-characterized chemotherapeutic drug doxorubicin. Vectorized doxorubicin proved to be more efficient than the free drug in a mouse glioma xenograft model after systemic administration of the drugs. In conclusion, we have characterized a novel glioma-homing peptide gHo, demonstrated development of a new and potential glioma-targeted drug delivery vector gHoPe2, and demonstrated the general feasibility of the current approach for constructing cell-penetrating peptide-based targeted delivery systems.

  • 232.
    Erlandsson, Mikael
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi och neurotoxikologi.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi och neurotoxikologi.
    Metallic zinc reduction of disulfide bonds between cysteine residues in peptides and proteins2005Ingår i: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 11, nr 4, s. 261-265Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The use of powdered metallic zinc in acidic solution for the reduction of disulfide bonds in peptides and proteins has been investigated. The method has several advantages over the traditional mereapto based reducing methods currently used; the reducing agent is readily available and inexpensive; reduction can be performed in weakly acidic solutions of water and/or acetonitrile; work up simply consists of a centrifugation step followed by pipeting the supernatant from the metal pellet, thereby greatly diminishing the risk of reoxidation as a more elaborate work up procedure could result in. As no mercapto compounds are added, there is no risk that the reducing agent will interfere in subsequent modification of the thiol functionality. Disulfides in a model peptide are reduced within 5 min in any mixture of water/acetonitrile containing 1% TFA, all disulfides in insulin is reduced within I h in any mixture of water/acetonitrile containing 5% acetic acid. To stress the convenience of the metallic zinc reduction method, the resulting thiol compound was subjected to two commonly employed reactions in peptide chemistry: Cys(Npys) directed disulfide formation (70% yield) and native chemical ligation between the reduced model peptide and Boc-Ala-p-metylthiobenzyl ester (65% yield of the ligation product plus disulfide formation between Cys and p-thiocresol).

  • 233. Ertem, Mehmed Z.
    et al.
    Cramer, Christopher J.
    Himo, Fahmi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Siegbahn, Per E. M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    N-O bond cleavage mechanism(s) in nitrous oxide reductase2012Ingår i: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 17, nr 5, s. 687-698Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Quantum chemical calculations of active-site models of nitrous oxide reductase (N2OR) have been undertaken to elucidate the mechanism of N-O bond cleavage mediated by the supported tetranuclear Cu4S core (Cu-Z) found in the enzymatic active site. Using either a minimal model previously employed by Gorelsky et al. (J. Am. Chem. Soc. 128:278-290, 2006) or a more extended model including key residue side chains in the active-site second shell, we found two distinct mechanisms. In the first model, N2O binds to the fully reduced Cu-Z in a bent mu-(1,3)-O,N bridging fashion between the Cu-I and Cu-IV centers and subsequently extrudes N-2 while generating the corresponding bridged mu-oxo species. In the second model, substrate N2O binds loosely to one of the coppers of Cu-Z in a terminal fashion, i.e., using only the oxygen atom; loss of N-2 generates the same mu-oxo copper core. The free energies of activation predicted for these two alternative pathways are sufficiently close to one another that theory does not provide decisive support for one over the other, posing an interesting problem with respect to experiments that might be designed to distinguish between the two. Effects of nearby residues and active-site water molecules are also explored.

  • 234.
    Ezzat, Kariem
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Karolinska Institutet, Sweden.
    Pernemalm, Maria
    Pålsson, Sandra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Roberts, Thomas C.
    Järver, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Dondalska, Aleksandra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Bestas, Burcu
    Sobkowiak, Michal J.
    Levanen, Bettina
    Skold, Magnus
    Thompson, Elizabeth A.
    Saher, Osama
    Kari, Otto K.
    Lajunen, Tatu
    Ekström, Eva Sverremark
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Nilsson, Caroline
    Ishchenko, Yevheniia
    Malm, Tarja
    Wood, Matthew J. A.
    Power, Ultan F.
    Masich, Sergej
    Linden, Anders
    Sandberg, Johan K.
    Lehtio, Janne
    Spetz, Anna-Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    EL Andaloussi, Samir
    The viral protein corona directs viral pathogenesis and amyloid aggregation2019Ingår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, artikel-id 2331Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid beta-peptide (A beta(42)), a major constituent of amyloid plaques in Alzheimer's disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.

