Ändra sökning
Avgränsa sökresultatet
16171819202122 901 - 950 av 1092
RefereraExporteraLänk till träfflistan
Permanent länk
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Träffar per sida
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sortering
  • Standard (Relevans)
  • Författare A-Ö
  • Författare Ö-A
  • Titel A-Ö
  • Titel Ö-A
  • Publikationstyp A-Ö
  • Publikationstyp Ö-A
  • Äldst först
  • Nyast först
  • Skapad (Äldst först)
  • Skapad (Nyast först)
  • Senast uppdaterad (Äldst först)
  • Senast uppdaterad (Nyast först)
  • Disputationsdatum (tidigaste först)
  • Disputationsdatum (senaste först)
  • Standard (Relevans)
  • Författare A-Ö
  • Författare Ö-A
  • Titel A-Ö
  • Titel Ö-A
  • Publikationstyp A-Ö
  • Publikationstyp Ö-A
  • Äldst först
  • Nyast först
  • Skapad (Äldst först)
  • Skapad (Nyast först)
  • Senast uppdaterad (Äldst först)
  • Senast uppdaterad (Nyast först)
  • Disputationsdatum (tidigaste först)
  • Disputationsdatum (senaste först)
Markera
Maxantalet träffar du kan exportera från sökgränssnittet är 250. Vid större uttag använd dig av utsökningar.
  • 901. Smit, Marjon A.
    et al.
    Maddalo, Gianluca
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Utrecht, Netherlands; Netherlands Proteomics Centre, Netherlands.
    Greig, Kylie
    Raaijmakers, Linsey M.
    Possik, Patricia A.
    van Breukelen, Bas
    Cappadona, Salvatore
    Heck, Albert J. R.
    Altelaar, A. F. Maarten
    Peeper, Daniel S.
    ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma2014Ingår i: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 10, nr 12, artikel-id 772Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.

  • 902. Sohlenius, A K
    et al.
    Lundgren, B
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    DePierre, J W
    Perfluorooctanoic acid has persistent effects on peroxisome proliferation and related parameters in mouse liver.1992Ingår i: Journal of biochemical toxicology, ISSN 0887-2082, Vol. 7, nr 4Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Male C57Bl/6 mice were treated for 5 days with 0.05% perfluorooctanoic acid (PFOA) in their diet. This treatment resulted in a potent induction of peroxisomal fatty acid beta-oxidation in the liver. In order to investigate recovery from treatment with PFOA, mice were given normal laboratory chow for up to 20 days after termination of PFOA administration. It was established that the activities of peroxisomal lauoryl-CoA oxidase and palmitoyl-CoA oxidation were still elevated 2-3 weeks after termination of treatment. The catalase activity recovered in the cytosolic fraction was also still significantly elevated after 20 days with normal laboratory chow. Furthermore, the protein content of the mitochondrial fraction was increased by PFOA and had not returned to control level at the end of the recovery period. Perfluorooctanoic acid also caused a persistent effect in omega hydroxylation of lauric acid (cytochrome P-452). The activities of cytosolic DT-diaphorase and glutathione transferase were also enhanced by PFOA. However, these two enzymes recovered relatively rapidly from the treatment (2-20 days). This study reveals two different patterns of recovery from PFOA treatment, one involving parameters that recovered completely, or almost completely, from PFOA treatment after 20 days and another involving parameters that were still elevated at the end of the recovery period.

  • 903.
    Sohlenius, Anna-Karin
    Stockholms universitet, Naturvetenskapliga fakulteten.
    In vivo and in vitro studies on peroxisome proliferation and associated effects1995Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 904.
    Soliman, Abdelhamid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ammonia assimilation and nitrogen fixation in phototrophic bacteria: studies on glutamine synthease and the metabolic regulation of nitrogenase in Rhodospirillum rubrum1991Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The phototrophic bacteria and a few other species, are able to control nitrogen fixation on the metabolic level. This is a fast and reversible mechanism, which “switches off and on” the nitrogenase activity in response to the accessibility of combined nitrogen, the energy supply and the level of oxygen in the medium. By the modification of dinitrogenase reductase the switch-off mechanism leads to the inhibition of the catalytic activity of the whole complex. On the physiological level the details of this mechanism are inadequately understood. The present investigation focus on two aspects of regulation; i) glutamine synthetase; a central enzyme in both ammonia assimilation and metabolic regulation of; nitrogenase activity, and ii) the identity of the signal that initiates the switch-off mechanism.

    Glutamine synthetase was purified to electrophoretic, homogeneity from Rsp.rubrum before and after switch-off by affinity chromatography as major step: The enzyme purified was in either high or low-activity form; due to using cultures growing, om N2. either before- or after switch-off respectively. The molecular mass of the enzyme as well as its kinetic properties are similar to other bacterial glutamine synthetases. Except the difference- in: activity between the high and: the tow active forms of Rsp. rubrum glutamine synthetase, the enzyme has- several properties which are different from glutamine synthetase of E. coli as well a» glutamine synthetase from other phototrophic bacteria: i) the different forms of the enzyme do not show an isoactivity pH in the transferase reaction, ii)in the presence of Mn^+, the transferase activity of both, forms is- inhibited by Mg2+, iii) snake venom phosphodiesterase has no effect on the low activity form;, iiw)) there are no differences in the sensitivity of the two forms of towards feedback inhibitors and* Vf the activity of glutamine synthetase from Rsp. rubrum shows switch-off behaviour similar to dinitrogenase reductase from the same organism. The present study could not reveal which type of modification regulates glutamine synthetase activity in Rsp. rubrum growing on N2.

    The metabolic regulation of nitrogen fixation in Rsp. rubrum, as presented in this thesis, involves both nitrogen and energy metabolism, therefore such a complex phenomenon can only, so far, be studied in vivo. The present studies demonstrated the following: i) The elevation of the cellular level of NAD+ could be the common signal molecule which triggers ADP-ribosylation of dinitrogenase reductase. Using exogenous NAD+, we found that it was taken up by the cells leading to switch-off, iijunlike glutamine as switch-off effector, exogenous NAD+ exerts its effect without participation of glutamate synthase. This result supports our assumption that, upon the addition of NFLf1-, extensive ammonia assimilation in the glutamine synthetase/glutamate synthase pathway leads to oxidation of NADPH. In the transhydrogenase reaction, the regeneration of NADPH would cause a sudden elevation of the cellular level NAD+, iii) switch-off by NAD+ was more pronounced under low light intensity and this observation made possible the use of this compound as a switch-off effector in Nstarved cells. The influence of light intensity on switch-off by exogenous NAD+, is also in line with the assumption that switch-off by darkness is due to direct elevation of the cellular level of this compound and v) the addition of reduced carbon compounds accelerate switch-on, whereas blocking the reduction of NAD+ favours switch-off. Furthermore, it was found that oxaloacetate and 2-oxoglutarate were able to trigger switch-off. Taken together with the results from other investigators, the present results are the basis for the formulation of a scheme which suggest that, the elevation of the cellular level of NAD+ could be the common signal between switch-off effectors and the regulatory cycle of nitrogenase activity in Rsp. rubrum.

  • 905.
    Sonnhammer, Erik L. L.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Östlund, Gabriel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    InParanoid 8: orthology analysis between 273 proteomes, mostly eukaryotic2015Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, nr D1, s. D234-D239Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The InParanoid database (http://InParanoid.sbc.su.se) provides a user interface to orthologs inferred by the InParanoid algorithm. As there are now international efforts to curate and standardize complete proteomes, we have switched to using these resources rather than gathering and curating the proteomes ourselves. InParanoid release 8 is based on the 66 reference proteomes that the 'Quest for Orthologs' community has agreed on using, plus 207 additional proteomes from the UniProt complete proteomes-in total 273 species. These represent 246 eukaryotes, 20 bacteria and seven archaea. Compared to the previous release, this increases the number of species by 173% and the number of pairwise species comparisons by 650%. In turn, the number of ortholog groups has increased by 423%. We present the contents and usages of InParanoid 8, and a detailed analysis of how the proteome content has changed since the previous release.

