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  • 251. Luecke, S.
    et al.
    Wincent, E.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Backlund, M.
    Rannug, U.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Rannug, A.
    Cytochrome P450 1A1 gene regulation by UVB involves crosstalk between the aryl hydrocarbon receptor and nuclear factor kappa B2010In: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 184, no 3, p. 466-473Article in journal (Refereed)
    Abstract [en]

    UVB induces the expression of genes controlled by the aryl hydrocarbon receptor (AhR). a transcription factor that has been implicated in the UV stress response. In this study, we used the human hepatoma cell line HepG2 to investigate in more detail the effects of UVB irradiation on AhR activation and induction of cytochrome P450 1A1 (CYP1A1), a highly AhR-responsive gene. The CYP1A1 enzyme efficiently degrades 6-formylindolo[3,2-b]carbazole (FICZ), a high affinity ligand and suggested endogenous activator of the AhR. We show that physiologically relevant doses of UVB suppress CYP1A1 gene expression immediately after irradiation, but induce its expression later in an AhR-dependent manner. The initial repression phase of CYP1A1 transcription was mediated by another UVB-inducible transcription factor, the nuclear factor kappa B (NF kappa B). Crosstalk between AhR and NF kappa B signaling has earlier been implicated to control CYP1A1 expression following stimulation by xenobiotics and cytokines. Now, our findings clearly indicate a role of NF kappa B also in UVB-dependent AhR signaling. We also observed that UVB reduced the catalytic activity of the CYP1A1 enzyme. Thereby. UVB attenuated the clearance of FICZ, which led to prolonged AhR activation. We further noted that repeated irradiation with UVB or H2O2 treatment shifted the cells into a refractory state in which AhR signaling could not be efficiently activated by UVB or H2O2, but by ligands. Together, our results suggest that the NF kappa B-mediated initial suppression of CYP1A1 as well as the unresponsiveness of AhR signaling to repeated irradiation may be part of a protective cellular UV stress response.

  • 252.
    Lundgren, Josefin
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Masson, Patrick
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Realini, Claudio
    Young, Patrick
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Use of RNA interference and Complementation to study the function of the Drosophila and Human 26S proteasome Subunit S132003In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 23, no 15, p. 5320-5330Article in journal (Refereed)
  • 253.
    Lundin, Cecilia
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Arnaudeau, Catherine
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Meuth, Mark
    The Institute for Cancer Studies, University of Sheffield.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Different Roles for Nonhomologous End Joining and Homologous Recombination following Replication Arrest in Mammalian Cells2002In: Molecular and Cellular Biology, ISSN 0270-7306, Vol. 22, no 16, p. 5869-78Article in journal (Refereed)
    Abstract [en]

    Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks (DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair of slowed replication forks in the absence of detectable DSBs.

  • 254. Lundin, Cecilia
    et al.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Jenssen, Dag
    Erixon, Klaus
    Helleday, Thomas
    Two recombination pathways involved in repair of alkylated lesionsManuscript (Other academic)
  • 255.
    Lundin, Cecilia
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Samuelsson, Magnus
    Department of Medical Nutrition Karolinska Institute Huddinge University Hospital.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Overexpression of Cyclin E Does not Affect Homologous Recombination in Chinese Hamster Cells2003In: Biochemical and Biophysical Research Communications, ISSN 0006-291X, Vol. 296, no 2, p. 363-7Article in journal (Refereed)
    Abstract [en]

    Overexpressed cyclin E in tumours is a prognosticator for poor patient outcome. Cells that overexpress cyclin E have been shown to be impaired in S-phase progression and exhibit genetic instability that may drive this subset of cancers. However, the origin for genetic instability caused by cyclin E overexpression is unknown. Homologous recombination plays an important role in S-phase progression and is also regulated by the same proteins that regulate cyclin E-associated kinase activity, i.e., p53 and p21. To test the hypothesis that overexpressed cyclin E causes genetic instability through homologous recombination, we investigated the effect of cyclin E overexpression on homologous recombination in the hprt gene in a Chinese hamster cell line. Although cyclin E overexpression shortened the G1 phase in the cell cycle as expected, we could see no change in neither spontaneous nor etoposide-induced recombination. Also, overexpression of cyclin E did not affect the repair of DNA double-strand breaks and failed to potentiate the cytotoxic effects of etoposide. Our data suggest that genetic instability caused by overexpression of cyclin E is not mediated by aberrant homologous recombination

  • 256.
    Lundin, Cecilia
    et al.
    Stockholm University, Faculty of Social Sciences, Department of Applied Communications Science - GI and IHR.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Arnaudeau, Catherine
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Mohindra, Atul
    2The Institute for Cancer Studies, University of Sheffield.
    Hansen, Lasse Tengbjerg
    3Institute of Molecular Pathology, University of Copenhagen.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    RAD51 is Involved in Repair of Damage Associated with DNA Replication in Mammalian Cells2003In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 328, no 3, p. 521-35Article in journal (Refereed)
    Abstract [en]

    The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than γ-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks.

  • 257.
    Löfmark, Sonja
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    de Klerk, Nele
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Aro, Helena
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Neisseria gonorrhoeae Infection Induces Altered Amphiregulin Processing and Release2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 1, article id e16369Article in journal (Refereed)
    Abstract [en]

    Adhesion of the human pathogen Neisseria gonorrhoeae has established effects on the host cell and evokes a variety of cellular events including growth factor activation. In the present study we report that infection with N. gonorrhoeae causes altered amphiregulin processing and release in human epithelial cells. Amphiregulin is a well-studied growth factor with functions in various cell processes and is upregulated in different forms cancer and proliferative diseases. The protein is prototypically cleaved on the cell surface in response to external stimuli. We demonstrate that upon infection, a massive upregulation of amphiregulin mRNA is seen. The protein changes its subcellular distribution and is also alternatively cleaved at the plasma membrane, which results in augmented release of an infection-specific 36 kDa amphiregulin product from the surface of human cervical epithelial cells. Further, using antibodies directed against different domains of the protein we could determine the impact of infection on pro-peptide processing. In summary, we present data showing that the infection of N. gonorrhoeae causes an alternative amphiregulin processing, subcellular distribution and release in human epithelial cervical cells that likely contribute to the predisposition cellular abnormalities and anti-apoptotic features of N. gonorrhoeae infections. 