  • 235.
    Falkevall, Annelie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Alikhani, Nyosha
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bhushan, Shashi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pavlov, Pavel F.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Busch, Katrin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Johnson, Kenneth A.
    Eneqvist, Therese
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Tjernberg, Lars
    Ankarcrona, Maria
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP2006Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, nr 39, s. 29096-29104Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid beta-protein (Abeta). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zinc-binding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against Abeta. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu(78) in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against Abeta-(1-42), Abeta-(1-40), and Abeta Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 A resolution of AtPreP allowed us to identify Cys(90) and Cys(527) that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial Abeta by hPreP may potentially be of importance in the pathology of Alzheimer disease.

  • 236.
    Faxén, Kristina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Active Transport of Ions across Biomembranes: A Kinetic Study of Cytochrome c Oxidase Reconstituted into Phospholipid Vesicles2007Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Ion transport across membranes is of uttermost importance for us. It is the foundation for signaling of various kinds e.g. in the nerve-system. Furthermore, energy from photosynthesis and metabolism is conserved in electrochemical gradients across membranes, maintained by ion pumps. In this thesis I discuss mechanisms of how protons and other ions are translocated across biomembranes against their concentration gradients. I have studied one specific proton pump, cytochrome c oxidase (CytcO) and in the summary I also compare CytcO with two other pumps for which a wealth of structural and functional information has recently been obtained. The data in the articles presented in this thesis support a model were proton pumping can be achieved without simultaneous oxidation of heme a or electron transfer (paper I); where a proton is transferred to the catalytic site before the pump site is protonated (paper IV); and where proton release is preceded by a conformational change (paper II). These observations could be explained by a model involving a conformational change of the pump element, recently proposed from our laboratory1. Furthermore the results from the papers in this thesis show that proton uptake precedes proton release in D2O (paper II). The kinetics of electron transfers linked to proton pumping is solely determined by the pH on the N-side of the membrane (paper III). Finally Zn2+ added on the P-side of the membrane inhibits a specific reaction step (paper IV). In the three pumps described here conformational changes, modulating ion affinities, and the opening and closing of gates, seem to be involved in driving the ions across the membrane.

    1. Brzezinski, P. & Larsson, G. (2003) Biochim. Biophys. Acta 1605, 1-13.

  • 237.
    Faxén, Kristina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The Inside pH Determines Rates of Electron and Proton Transfer in Vesicle-Reconstituted Cytochrome c Oxidase2007Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1767, nr 5, s. 381-386Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane. The results show that the pH on the inside of the membrane fully determines the kinetics of internal electron transfers that are linked to proton pumping. Thus, even though proton release is rate limiting for these reaction steps (Salomonsson et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 17624), the transition kinetics is insensitive to the outside pH (in the range 6–9.5).

  • 238.
    Ferreira Vasconcelos, Luis Daniel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Oligonucleotide Complexes with Cell-Penetrating Peptides: Structure, Binding, Translocation and Flux in Lipid Membranes2014Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The ability of cell-penetrating peptides to cross plasma membranes has been explored for various applications, including the delivery of bioactive molecules to inhibit disease-causing cellular processes. The uptake mechanisms by which cell-penetrating peptides enter cells depend on the conditions, such as the cell line the concentration and the temperature. To be used as therapeutics, each novel cell-penetrating peptide needs to be fully characterized, including their physicochemical properties, their biological activity and their uptake mechanism. Our group has developed a series of highly performing, non-toxic cell-penetrating peptides, all derived from the original sequence of transportan 10. These analogs are called PepFects and NickFects and they are now a diverse family of N-terminally stearylated peptides. These peptides are known to form noncovalent, nano-sized complexes with diverse oligonucleotide cargoes. One bottleneck that limits the use of this technology for gene therapy applications is the efficient release of the internalized complexes from endosomal vesicles.

    The general purpose of this thesis is to reveal the mechanisms by which our in house designed peptides enter cells and allow the successful transport of biofunctional oligonucleotide cargo. To reach this goal, we used both biophysical and cell biology methods. We used spectroscopy methods, including fluorescence, circular dichroism and dynamic light scattering to reveal the physicochemical properties. Using confocal and transmission electron microscopy we observed and tracked the internalization and intracellular trafficking. Additionally we tested the biological activity in vitro and the cellular toxicity of the delivery systems.

    We conclude that the transport vectors involved in this study are efficient at perturbing lipid membranes, which correlates with their remarkable capacity to transport oligonucleotides into cells. The improved and distinct capacities to escape from endosomal vesicles can be the result of their different structures and hydrophobicity. These findings extend the knowledge of the variables that condition intracellular Cell-penetrating peptide mediated transport of nucleic acids, which ultimately translates into a small step towards successful non-viral gene therapy.