  • 906. Sork, Helena
    et al.
    Nordin, Joel Z.
    Turunen, Janne J.
    Wiklander, Oscar P. B.
    Bestas, Burcu
    Zaghloul, Eman M.
    Margus, Helerin
    Padari, Kärt
    Duru, Adil D.
    Corso, Giulia
    Bost, Jeremy
    Vader, Pieter
    Pooga, Margus
    Smith, C. I. Edvard
    Wood, Matthew J. A.
    Schiffelers, Raymond M.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Andaloussi, Samir E. L.
    Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency2016Ingår i: Molecular Therapy - Nucleic Acids, ISSN 2162-2531, E-ISSN 2162-2531, Vol. 5, artikel-id e290Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.

  • 907.
    Spånning, Erika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dual targeting of proteins to mitochondria and chloroplasts: Characterization of dual targeting peptides and their interaction with organellar receptors2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Most mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins with an N-terminal targeting peptide (TP), which directs them to the correct organelle. There is also a group of proteins that are dual targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide (dTP). The aim of this thesis was to characterize dTPs with respect to physicochemical features, sequence patterns, structural properties and interaction with the mitochondrial and chloroplastic receptors.

    We have used different statistical methods, including a multivariate data analysis (MVDA) to analyse all available dTPs and compare them to organelle-specific TPs of proteome-identified mitochondrial and chloroplastic proteins from Arabidopsis thaliana. The overall amino acid sequence patterns of dTPs were intermediate between mitochondrial targeting peptides (mTPs) and chloroplastic targeting peptides (cTPs) but the greatest differences in amino acid composition were found within the very N-terminal region of dTPs, where especially arginines are highly overrepresented in relation to cTPs. Interestingly, introducing arginines to the dTPs showed clustering towards the mTPs in silico and resulted in inhibition of chloroplast import in vitro, suggesting that positive charges in the N-terminal region of TPs may function as an 'avoidance signal' for chloroplast import.

    Studies with the dTP of threonyl-tRNA synthetase (ThrRS-dTP) revealed that 60 amino acids were required to confer dual targeting. The purified ThrRS-dTP(2-60) inhibited import of organelle-specific proteins, providing evidence that dual and organelle-specific proteins use the same organellar import pathways. CD spectra indicated that ThrRS-dTP(2-60) has the propensity to form a-helical structure in membrane mimetic environments. Further, NMR investigations of interaction profiles of ThrRS-dTP(2-60) with the mitochondrial Tom20 and the chloroplastic Toc34 receptor demonstrated that the mode of the recognition of a dual targeting peptide by mitochondrial and chloroplastic receptors is different. Our studies provide thorough characterization of dTPs and present for the first time dTP-organellar receptor interactions on the molecular level.

  • 908.
    Srimanee, Artita
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Mahidol University, Thailand.
    Regberg, Jakob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Vajragupta, Opa
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Role of scavenger receptors in peptide-based delivery of plasmid DNA across a blood-brain barrier model2016Ingår i: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 500, nr 1-2, s. 128-135Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Receptor-mediated transcytosis remains a major route for drug delivery across the blood-brain barrier (BBB). PepFect 32 (PF32), a peptide-based vector modified with targeting ligand (Angiopep-2) binding to low-density lipoprotein receptor-related protein-1 (LRP-1), was previously found to be a promising vector for plasmid delivery across an in vitro model of the BBB. Cellular uptake of PF32/plasmid DNA (pDNA) complexes was speculated the internalization via LRP-1 receptor. In this study, we prove that PF32/pDNA nanocomplexes are not only transported into brain endothelial cells via LRP-1 receptor-mediated endocytosis, but also via scavenger receptor class A and B (SCARA3, SCARA5, and SR-BI)-mediated endocytosis. SCARA3, SCARA5, and SR-BI are found to be expressed in the brain endothelial cells. Inhibition of these receptors leads to a reduction of the transfection. In conclusion, this study shows that scavenger receptors also play an essential role in the cellular uptake of the PF32/pDNA nanocomplexes.

  • 909.
    Srimanee, Artita
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Regberg, Jakob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Tartu University, Estonia.
    Application of CPPs for Brain Delivery2015Ingår i: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, Springer-Verlag New York, 2015, Vol. 1324, s. 349-356Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Cell-penetrating peptides provide a promising strategy for delivery of drugs across the blood-brain barrier. Here, we present an overview of CPP and peptide-mediated delivery to the central nervous system as well as a Transwell in vitro model to evaluate passage across an endothelial cell layer mimic of the blood-brain barrier.

  • 910.
    Srinivas, Vivek
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    To metal, or not to metal: Diverse mechanisms of O2-activation and radical storage in the ferritin superfamily2019Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Proteins in the Ferritin-like superfamily are characterized by a four alpha-helical structural motif. These proteins are distributed across all three kingdoms of life and perform a wide range of functions. Several members in this protein superfamily can activate dioxygen using a di-metal active site coordinated by four carboxylate and two histidine amino acid residues. The resulting diverse set of dioxygen activated intermediates is used in nature to perform complex redox chemical reaction in cells. The R2 subunit of class I Ribonucleotide reductase and soluble Methane monooxygenase are the most well-characterized groups of proteins in this superfamily. Upon oxygen (or reduced-oxygen) activation of the di-metal site, the R2 subunit can generate a catalytic radical required for the conversion of ribonucleotides to deoxyribonucleotides, while soluble Methane monooxygenase can oxidize methane to methanol in an alternative form of carbon assimilation.

    The work presented in this thesis aims to better understand metal selectivity, working and the regulation of substrate specificity in various proteins of the Ferritin-like superfamily, and the development of a novel method to study radiation-sensitive intermediates. The papers discussed in this thesis present crystallographic and spectroscopic studies of several Ferritin-like superfamily proteins.

    In paper I, the assembly mechanisms of the heterodinuclear manganese-iron cofactor in a class Ic R2 protein and an R2-like ligand-binding oxidase are compared. Paper II presents the discovery of a novel radical-generating subunit subclass of Ribonucleotide reductase in Mollicutes, including mycoplasma pathogens, that breaks the paradigm of metal requirement for radical translocation and catalysis. This new subclass, denoted class Ie, is shown to instead use an unprecedented modified tyrosine DOPA residue in its four-helix bundle for radical translocation and storage. Paper III presents a new X-ray free-electron laser sample delivery system that combines acoustic droplet ejection with a drop-on-tape setup, allowing simultaneous multimodal X-ray diffraction and X-ray emission data collection. This setup is also shown to support photochemical and chemical activation of catalysis in crystals, allowing the study of radiation-sensitive transient reaction intermediates. We used this setup in paper IV to solve the first radiation damage-free crystallographic structures of the soluble methane monooxygenase hydroxylase and its regulatory subunit complex from Methylosinus trichosporium OB3b. The high-resolution crystal structures of the complex, in both di-ferrous and di-ferric oxidation states, illustrate the structural reorganization in the hydroxylase subunit upon binding to the regulatory subunit.

    These results illustrate the functional range and flexibility in the Ferritin-like protein superfamily. Including the distinctive metal discrimination in heterodinuclear metalloproteins, influencing substrate specificity in sMMO, and using a novel metal-free DOPA radical to catalyze ribonucleotide reduction in the class Ie R2 subclass. Experiments using the novel ADE-DOT setup also showed promising progress towards determining the highly sought-after structures of di-metal oxygen activated intermediates such as X and Q in subclass Ia R2 and sMMO, respectively.