  • 258. MacKay, Craig
    et al.
    Declais, Anne-Cecile
    Lundin, Cecilia
    Agostinho, Ana
    Deans, Andrew J.
    MacArtney, Thomas J.
    Hofmann, Kay
    Gartner, Anton
    West, Stephen C.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lilley, David M. J.
    Rouse, John
    Identification of KIAA1018/FAN1, a DNA Repair Nuclease Recruited to DNA Damage by Monoubiquitinated FANCD22010In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 142, no 1, p. 65-76Article in journal (Refereed)
    Abstract [en]

    DNA interstrand crosslinks (ICLs) are highly toxic because they block the progression of replisomes. The Fanconi Anemia (FA) proteins, encoded by genes that are mutated in FA, are important for repair of ICLs. The FA core complex catalyzes the monoubiquitination of FANCD2, and this event is essential for several steps of ICL repair. However, how monoubiquitination of FANCD2 promotes ICL repair at the molecular level is unknown. Here, we describe a highly conserved protein, KIAA1018/MTMR15/FAN1, that interacts with, and is recruited to sites of DNA damage by, the monoubiquitinated form of FANCD2. FAN1 exhibits endonuclease activity toward 50 flaps and has 5' exonuclease activity, and these activities are mediated by an ancient VRR_nuc domain. Depletion of FAN1 from human cells causes hypersensitivity to ICLs, defects in ICL repair, and genome instability. These data at least partly explain how ubiquitination of FANCD2 promotes DNA repair.

  • 259.
    Macvanin, Mirjana
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    de Valdivia, Ernesto I. Gonzalez
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Ardell, David H.
    Isaksson, Leif A.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Transient erythromycin resistance phenotype associated with peptidyl-tRNA drop-off on early UGG and GGG codons2007In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 189, no 24, p. 8993-9000Article in journal (Refereed)
    Abstract [en]

    Expression of minigenes encoding tetra- or pentapeptides MXLX or MXLXV (E peptides), where X is a nonpolar amino acid, renders cells erythromycin resistant whereas expression of minigenes encoding tripeptide MXL does not. By using a 3A' reporter gene system beginning with an E-peptide-encoding sequence, we asked whether the codons UGG and GGG, which are known to promote peptidyl-tRNA drop-off at early positions in mRNA, would result in a phenotype of erythromycin resistance if located after this sequence. We find that UGG or GGG, at either position +4 or +5, without a following stop codon, is associated with an erythromycin resistance phenotype upon gene induction. Our results suggest that, while a stop codon at +4 gives a tripeptide product (MIL) and erythromycin sensitivity, UGG or GGG codons at the same position give a tetrapeptide product (MILW or MILG) and phenotype of erythromycin resistance. Thus, the drop-off event on GGG or UGG codons occurs after incorporation of the corresponding amino acid into the growing peptide chain. Drop-off gives rise to a peptidyl-tRNA where the peptide moiety functionally mimics a minigene peptide product of the type previously associated with erythromycin resistance. Several genes in Escherichia coli fulfill the requirements of high mRNA expression and an E-peptide sequence followed by UGG or GGG at position +4 or +5 and should potentially be able to give an erythromycin resistance phenotype.

  • 260. Mahdavi, Jafar
    et al.
    Royer, Pierre-Joseph
    Sjölinder, Hong S.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Azimi, Sheyda
    Self, Tim
    Stoof, Jeroen
    Wheldon, Lee M.
    Brännström, Kristoffer
    Wilson, Raymond
    Moreton, Joanna
    Moir, James W. B.
    Sihlbom, Carina
    Borén, Thomas
    Jonsson, Ann-Beth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Soultanas, Panos
    Ala'Aldeen, Dlawer A. A.
    Pro-inflammatory cytokines can act as intracellular modulators of commensal bacterial virulence2013In: Open Biology, ISSN 2046-2441, E-ISSN 2046-2441, Vol. 3, no 10, article id 130048Article in journal (Refereed)
    Abstract [en]

    Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.

  • 261.
    Mandali, Sridhar
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Interactive studies of attachment sites with proteins involved in site-specific recombination of P2-like phages2009Licentiate thesis, monograph (Other academic)
  • 262.
    Mandali, Sridhar
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Site-specific recombination of P2-like phages; possible tools for safe gene therapy: A focus on phage ΦD1452010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    P2-like bacteriophages integrate their genome into the E. coli host cell by a site-specific recombination event upon lysogenization. The integrative recombination occurs between a specific sequence in the phage genome, attP, and a specific sequence in the host genome, attB, generating the host-phage junctions attL and attR. The integration is mediated by the phage enzyme integrase (Int) and the host factor IHF. The excisive recombination takes place between attL and attR, and is mediated by Int, IHF and phage encoded protein Cox. For safe integration of foreign genes into eukaryotic chromosome a recombinases is necessary which can perform the integration site-specifically. P2-like phage integrases have the potential to become tools for safe gene therapy. Their target is simple but specific, and once integration has occurred it is very stable in the absence of the Cox protein. The site-specific recombination mechanism has to be understood at the molecular level. Therefore, I have initiated the characterization of the site-specific recombination system of the P2-like phage ΦD145. In this work, Int and IHF are shown to bind to the different attachment sites cooperatively. One of two possible inverted repeats in attP is shown to be the Int core recognition site. The attP core of this phage has high identity with a site on human chromosome, denoted as ΨattB. In this study we have shown that in in vivo recombination ΦD145 Int can accept ΨattB in both bacteria and in eukaryotic cells. Also shown that Int consists of an intrinsic nuclear localization signal. A study also reveled that ΦD145 Int activity was affected by the Tyr-phosphorylation. Attempts have been made to change the specificity of the other P2-like phage P2 and WΦ integrases and also structural and functional analysis was done. A study on comparative analysis of Cox proteins and Cox binding sites gave us the basic information about the recombination mechanism.

  • 263.
    Mandali, Sridhar
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cardoso-Palacios, Carlos
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sylwan, Lina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Characterization of the site-specific recombination system of phage ΦD145, and its capacity to promote recombination in human cells2010In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 408, no 1, p. 64-70Article in journal (Refereed)
    Abstract [en]

    Phage integrases have the potential of becoming tools for safe site-specific integration of genes into unmodified human genomes. The P2-like phages have been found to have different bacterial host integration sites and consequently they have related integrases with different sequence specificities. In this work the site-specific recombination system of the P2-like phage ΦD145 is characterized. The minimal attB site is determined to 22 nt with 18 nt identity to the core region of attP. A non-coding sequence on the human chromosome 13 is shown to be a rather good substrate for recombination in vivo in bacteria as well as in a plasmid system in HeLa cells when HMG protein recognition sequences are inserted between the left arm-binding site and the core in the complex phage attachment site attP. Thus ΦD145 integrase that belongs to the tyrosine family shows potential as a tool for site-specific integration into the human genome.