  • 239.
    Figueroa, Ricardo A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The functional organization of nuclear membrane proteins and development of new technology for studies of cell signaling2011Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The eukaryotic cell is defined by the nucleus, which is delimited by a double membrane structure termed the nuclear envelope (NE). The NE is implicated in a multitude of different processes, for example chromatin organization. During mitosis in higher eukaryotes the nucleus is disassembled to allow the formation of the mitotic spindle, which segregates the duplicated chromosomes between daughter cells. We have characterized a novel transmembrane protein of the inner nuclear membrane. Because of its distribution along spindle microtubule during mitosis, we termed the protein Samp1 (Spindle associated membrane protein 1). Samp1 is the founding member of transmembrane proteins that define a novel membrane domain that we have termed the SE (spindle endomembrane). Furthermore, we have shown that in interphase Samp1 specifically interacts with the centrosome and A-type lamina network proteins. Moreover, Samp1 contains an evolutionary highly conserved N-terminal tail containing two putative zinc fingers.

    Recent studies indicate local caspase activity in dendrites or axons during development and in neurodegenerative disorders. Here I present the development of a novel and unique system to monitor protease activity at sub-cellular resolution in live cells. This system relies on a cleavable FRET sensor that is anchored to the cytoskeleton. Using this system we demonstrate local caspase activation of the soma in neuronaly differentiated cells. We also used the anchored FRET sensors to monitor caspase activation after treatment with the Alzheimer’s decease related amyloid-β peptide.

    Moreover we have improved a NF-ĸB decoy delivery system. The system consists of a cell penetrating peptide, transportan-10, covalently linked to a peptide nucleic acid sequence that hybridizes with a nonanucleotide sequence in the decoy. We show that this system effectively delivered the decoy and inhibited an inflammatory response in primary rat glial cells.

  • 240. Finn, Robert D.
    et al.
    Bateman, Alex
    Clements, Jody
    Coggill, Penelope
    Eberhardt, Ruth Y.
    Eddy, Sean R.
    Heger, Andreas
    Hetherington, Kirstie
    Holm, Liisa
    Mistry, Jaina
    Sonnhammer, Erik L. L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Tate, John
    Punta, Marco
    Pfam: the protein families database2014Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, nr D1, s. d222-D230Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pfam, available via servers in the UK (http://pfam.sanger.ac.uk/) and the USA (http://pfam.janelia.org/), is a widely used database of protein families, containing 14 831 manually curated entries in the current release, version 27.0. Since the last update article 2 years ago, we have generated 1182 new families and maintained sequence coverage of the UniProt Knowledgebase (UniProtKB) at nearly 80%, despite a 50% increase in the size of the underlying sequence database. Since our 2012 article describing Pfam, we have also undertaken a comprehensive review of the features that are provided by Pfam over and above the basic family data. For each feature, we determined the relevance, computational burden, usage statistics and the functionality of the feature in a website context. As a consequence of this review, we have removed some features, enhanced others and developed new ones to meet the changing demands of computational biology. Here, we describe the changes to Pfam content. Notably, we now provide family alignments based on four different representative proteome sequence data sets and a new interactive DNA search interface. We also discuss the mapping between Pfam and known 3D structures.

  • 241.
    Fischer, Alexander W.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. University Medical Center Hamburg-Eppendorf, Germany.
    Shabalina, Irina G.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Mattsson, Charlotte L.
    Abreu-Vieira, Gustavo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Cannon, Barbara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Nedergaard, Jan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Petrovic, Natasa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    UCP1 inhibition in Cidea-overexpressing mice is physiologically counteracted by brown adipose tissue hyperrecruitment2017Ingår i: American Journal of Physiology. Endocrinology and Metabolism, ISSN 0193-1849, E-ISSN 1522-1555, Vol. 312, nr 1, s. e72-E87Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cidea is a gene highly expressed in thermogenesis- competent (UCP1-containing) adipose cells, both brown and brite/beige. Here, we initially demonstrate a remarkable adipose-depot specific regulation of Cidea expression. In classical brown fat, Cidea mRNA is expressed continuously and invariably, irrespective of tissue recruitment. However, Cidea protein levels are regulated posttranscriptionally, being conspicuously induced in the thermogenically recruited state. In contrast, in brite fat, Cidea protein levels are regulated at the transcriptional level, and Cidea mRNA and protein levels are proportional to tissue briteness. Although routinely followed as a thermogenic molecular marker, Cidea function is not clarified. Here, we employed a gain-of-function approach to examine a possible role of Cidea in the regulation of thermogenesis. We utilized transgenic aP2-hCidea mice that overexpress human Cidea in all adipose tissues. We demonstrate that UCP1 activity is markedly suppressed in brown-fat mitochondria isolated from aP2-hCidea mice. However, mitochondrial UCP1 protein levels were identical in wildtype and transgenic mice. This implies a regulatory effect of Cidea on UCP1 activity, but as we demonstrate that Cidea itself is not localized to mitochondria, we propose an indirect inhibitory effect. The Cidea-induced inhibition of UCP1 activity (observed in isolated mitochondria) is physiologically relevant since the mice, through an appropriate homeostatic compensatory mechanism, increased the total amount of UCP1 in the tissue to exactly match the diminished thermogenic capacity of the UCP1 protein and retain unaltered nonshivering thermogenic capacity. Thus, we verified Cidea as being a marker of thermogenesis-competent adipose tissues, but we conclude that Cidea, unexpectedly, functions molecularly as an indirect inhibitor of thermogenesis.