  • 911.
    Srinivas, Vivek
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Banerjee, Rahul
    Lebrette, Hugo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jones, Jason
    Aurelius, Oskar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    John, Juliane
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kim, In-Sik
    Pham, Cindy
    Gul, Sheraz
    Sutherlin, Kyle
    Bhowmick, Asmit
    Fransson, Thomas
    Aller, Pierre
    Butryn, Agata
    Tono, Kensuke
    Alonso-Mori, Roberto
    Fuller, Franklin
    Batyuk, Alexander
    Brewster, Aaron
    Sauter, Nicholas
    Orville, Allen
    Yachandra, Vittal
    Yano, Junko
    Lipscomb, John
    Kern, Jan
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    High Resolution XFEL Structure of the Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation StatesManuskript (preprint) (Övrigt vetenskapligt)
  • 912.
    Srinivas, Vivek
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lebrette, Hugo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundin, Daniel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kutin, Yuri
    Sahlin, Margareta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lerche, Michael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Eirich, Jürgen
    Branca, Rui M. M.
    Cox, Nicholas
    Sjöberg, Britt-Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stanford University School of Medicine, USA.
    Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens2018Ingår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 563, s. 416-420Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5,6,7. Here we describe a group of RNR proteins in Mollicutes—including Mycoplasma pathogens—that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR—some of which are developing resistance to antibiotics—are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.

  • 913. Sroka-Markovic, Janina
    et al.
    Johansson, Linda
    Andersson, Magnus B. O.
    Granelli, Ingrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
    Development of an HPLC Method for Determination of Related Impurities in Prilocaine Substance2012Ingår i: Chromatographia, ISSN 0009-5893, E-ISSN 1612-1112, Vol. 75, nr 7-8, s. 329-336Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Development of a reversed phase high performance liquid chromatographic method for determination of six related impurities in prilocaine substance is reported. The test of related impurities in European Pharmacopoeia (Ph. Eur.) cannot meet the demands with the chromatographic parameters given, therefore different types of chromatographic systems and eight columns have been evaluated in the present study. A new method with a Hypercarb column was developed and validated. This method fulfils the demands in the Ph. Eur., and the validation shows that the method is selective, reproducible, linear, accurate and robust with sufficient limits of detection (0.001-0.004% of 2.5 mg prilocaine mL(-1)) and quantification (0.002-0.009% of 2.5 mg prilocaine mL(-1)).

  • 914.
    Staaf, Elina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Cellular effects after exposure to mixed beams of ionizing radiation2012Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Mixed beams of ionizing radiation in our environment originate from space, the bedrock and our own houses. Radiotherapy patients treated with boron neutron capture therapy or with high energy photons are also exposed to mixed beams of gamma radiation and neutrons. Earlier investigations have reported additivity as well as synergism (a greater than additive response) when combining radiations of different linear energy transfer. However, the outcome seemed to be dependent on the experimental setup, especially the order of irradiation and the temperature at exposure.

    A unique facility allowing simultaneously exposure of cells to X-rays and 241Am alpha particles at 37 ºC was constructed and characterized at the Stockholm University (Paper I). To investigate the cytogenetic response to mixed beam irradiation (graded doses of alpha particles, X-rays or a mixture of both) several different cell types were utilized. AA8 Chinese Hamster Ovary cells were analyzed for clonogenic survival (Paper I), human peripheral blood lymphocytes were analyzed for micronuclei and chromosomal aberrations (Paper II and Paper III respectively) and VH10 normal human fibroblasts were scored for gamma-H2AX foci (Paper IV).

    For clonogenic survival, mixed beam results were additive, while a significant synergistic effect was observed for micronuclei and chromosomal aberrations. The micronuclei dose responses were linear, and a significant synergistic effect was present at all investigated doses. From the analysis of micronuclei distributions we speculated that the synergistic effect was due to an impaired repair of X-ray induced DNA damage, a conclusion that was supported by chromosomal aberration results. Gamma-H2AX foci dose responses were additive 1 h after exposure, but the kinetics indicated that the presence of low LET-induced damage engages the DNA repair machinery, leading to a delayed repair of the more complex DNA damage induced by alpha particles. These conclusions are not necessary contradictory since fast repair does not necessarily equal correct repair. Taken together, the observed synergistic effects indicate that the risks of stochastic effects from mixed beam exposure may be higher than expected from adding the individual dose components.

  • 915.
    Stedingk, Lars-Victor von
    Stockholms universitet.
    Ion movements related to electron transport and energy conservation: A study of the chromatophore system from the photosynthetic bacterium Rhodospirillum rubrum1968Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 916.
    Stednigk, Erik von
    Stockholms universitet.
    Sorting and Import of Plant Mitochondrial Precursor Proteins1999Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The mitochondrial genome encodes only a handful of the mitochondrial proteins; hundreds of mitochondrial proteins are encoded in the nucleus, synthesised on cytosolic polyribosomes and subsequently imported into the organelle. Most of the proteins targeted to the mitochondria are synthesised as precursors with an N-terminal extension called presequence or targeting sequence. Following synthesis in the cytosol, general molecular chaperones keep the precursor protein in a loosely folded and import competent conformation. More specific cytosolic factors might be involved in assisting protein transport to the correct organelle. At the surface of mitochondria, the precursor interacts with specific receptors and is subsequently translocated across the membranes through the translocases of the outer and inner membranes.

    The aim of this thesis has been to illuminate key steps of protein import into plant mitochondria by studying protein sorting, translocation and the involvement of molecular chaperones in the import process.

    Do there exist molecular chaperones in the plant cell, which affect mitochondrial protein import? We isolated a post-microsomal cytosolic fraction from Spinacia oleracea and showed the presence of a proteinaceous cytosolic factor with a capacity to stimulate protein import by enhancing the kinetics of translocation rather than the binding capacity. We investigated the occurrence and induction of two mitochondrial molecular chaperones, heat shock protein 70 (Hsp70) and Hsp60, in normal and heat-stressed spinach plants. We found that whereas Hsp60 was totally localised to the matrix, 50% of the Hsp70 was associated with the mitochondrial inner membrane. In stressed plants, the Hsp70 was induced 2 to 3-fold and the total amount of the induced Hsp70 was associated with the membrane.

    We have shown a unique property of higher plant mitochondria: the precursor of the F1b subunit of the ATP synthase was covalently modified upon binding to mitochondria. The modification was catalysed by an enzyme of the mitochondrial outer membrane and occured at the N-terminal region of the cleavable presequence of the precursor. The modification was ATP and Ca2+ dependent. This is the first report of a covalent modification of a mitochondrial precursor. The unmodified precursor protein was favoured for import, implicating that the modification might be removed prior to translocation across the outer membrane.

    We have studied the mechanism of protein translocation into plant mitochondria. Sulfhydryl group reagents affected different events of the protein import process, such as the binding and conformational status of the precursor, the receptor independent bypass import pathway and protein transport across the mitochondrial inner membrane. The effect of sulfhydryl group reagents was dependent on energisation state of mitochondria. We concluded that the redox and the conformational status of the sulfhydryl groups located on the outer side of the inner membrane translocase components were essential for protein import.

  • 917. Stefansson, Anne
    et al.
    Armulik, Annika
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Johansson, Staffan
    Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits2004Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, nr 20, s. 21200-21205Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Previous studies on the membrane-cytoplasm interphase of human integrin subunits have shown that a conserved lysine in subunits alpha(2), alpha(5), beta(1), and beta(2) is embedded in the plasma membrane in the absence of interacting proteins (Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. (1999) in J. Biol. Chem. 274, 37030-37034). Using a glycosylation mapping technique, we here show that alpha(10) and beta(8), two subunits that deviate significantly from the integrin consensus sequences in the membrane-proximal region, were found to have the conserved lysine at a similar position in the lipid bilayer. Thus, this organization at the C-terminal end of the transmembrane (TM) domain seems likely to be general for all 24 integrin subunits. Furthermore, we have determined the N-terminal border of the TM domains of the alpha(2), alpha(5), alpha(10), beta(1), and beta(8) subunits. The TM domain of subunit beta(8) is found to be 22 amino acids long, with a second basic residue (Arg(684)) positioned just inside the membrane at the exoplasmic side, whereas the lipid-embedded domains of the other subunits are longer, varying from 25 (alpha(2)) to 29 amino acids (alpha(10)). These numbers implicate that the TM region of the analyzed integrins (except beta(8)) would be tilted or bent in the membrane. Integrin signaling by transmembrane conformational change may involve alteration of the position of the segment adjacent to the conserved lysine. To test the proposed "piston" model for signaling, we forced this region at the C-terminal end of the alpha(5) and beta(1) TM domains out of the membrane into the cytosol by replacing Lys-Leu with Lys-Lys. The mutation was found to not alter the position of the N-terminal end of the TM domain in the membrane, indicating that the TM domain is not moving as a piston. Instead the shift results in a shorter and therefore less tilted or bent TM alpha-helix.