  • 264.
    Mandali, Sridhar
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Phosphorylation affects the biological activity of the integrase of the P2-likephage ΦD145Manuscript (preprint) (Other academic)
  • 265.
    Markova, Eva
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Malmgren, Lars O. G.
    Belyaev, Igor Y.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Microwaves from Mobile Phones Inhibit 53BP1 Focus Formation in Human Stem Cells More Strongly Than in Differentiated Cells: Possible Mechanistic Link to Cancer Risk2010In: Journal of Environmental Health Perspectives, ISSN 0091-6765, E-ISSN 1552-9924, Vol. 118, no 3, p. 394-399Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: It is widely accepted that DNA double-strand breaks (DSBs) and their misrepair in stem cells are critical events in the multistage origination-of various leukemias and tumors, including gliomas. OBJECTIVES: We studied whether microwaves from mobile telephones of the Global System for Mobile Communication (GSM) and the Universal Global Telecommunications System (UMTS) induce DSBs or affect DSB repair in stem cells. METHODS: We analyzed tumor suppressor TP53 binding protein 1 (53BP1) foci that are typically formed at the sites of DSB location (referred to as DNA repair foci) by laser confocal microscopy. RESULTS: Microwaves from mobile phones inhibited formation of 53BP1 foci in human primary fibroblasts and mesenchymal stem cells. These data parallel our previous findings for human lymphocytes. Importantly, the same GSM carrier frequency (915 MHz) and UMTS frequency band (1947.4 MHz) were effective for all cell types. Exposure at 905 MHz did not inhibit 53BP1 foci in differentiated cells, either fibroblasts or lymphocytes, whereas some effects were seen in stem cells at 905 MHz. Contrary to fibroblasts, stem cells did not adapt to chronic exposure during 2 weeks. CONCLUSIONS: The strongest microwave effects were always observed in stem cells. This result may suggest both significant misbalance in DSB repair and severe stress response. Our findings that stem cells are most sensitive to microwave exposure and react to more frequencies than do differentiated cells may be important for cancer risk assessment and indicate that stem cells are the most relevant cellular model for validating safe mobile communication signals.

  • 266.
    Markova, Eva
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Torudd, Jesper
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Belyaev, Igor
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Long time persistence of residual 53BP1/gamma-H2AX foci in human lymphocytes in relationship to apoptosis, chromatin condensation and biological dosimetry2011In: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, Vol. 87, no 7, p. 736-745Article in journal (Refereed)
    Abstract [en]

    Purpose: Novel assay for radiosensitivity is based on measurements of residual DNA repair foci produced by several proteins including phosphorylated H2AX (gamma-H2AX), recombinase Rad51 (Rad51) and tumour suppressor p53 binding protein 1 (53BP1), which co-localise with radiation-induced DNA double-strand breaks (DSB). Here, we studied dose-response for residual 53BP1, Rad51, and gamma-H2AX foci in relationship to apoptosis and chromatin condensation in human G(0)-lymphocytes. Materials and methods: Residual foci, apoptosis and condensation of chromatin were studied following irradiation with gamma-rays at doses of 0.5-10 Gy. Results: No clear dose response for residual Rad51 was seen. Residual 53BP1/gamma-H2AX foci remained in human lymphocytes up to four weeks after irradiation. No foci formed during radiation-induced apoptosis. We provide evidence that irreversible apoptotic condensation of chromatin is responsible for arrest of residual foci and preventing de novo focus formation. Similar linear dose dependences up to 2 Gy were observed for the 53BP1/gamma-H2AX foci at all studied time points. At higher doses, saturation and decline were caused by preferential elimination of apoptotic lymphocytes with residual foci. While primary 53BP1 and gamma-H2AX foci almost completely co-localised, co-localisation of residual foci did not exceed 17%, indicating that 53BP1 and gamma-H2AX proteins may remain for different times at the locations of DSB repair. Conclusions: Prolonged persistence of residual 53BP1/gamma-H2AX foci may be used for biological dosimetry within the dose range up to 2 Gy. While foci are not formed during radiation-induced apoptosis in human lymphocytes, elimination of apoptotic cells with residual foci may affect the dose response.

  • 267.
    Marková, E
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, N
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Belyaev, I Y
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Kinetics and dose-response of residual 53BP1/gamma-H2AX foci: co-localization, relationship with DSB repair and clonogenic survival.2007In: Int J Radiat Biol, ISSN 0955-3002, Vol. 83, no 5, p. 319-329Article in journal (Refereed)
  • 268. Marková, E
    et al.
    Schultz, N
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Belyaev, I Y
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Kinetics and dose-response of residual 53BP1/gamma-H2AX foci: co-localization, relationship with DSB repair and clonogenic survival.2007In: Int J Radiat Biol, ISSN 0955-3002, Vol. 83, no 5, p. 319-29Article in journal (Refereed)
  • 269.
    Masala, Silvia
    et al.
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Rannug, Ulf
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Westerholm, Roger
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Pressurized liquid extraction as an alternative to the Soxhlet extraction procedure stated in the US EPA method TO-13A for the recovery of polycyclic aromatic hydrocarbons adsorbed on polyurethane foam plugs2014In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 6, no 20, p. 8420-8425Article in journal (Refereed)
    Abstract [en]

    The aim of the present study was to develop a pressurized liquid extraction (PLE) method as an alternative to the relatively time consuming Soxhlet extraction procedure described in the United States Environmental Protection Agency (US EPA) method TO-13A for the extraction of PAHs adsorbed onto polyurethane foam plugs (PUFs). For this purpose PUF air samples were collected and split into two parts: one part extracted using PLE and the other one using Soxhlet extraction. Comparable PAH concentrations were obtained upon analysis of the extracts showing that the PLE method developed in this work is a more convenient choice than the commonly used Soxhlet extraction technique proposed by US EPA for the determination of PAHs in air samples. In fact, the developed PLE method required shorter assay times (minutes versus hours), less solvent consumption and simpler operational methods. The exhaustiveness of the developed PLE method was evaluated using repeat static extraction cycles, demonstrating an extraction efficiency for the PAHs of greater than 99%. The PLE method was then applied to diesel exhaust and wood smoke PUF samples showing an extraction efficiency for the PAHs of greater than 93% and 96%, respectively. Furthermore, a PLE method for PUF cleaning was developed as well and employed as an alternative to Soxhlet extraction. The PLE methods developed for cleaning and extracting PUFs presented in this work are suitable to be used in mutagenicity studies using the Ames Salmonella assay as no mutagenicity was found in the PLE generated blanks