  • 242. Fischer, Katrin
    et al.
    Ruiz, Henry H.
    Jhun, Kevin
    Finan, Brian
    Oberlin, Douglas J.
    van der Heide, Verena
    Kalinovich, Anastasia V.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Petrovic, Natasa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Wolf, Yochai
    Clemmensen, Christoffer
    Shin, Andrew C.
    Divanovic, Senad
    Brombacher, Frank
    Glasmacher, Elke
    Keipert, Susanne
    Jastroch, Martin
    Nagler, Joachim
    Schramm, Karl-Werner
    Medrikova, Dasa
    Collden, Gustav
    Woods, Stephen C.
    Herzig, Stephan
    Homann, Dirk
    Jung, Steffen
    Nedergaard, Jan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Cannon, Barbara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Tschoep, Matthias H.
    Mueller, Timo D.
    Buettner, Christoph
    Alternatively activated macrophages do not synthesize catecholamines or contribute to adipose tissue adaptive thermogenesis2017Ingår i: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 23, nr 5, s. 623-630Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Adaptive thermogenesis is the process of heat generation in response to cold stimulation. It is under the control of the sympathetic nervous system, whose chief effector is the catecholamine norepinephrine (NE). NE enhances thermogenesis through beta 3-adrenergic receptors to activate brown adipose tissue and by 'browning' white adipose tissue. Recent studies have reported that alternative activation of macrophages in response to interleukin (IL)-4 stimulation induces the expression of tyrosine hydroxylase (TH), a key enzyme in the catecholamine synthesis pathway, and that this activation provides an alternative source of locally produced catecholamines during the thermogenic process. Here we report that the deletion of Th in hematopoietic cells of adult mice neither alters energy expenditure upon cold exposure nor reduces browning in inguinal adipose tissue. Bone marrow-derived macrophages did not release NE in response to stimulation with IL-4, and conditioned media from IL-4-stimulated macrophages failed to induce expression of thermogenic genes, such as uncoupling protein 1 (Ucp1), in adipocytes cultured with the conditioned media. Furthermore, chronic treatment with IL-4 failed to increase energy expenditure in wild-type, Ucp1(-/-) and interleukin-4 receptor-alpha double-negative (Il4ra(-/-)) mice. In agreement with these findings, adipose-tissue-resident macrophages did not express TH. Thus, we conclude that alternatively activated macrophages do not synthesize relevant amounts of catecholamines, and hence, are not likely to have a direct role in adipocyte metabolism or adaptive thermogenesis.

  • 243.
    Fisone, Gilberto
    Stockholms universitet.
    Galanin and acetylcholine in the rat hippocampus: a functional study1990Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 244.
    Flock, Ulrika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nitric Oxide Reductase from Paracoccus denitrificans: A Proton Transfer Pathway from the “Wrong” Side2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Denitrification is an anaerobic process performed by several soil bacteria as an alternative to aerobic respiration. A key-step in denitrification (the N-N-bond is made) is catalyzed by nitric oxide reductase (NOR); 2NO + 2e- + 2H+ → N2O + H2O. NOR from Paracoccus denitrificans is a member of the heme copper oxidase superfamily (HCuOs), where the mitochondrial cytochrome c oxidase is the classical example. NOR is situated in the cytoplasmic membrane and can, as a side reaction, catalyze the reduction of oxygen to water.