  • 918. Stella, Roberto
    et al.
    Cifani, Paolo
    Peggion, Caterina
    Hansson, Karin
    Lazzari, Cristian
    Bendz, Maria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Levander, Fredrik
    Sorgato, Maria Catia
    Bertoli, Alessandro
    James, Peter
    Relative Quantification of Membrane Proteins in Wild-Type and Prion Protein (PrP)-Knockout Cerebellar Granule Neurons2012Ingår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, nr 2, s. 523-536Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Approximately 25% of eukaryotic proteins possessing homology to at least two trans membrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme a-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.

  • 919.
    Stenberg Bruzell, Filippa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Protein complexes of the Escherichia coli cell envelope2010Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The cell envelope of Escherichia coli, as for all living cells, is a magnificent semi-permeable membrane barrier that facilitates protection as well as enables fundamental contact with the exterior world. The envelope comprises a mixture of phospholipids, organized in two bilayers, which are stabilized by a rigid peptidoglycan layer. There are also a large number of proteins, which can be lipid-integrated or attached. Infact, it is anticipated that approximately 30-40% of the cellular proteome of E. coli could be associated with the envelope. These proteins are involved in the transport of small molecules and nutrients, the biogenesis of the envelope, metabolism, signaling, channeling and cellular movement and attachment.

    The focus of this thesis is to understand the cell envelope of E. coli by understanding the proteins it holds. Three main questions have been addressed: 1) Which proteins are present? 2) How do these proteins interact? 3) How are the interactions brought about? To answer these questions we have designed and optimized methods suitable for proteome-wide separation, visualization and characterization of membrane proteins and protein complexes. We present reference proteome and interactome maps of the envelope, which further our understanding of the assembly and composition of the cell envelope. In many instances our studies have provided a first step towards understanding protein function(s) and for carrying out meaningful biochemical and structural analysis. We have also developed parallel approaches, which have enabled us to dissect the assembly process for two specific membrane protein complexes, a homo-dimer of penicillin binding protein 5 and the respiratory oxidase cytochrome bo3. These studies have extended our understanding of the relationship between structure and function of protein complexes.

  • 920.
    Stenberg, Filippa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Chovanec, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Maslen, Sarah L.
    Robinson, Carol V.
    Ilag, Leopold L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daley, Daniel O,
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Protein complexes of the Escherichia coli cell envelope2005Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, nr 41, s. 34409-34419Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Protein complexes are an intrinsic aspect of life in the membrane. Knowing which proteins are assembled in these complexes is therefore essential to understanding protein function(s). Unfortunately, recent high throughput protein interaction studies have failed to deliver any significant information on proteins embedded in the membrane, and many membrane protein complexes remain ill defined. In this study, we have optimized the blue native-PAGE technique for the study of membrane protein complexes in the inner and outer membranes of Escherichia coli. In combination with second dimension SDS-PAGE and mass spectrometry, we have been able to identify 43 distinct protein complexes. In addition to a number of well characterized complexes, we have identified known and orphan proteins in novel oligomeric states. For two orphan proteins, YhcB and YjdB, our findings enable a tentative functional assignment. We propose that YhcB is a hitherto unidentified additional subunit of the cytochrome bd oxidase and that YjdB, which co-localizes with the ZipA protein, is involved in cell division. Our reference two-dimensional blue native-SDS-polyacrylamide gels will facilitate future studies of the assembly and composition of E. coli membrane protein complexes during different growth conditions and in different mutant backgrounds.

  • 921.
    Stenberg, Filippa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daley, Daniel O
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Assembly of the cytochrome bo3 complex2007Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 371, nr 3, s. 765-773Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An understanding of the mechanisms that govern the assembly of macromolecular protein complexes is fundamental for studying their function and regulation. With this in mind, we have determined the assembly pathway for the membrane-embedded cytochrome bo3 of Escherichia coli. We show that there is a preferred order of assembly, where subunits III and IV assemble first, followed by subunit I and finally subunit II. We also show that cofactor insertion catalyses assembly. These findings provide novel insights into the biogenesis of this model membrane protein complex.

  • 922.
    Stenberg, Gun
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Glutathione transferases: molecular cloning, site-directed mutagenesis and structure-function studies1991Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Three distinct glutathione transferases (GST) cDNA clones were obtained by screening of tumour cell line cDNA libraries. The cDNA clones were expressed in Escherichia coli and the enzymes characterized. Rat GST 8-8 is the only member in the multifunctional GST family for which a specific cellular function has been proposed. Several new substrates among toxic unsaturated compounds were found, supporting the idea that rat GST 8-8 is involved in the cellular protection against oxidative stress.

    A partial cDNA clone encoding human class Pi GST was extended with synthetic DNA, designed to optimize expression in E. coli. A ten-fold increased yield of protein was obtained as compared to previous expression constructs.

    Human GST Al-1 was subjected to site-directed mutagenesis for structure-function analysis. A tyrosine residue was identified as an important component of the active site, by demonstrating that a Tyr7—>Phe mutant displayed strongly reduced catalytic activity and affinity for GSH.

    The role of evolutionary conserved arginine residues for structural stability and catalytic function was investigated, by construction of four Arg—> Ala mutants. The results demonstrate that three of these arginine residues are important for maintaining a funtional enzyme. The results are discussed in relation to a recently determined 3D structure of a pig lung GST.

  • 923.
    Stenmark, Pål
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dong, Min
    Dupuy, Jerome
    Chapman, Edwin R.
    Stevens, Raymond C.
    Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding2010Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, nr 5, s. 1287-1297Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-angstrom X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  • 924.
    Stenström, Magnus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Regulation of gene expression by translation initiation efficiency in E. coli2001Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 925.
    Stephan, Katharina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ott, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Timing of dimerization of the bc1 complex during mitochondrial respiratory chain assembly2020Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1861, nr 5-6, artikel-id 148177Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mitochondrial bc1 complex plays an important role in mitochondrial respiration. It transfers electrons from ubiquinol to the soluble electron shuttle cytochrome c and thereby contributes to the proton motive force across the inner mitochondrial membrane. In the yeast Saccharomyces cerevisiae, each monomer consists of three catalytic and seven accessory subunits. The bc1 complex is an obligate homo-dimer in all systems. It is currently not known when exactly during the assembly dimerization occurs. In this study, we determined that the dimer formation is an early event. Specifically, dimerization is mediated by the interaction of a stable tetramer formed by the two Cor subunits, Cor1 and Cor2, that joins assembly intermediate II, containing the fully hemylated cytochrome b and the two small accessory proteins, Qcr7 and Qcr8. Addition of cytochrome c1 and Qcr6 can either occur concomitantly or independently of dimerization. These results reveal a strict order in assembly, where dimerization occurs after stabilization of co-factor acquisition by cytochrome b. Finally, assembly is completed by addition of the remaining subunits.

  • 926.
    Stephan, Katharina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ott, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Singh, Abeer
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The role of the assembly factor Bca1 and the structural subunit Qcr7 during bc1 complex biogenesisManuskript (preprint) (Övrigt vetenskapligt)
  • 927.
    Stoimenov, Ivaylo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Interplay between Transcription and Homologous Recombination in the Presence of DNA Damage2011Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The biochemical processes of DNA repair, replication and recombination compete for the same substrate, the DNA molecule. This competition is natural, as each process requires the same template. In order to resolve possible conflicts between these processes, when they take place on a particular stretch of DNA, certain crosstalk is expected. The complexity is additionally increased by the existence of another DNA dependent process, which occurs in all phases of the cell cycle: transcription.

    This thesis aims to investigate the link between transcription inhibition and homologous recombination, especially in the context of UV-induced DNA damage. The results show that the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) induces homologous recombination. The DNA damage caused by UVC irradiation is repaired mainly via nucleotide excision repair, however, it is also known to trigger recombinational repair. In the presence of UV-induced damage, transcription inhibition by DRB potentiates the induction of homologous recombination as a necessary mechanism of cell survival.