  • 270.
    Massad, Tariq
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Papadopoulos, Evangelos
    Beth Israel Deaconess Med. Center Harvard Institute of Medicin.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Damberg, Peter
    Department of Neurobiology, Care Sciences and Society, Karolinska Institutet .
    NMR Structure Note: The C Repressor of the P2 BacteriophageManuscript (preprint) (Other academic)
  • 271.
    Massad, Tariq
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Papadopoulos, Evangelos
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Henriksson-Peltola, Petri
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Damberg, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Assignment of 1H, 13C, and 15N chemical shift resonances of P2 C-repressor protein2008In: Biomolecular NMR Assignments, ISSN 1874-270X, Vol. 2, no 2, p. 215-217Article in journal (Refereed)
  • 272.
    Massad, Tariq
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Skaar, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, Hanna
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Damberg, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Henriksson-Peltola, Petri
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal structure of the P2 C-repressor: a binder of nonpalindromic direct DNA repeats2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 21, p. 7778-7790Article in journal (Refereed)
    Abstract [en]

    As opposed to the vast majority of prokaryoticrepressors, the immunity repressor of temperateEscherichia coli phage P2 (C) recognizes nonpalindromicdirect repeats of DNA rather thaninverted repeats. We have determined the crystalstructure of P2 C at 1.8A ° . This constitutes the firststructure solved from the family of C proteins fromP2-like bacteriophages. The structure reveals thatthe P2 C protein forms a symmetric dimer orientedto bind the major groove of two consecutive turns ofthe DNA. Surprisingly, P2 C has great similarities tobinders of palindromic sequences. Nevertheless, thetwo identical DNA-binding helixes of the symmetricP2 C dimer have to bind different DNA sequences.Helix 3 is identified as the DNA-recognition motif inP2 C by alanine scanning and the importance for theindividual residues in DNA recognition is defined.A truncation mutant shows that the disorderedC-terminus is dispensable for repressor function.The short distance between the DNA-bindinghelices together with a possible interaction betweentwo P2 C dimers are proposed to be responsible forextensive bending of the DNA. The structure providesinsight into the mechanisms behind the mutants ofP2 C causing dimer disruption, temperature sensitivityand insensitivity to the P4 antirepressor.

  • 273.
    Masson, Patrick
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Andersson, Oskar
    Petersen, Ulla-Maya
    Young, Patrick
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Identification and characterization of a Drosophila nuclear proteasome activator: a homolog of human 11S REggamma (PA28gamma)2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 2, p. 1383-1390Article in journal (Refereed)
    Abstract [en]

    We report the cloning and characterization of aDrosophila proteasome 11 S REGγ (PA28) homolog. The 28-kDa protein shows 47% identity to the human REGγ and strongly enhances the trypsin-like activities of both Drosophila and mammalian 20 S proteasomes. Surprisingly, the DrosophilaREG was found to inhibit the proteasome's chymotrypsin-like activity against the fluorogenic peptide succinyl-LLVY-7-amino-4-methylcoumarin. Immunocytological analysis reveals that the Drosophila REG is localized to the nucleus but is distributed throughout the cell when nuclear envelope breakdown occurs during mitosis. Through site-directed mutagenesis studies, we have identified a functional nuclear localization signal present in the homolog-specific insert region. TheDrosophila PA28 NLS is similar to the oncogene c-Myc nuclear localization motif. Comparison between uninduced and innate immune induced Drosophila cells suggests that the REGγ proteasome activator has a role independent of the invertebrate immune system. Our results support the idea that γ class proteasome activators have an ancient conserved function within metazoans and were present prior to the emergence of the α and β REG classes.

  • 274.
    Masson, Patrick
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lundgren, Josefin
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Young, Patrick
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Drosophila Proteasome Regulator REGγ: Transcriptional Activation by DNA Replication-related Factor DREF and Evidence for a Role in Cell Cycle Progression2003In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 327, no 5, p. 1001-1012Article in journal (Refereed)
    Abstract [en]

    The proteasome regulator REG (PA28γ) is a conserved complex present in metazoan nuclei and is able to stimulate the trypsin-like activity of the proteasome in a non-ATP dependent manner. However, the in vivo function for REGγ in metazoan cells is currently unknown. To understand the role of Drosophila REGγ we have attempted to identify the type of promoter elements regulating its transcription. Mapping the site of the transcription initiation revealed a TATA-less promoter, and a sequence search identified elements found typically in Drosophila genes involved in cell cycle progression and DNA replication. In order to test the relevance of the motifs, REGγ transcriptional assays were carried out with mutations in the proposed promoter. Our results indicate that a single Drosophila replication-related element sequence, DRE, is essential for REGγ transcription. To confirm that REGγ has a role in cell cycle progression, the effect of removing REGγ from S2 cells was tested using RNA interference. Drosophila cells depleted of REGγ showed partial arrests in G1/S cell cycle transition. Immuno-staining of Drosophila embryos revealed that REGγ is typically localized to the nucleus during embryogenesis with increased levels present in invaginating cells during gastrulation. The REGγ was found dispersed throughout the cell volume within mitotic domains undergoing cell division. Finally, database searches suggest that the DRE system may regulate key members of the proteasome system in Drosophila.

  • 275. Masson, Patrick
    et al.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Söderbom, Fredrik
    Young, Patrick
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Characterization of a REG/PA28 Proteasome Activator Homolog in Dictyostelium discoideum Indicates that the Ubiquitin- and ATP-Independent REG gamma Proteasome Is an Ancient Nuclear Protease2009In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 8, no 6, p. 844-851Article in journal (Refereed)
    Abstract [en]

    The nuclear proteasome activator REG gamma/PA28 gamma is an ATP- and ubiquitin-independent activator of the 20S proteasome and has been proposed to degrade and thereby regulate both a key human oncogene, encoding the coactivator SRC-3/AIB1, and the cyclin-dependent kinase inhibitor p21 (Waf/Cip1). We report the identification and characterization of a PA28/REG homolog in Dictyostelium. Association of a recombinant Dictyostelium REG with the purified Dictyostelium 20S proteasome led to the preferential stimulation of the trypsin-like proteasome peptidase activity. Immunolocalization studies demonstrated that the proteasome activator is localized to the nucleus and is present in growing as well as starving Dictyostelium cells. Our results indicate that the Dictyostelium PA28/REG activator can stimulate both the trypsin-like and chymotrypsin-like activities of the 20S proteasome and supports the idea that the REG gamma-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.