    NORs have properties that make them divergent members of the HCuOs; the reactions they catalyze are not electrogenic and they do not pump protons. They also have five strictly conserved glutamates in their catalytic subunit (NorB) that are not conserved in the ‘classical’ HCuOs. It has been asked whether the protons used in the reaction really come from the periplasm and if so how do the protons proceed through the protein into the catalytic site?

    In order to find out whether the protons are taken from the periplasm or the cytoplasm and in order to pinpoint the proton-route in NorB, we studied electron- and proton transfer during a single- as well as multiple turnovers, using time resolved optical spectroscopy. Wild type NOR and several variants of the five conserved glutamates were investigated in their solubilised form or/and reconstituted into vesicles.

    The results demonstrate that protons needed for the reaction indeed are taken from the periplasm and that all but one of the conserved glutamates are crucial for the oxidative phase of the reaction that is limited by proton uptake to the active site.

    In this thesis it is proposed, using a model of NorB, that two of the glutamates are located at the entrance of the proton pathway which also contains two of the other glutamates close to the active site.

  • 245.
    Fluman, Nir
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Tobiasson, Victor
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Stable membrane orientations of small dual-topology membrane proteins2017Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 30, s. 7987-7992Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The topologies of alpha-helical membrane proteins are generally thought to be determined during their cotranslational insertion into the membrane. It is typically assumed that membrane topologies remain static after this process has ended. Recent findings, however, question this static view by suggesting that some parts of, or even the whole protein, can reorient in the membrane on a biologically relevant time scale. Here, we focus on antiparallel homo- or heterodimeric small multidrug resistance proteins and examine whether the individual monomers can undergo reversible topological inversion (flip flop) in the membrane until they are trapped in a fixed orientation by dimerization. By perturbing dimerization using various means, we show that the membrane orientation of a monomer is unaffected by the presence or absence of its dimerization partner. Thus, membrane-inserted monomers attain their final orientations independently of dimerization, suggesting that wholesale topological inversion is an unlikely event in vivo.

  • 246. Fong, Nova
    et al.
    Öhman, Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Bentley, David L
    Fast ribozyme cleavage releases transcripts from RNA polymerase II and aborts co-transcriptional pre-mRNA processing2009Ingår i: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 16, nr 9, s. 916-923Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Adenosine-to-inosine (A-to-I) editing has been shown to be an important mechanism that increases protein diversity in the brain of organisms from human to fly. The family of ADAR enzymes converts some adenosines of RNA duplexes to inosines through hydrolytic deamination. The adenosine recognition mechanism is still largely unknown. Here, to investigate it, we analyzed a set of selectively edited substrates with a cluster of edited sites. We used a large set of individual transcripts sequenced by the 454 sequencing technique. On average, we analyzed 570 single transcripts per edited region at four different developmental stages from embryogenesis to adulthood. To our knowledge, this is the first time, large-scale sequencing has been used to determine synchronous editing events. We demonstrate that edited sites are only coupled within specific distances from each other. Furthermore, our results show that the coupled sites of editing are positioned on the same side of a helix, indicating that the three-dimensional structure is key in ADAR enzyme substrate recognition. Finally, we propose that editing by the ADAR enzymes is initiated by their attraction to one principal site in the substrate.

  • 247.
    Fontana, Carolina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Conde-Alvarez, Raquel
    Ståhle, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Holst, Otto
    Iriarte, Maite
    Zhao, Yun
    Arce-Gorvel, Vilma
    Hanniffy, Sean
    Gorvel, Jean-Pierre
    Moriyon, Ignacio
    Widmalm, Göran
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Structural Studies of Lipopolysaccharide-defective Mutants from Brucella melitensis Identify a Core Oligosaccharide Critical in Virulence2016Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, nr 14, s. 7727-7741Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and ManB(core) proteins have been fully characterized using NMR spectroscopy. The results revealed that disruption of wbkD gives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (beta-D-Glcp-(1 -> 4)-alpha-Kdop-(2 -> 4)[beta-D-GlcpN-(1 -> 6)-beta-D-GlcpN-(1 -> 4)[beta-D-GlcpN-(1 -> 6)]-beta-D-GlcpN-(1 -> 3)-alpha-D-Manp-(1 -> 5)]-alpha-Kdop-(2 -> 6)-beta-D-GlcpN3N4P-(1 -> 6)-alpha-D-GlcpN3N1P), in addition to components lacking one of the terminal beta-D-GlcpN and/or the beta-D-Glcp residues (48 and 17%, respectively). These structures were identical to those of the R-LPS from B. melitensis EP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption of man-B-core gives rise to a deep-rough pentasaccharide core (beta-D-Glcp-(1 -> 4)-alpha-Kdop-(2 -> 4)-alpha-Kdop-(2 -> 6)-beta-D-GlcpN3N4P-(1 -> 6)-alpha-D-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal beta-D-Glcp residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManB(core) proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion of B. melitensis wadC removes the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyltransferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential in B. melitensis virulence, the core deficiency in the wadC mutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the beta-D-GlcpN-(1 -> 6)-beta-D-GlcpN-(1 -> 4)[beta-D-GlcpN-(1 -> 6)]-beta-D-GlcpN-(1 -> 3)-alpha-D-Manp-(1 -> 5) structure in virulence.