    The thesis also focuses on the toxicity mechanisms of the chemotherapeutic compound 6-thioguanine (6TG). The work in the thesis suggests application of 6TG as a treatment for hereditary forms of breast cancer, with genetically altered BRCA1 or BRCA2 functions. Most importantly, the treatment with 6TG is applicable to breast cancers, which have developed resistance to another class of chemotherapeutic drugs, poly-(ADP-ribose) polymerase (PARP) inhibitors. Repair of the DNA damage induced by 6TG treatment is investigated further with a particular focus the pathway of DNA damage avoidance involving DNA polymerase η. The function of DNA polymerase η seems to be an important factor for the outcome of DNA repair after 6TG exposure. The deficiency of DNA polymerase η is also investigated in connection with normal replication and the repair of UV-induced DNA damage.

    In summary, the work in the thesis sheds more light onto the fundamental connections between DNA replication, recombination, transcription, repair and damage avoidance. On a more practical side, the information obtained about these fundamental connections is used to suggest a possible therapy for several forms of breast cancer.

  • 928.
    Stoimenov, Ivaylo
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Elvers, Ingegerd
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Helleday, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Polymerase η proficiency sensitises cells to 6-thioguanineManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Severe photosensitivity of the skin and predisposition to cancer development are two important features whichcharacterising a genetic syndrome known as Xeroderma pigmentosum. An interesting class of patients has beendescribed, characterised by a proficiency in nucleotide excision repair and a defect in the DNA damage avoidancepathways. This class is termed Xeroderma pigmentosum variant (XP-V) and is known to be caused by a deficiencyin the function of the specific DNA Polymerase η. Cells derived from XP-V patients are sensitivie not only to UVlight but also to crosslinkers such as cisplatin, and Polη overexpression is potentially relevant to development ofcisplatin resistance. In this paper we investigate the importance of the Polη status in the response to treatment withthe chemotherapeutic agent 6-thioguanine (6TG). Our results show that Polη deficient cells are more resistant totreatment with 6TG in comparison to Polη complemented cells. This is in contrast to the typical UV sensitivity ofPolη deficient cells, which is confirmed in the same cells. We also show that 6TG has a growth retardation effect,regardless of the Polη status. There were no DNA double-strand breaks detected in a short period after theexposure to 6TG in physiologically relevant doses, although a DNA damage response was observed in high doses.It was previously demonstrated that 6TG can be used to kill cisplatin resistant cells and these data may indicateone potential explanation.

  • 929.
    Stoimenov, Ivaylo
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Gottipati, Ponnari
    Schultz, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Helleday, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) causes DNA damage and triggers homologous recombination repair in mammalian cells2011Ingår i: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 706, nr 1-2, s. 1-6Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transcription, replication and homologous recombination are intrinsically connected and it is well established that an increase of transcription is associated with an increase in homologous recombination. Here, we have studied how homologous recombination is affected during transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a compound that prevents activating phosphorylations of the RNA Pol II C-terminal domain. We identify that DRB triggers an increase in homologous recombination within the hprt gene as well as increasing RAD51 foci formation in mammalian cells. Furthermore, we find that DRB-induced transcriptional stress is associated with formation of the nuclear foci of the phosphorylated form of H2AX (γH2AX). We accounted that about 72% of RAD51 foci co-localized with the observed γH2AX foci. Interestingly, we find that XRCC3 mutated, homologous recombination defective cells are hypersensitive to the toxic effect of DRB and fail to form RAD51 foci. In conclusion, we show that DRB-induced transcription inhibition is associated with the formation of a lesion that triggers RAD51-dependent homologous recombination repair, required for survival under transcriptional stress.

  • 930.
    Stoimenov, Ivaylo
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Schultz, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Gottipati, Ponnari
    Helleday, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Transcription inhibition by DRB potentiates recombinational repair of UVC lesions in mammalian cellsManuskript (preprint) (Övrigt vetenskapligt)
  • 931. Stone, Tracy A.
    et al.
    Schiller, Nina
    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).
    Deber, Charles M.
    Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences2015Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, nr 7, s. 1465-1473Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Biophysical hydrophobicity scales suggest that partitioning of a protein segment from an aqueous phase into a membrane is governed by its perceived segmental hydrophobicity but do not establish specifically (i) how the segment is identified in vivo for translocon-mediated insertion or (ii) whether the destination lipid bilayer is biochemically receptive to the inserted sequence. To examine the congruence between these dual requirements, we designed and synthesized a library of Lys-tagged peptides of a core length sufficient to span a bilayer but with varying patterns of sequence, each composed of nine Leu residues, nine Ser residues, and one (central) Trp residue. We found that peptides containing contiguous Leu residues (Leu-block peptides, e.g., LLLLLLLLLWSSSSSSSSS), in comparison to those containing discontinuous stretches of Leu residues (non-Leu-block peptides, e.g., SLSLLSLSSWSLLSLSLLS), displayed greater helicity (circular dichroism spectroscopy), traveled slower during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had longer reverse phase high-performance liquid chromatography retention times on a C-18 column, and were helical when reconstituted into 1-palmitoyl-2-oleoylglycero-3-phosphocholine liposomes, each observation indicating superior lipid compatibility when a Leu-block is present. These parameters were largely paralleled in a biological membrane insertion assay using microsomal membranes from dog pancreas endoplasmic reticulum, where we found only the Leu-block sequences successfully inserted; intriguingly, an amphipathic peptide (SLLSSLLSSWLLSSLLSSL; Leu face, Ser face) with biophysical properties similar to those of Leu-block peptides failed to insert. Our overall results identify local sequence lipid compatibility rather than average hydrophobicity as a principal determinant of transmembrane segment potential, while demonstrating that further subtleties of hydrophobic and helical patterning, such as circumferential hydrophobicity in Leu-block segments, promote translocon-mediated insertion.

  • 932.
    Strauss, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för funktionell zoomorfologi.
    Zhang, Qian
    Verleyen, Peter
    Huybrechts, Jurgen
    Neupert, Susanne
    Predel, Reinhard
    Pauwels, Kevin
    Dircksen, Heinrich
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för funktionell zoomorfologi.
    Pigment-dispersing hormone in Daphnia interneurons, one type homologous to insect clock neurons displaying circadian rhythmicity2011Ingår i: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 68, nr 20, s. 3403-3423Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We report identification of a beta-type pig-ment-dispersing hormone (PDH) identical in two water fleaspecies, Daphnia magna and Daphnia pulex. It has been identified by cloning of precursors, chromatographic iso-lation from tissue extracts followed by immunoassays and de novo-mass spectrometric sequencing. The peptide is restricted to a complex system of distinct interneurons in the brain and visual ganglia, but does not occur in neurosecretory cells projecting to neurohemal organs as in decapod crustaceans. Thirteen neuron types individually identified and reconstructed by immunohistochemistry were almost identical in terms of positions and projection patterns in both species. Several neurons invade and form plexuses in visual ganglia and major brain neuropils including the central body. Five neuron types show con-tralateral pathways and form plexuses in the lateral, dorsal,or postlateral brain neuropils. Others are local interneurons,and a tritocerebral neuron connects the protocerebrum with the neuropil of the locomotory second antenna. Two visual ganglia neuron types lateral to the medulla closely resemble insect medulla lateral circadian clock neurons containing pigment-dispersing factor based upon positional and projectional criteria. Experiments under 12:12 h light/dark cycles and constant light or darkness conditions showed significant circadian changes in numbers and activities of one type of medulla lateral PDH neuron with an acrophase in the evening. This simple PDH system shows striking homologies to PDH systems in decapod crustaceans and well-known clock neurons in several insects, which suggests evolutionary conservation of an ancient peptidergic interneuronal system that is part of biological clocks.