  • 276. Matronchik, A. Y.
    et al.
    Belyaev, I. Y.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Mechanism for Combined Action of Microwaves and Static Magnetic Field: Slow Non Uniform Rotation of Charged Nucleoid2008In: Electromagnetic Biology and Medicine, ISSN 1536-8378, E-ISSN 1536-8386, Vol. 27, no 4, p. 340-354Article in journal (Refereed)
    Abstract [en]

    Recent data show that microwaves (MW) and extremely low-frequency (ELF) magnetic fields at low intensities affect conformation of nucleoids in bacterial E. coli cells and human lymphocytes. Experimental data suggest that magnitude of the effects of both MW and ELF depend on frequency and static magnetic field. We have previously proposed the physical model for the effects of combined ELF/static magnetic fields on the charged DNA-domain/nucleoid. In this article, we present the model of slow non uniform rotation of the charged DNA-domain/nucleoid for the combined effects of MW and static magnetic field. The solution of this model suggests that the combined action of MW and static magnetic field results in slow non uniform rotation of nucleoid with angular speed that depends on Larmor frequency. The model predicts that non thermal effects of MW are dependent on carrier frequency and also static magnetic field in the area of exposure.

  • 277.
    Maudsdotter, Lisa
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Interactions between pathogenic bacteria and the innate immune system2011Licentiate thesis, monograph (Other academic)
  • 278.
    Maudsdotter, Lisa
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jonsson, Hans
    Roos, Stefan
    Jonsson, Ann-Beth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lactobacilli reduce cell cytotoxicity caused by Streptococcus pyogenes by producing lactic acid that degrades the toxic component lipoteichoic acid2011In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 55, no 4, p. 1622-1628Article in journal (Refereed)
    Abstract [en]

    Lactobacilli are known to prevent colonization by many pathogens; nevertheless, the mechanisms of their protective effect are largely unknown. In this work, we investigated the role of lactobacilli during infection of epithelial cells with group A streptococci (GAS). GAS cause a variety of illnesses ranging from noninvasive disease to more severe invasive infections, such as necrotizing fasciitis and toxic shock-like syndrome. Invasion of deeper tissues is facilitated by GAS-induced apoptosis and cell death. We found that lactobacilli inhibit GAS-induced host cell cytotoxicity and shedding of the complement regulator CD46. Further, survival assays demonstrated that lactic acid secreted by lactobacilli is highly bactericidal toward GAS. In addition, lactic acid treatment of GAS, but not heat killing, prior to infection abolishes the cytotoxic effects against human cells. Since lipoteichoic acid (LTA) of GAS is heat resistant and cytotoxic, we explored the effects of lactic acid on LTA. By applying such an approach, we demonstrate that lactic acid reduces epithelial cell damage caused by GAS by degrading both secreted and cell-bound LTA. Taken together, our experiments reveal a mechanism by which lactobacilli prevent pathogen-induced host cell damage.

  • 279. McLachlan, Jennifer
    et al.
    Fernandez, Serena
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bryant, Helen E
    Specific targeted gene repair using single-stranded DNA oligonucleotides at an endogenous locus in mammalian cells uses homologous recombination.2009In: DNA repair, ISSN 1568-7856Article in journal (Refereed)
    Abstract [en]

    The feasibility of introducing point mutations in vivo using single-stranded DNA oligonucleotides (ssON) has been demonstrated but the efficiency and mechanism remain elusive and potential side effects have not been fully evaluated. Understanding the mechanism behind this potential therapy may help its development. Here, we demonstrate the specific repair of an endogenous non-functional hprt gene by a ssON in mammalian cells, and show that the frequency of such an event is enhanced when cells are in S-phase of the cell cycle. A potential barrier in using ssONs as gene therapy could be non-targeted mutations or gene rearrangements triggered by the ssON. Both the non-specific mutation frequencies and the frequency of gene rearrangements were largely unaffected by ssONs. Furthermore, we find that the introduction of a mutation causing the loss of a functional endogenous hprt gene by a ssON occurred at a similarly low but statistically significant frequency in wild type cells and in cells deficient in single strand break repair, nucleotide excision repair and mismatch repair. However, this mutation was not induced in XRCC3 mutant cells deficient in homologous recombination. Thus, our data suggest ssON-mediated targeted gene repair is more efficient in S-phase and involves homologous recombination.

  • 280.
    Melik, Wessam
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Molecular characterization of the Tick-borne encephalitis virus: Environments and replication2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The flavivirus genus is of major concern for world morbidity and mortality and includes viruses causing both encephalitic as well as hemorrhagic diseases. The incidence of Tick-borne encephalitis is increasing in many European countries and several reports have emphasized the expansion of the main vector, Ixodes ricinus. The pattern of vector distribution is also changing in Sweden, which makes it important to set up solid and successful strategies for detection and genetic characterization of novel Swedish TBEV strains.

    In this study we have generated strategies for detection of broad types of tick-borne flaviviruses in pools of I. ricinus sampled in Sweden.

    The positive collection on the island of Torö was used to generate a sequence of a complete TBEV genome straight from the arthropod reservoir. This cloned virus was used to construct a self-replicating DNA based sub-genomic TBEV replicon capable of expressing reporter genes. The replicon was used to study the effect of TBEV on neurite outgrowth, which revealed that the MTase domain of NS5 block the formation of the Scribble/Rac1/βPIX protein complex, impairing neurite outgrowth in neuronal growth factor induced PC12 cells.

    We also demonstrate that TBEV replication is affected by two PDZ binding motifs within NS5 and reveal putative PDZ binding proteins. These interactions might affect cellular pathways and might have a role in flavivirus replication.

    We also characterize the variable 3´ non-coding region (V3’-NCR) by in silico studies on TBEV. Analysis brings new evidence that V3’-NCR region carries an enhancer element important for different replication/translation dynamics during the viral lifecycle in mammalian and tick cells. We also propose a temperature-sensitive trans-acting riboswitch mechanism; altering the secondary RNA structures of a closed form at lower temperatures and a form open for translation at higher temperatures. This mechanism may explain the low TBEV level observed in sampled ticks.

  • 281.
    Melik, Wessam
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Ellencrona, Karin
    Wigerius, Michael
    Elväng, Annelie
    Johansson, Magnus
    Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replicationManuscript (Other academic)
    Abstract [en]

    The flavivirus genus includes important human pathogens like Tick-borne encephalitis virus (TBEV), Dengue virus (DV) and West-Nile virus (WNV), that can cause severe disease e.g. encephalitis or hemorrhagic fever. The NS5 protein is a multifunctional RNA dependent RNA polymerase indispensable for the flavivirus replication. We have previously shown that TBEVNS5 contains a unique internal PDZ binding motif (YS223) for specific targeting of the PDZ protein Scribble. This interaction has impact on both viral down regulation of host cellular defense systems and neurite outgrowth. Putative C-terminal PDZ binding motifs present in TBEVNS5 (-SII903) and WNVNS5 (-TVL905) have also previously been highlighted.