  • 248.
    Fontana, Carolina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Kovacs, Helena
    Widmalm, Göran
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    NMR structure analysis of uniformly 13C-labeled carbohydrates2014Ingår i: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 59, nr 2, s. 95-110Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of C-13-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly C-13-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-C-13)-sucrose, 342 Da] and one compound of medium molecular weight (C-13-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, similar to 10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The C-13 resonances are traced using C-13-C-13 correlations from homonuclear experiments, such as (H)CC-CT-COSY, (H)CC-NOESY, CC-CT-TOCSY and/or virtually decoupled (H)CC-TOCSY. Based on the assignment of the C-13 resonances, the H-1 chemical shifts are derived in a straightforward manner using one-bond H-1-C-13 correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J (CC) splitting of the C-13 resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either C-13 or H-1 detected experiments, namely CC-CT-COSY, band-selective (H)CC-TOCSY, HC-CT-HSQC-NOESY or long-range HC-CT-HSQC. However, due to the short T-2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the H-1-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the C-13-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with N-15 at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and C-13-detected (H)CACO spectra.

  • 249.
    Fontana, Carolina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Lundborg, Magnus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Weintraub, Andrej
    Widmalm, Göran
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Structural studies and biosynthetic aspects of the o antigen polysaccharide from Escherichia coli o1742012Ingår i: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 354, s. 102-105Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The structure of the repeating unit of the O-antigenic polysaccharide (PS) from Escherichia coli O174 has been determined. Component analysis together with H-1 and C-13 NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by H-1, C-13-heteronuclear multiple-bond correlation and H-1, H-1-NOESY experiments. The PS is composed of tetrasaccharide repeating units with the following structure: -> 4)-beta-D-GlcpA-(1 -> 3)-beta-D-Galp-(1 -> 3)-beta-D-GalpNAc-(1 -> vertical bar beta-D-GlcpNAc-(1 -> 2) Cross-peaks of low intensity were present in the NMR spectra consistent with a beta-D-GlcpNAc-(1 -> 2)-beta-D-GlcpA(1 -> structural element at the terminal part of the polysaccharide, which on average is composed of similar to 15 repeating units. Consequently the biological repeating unit has a 3-substituted N-acetyl-D-galactosamine residue at its reducing end.

  • 250.
    Forsberg, Björn
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The antimicrobial effect of dermcidin investigated by computational electrophysiology molecular dynamics simulations2013Självständigt arbete på avancerad nivå (masterexamen), 40 poäng / 60 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Eukaryotic organisms rely on several mechanisms to inhibit bacterial growth and infection, which mankind has sought to mimic to their specific and more targeted use. Due to the effect of adaptation by bacteria in response to new antibiotics, known as antibiotic resistance, there will be an ever-present need to develop new antibiotics to maintain their high efficiency. Peptide antibiotics appear to target a general property of the bacterial membrane, and should therefore constitute a mechanism which is highly robust to mutational adaptation. We employ a specialized implementation of molecular dynamics simulations to examine the membrane-interactions and -permeabilization caused by human dermcidin in bacterial membranes, which shows antibacterial properties in experiment and forms a transmembrane channel-like structure according to a solved crystal structure. We are able to conclude that charged sidechains maintain a structural rigidity of the oligomeric assembly which in turn enables it to maintain anion selectivity, and that lower oligomeric states constitute an potentially functional oligomeric precursor to the crystallized hexamer. Further we are able to improve channel conductance, and suggest experimental observables to corroborate the given conclusions. The knowledge gained forwards the knowledge-base needed to establish a categorization of the class of AntiMicrobial Peptides, and holds promise for further development as a possible broad-spectrum antibiotic.

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