  • 933.
    Strell, Carina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Hilscher, Markus M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Laxman, Navya
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Svedlund, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Wu, Chenglin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Yokota, Chika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Placing RNA in context and space - methods for spatially resolved transcriptomics2019Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 286, nr 8, s. 1468-1481Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Single-cell transcriptomics provides us with completely new insights into the molecular diversity of different cell types and the different states they can adopt. The technique generates inventories of cells that constitute the building blocks of multicellular organisms. However, since the method requires isolation of discrete cells, information about the original location within tissue is lost. Therefore, it is not possible to draw detailed cellular maps of tissue architecture and their positioning in relation to other cells. In order to better understand the cellular and tissue function of multicellular organisms, we need to map the cells within their physiological, morphological, and anatomical context and space. In this review, we will summarize and compare the different methods of in situ RNA analysis and the most recent developments leading to more comprehensive and highly multiplexed spatially resolved transcriptomic approaches. We will discuss their highlights and advantages as well as their limitations and challenges and give an outlook on promising future applications and directions both within basic research as well as clinical integration.

  • 934.
    Ström, Anna-Lena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hayward, Lawrence J.
    Kasarskis, Edward J.
    Zhu, Haining
    Axonal transport and motor neuron disease2011Ingår i: Cytoskeleton of the Nervous System / [ed] Nixon, Ralph A., Aidong Yuan, New York: Springer-Verlag New York, 2011, s. 529-544Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Describes cytoskeleton in axonal development and pathology, microtubules and associated proteins, neurofilaments and interacting proteins, actin and its binding proteins, and glial fibrillary acidic protein 2. Focuses on functional significance of neuronal cytoskeleton in axonal transport 3. Encourages further development of unifying principles, stimulates new conceptual and technical approaches toward a better understanding of cytoskeleton functions in health and disease Without a cytoskeleton, a neuron or glial cell would be a shapeless jelly mass unable to function in the milieu of the brain. If we are to understand neuronal cells function in health and disease, we must determine how the cytoskeleton forms and contributes to neural physiology and pathobiology. Cytoskeleton of the Nervous System provides a comprehensive, authoritative and up-to-date account of what we now know and what we want to know in the near future--about the functioning of the cytoskeleton of neuronal cells at the molecular level. In lively accounts, which are unafraid to address controversy, Cytoskeleton of the Nervous System introduces readers to the most sophisticated concepts and latest discoveries: from overexpression systems to knock-out models for specific cytoskeletal proteins, from continuous transport assays in vivo to live-cell imaging in primary neurons, and from factors regulating cytoskeleton behavior to the dysregulation of these processes leading to neurological disease.

  • 935.
    Ström, Cecilia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Post-translational modifications in DNA base excision repair: The roles of CK2 and PARP-12011Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Base lesions and DNA single-strand breaks (SSBs) are very common types of DNA damage. The base excision repair (BER) and single-strand break repair (SSBR) machineries both require a succession of enzymatic events in order to remove these types of endogenous lesions and to restore the DNA. Coordinated repair involves signalling between the proteins concerned and is achieved by post-translational modification. Here, we study two types of modifications in the context of BER and SSBR.

    Poly(ADP-ribose) polymerase-1 (PARP-1) is a known SSB sensor, which utilizes NAD+ and converts these to ADP-ribose polymers as a post-translational modification of primarily itself, to accelerate repair. However, its role in BER is not as clear. By quantification of SSBs in vivo, we find that PARP inhibition prevents the completion of BER, while siRNA knockdown of PARP-1 leaves repair unaffected. Our results indicate that PARP-1 is not required for BER to progress, but that the enzyme interferes with the SSB intermediate.

    Another known post-translational modification in SSBR is the phosphorylation of XRCC1 by CK2. Here, we show that the majority of the cellular XRCC1 is phosphorylated and that CK2 is the main kinase responsible for this. We find that this modification prevents degradation of XRCC1 by the proteasome, resulting in faster repair of oxidative damage in the DNA. In addition, we propose a new role for CK2 modifications of XRCC1 in BER. We demonstrate that, even though the presence of XRCC1 or the activity of PARP are not required for SSB intermediate formation, the expression of a non-phosphorylated form of XRCC1 results in reduced SSB levels. Furthermore, the affinity of XRCC1 for a nicked DNA substrate increases when the CK2 phosphorylation sites are mutated.

    To summarise, our findings increase the knowledge of the BER and SSBR processes and demonstrate that the impact of post-translational modifications is more complex than it originally appeared.

  • 936.
    Strömqvist, Johan
    et al.
    Kungliga Tekniska Högskolan, Stockholm.
    Skoog, Karl
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daley, Daniel O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Widengren, Jerker
    Kungliga Tekniska Högskolan, Stockholm.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Estimating Z-ring radius and contraction in dividing Escherichia coli2010Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 76, nr 1, s. 151-158Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We present a fluorescence recovery after photobleaching-based method for monitoring the progression of septal Z-ring contraction in dividing Escherichia coli cells. In a large number of cells undergoing division, we irreversibly bleached cytosolically expressed Enhanced Green Fluorescent Protein on one side of the septal invagination and followed the fluorescence relaxation on both sides of the septum. Since the relaxation time depends on the cross-sectional area of the septum, it can be used to determine the septal radius r. Assuming that the fraction of the observed cells with r-values in a given interval reflects the duration of that interval in the division process we could derive an approximate time-course for the contraction event, as a population average. By applying the method repeatedly on individual cells, the contraction process was also followed in real time. On a population average level, our data are best described by a linear contraction process in time. However, on the single cell level the contraction processes display a complex behaviour, with varying levels of activity. The proposed approach provides a simple yet versatile method for studying Z-ring contraction in vivo, and will help to elucidate its underlying mechanisms.

  • 937.
    Studham, Matthew E.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Tjärnberg, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nordling, Torbjörn E. M.
    Nelander, Sven
    Sonnhammer, Erik L. L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Swedish e-Science Research Center, Sweden.
    Functional association networks as priors for gene regulatory network inference2014Ingår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 30, nr 12, s. 130-138Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Motivation: Gene regulatory network (GRN) inference reveals the influences genes have on one another in cellular regulatory systems. If the experimental data are inadequate for reliable inference of the network, informative priors have been shown to improve the accuracy of inferences. Results: This study explores the potential of undirected, confidence-weighted networks, such as those in functional association databases, as a prior source for GRN inference. Such networks often erroneously indicate symmetric interaction between genes and may contain mostly correlation-based interaction information. Despite these drawbacks, our testing on synthetic datasets indicates that even noisy priors reflect some causal information that can improve GRN inference accuracy. Our analysis on yeast data indicates that using the functional association databases FunCoup and STRING as priors can give a small improvement in GRN inference accuracy with biological data.

  • 938.
    Ståhl, Annelie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    PreP - a novel metalloprotease that degrades targeting peptides in mitochondria and chloroplasts2003Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 939. Ståhl, Patrik L.
    et al.
    Salmen, Fredrik
    Vickovic, Sanja
    Lundmark, Anna
    Navarro, Jose Fernandez
    Magnusson, Jens
    Giacomello, Stefania
    Asp, Michaela
    Orzechowski Westholm, Jakub
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Huss, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Mollbrink, Annelie
    Linnarsson, Sten
    Codeluppi, Simone
    Borg, Åke
    Ponten, Fredrik
    Costea, Paul Igor
    Sahlen, Pelin
    Mulder, Jan
    Bergmann, Olaf
    Lundeberg, Joakim
    Frisen, Jonas
    Visualization and analysis of gene expression in tissue sections by spatial transcriptomics2016Ingår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 353, nr 6294, s. 78-82Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Analysis of the pattern of proteins or messenger RNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call spatial transcriptomics, that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.

  • 940.
    Suhm, Tamara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mitochondrial translation and its impact on protein homeostasis and aging2019Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Besides their famous role as powerhouse of the cell, mitochondria are also involved in many signaling processes and metabolism. Therefore, it is unsurprising that mitochondria are no isolated organelles but are in constant crosstalk with other parts of the cell. Due to the endosymbiotic origin of mitochondria, they still contain their own genome and gene expression machinery. The mitochondrial genome of yeast encodes eight proteins whereof seven are core subunits of the respiratory chain and ATP synthase. These subunits need to be assembled with subunits imported from the cytosol to ensure energy supply of the cell. Hence, coordination, timing and accuracy of mitochondrial gene expression is crucial for cellular energy production and homeostasis. Despite the central role of mitochondrial translation surprisingly little is known about the molecular mechanisms.