    To determine whether the PDZ binding motifs of TBEVNS5 has an effect on virus replication we constructed a DNA based sub-genomic TBEV replicon expressing firefly luciferase. The motifs within NS5 were mutated individually and in concert and the replicons were assayed in cell culture. Our results show that the replication rate was impaired in all mutants, which indicates that PDZ dependent host interactions influence flavivirus replication.We also find that the C-terminal PDZ binding motif present in TBEVNS5 and WNVNS5 are targeting various human PDZ domain proteins. TBEVNS5 has high affinity to Zonulaoccludens-2 (ZO-2),GIAP C-terminus interacting protein (GIPC), Calcium/calmodulin-dependent serine protein kinase (CASK) and Interleukin 16 (IL-16).A different pattern was observed for WNVNS5 as it associated with IL-16, and several other putative interaction partners.

  • 282.
    Melik, Wessam
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Nilsson, Anders S.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Magnus
    Detection strategies of tick-borne encephalitis virus in Swedish Ixodes ricinus reveal evolutionary characteristics of emerging tick-borne flaviviruses2007In: Archives of Virology, ISSN 0304-8608, E-ISSN 1432-8798, Vol. 152, no 5, p. 1027-1034Article in journal (Refereed)
    Abstract [en]

    The flaviviral tick-borne encephalitis virus (TBEV) is a human pathogen having significant impact on public health. The geographical distribution of TBEV and TBEV-like viruses is increasing, which makes it important to characterise the natural virus populations. Here we present four RT-PCR strategies designed for detection of broad types of tick-borne flaviviruses. Sequence information on more than 32% of a TBEV genome was generated from a small pool of ticks collected in the Stockholm archipelago on the island of Torö. The sequences were characterised and compared with those of other tick-borne flaviviruses, which classified the virus as Western European TBEV.

  • 283.
    Mellroth, Peter
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Peptidoglycan Recognition Proteins: Major Regulators of Drosophila Immunity2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    All eukaryotic organisms have an innate immune system characterized by germ-line encoded receptors and effector molecules, which mediate detection and clearance of microbes such as bacteria, fungi, and parasites. VertebrateDrosophila as a genetically tractable organism with a

    This thesis concerns the peptidoglycan recognition protein (PGRP) gene family in the fruit fly. The family consists of thirteen genes, of which a few have been reported to be part of the signaling pathways that regulates immune

    Data presented show that the putative receptors have affinity for peptidoglycan, but not for lipopolysaccharide, or the fungal cell wall polymer beta-glucan. PGRP-SA, receptor of the Toll pathway, has a preference for

    In a search for novel PGRP receptors I found two PGRP proteins that instead displayed enzymatic activity towards peptidoglycan. They are of the N-actylmuramoyl L-alanine amidase type, which degrades peptidoglycan by splittingStaphylococcus aureus peptidoglycan looses its immune elicitor capacity. This is in contrast to lysozyme-degraded peptidoglycan, which isDrosophila PGRPs to be potential enzymes. PGRP-SB1 is the other enzymatic PGRP described within this thesis. It has a moreBacillus megaterium.

    In conclusion, receptor PGRP proteins binds bacterial peptidoglycan and triggers immune gene pathways and enzymatic PGRPs have the capacity to reduce the elicitor property of peptidoglycan.

  • 284. Mellroth, Peter
    et al.
    Karlsson, Jenny
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Håkansson, Janet
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Goldman, William E
    Steiner, Håkan
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Ligand-induced dimerization of Drosophila peptidoglycan recognition proteins in vitro2005In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 102, no 18, p. 6455-6460Article in journal (Refereed)
    Abstract [en]

    Drosophila knockout mutants have placed peptidoglycan recognition proteins (PGRPs) in the two major pathways controlling immune gene expression. We now examine PGRP affinities for peptidoglycan. PGRP-SA and PGRP-LCx are bona fide pattern recognition receptors, and PGRP-SA, the peptidoglycan receptor of the Toll/Dif pathway, has selective affinity for different peptidoglycans. PGRP-LCx, the default peptidoglycan receptor of the Imd/Relish pathway, has strong affinity for all polymeric peptidoglycans tested and for monomeric peptidoglycan. PGRP-LCa does not have affinity for polymeric or monomeric peptidoglycan. Instead, PGRP-LCa can form heterodimers with LCx when the latter is bound to monomeric peptidoglycan. Hence, PGRP-LCa can be said to function as an adaptor, thus adding a new function to a member of the PGRP family.

  • 285. Mellroth, Peter
    et al.
    Karlsson, Jenny
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Steiner, Hakan
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    A scavenger function for a Drosophila peptidoglycan recognition protein2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 9, p. 7059-7064Article in journal (Refereed)
    Abstract [en]

    Recent studies of peptidoglycan recognition protein (PGRP) have shown that 2 of the 13 Drosophila PGRP genes encode proteins that function as receptors mediating immune responses to bacteria. We show here that another member, PGRP-SC1B, has a totally different function because it has enzymatic activity and thereby can degrade peptidoglycan. A mass spectrometric analysis of the cleavage products demonstrates that the enzyme hydrolyzes the lactylamide bond between the glycan strand and the cross-linking peptides. This result assigns the protein as anN-acetylmuramoyl-l-alanine amidase (EC3.5.1.28), and the corresponding gene is thus the first of this class to be described from a eukaryotic organism. Mutant forms of PGRP-SC1B lacking a potential zinc ligand are enzymatically inactive but retain their peptidoglycan affinity. The immunostimulatory properties of PGRP-SC1B-degraded peptidoglycan are much reduced. This is in striking contrast to lysozyme-digested peptidoglycan, which retains most of its elicitor activity. This points toward a scavenger function for PGRP-SC1B. Furthermore, a sequence homology comparison with phage T7 lysozyme, also an N-acetylmuramoyl-l-alanine amidase, shows that as many as six of the Drosophila PGRPs could belong to this class of proteins.

  • 286.
    Mellroth, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Steiner, Håkan
    PGRP-SB1 - an N-acetylmuramoyl L-alanine amidase with antibacterial activityManuscript (Other academic)
  • 287. Mohammadi-Bardbori, Afshin
    et al.
    Bengtsson, Johanna
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Rannug, Ulf
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Rannug, Agneta
    Wincent, Emma
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Quercetin, Resveratrol, and Curcumin Are Indirect Activators of the Aryl Hydrocarbon Receptor (AHR)2012In: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 25, no 9, p. 1878-1884Article in journal (Refereed)
    Abstract [en]

    Several polyphenols have been shown to activate the aryl hydrocarbon receptor (AHR) in spite of the fact that they bind to the receptor with low affinity. The aim of this study was to investigate whether quercetin (QUE), resveratrol (RES), and curcumin (CUR) interfere with the metabolic degradation of the suggested endogenous AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ) and thereby indirectly activate the AHR. Using recombinant human enzyme, we confirmed earlier reported inhibitory effects of the polyphenols on cytochrome P4501A1 (CYP1A1) activity, and inhibition of metabolic clearance of FICZ was documented in FICZ-treated immortalized human keratinocytes (HaCaT). CYP1A1 activity was induced in HaCaT cells by all three compounds, and when they were added together with FICZ, a prolonged activation was observed after a dose-dependent inhibition period. The same pattern of responses was seen at the transcriptional level as determined with a CYP1A1 reporter assay in human liver hepatoma (HepG2) cells. To test the ability of the polyphenols to activate the AHR in the absence of FICZ, the cells were treated in medium, which in contrast to commercial batches of medium did not contain background levels of FICZ. Importantly, AHR activation was only observed in the commercial medium. Taken together, these findings suggest that QUE, RES, and CUR induce CYP1A1 in an indirect manner by inhibiting the metabolic turnover of FICZ. Humans are exposed to these compounds through the diet and nutritional supplements, and we propose that altered systemic levels of FICZ caused by such compounds may have physiological consequences.

  • 288. Mosig, Gisela
    et al.
    Yu, Sidney
    Myung, Heejoon
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Davenport, Laura
    Carlson, Karin
    Calendar, Richard
    A novel mechanism of virus-virus interactions: bacteriophage P2 Tin protein inhibits phage T4 DNA synthesis by poisoning the T4 single-stranded DNA binding protein, gp32.1997In: Virology, ISSN 0042-6822, Vol. 230, no 1, p. 72-81Article in journal (Other academic)
  • 289. Moskwa, Patryk
    et al.
    Buffa, Francesca M.
    Pan, Yunfeng
    Panchakshari, Rohit
    Gottipati, Ponnari
    Muschel, Ruth J.
    Beech, John
    Kulshrestha, Ritu
    Abdelmohsen, Kotb
    Weinstock, David M.
    Gorospe, Myriam
    Harris, Adrian L.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Chowdhury, Dipanjan
    miR-182-Mediated Downregulation of BRCA1 Impacts DNA Repair and Sensitivity to PARP Inhibitors2011In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 41, no 2, p. 210-220Article in journal (Refereed)
    Abstract [en]

    Expression of BRCA1 is commonly decreased in sporadic breast tumors, and this correlates with poor prognosis of breast cancer patients. Here we show that BRCA1 transcripts are selectively enriched in the Argonaute/miR-182 complex and miR-182 downregulates BRCA1 expression. Antagonizing miR-182 enhances BRCA1 protein levels and protects them from IR-induced cell death, while overexpressing miR-182 reduces BRCA1 protein, impairs homologous recombination-mediated repair, and render cells hypersensitive to IR. The impaired DNA repair phenotype induced by mill 182 overexpression can be fully rescued by overexpressing miR-182-insensitive BRCA1. Consistent with a BRCA1-deficiency phenotype, miR-182-over-expressing breast tumor cells are hypersensitive to inhibitors of poly (ADP-ribose) polymerase 1 (PARP1). Conversely, antagonizing miR-182 enhances BRCA1 levels and induces resistance to PARP1 inhibitor. Finally, a clinical-grade PARP1 inhibitor impacts outgrowth of miR-182-expressing tumors in animal models. Together these results suggest that miR-182-mediated downregulation of BRCA1 impedes DNA repair and may impact breast cancer therapy.

  • 290.
    Mottagui-Tabar, Salim
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The influences of 5' codon context on translation termination1997Doctoral thesis, comprehensive summary (Other academic)
  • 291. Natarajan, Adayapalam T.
    et al.
    Palitti, Fabrizio
    Hill, Mark A.
    Stevens, David L.
    Ahnström, Gunnar
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Influence of DMSO on Carbon K ultrasoft X-rays induced chromosome aberrations in V79 Chinese hamster cells2010In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 691, no 02-jan, p. 23-26Article in journal (Refereed)
    Abstract [en]

    Ultrasoft X-rays have been shown to be very efficient in inducing chromosomal aberrations in mammalian cells. The present study was aimed to evaluate the modifying effects of DMSO (a potent scavenger of free radicals) on the frequencies of chromosome aberrations induced by soft X-rays. Confluent held G1 Chinese hamster cells (V79) were irradiated with Carbon K ultrasoft X-rays in the presence and absence of 1 M DMSO and frequencies of chromosome aberrations in the first division cells were determined. DMSO reduced the frequencies of exchange types of aberrations (dicentrics and centric rings) by a factor of 2.1-3.5. The results indicate that free radicals induced by ultrasoft X-rays contribute to a great extent to the induction of chromosome aberrations. The possible implications of these results in interpreting the mechanisms involved in the high efficiency of ultrasoft X-rays in the induction of chromosome aberrations are discussed.

  • 292.
    Nilsson, Anders
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Evolutionary patterns and processes of bacteriophages, particularly P2-like phages2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    DNA sequencing of structural genes of temperate P2-like prophages from isolates of Escherichia coli bacteria showed that the difference between these genes was very small. Separate phylogenetic analyses on the genes resulted in unresolved trees, caused by a high level of homologous recombination, which implied that homologous recombination is the most important source of new genetic variation for P2-like phages infecting E. coli. The genomes of seven P2-like phages from different bacterial host families were also compared in phylogenetic analyses, that were based on the amino acid sequences of nine structural genes, the gene content of the genome, and the gene order. These analyses resulted in only one tree with high support for all branches in all analyses with one exception. It was shown that some of the genes in phage 186 had been transferred between genomes in separate branches. The results of the analyses on P2-like phages from E. coli were in sharp contrast to the results of analyses of P2-like phages from different hosts. This implies that P2-like phages from the same host belong to clonal lines that occasionally meet in the same host cell and that compete for the same resources, whereas P2-like phages from different hosts constitute discrete and separate clusters of clones that track the evolution of respective hosts. The evolutionary pattern in P2-like phages was compared to the pattern in the temperate phage P1. Phage P1 is structurally similar to P2 and use the same host, but it has genes with additional functions, similar to the genes of the host. The results from experiments of host bacteria infected with P1 with and without one of these additional genes, the ssb gene, showed that it was nonessential for the host, but a selective advantage for the phage since reproduction is guaranteed even if the host is dying. Phylogenetic analyses showed that phage P1 have had an ssb gene for a long time and that the gene has not been transferred from any known bacteria or plasmid. P2-like prophages are present in about 28% of E. coli isolates. The genetic variation among these strains seems to be low. The region corresponding to the P2 Z/fun locus was showed to contain DNA of different length at this site in many prophages. The sequences consisted of a central, highly variable AT-rich part, flanked by almost identical upstream and downstream sequences that were inverted repeats. The explanation to the diversity of genes at this site was that a site-specific recombination mechanism was acting on these sequences. The central part contained at least one open reading frame per insert, coding for proteins that in only a few cases were found in other organisms. The pattern was also found in genetically unstable regions of pathogenic bacteria. In view of all investigations, the phylogeny of P2-like phages could be described as a tree with reticulated branches. Each branch is made up by bundles of clonal lines that exchange genetic material through homologous recombination. Occasionally there is a transfer of genes between branches, but this has probably not happened many times in the last 100 million years. Each clonal line is mainly defined by the lysogenic conversion genes that it harbours, and by the ability to repress other clonal lines, but has also differentiated by selectively neutral changes in the nucleotide sequence.