    In this work, I used baker’s yeast Saccharomyces cerevisiae to study different aspects of mitochondrial translation. Exploiting the unique possibility to make directed modifications in the mitochondrial genome of yeast, I established a mitochondrial encoded GFP reporter. This reporter allows monitoring of mitochondrial translation with different detection methods and enables more detailed studies focusing on timing and regulation of mitochondrial translation. Furthermore, employing insights gained from bacterial translation, we showed that mitochondrial translation efficiency directly impacts on protein homeostasis of the cytoplasm and lifespan by affecting stress handling. Lastly, we provided first evidence that mitochondrial protein quality control happens at a very early stage directly after or during protein synthesis at the ribosome. Surveillance of protein synthesis and assembly into complexes is important to avoid accumulation of misfolded or unassembled respiratory chain subunits which would disturb mitochondrial function.

  • 941. Suhorutsenko, Julia
    et al.
    Eriste, Elo
    Copolovici, Dana-Maria
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Human protein 53 derived cell penetrating peptides2012Ingår i: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 18, nr 4, s. 291-297Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tumor suppressor protein 53 plays an important role in the initiation of cell cycle arrest and apoptosis. Being highly mutated in several different cancer types, p53 is a good target for anticancer therapeutics. It has been shown that a peptide derived from the C-terminus of p53 activates specific DNA-binding of endogenous mutated p53, restoring its original activity. Detection of short cell-penetrating peptide sequences using quantitative structure-activity relationship algorithm gives new opportunities for developing novel peptide-based platforms for modulation of biological activity inside the cell. Here we present novel human protein 53 C-terminal domain-derived peptides, Peptide4 and Peptide5 that were designed using cell-penetrating peptide prediction algorithm and synthesised by Fmoc chemistry. Peptide4 and Peptide5 showed to be capable for translocation inside the breast cancer cells. Subsequent introduction of stearic acid moiety in the backbone of these peptides at N-terminal or lysine 3-orthogonal positions enhanced their cell-penetrating ability. Moreover Peptide4 and Peptide5 showed certain cytotoxic activity and were able to induce apoptosis in MDA-MB-231 cell line in the absence of serum. We suggest that human protein 53 C-terminal domain-derived cell-penetrating peptides Peptide4 and Peptide5 have promising perspectives for the future anticancer applications.

  • 942.
    Suhorutsenko, Julia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Oskolkov, Nikita
    Arukuusk, Piret
    Kurrikoff, Kaido
    Eriste, Elo
    Copolovici, Dana-Maria
    Langel, Ulo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-Penetrating Peptides, PepFects, Show No Evidence of Toxicity and Immunogenicity In Vitro and In Vivo2011Ingår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 22, nr 11, s. 2255-2262Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cell-penetrating peptide based vehicles have been developed for the delivery of different payloads into the cells in culture and in animals. However, several biological features, among which is the tendency to trigger innate immune response, limit the development of highly efficient peptide-based drug delivery vectors. This study aims to evaluate the influence of transportan 10 (TP10) and its chemically modified derivatives, PepFects (PFs), on the innate immune response of the host system. PFs have shown high efficiency in nucleic acid delivery in vitro and in vivo; hence, the estimation of their possible toxic side effects would be of particular interest. In this study, we analyzed cytotoxic and immunogenic response of PF3, PF4, and PF6 peptides in monocytic leukemia and peripheral blood mononuclear cell lines. In comparison with amphipathic PFs, TP10, TAT, stearyl-(RxR)(4) peptides, and the most widely used transfection reagents Lipofectamine 2000 and Lipofectamine RNAiMAX were also analyzed in this study. IL-1 beta, IL-18, and TNF-alpha cytokine release was detected using highly sensitive enzyme-linked immunosorbent assay (ELISA). Cell viability was detected by measuring the activity of cellular enzymes that reduce water-soluble tetrazolium salts to formazan dyes and apoptosis was evaluated by measuring the levels of caspase-1 and caspase-3/7 over untreated cells. All peptides were found to be nontoxic and nonimmunogenic in vitro at the concentrations of 10 mu M and 5 mu M, respectively, and at a dose of 5 mg/kg in vivo, suggesting that these CPPs exhibit a promising potential in the delivery of therapeutic molecules into the cell without risks of toxicity and inflammatory reactions.

  • 943. Sun, Song
    et al.
    Zhang, Wei
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Andersson, Dan I.
    Evolution of Broad Spectrum beta-Lactam Resistance in an Engineered Metallo-beta-lactamase2013Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 4, s. 2314-2324Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The extensive use and misuse of antibiotics during the last seven decades has led to the evolution and global spread of a variety of resistance mechanisms in bacteria. Of high medical importance are beta-lactamases, a group of enzymes inactivating beta-lactam antibiotics. Metallo-beta-lactamases (MBLs) are particularly problematic because of their ability to act on virtually all classes of beta-lactam antibiotics. An engineered MBL (evMBL9) characterized by low level activity with several beta-lactam antibiotics was constructed and employed as a parental MBL in an experiment to examine how an enzyme can evolve toward increased activity with a variety of beta-lactam antibiotics. We designed and synthesized a mutant library in which the substrate activity profile was varied by randomizing six active site amino acid residues. The library was expressed in Salmonella typhimurium, clones with increased resistance against seven different beta-lactam antibiotics (penicillin G, ampicillin, cephalothin, cefaclor, cefuroxime, cefoperazone, and cefotaxime) were isolated, and the MBL variants were characterized. For the majority of the mutants, bacterial resistance was significantly increased despite marked reductions in both mRNA and protein levels relative to those of parental evMBL9, indicating that the catalytic activities of these mutant MBLs were highly increased. Multivariate analysis showed that the majority of the mutant enzymes were generalists, conferring increased resistance against most of the examined beta-lactams.

  • 944. Sundberg, C
    et al.
    Wachtmeister, C A
    Lundgren, Bo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    DePierre, J W
    Comparison of the potencies of (+)- and (-)-2-ethylhexanoic acid in causing peroxisome proliferation and related biological effects in mouse liver.1994Ingår i: Chirality, ISSN 0899-0042, E-ISSN 1520-636X, Vol. 6, nr 1Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Male C57BL/6 mice were exposed to 1% (w/w) (+)- or (-)-2-ethylhexanoic acid or an equimolar mixture of these enantiomers in their diet for 4 or 10 days. A significant increase in liver weight and a 2- to 3-fold increase in the protein content of the mitochondrial fraction were seen in all cases. Peroxisomal palmitoyl-CoA oxidation was increased 2- to 3.5-fold after 4 days of treatment and 4- to 5-fold after 10 days, while the corresponding increases in peroxisomal lauroyl-CoA oxidase activity were 2- to 3-fold and 9- to 12-fold, respectively. Peroxisomal catalase activity was unchanged, whereas the microsomal and cytosolic activities were increased 2- to 3-fold and 6- to 16-fold, respectively. These treatments also induced microsomal omega-hydroxylation of lauric acid 7-fold and soluble epoxide hydrolase activity in the mitochondrial and cytosolic fractions, as well as microsomal epoxide hydrolase activity about 50-100%. The only significant differences observed between the effects of (+)-2-ethylhexanoic acid and its (-)-enantiomer were on peroxisomal palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity after 4 days of treatment. In both these cases the (+)-enantiomer resulted in increases which were 50-75% greater than those seen with the (-)-form.