  • 293.
    Nilsson, Anders S
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Detection of homologous recombination among bacteriophage P2 relatives.2001In: Mol Phylogenet Evol, ISSN 1055-7903, Vol. 21, no 2, p. 259-69Article in journal (Other academic)
  • 294.
    Nilsson, Anders S
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Evolution of P2-like phages and their impact on bacterial evolution.2007In: Res Microbiol, ISSN 0923-2508, Vol. 158, no 4, p. 311-7Article in journal (Other academic)
  • 295.
    Nilsson, Anders S
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The P2-like bacteriophages2006In: The Bacteriophages / [ed] Richard Calendar, New York: Oxford University Press, 2006, 2, p. 365-390Chapter in book (Refereed)
  • 296.
    Nilsson, Anders S
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The P2-like bacteriophages2006In: The Bacteriophages, Oxford University Press , 2006Chapter in book (Other academic)
  • 297.
    Nilsson, Anders S
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Karlsson, Joakim L
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Site-specific recombination links the evolution of P2-like coliphages and pathogenic enterobacteria.2004In: Mol Biol Evol, ISSN 0737-4038, Vol. 21, no 1, p. 1-13Article in journal (Other academic)
  • 298. Nilsson, Charlotta
    et al.
    Jönsson, Ingemar
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. Kristianstad University, Sweden.
    Pallon, Jan
    Element analysis of the eutardigrades Richtersius coronifer and Milnesium cf. asiaticum using particle induced X-ray emission (PIXE)2013In: Journal of limnology, ISSN 1129-5767, E-ISSN 1723-8633, Vol. 72, p. 92-101Article in journal (Refereed)
    Abstract [en]

    Semi-terrestrial tardigrades are well-known for their tolerance to a variety of environmental extremes, including desiccation, freezing and radiation. Despite several attempts to reveal the genetic and molecular mechanisms behind the resilience of tardigrades, it is still unknown how these animals are able to maintain the integrity of their cellular components under severe stress. Quantitative or qualitative changes in molecular compounds (e.g., carbohydrates, proteins) are expected, and have been the main line of research towards understanding the tolerance of tardigrades. In radiation tolerant bacteria, a tolerance mechanism based on manganese has been proposed. We evaluate this hypothesis in tardigrades and provide the first data on element composition in desiccated and non-desiccated specimens of two eutardigrade species, Richtersius coronifer and Milnesium cf. asiaticum. A focused 2 MeV proton microbeam was utilised to determine the elemental content, distributions and concentrations, using the ion beam analytical technique particle induced X-ray emission (PIXE). The presence of six elements - phosphorus, sulphur, chlorine, potassium, calcium and iron - were confirmed in all tardigrade specimens, at levels up to a few mg g(-1). However, manganese was found in less than 10% of the analysed specimens, and in low amounts, thus our study provides no evidence for the manganese hypothesis. We also show that the distributions and/or concentrations of some elements differ between the two species as well as between the dehydrated and hydrated state. In particular, very low levels of iron were found in dehydrated M. cf. asiaticum. Our analysis shows that the PIXE technique is a useful tool for investigating questions on the distribution of elements both in dehydrated and hydrated tardigrades.

  • 299. Nilsson, E. J. Charlotta
    et al.
    Jonsson, K. Ingemar
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Pallon, Jan
    Tolerance to proton irradiation in the eutardigrade Richtersius coronifer - a nuclear microprobe study2010In: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, Vol. 86, no 5, p. 420-427Article in journal (Refereed)
    Abstract [en]

    Materials and methods: Dehydrated tardigrades of the species R. coronifer were irradiated with 2.55 MeV (megaelectronvolts) protons at doses ranging from 500 gray (Gy) to 15,000 Gy, to investigate the dose-viability relationship. In addition, a focused proton microbeam was utilised to determine the areal mass distribution, using the ion beam analytical technique STIM (Scanning Transmission Ion Microscopy). Results: The experiment suggests that R. coronifer is unaffected by doses of proton irradiation up to 10,000 Gy, but shows very little viability at higher doses. The STIM analysis revealed that the thickness of the dehydrated tardigrades exceeds 150 mu m, and that a fraction of the protons may not be fully absorbed. Conclusion: Our results are in line with previous studies of exposure to high-LET radiation in tardigrades, indicating that these animals are equally or even more tolerant to high-LET compared to low-LET gamma radiation. The physiological background to this remarkable result is currently unknown, but deserves investigation.

  • 300.
    Nilsson, Hanna
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    A Genetic Switch in Bacteriophages within the Peduovirinae Subfamily: Structure, Function and Evolution2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The temperate bacteriophages in the Peduovirinae subfamily can either grow lytically or integrate into their bacterial host and form lysogeny. Which one of the two life cycles the phage will enter after infection is controlled by a transcriptional switch. The switch also controls the induction of genes necessary for an integrated phage, a prophage, to excise out of the host genome and propagate lytically. In its most simple form, the transcriptional switch consists of two proteins repressing each other’s promoters, which are oriented face to face in close proximity.

    The Peduovirinae phages contain two types of transcriptional switches. They were studied with phylogenetic methods to determine their evolution and distribution. Bioinformatic analyses showed that there were several new E. coli integration sites and new inferred immunity classes among the Peduovirinae phages. The two switch types fell into two distinct groups, with no overlap in any of the proteins, but these groups were not defined by host barriers. But in vivo distribution did show a host preference.

    The P2 C protein was crystallized and its 3D structure determined. It forms a symmetrical dimer in vitro, with an unstructured C-terminal end. The DNA binding domain was determined to lie in alpha helix three and narrowed down to three residues. The C terminal end of the protein is suggested to be part of tetramerization, but a nine amino acid truncation does not affect activity in vitro.

    In an attempt to discover the mechanism between the switch from lysogeny to lysis in phage P2 the interactions between the two switch proteins and the proteins of its host E. coli was analyzed. Eight E. coli proteins interacted with protein C or Cox, but no interaction between the two switch proteins was detected. Two E. coli proteins showed a distinct effect on the expression of C, and several affected the level of phage lysis. The mechanisms behind these effects are still unclear.

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