  • 945.
    Surkov, Serhiy
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Studies on Translation Initiation and trans-Translation2010Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

     Translation initiation factor 1 (IF1) is an essential bacterial protein, without any clearly defined function in translation initiation. Same point mutants in this protein exhibit cold-sensitivity. In addition, a sequence specific increase in test protein expression has been observed for these mutants. Here in Paper I we analyze the parts of mRNA translation initiation region (TIR) involved in this effect, showing that Shine-Dalgarno sequence (SD), upstream enhancer region as well as linker between the initiation codon and SD are important. The similarity in action between the mutated IF1 and the antibiotic kasugamycin leads us to the suggestion that it occurs during the recently discovered proof-reading stage after the subunit joining step in the translation initiation. Deletion of yggJ and cspA genes involved in mRNA translation partially suppresses the cold-sensitivity phenotype of the R40D IF1 mutant. Deletion of the bipA gene confers even higher suppression confirming the previously assigned function of this protein in initiation of translation. In Paper II of this thesis proteome analysis of the IF1 mutation and kasugamycin action reveals some interesting features, such as increase in S6 polyglutamylation in the IF1 mutant cells or high level of GroEL-GroES protein in the cells treated with kasugamycin. A subset of genes having a similar TIR-specific increase in the expression under both conditions confirms previously made observations. Finally, in the Paper III we use cryo-electron tomography as well as chemical probing and computational modeling to address the question, how tmRNA moves on the ribosome during trans-translation. We observe that the tmRNA pseudoknots remain intact, while the mRNA part moves in the mRNA channel, allowing translation elongation to proceed.

  • 946.
    Surkov, Serhiy
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Isak, Georgina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Isaksson, Leif
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Influences of a mutated translation initiation factor IF1 or kasugamycin on  Escherichia coli gene expressionManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The infA (R40D) mutation or the addition of antibiotic kasugamycin to a wild type strain has been suggested to affect the same related step in translation initiation. Two-dimensional gel electrophoresis of radioactively labeled proteins was used to investigate this effect and to gain an overview of the proteins expressed under these conditions. A number of protein spots with increased or decreased expression levels were identified. The most pronounced increased expression in the IF1 mutant strain were the proteins YbgF (> 5 times) and in the relative abundance of the ribosomal protein S6 (rpsF) isoforms.  In the case of kasugamycin treatment strongly increased expression of natural proteins was seen for OppA, GroL, YbgF and several others. Expression of several proteins was affected similarly as a response to either the infA mutation or to the addition of kasugamycin. The translation initiation regions (TIR) comprising a region upstream and downstream of AUG from several of these genes were cloned into the protein A’ reporter gene system for further analysis. TIR sequences of several natural genes that showed elevated expression levels gave a corresponding increase in gene expression as reflected by the protein A’ expression assay system. This is especially true in the case of that the TIR sequence of ybgF gives a strongly increased expression in agreement with the increased expression observed for the native YbgF protein in the infA mutant or upon kasugamycin addition. The results suggest that the TIR region of ybgF has some unique properties that influence its expression at the early translational phase.

  • 947.
    Surkov, Serhiy
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Nilsson, Hanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Rasmussen, Louise CV
    Department of Molecular Biology, Aarhus university.
    Sperling-Petersen, Hans U
    Department of Molecular Biology, Aarhus university.
    Isaksson, Leif
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Translation initiation region dependency of translation initiation in Escherichia coli by IF1 and kasugamycin2010Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, nr 11, s. 2428-2439Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Translation initiation factor 1 (IF1) is an essential protein in prokaryotes. The nature of IF1 interactions with the mRNA during translation initiation on the ribosome remains unclear, even though the factor has several known functions, one of them being RNA chaperone activity. In this study, we analyzed translational gene expression mutant variants of IF1 with amino acid substitutions, R40D and R69L, using two different reporter gene systems. The strains with them mutant IF1 gave higher reporter gene expression than the control strain. The extent of this effect was dependent on the composition of the translation initiation region. The Shine–Dalgarno (SD) sequence, AU-rich elements upstream of the SD sequence and the region between the SD sequence and the initiation codon are important for the magnitude of this effect. The data suggest that the wild-type form of IF1 has a translation initiation region-dependent inhibitory effect on translation initiation. Kasugamycin is an antibiotic that blocks translation initiation. Addition of kasugamycin to growing wild-type cells increases reporter gene expression in a very similar way to the altered IF1, suggesting that the and kasugamycin affect some related step in translation initiation. Genetic knockout of three proteins (YggJ, BipA, and CspA) that are known to interact with RNA causes partial suppression of the IF1-dependent cold sensitivity.

  • 948. Suski, Jan M.
    et al.
    Schoenfeld, Peter
    Bonora, Massimo
    Shabalina, Irina
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Pinton, Paolo
    Wieckowski, Mariusz R.
    Guanosine diphosphate exerts a lower effect on superoxide release from mitochondrial matrix in the brains of uncoupling protein-2 knockout mice: New evidence for a putative novel function of uncoupling proteins as superoxide anion transporters2012Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 428, nr 2, s. 234-238Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In this report, we show new experimental evidence that, in mouse brain mitochondria, uncoupling protein-2 (UCP2) can be involved in superoxide (O-2(center dot-)) removal from the mitochondrial matrix. We found that the effect of guanosine 5'-diphosphate (GDP) on the rate of reactive oxygen species (ROS) release from brain mitochondria of UCP2 knockout mice was less Pronounced compared to the wild type animals. This putative novel UCP2 activity, evaluated by the use of UCP2-knockout transgenic animals, along with the known antioxidant defence systems, may provide additional protection from ROS in brain mitochondria.

  • 949.
    Svahn, Emelie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Molecular machinery of a membrane-bound proton pump: Studies of charge transfer reactions in cytochrome c oxidase2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In cellular respiration, electron transfer from the breakdown of foodstuff is coupled to the formation of an electrochemical proton gradient. This is accomplished through proton translocation by respiratory complexes, and the proton gradient is subsequently used e.g. to drive ATP production. Consequently, proton- and electron-transfer reactions through the hydrophobic interior of membrane proteins are central to cellular respiration. In this thesis, proton- and electron transfer through an aa3-type terminal oxidase, cytochrome c oxidase (CytcO) from Rhodobacter sphaeroides, have been studied with the aim of understanding the molecular proton-transfer machinery of this proton pump.

    In the catalytic site of CytcO the electrons combine with protons and the terminal electron acceptor O2 to form water in an exergonic reaction that drives proton pumping. Therefore, CytcO must transfer both protons that are pumped and protons for the oxygen chemistry through its interior. This is done through its two proton-transfer pathways, termed the D pathway and the K pathway. Our studies have shown that the protons pumped during oxidation of CytcO are taken through the D pathway, and that this process does not require a functional K pathway. Furthermore, our data suggests that the K pathway is used for charge compensation of electron transfer to the catalytic site, but only in the A2  P3 state transition. Our data also show that the water molecules identified in the crystal structures of CytcO play an important role in proton transfer through the D pathway. Finally, the effects of liposome reconstitution of CytcO on D-pathway proton transfer were investigated. The results suggest that the membrane modulates the rates of proton transfer through the D pathway, and also influences the extent of electron transfer between redox-active sites CuA and heme a.

  • 950.
    Svahn, Emelie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Faxén, Kristina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Proton pumping by an inactive structural variant of cytochrome c oxidase: 2014Ingår i: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 140, s. 6-11Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The aa3-type cytochrome c oxidases (CytcOs) from e.g. Rhodobacter sphaeroides and Paracoccus denitrificans harbor two proton-transfer pathways. The K pathway is used for proton uptake upon reduction of the CytcO, while the D pathway is used after binding of O2 to the catalytic site. The aim of the present study was to determine whether or not CytcO in which the K pathway is blocked (by e.g. the Lys362Met replacement) is capable of pumping protons. The process can not be studied using conventional assays because the O2-reduction activity is too low when the K pathway is blocked. Consequently, proton pumping with a blocked K pathway has not been demonstrated directly. Here, the Lys362Met and Ser299Glu structural variants were reconstituted in liposomes and allowed to (slowly) become completely reduced. Then, the reaction with O2 was studied with μs time resolution after flash photolysis of a blocking CO ligand bound to heme a3. The data show that with both the inactive Lys362Met and partly active Ser299Glu variants proton release occurred with the same time constants as with the wild-type oxidase, i.e. ~ 200 μs and ~ 3 ms, corresponding in time to formation of the ferryl and oxidized states, respectively. Thus, the data show that the K pathway is not required for proton pumping, suggesting that D and K pathways operate independently of each other after binding of O2 to the catalytic site.

16171819202122 901 - 950 av 1092
RefereraExporteraLänk till träfflistan
Permanent länk
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf