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  • 401.
    Stoimenov, Ivaylo
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Gottipati, Ponnari
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Transcription Inhibition by DRB Potentiates Recombinational Repair of UV Lesions in Mammalian Cells2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 5, p. e19492-Article in journal (Refereed)
    Abstract [en]

    Homologous recombination (HR) is intricately associated with replication, transcription and DNA repair in all organisms studied. However, the interplay between all these processes occurring simultaneously on the same DNA molecule is still poorly understood. Here, we study the interplay between transcription and HR during ultraviolet light (UV)-induced DNA damage in mammalian cells. Our results show that inhibition of transcription with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) increases the number of UV-induced DNA lesions (gamma H2AX, 53BP1 foci formation), which correlates with a decrease in the survival of wild type or nucleotide excision repair defective cells. Furthermore, we observe an increase in RAD51 foci formation, suggesting HR is triggered in response to an increase in UV-induced DSBs, while inhibiting transcription. Unexpectedly, we observe that DRB fails to sensitise HR defective cells to UV treatment. Thus, increased RAD51 foci formation correlates with increased cell death, suggesting the existence of a futile HR repair of UV-induced DSBs which is linked to transcription inhibition.

  • 402.
    Stoimenov, Ivaylo
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Gottipati, Ponnari
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Transcription inhibition by DRB potentiates recombinational repair of UVC lesions in mammalian cellsManuscript (preprint) (Other academic)
  • 403.
    Ström, Cecilia
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Post-translational modifications in DNA base excision repair: The roles of CK2 and PARP-12011Doctoral thesis, comprehensive summary (Other academic)
    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.

  • 404.
    Ström, Cecilia
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Post-translational modifications of XRCC1 by the protein kinase CK22009Licentiate thesis, monograph (Other academic)
  • 405.
    Ström, Cecilia E.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Uhlén, Mathias
    Al-Khalili Szigyarto, Cristina
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 8, p. 3166-3175Article in journal (Refereed)
    Abstract [en]

    Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.

  • 406.
    Ström, Cecilia E.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Mortusewicz, Oliver
    Finch, David
    Parsons, Jason L.
    Lagerqvist, Anne
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Dianov, Grigory L.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    CK2 phosphorylation of XRCC1 facilitates dissociation from DNA and single-strand break formation during base excision repair2011In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 10, no 9, p. 961-969Article in journal (Refereed)
    Abstract [en]

    CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1(ckm)) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the allcylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1(ckm) (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1(ckm) protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1(ckm) and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting.

  • 407.
    Ström, Cecilia
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lagerqvist, Anne
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Finch, David
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Dianov, Grigory
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    CK2 phosphorylation of XRCC1 facilitate single-strand break formation during base excision repairManuscript (preprint) (Other academic)
    Abstract [en]

    Casein kinase 2 (CK2) phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express a XRCC1 protein (XRCC1ckm) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification in SSB repair is distinct from its role in base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1ckm (CKM cells) or following inhibition with CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1ckm protein has a higher affinity for nicked DNA substrate than wild type XRCC1 protein and we propose a model whereby the increased affinity for DNA sequesters XRCC1ckm and the repair enzymes associated with it, at the repair site. In conclusion, our results indicate that CK2-phosphorylations of XRCC1 affect the kinetics of SSB repair and BER differentially, and that the modifications on XRCC1 facilitate the BER incision step.

  • 408. Sun, Jiao
    et al.
    Lou, Xudan
    Wang, Haidong
    Sollazzo, Alice
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Harms-Ringdahl, Mats
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Skog, Sven
    He, Ellen
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Serum 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) levels are elevated in diabetes patients2015In: International Journal of Diabetes in Developing Countries, ISSN 0973-3930, E-ISSN 1998-3832, Vol. 35, no 3, p. 368-373Article in journal (Refereed)
    Abstract [en]

    The increased oxidative stress in diabetes is known to contribute to the development of diabetes. We investigate whether serum 8-hydro-2'-deoxyguanosine (8-oxo-dG) is associated with diabetes at the time of first diagnosis and evaluate whether it can be used as a reliable biomarker for the oxidative stress in diabetes. The study was designed as a case control study with two groups: patient with diabetes and control. The diabetes group consisted of a total of 28 patients consulting the hospital for the first time and definitely diagnosed for diabetes, and the control group was composed of 65 healthy subjects. Serum 8-oxo-dG was measured by a competitive enzyme-linked immunosorbent assay (ELISA) kit, specially developed to minimize cross-reaction of 8-oxo-dG antibody with serum guanosine. The average serum 8-oxo-dG levels in patients with diabetes and controls were 0.72 +/- 0.41 and 0.24 +/- 0.14 ng/mL, respectively, statistically significant (p < 0.001). The 8-oxo-dG value was significantly higher in women with diabetes, compared with men with diabetes (p = 0.028). The sensitivity and the specificity of the 8-oxo-dG ELISA assay were 0.80 and 0.96, respectively, and the ROC value was 0.93. This study suggests that increased oxidative stress has an important role in the pathogenesis of diabetes. Serum 8-oxo-dG may be a useful clinical biomarker for the early diagnosis of stress-related diseases, e.g. diabetes and its management.

  • 409.
    Surkov, Serhiy
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Studies on Translation Initiation and trans-Translation2010Doctoral thesis, comprehensive summary (Other academic)
    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.

  • 410.
    Surkov, Serhiy
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Isak, Georgina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Isaksson, Leif
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Influences of a mutated translation initiation factor IF1 or kasugamycin on  Escherichia coli gene expressionManuscript (preprint) (Other academic)
    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.

  • 411.
    Surkov, Serhiy
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Nilsson, Hanna
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Rasmussen, Louise CV
    Department of Molecular Biology, Aarhus university.
    Sperling-Petersen, Hans U
    Department of Molecular Biology, Aarhus university.
    Isaksson, Leif
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Translation initiation region dependency of translation initiation in Escherichia coli by IF1 and kasugamycin2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 11, p. 2428-2439Article in journal (Refereed)
    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.

  • 412.
    Svensson, Linda M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jemth, Ann-Sofie
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Desroses, Matthieu
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Loseva, Olga
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    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 human MTH1 and the 8-oxo-dGMP product complex2011In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 585, no 16, p. 2617-2621Article in journal (Refereed)
    Abstract [en]

    MTH1 hydrolyzes oxidized nucleotide triphosphates, thereby preventing them from being incorporated into DNA. We here present the structures of human MTH1 (1.9 angstrom) and its complex with the product 8-oxo-dGMP (1.8 angstrom). Unexpectedly MTH1 binds the nucleotide in the anti conformation with no direct interaction between the 8-oxo group and the protein. We suggest that the specificity depends on the stabilization of an enol tautomer of the 8-oxo form of dGTP. The binding of the product induces no major structural changes. The structures reveal the mode of nucleotide binding in MTH1 and provide the structural basis for inhibitor design.

    Download full text (pdf)
    fulltext
  • 413.
    Sylwan, Lina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Site-Specific Recombination: Integrases, Accessory Factors and DNA Targets of P2-like Coliphages2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The temperate coliphage P2 and its family members integrate their genomes into the host Escherichia coli chromosome by a site-specific recombination mechanism to form lysogeny. Integration takes place between the complex phage attP site and the simple bacterial attB site and is catalyzed by the phage encoded integrase (Int). Similar to the archetype λ Int, the P2-like phage integrases are heterobivalent tyrosine recombinases which possess the ability to simultaneously bind two different and distant types of DNA sequences within the attP region. To bridge the core and the flanking arm-binding sites in attP, the integrase requires the assistance of accessory factors that bend the DNA; the host encoded IHF and the phage encoded Cox protein. Cox acts as a directionality factor by being required for integration but is inhibitory for the excisive reaction.

    The purpose of this doctoral thesis has been to gain a more detailed knowledge of the site-specific recombination systems of phages P2 and WΦ, which are close relatives but integrate into different host targets. The future aim is to develop these systems for targeted integration into the genome of higher eukaryotes.

    The P2 Int and an N-terminal truncation of the integrase were shown to bind cooperatively together with IHF or Cox to the DNA targets, however the N-truncated protein lost its ability to bind to the arm sequence. WΦ Cox was shown to bind cooperatively with WΦ Int to attP whereas the opposite was evident for WΦ Cox and IHF. The 27 nucleotides that are identical between the core and attB of phage P2 were investigated for their importance in binding and recombination. The right part of the core was shown to be the primary Int binding site where one single base substitution was shown to abolish P2 Int binding and recombination. An alanine scanning of the two predicted alpha-helices in the presumed core-binding domain of P2 Int was carried out in order to identify amino acids involved in binding to the core. An in vivo excisive assay and an in vivo integrative assay were used resulting in the identification of four amino acids as candidates for core-binding. The fact that the recombination reaction shows directionality renders the site-specific recombination systems of the P2-like phages attractive to develop as tools for safe and efficient non-viral gene delivery in humans. The wild-type P2 integrase was shown to accept a human attB sequence and localizes to the nucleus in human cell lines.

    The work presented in this thesis has increased our understanding of the site-specific recombination systems of the phages P2 and WΦ and provides a basis for further characterization and development for future use in a eukaryotic context.

  • 414.
    Sylwan, Lina
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Frumerie, Clara
    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.
    Identification of bases required for P2 integrase core binding and recombination2010In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 404, p. 240-245Article in journal (Refereed)
    Abstract [en]

    Temperate coliphage P2 integrates its genome into the host chromosome upon lysogenization via a site-specific recombination event mediated by an integrase belonging to the complex family of tyrosine recombinases. The host integration site attB (BOB′) is localized in the end of the cyaR gene and shares 27 nucleotides with the core of attP (COC′). In the present study we determine the minimal attB site using an in vivo recombination assay. Ten nt on the left side (B) are found to be nonessential for recombination. We show that the integrase has higher affinity for the right side (B′) compared to B and that artificial B′OB′ and an attP site with a matching core (C′OC′) are efficient substrates for recombination in vitro. We have analyzed single nucleotides in attB and find that sequence homology within a non-centrally located quadruplet in the hypothetical overlap region is essential for efficient recombination in vivo.

  • 415.
    Sylwan, Lina
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Frumerie, Clara
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Identification of bases required for P2 Integrase core-binding and recombinationManuscript (preprint) (Other academic)
    Abstract [en]

    Temperate coliphage P2 integrates its genome into the host chromosome upon lysogenization via a site-specific recombination event mediated by an integrase belonging to the complex family of tyrosine recombinases. The host integration site attB (BOB´) is localized in the end of the cyaR gene and share 27 nucleotides with the core of attP (COC´). In the present study we determine the minimal attB site using an in vivo recombination assay and 10 nt on the left side (B) are found to be nonessential for recombination. We show that the integrase has higher affinity for the right side (B´) compared to B and that artificial B´OB´ and an attP site with a matching core (C´OC´) are efficient substrates for recombination in vitro. We have analyzed single nucleotides in attB and find that sequence homology within a non-centrally located quadruplet in the hypotetical overlap region is essential for efficient recombination in vivo.

  • 416.
    Söderholm, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Molecular interactions between pathogenic Neisseria and polymophonuclear leukocytes2011Licentiate thesis, monograph (Other academic)
  • 417.
    Söderholm, Niklas
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Vielfort, Katarina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hultenby, Kjell
    Aro, Helena
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Pathogenic Neisseria Hitchhike on the Uropod of Human Neutrophils2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, p. e24353-Article in journal (Refereed)
    Abstract [en]

    Polymorphonuclear neutrophils (PMNs) are important components of the human innate immune system and are rapidly recruited at the site of bacterial infection. Despite the effective phagocytic activity of PMNs, Neisseria gonorrhoeae infections are characterized by high survival within PMNs. We reveal a novel type IV pilus-mediated adherence of pathogenic Neisseria to the uropod (the rear) of polarized PMNs. The direct pilus-uropod interaction was visualized by scanning electron microscopy and total internal reflection fluorescence (TIRF) microscopy. We showed that N. meningitidis adhesion to the PMN uropod depended on both pilus-associated proteins PilC1 and PilC2, while N. gonorrhoeae adhesion did not. Bacterial adhesion elicited accumulation of the complement regulator CD46, but not I-domain-containing integrins, beneath the adherent bacterial microcolony. Electrographs and live-cell imaging of PMNs suggested that bacterial adherence to the uropod is followed by internalization into PMNs via the uropod. We also present data showing that pathogenic Neisseria can hitchhike on PMNs to hide from their phagocytic activity as well as to facilitate the spread of the pathogen through the epithelial cell layer. 

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    Söderholm_Aro 2011
  • 418.
    Söderpalm-Berndes, Cecilia
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Does acetylcholin play a role in mitosis of V79 Chinese hamster cells?1997Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The first stages of mitosis are governed by a cascade of phosphorylations brought about by the universal MPF kinase complex. From a regulatory point of view, less is known about late mitotic events. The present study demonstrates that, firstly, V79 Chinese hamster cells produce acetylcholine and, secondly, that a set of acetylcholine receptor ligands can affect progression throughout mitosis.

    This was suggested using different approaches. Carbaryl and a-naphthol were used to induce a particular mitotic population consisting of cells with dislocated, unseparated chromosomes. Five distinct cell types can be scored, each representing different mitotic stages. Acetylcholine, as well as other acetylcholine receptor ligands, were found to change the frequencies of the five cell types, implying that mitotic progression be altered.

    Mitotic cell elongation was studied in more detail, using the experimental system with carbaryl and a-naphthol. The results indicate that cell elongation likely depends on actin filament formation and regulatory proteins functionally coupled to actin, as well as a maintained energy status. Also, some acetylcholine receptor might be involved in this cytoskeletal rearrangement. This was suggested by the finding that the acetylcholine analogue carbachol speeds up the formation of elongated cells and, in addition, that two acetylcholine receptor antagonists can decrease the frequency of elongated cells. Recording cells with a video time-lapse equipment demonstrated that acetylcholine receptor ligands can accelerate or decelerate progression through metaphase as well as anaphase, in line with the previous findings.

    Three different approaches were used to assess and evaluate the effects of acetylcholine receptor ligands on cell cleavage. Firstly, in the experimental system with carbaryl or a-naphthol complex concentration response curves were obtained for cleavage. Secondly, binuclear cells were induced by a two-hour treatment with five different acetylcholine receptor antagonists. Thirdly, video time-lapse recordings demonstrated that two acetylcholine receptor agonists, carbachol and nicotine, accelerates onset and progression of cleavage, respectively.

    Since the tested ligands affect different parts of mitosis it might be that there are a number of acetylcholine receptor types or affinity states which elicit signals in a time sequence. The role of acetylcholine could be to fine-tune mitotic progression.

  • 419.
    Tange Olsen, Morten
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Evolutionary Genetics of Marine Mammals.2010Licentiate thesis, monograph (Other academic)
  • 420.
    Tekle, Michael
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gromadzinska, Jolanta
    Joksic, Gordana
    Antic, Ruza
    Nilsson, Robert
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Dallner, Gustav
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Undén, Anna-Lena
    Brismar, Kerstin
    Plasma levels of insulin-like growth factor-I, insulin-like growth factor binding protein-1, coenzyme Q10 and vitamin E in female populations from Poland, Serbia and Sweden2010In: Environment International, ISSN 0160-4120, E-ISSN 1873-6750, Vol. 36, no 2, p. 188-194Article in journal (Refereed)
    Abstract [en]

    Exposure to environmental contaminants such as polycyclic aromatic hydrocarbons (PAHs), life style and nutritional status of a population are important factors that may influence normal serum levels of antioxidants and the insulin-like growth factor system. In this study we examined serum levels of insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-1 (IGFBP-1), coenzyme Q10 (CoQ) and vitamin E in healthy female populations (n = 4 x 100) aged 19-59 years from Poland (PL), Sweden (SE), Serbia I (SR I) and Serbia II (SR II). The last group lived in an environmental emergency area affected by the bombings of 1999 in Serbia. The Polish and SR I cohorts exhibited low IGFSD-score levels, (-2 to +/-0), compared to females from SE with IGFSD-score 0. In the SIR II population, the IGFSD range was between -1 and 1. The IGFBP-1 levels of the Polish and SIR I groups were lower than in the Swedish population, while the SR II levels showed a broader distribution, 20-80 mu g/l. The CoQ values in the Swedish and Polish samples were around 1 nmol/ml. In contrast. the SIR I cohorts exhibited higher concentrations, 1.5-3.5 nmol/ml and the SIR II group had extremely low levels, <0.5 nmol/ml. The vitamin E concentrations were similar in the Polish and Swedish populations, 20-40 nmol/ml, while it was twice as high, 40-80 nmol/ml in the SR I and very low in the SIR II group, which is half of the Polish and Swedish cohorts. These results suggest that different lifestyles and environmental factors affect both the IGF system and the antioxidants CoQ10 and vitamin E in female populations in Europe. The females living in the polluted area had different patterns of both the IGF and antioxidant systems. These findings may explain differences in morbidity and mortality in these countries.

  • 421.
    Terenius, Olle
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Anti-parasitic and anti-viral immune responses in insects2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Insects encounter many microorganisms in nature and to survive they have developed counter measures against the invading pathogens. In Drosophila melanogaster research on insect immunity has mainly been focused on infections by bacteria and fungi. We have explored the immune response against natural infections of the parasite Octosporea muscaedomesticae and the Drosophila C virus as compared to natural infections of bacteria and fungi. By using Affymetrix Drosophila GeneChips, we were able to obtain 48 genes uniquely induced after parasitic infection. It was also clearly shown that natural infections led to different results than when injecting the pathogens.

    In order to search for the ultimate role of the lepidopteran protein hemolin, we used RNA interference (RNAi). We could show that injection of double stranded RNA (dsRNA) of Hemolin in pupae of Hyalophora cecropia led to embryonic malformation and lethality and that there was a sex specific difference. We continued the RNAi investigation of hemolin in another lepidopteran species, Antheraea pernyi, and discovered that hemolin was induced by dsRNA per se. A similar induction of hemolin was seen after infection with baculovirus and we therefore performed in vivo experiments on baculovirus infected pupae. We could show that a low dose of dsHemolin prolonged the period before the A. pernyi pupae showed any symptoms of infection, while a high dose led to a more rapid onset of symptoms. By performing in silico analysis of the hemolin sequence from A. pernyi in comparison with other Hemolin sequences, it was possible to select a number of sites that either by being strongly conserved or variable could be important targets for future studies of hemolin function.

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  • 422.
    Terenius, Olle
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bettencourt, Raul
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lee, So Young
    Li, Wenli
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Soderhall, Kenneth
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    RNA interference of Hemolin causes depletion of phenoloxidase activity in Hyalophora cecropia2007In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 31, no 6, p. 571-575Article in journal (Refereed)
    Abstract [en]

    Melanization is regulated by the prophenoloxidase cascade and functions as a response to intruding microorganisms in invertebrates. When injecting dsRNA of the lepidopteran immune protein hemolin in pupae of Hyalophora cecropia (Lepidoptera: Saturniidae), we observed a significant reduction in phenoloxidase activity after 24 h, but not after 72 h. The link between hemolin and the prophenoloxidase system suggests that hemolin is a pattern recognition protein important for the triggering of the prophenoloxidase cascade in the defence against bacterial infections. 

  • 423.
    Terenius, Olle
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lindh, Jenny M.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Eriksson-Gonzales, Karolina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bussiere, Luc
    Laugen, Ane T.
    Bergquist, Helen
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Titanji, Kehmia
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Midgut bacterial dynamics in aedes aegypti2012In: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 80, no 3, p. 556-565Article in journal (Refereed)
    Abstract [en]

    In vector mosquitoes, the presence of midgut bacteria may affect the ability to transmit pathogens. We have used a laboratory colony of Aedes aegypti as a model for bacterial interspecies competition and show that after a blood meal, the number of species (culturable on LuriaBertani agar) that coexist in the midgut is low and that about 40% of the females do not harbor any cultivable bacteria. We isolated species belonging to the genera Bacillus, Elizabethkingia, Enterococcus, Klebsiella, Pantoea, Serratia, and Sphingomonas, and we also determined their growth rates, antibiotic resistance, and ex vivo inhibition of each other. To investigate the possible existence of coadaptation between midgut bacteria and their host, we fed Ae.similar to aegypti cohorts with gut bacteria from human, a frog, and two mosquito species and followed the bacterial population growth over time. The dynamics of the different species suggests coadaptation between host and bacteria, and interestingly, we found that Pantoea stewartii isolated from Ae.similar to aegypti survive better in Ae.similar to aegypti as compared to P.similar to stewartii isolated from the malaria mosquito Anopheles gambiae.

  • 424. Terenius, Olle
    et al.
    Papanicolaou, Alexie
    Garbutt, Jennie S.
    Eleftherianos, Ioannis
    Huvenne, Hanneke
    Kanginakudru, Sriramana
    Albrechtsen, Merete
    An, Chunju
    Aymeric, Jean-Luc
    Barthel, Andrea
    Bebas, Piotr
    Bitra, Kavita
    Bravo, Alejandra
    Chevalier, François
    Collinge, Derek P.
    Crava, Cristina M.
    de Maagd, Ruud A.
    Duvic, Bernard
    Erlandson, Martin
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Felföldi, Gabriella
    Fujiwara, Haruhiko
    Futahashi, Ryo
    Gandhe, Archana S.
    Gatehouse, Heather S.
    Gatehouse, Laurence N.
    Giebultowicz, Jadwiga M.
    Gómez, Isabel
    Grimmelikhuijzen, Cornelis J. P.
    Groot, Astrid T.
    Hauser, Frank
    Heckel, David G.
    Hegedus, Dwayne D.
    Hrycaj, Steven
    Huang, Lihua
    Hull, J. Joe
    Iatrou, Kostas
    Iga, Masatoshi
    Kanost, Michael R.
    Kotwica, Joanna
    Li, Changyou
    Li, Jianghong
    Liu, Jisheng
    Lundmark, Magnus
    Matsumoto, Shogo
    Meyering-Vos, Martina
    Millichap, Peter J.
    Monteiro, Antónia
    Mrinal, Nirotpal
    Niimi, Teruyuki
    Nowara, Daniela
    Ohnishi, Atsushi
    Oostra, Vicencio
    Ozaki, Katsuhisa
    Papakonstantinou, Maria
    Popadic, Aleksandar
    Rajam, Manchikatla V.
    Saenko, Suzanne
    Simpson, Robert M.
    Soberón, Mario
    Strand, Michael R.
    Tomita, Shuichiro
    Toprak, Umut
    Wang, Ping
    Wee, Choon Wei
    Whyard, Steven
    Zhang, Wenqing
    Nagaraju, Javaregowda
    Ffrench-Constant, Richard H.
    Herrero, Salvador
    Gordon, Karl
    Swevers, Luc
    Smagghe, Guy
    RNA interference in Lepidoptera: An overview of successful and unsuccessful studies and implications for experimental design2011In: Journal of insect physiology, ISSN 0022-1910, E-ISSN 1879-1611, Vol. 57, no 2, p. 231-245Article in journal (Refereed)
    Abstract [en]

    Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.

  • 425. Thiel, Tomas
    et al.
    Ryk, Charlotta
    Chatzakos, Vicky
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Grufman, Katarina Hallen
    Bavand-Chobot, Nasrin
    Flygare, Jenny
    Wiklund, N. Peter
    de Verdier, Petra J.
    Secondary stimulation from Bacillus Calmette-Guerin induced macrophages induce nitric oxide independent cell-death in bladder cancer cells2014In: Cancer Letters, ISSN 0304-3835, E-ISSN 1872-7980, Vol. 348, no 1-2, p. 119-125Article in journal (Refereed)
    Abstract [en]

    The anti-tumour mechanisms following Bacillus Calmette-Guerin (BCG) treatment of bladder-cancer remain largely unknown. Previous studies have shown involvement of nitric-oxide (NO) formation in the BCG-mediated effect. We analyzed the effects of macrophage secreted factors (MSFs) from BCG-stimulated RAW264.7 cells on the bladder-cancer cell line MBT2. Direct treatment with BCG did not induce NO in MBT2-cells whereas supernatant from BCG-stimulated macrophages increased NOS2 mRNA and protein expression, NO concentrations and cell-death. Blocking NO-synthesis with the NOS-inhibitor L-NAME did not affect levels of cell-death suggesting cytotoxic pathways involving other signalling molecules than NO. Several such candidate genes were identified in a microarray.

  • 426.
    Thiel, Tomas
    et al.
    Karolinska Institutet.
    Ryk, Charlotta
    Chatzakos, Vicky
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hallén Grufman, Katarina
    Karolinska Institutet.
    Bavand-Chobot, Nasrin
    Karolinska Institutet.
    Flygare, Jenny
    Karolinska Institutet.
    Wiklund, N. Peter
    Karolinska Institutet.
    De Verdier, Petra J.
    Karolinska Institutet.
    Secondary stimulation from Bacillus Calmette-Guérin induced macrophages upregulatesNOS2 protein in bladder cancer cellsManuscript (preprint) (Other academic)
    Abstract [en]

    Treatment with Bacillus Calmette-Guérin (BCG) bladder instillations is an established treatment modality for superficial urinary bladder cancer and carcinoma in situ (CIS), but the anti-tumor mechanisms following BCG-instillations remain largely unknown. Previous data show increased nitric oxide (NO) concentrations in the urinary bladder from patients treated with BCG suggesting that NO-formation may be involved in the BCG-mediated effect. Using immunohistochemistry we have previously shown nitric oxide synthase 2 (NOS2/iNOS) protein expression in both immune cells and in urothelial cells in bladder cancer patient biopsies. In this study we analysed the influence of macrophage- (RAW 264.7) secreted factors on NO production by stimulating urothelial carcinoma cells (MBT2) with supernatant from BCG-treated macrophages as well as supernatant from untreated macrophages. Using real-time PCR, western blot and chemiluminescence, we found no effect of BCG when added straight to the culture medium of urothelial carcinoma cells. However, when 40% of the culture medium of the bladder cancer cells was substituted with supernatant from BCGstimulated macrophages, we found increased NOS2 mRNA and protein expression as well as increased levels of NO. In addition we found increased cell death only in bladder cancer cells stimulated with supernatant from BCG-treated macrophages, as visualized by cell cycle analysis and PARP cleavage. These results suggest that simultaneous targeting of the microenvironment and subsequent stimulation of adaptive responses can improve conventional BCG-therapy.

  • 427.
    Torudd, J.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Issaeva, N.
    Ström, C.
    Harms-Ringdahl, M.
    Jenssen, D.
    Helleday, T.
    Erixon, K.
    Replication progression is retarded only at very high doses of ionizing radiation independently of check-point signallingIn: Oncogene, ISSN 0950-9232Article in journal (Refereed)
  • 428.
    Torudd, J.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Protopopova, M.
    Sarimov, R.
    Nygren, J.
    Eriksson, S.
    Markova, E.
    Chovanec, M.
    Selivanova, G.
    Belyaev, I. Y.
    Dose-response for radiation-induced apoptosis, residual 53BP1 foci and DNA-loop relaxation in human lymphocytes2005In: International Journal of Radiation Biology, ISSN 0955-3002, Vol. 81, no 2, p. 125-138Article in journal (Refereed)
  • 429.
    Torudd, Jesper
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Induction of apoptosis in relation to chromatin structure and inhibition of replication by DNA damage from ionizing radiation2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The theme of this thesis has been chromatin organization ranging from methodological studies to involvement in apoptotic response. The aims have been: (i) to compare the information obtained by the AVTD method and comet assay concerning DNA-loop organization, (ii) to test the hypothesis that DNA-loop relaxation could be the triggering signal for induction of apoptosis in G0-lymphocytes, and (iii) to study the dose response for inhibition of the replication fork movement and pathways for the DNA repair process at the replication fork. AVTD was evaluated and compared with the established comet assay by studying changes in DNA-loop structure, induced by ethidium bromide. DNA-loops either relaxed or condensed in a dose dependent manner and changes in viscosity correlated with the length of comet tails. The dose response relations for induction of apoptosis in G0-lymphocytes were determined and compared with the dose response relations for relaxation of DNA-loops. Relaxation was shown to saturate at doses of 2-3 Gy after γ-irradiation, a dose in which approximately one SSB per chromatin loop of DNA was induced. Apoptotic markers such as chromatin condensation, p53 stabilization and DNA fragmentation also saturated at 2-3 Gy, suggesting that SSB dependent loop relaxation may trigger apoptosis. Radiation induced inhibition of replication fork movement was studied in proliferating Chinese hamster ovary cells. Doses over 100 Gy were needed to inhibit the fork elongation, as verified by both the ADU and by the DNA combing assay. Checkpoint signalling was shown not to be involved in this retarded elongation. On the other hand, the initiation of replication was sensitive to low doses of ionizing radiation. A dose of 12.5 Gy was enough to stop firing of new replicons and caffeine attenuated this inhibition. By measuring the speed of replication fork progression in repair deficient cell lines we concluded that replication forks are retarded by un-repaired DSBs, SSBs and/or base lesions.

  • 430.
    Urbin, Salustra
    et al.
    Lawrence Livermore Natl. Laboratory, Biosciences and Biotechnology Division.
    Elvers, Ingegerd
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hinz, John
    Washington State University, School of Molecular biosciences.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Thompson, Larry
    Lawrence Livermore Natl. Laboratory, Biosciences and Biotechnoogy Division.
    Uncoupling of RAD51 focus formation and cell survival after replication fork stalling in RAD51D null CHO cellsArticle in journal (Refereed)
    Abstract [en]

    In vertebrate cells the five RAD51 paralogs (XRCC2/3, RAD51B/C/D) enhance the efficiency of homologous reocmbination repair (HRR). Stalling and breakage of DNA replication forks is a common event in the large genomes of higher eukaryotes. When cells are exposed to agents that arrest DNA replicaiton, such as hydroxyurea or aphidicolin, fork breakage can lead to chromosomal aberrations and cell killing. We assessed the contribution of the HRR protein RAD51D in resistance to killing by replication-associated DSBs. In repsonse to hydroxyurea, the isogenic rad51d null CHO mutant fails to show any indication of HRR initiation, as assessed by induction of RAD51 foci, as expected. Surprisingly, these cells have normal resistance to killing by replication inhibition from either hydroxyurea or aphidicolin, but show the expected sensitivity to camptothecin, which also generates replication-dependent DSBs. In contrast, we confirm that the V79 xrcc2 mutant does show increased sensitivity to hydroxyurea under some conditinos, which was correlated to its attenuated RAD51 focus response. In response to a PARP1 inhibitor PARP1, rad51d cells, like other HRR mutants, show exquisite sensitivity (>1000 fold), which is also associated with defective RAD51 focus formation. Thus, rad51d cells are broadly deficient in RAD51 focus formation in response to various agents, but this defect is not invariably associated with increased sensitivity. Our results indicate that RAD51 paralogs do not contribute equally to cellular resistance of inhibitors of DNA replication, and that the RAD51 foci associated with replication inhibition may not be a reliable indicator of cellular resistance to such agents.

  • 431. Urbin, Salustra S.
    et al.
    Elvers, Ingegerd
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hinz, John M.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Thompson, Larry H.
    Uncoupling of RAD51 focus formation and cell survival after replication fork stalling in RAD51D null CHO cells2012In: Environmental and Molecular Mutagenesis, ISSN 0893-6692, E-ISSN 1098-2280, Vol. 53, no 2, p. 114-124Article in journal (Refereed)
    Abstract [en]

    In vertebrate cells, the five RAD51 paralogs (XRCC2/3 and RAD51B/C/D) enhance the efficiency of homologous recombination repair (HRR). Stalling and breakage of DNA replication forks is a common event, especially in the large genomes of higher eukaryotes. When cells are exposed to agents that arrest DNA replication, such as hydroxyurea or aphidicolin, fork breakage can lead to chromosomal aberrations and cell killing. We assessed the contribution of the HRR protein RAD51D in resistance to killing by replication-associated DSBs. In response to hydroxyurea, the isogenic rad51d null CHO mutant fails to show any indication of HRR initiation, as assessed by induction RAD51 foci, as expected. Surprisingly, these cells have normal resistance to killing by replication inhibition from either hydroxyurea or aphidicolin, but show the expected sensitivity to camptothecin, which also generates replication-dependent DSBs. In contrast, we confirm that the V79 xrcc2 mutant does show increased sensitivity to hydroxyurea under some conditions, which was correlated to its attenuated RAD51 focus response. In response to the PARP1 inhibitor KU58684, rad51d cells, like other HRR mutants, show exquisite sensitivity (>1000-fold), which is also associated with defective RAD51 focus formation. Thus, rad51d cells are broadly deficient in RAD51 focus formation in response to various agents, but this defect is not invariably associated with increased sensitivity. Our results indicate that RAD51 paralogs do not contribute equally to cellular resistance of inhibitors of DNAreplication, and that the RAD51 foci associated with replication inhibition may not be a reliable indicator of cellular resistance to such agents.

  • 432.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Interstrand Crosslinks - Induction and repair2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    DNA crosslinking agents exhibit a variety of DNA lesions, such as monoadducts, DNA-DNA interstrand or intrastrand crosslinks or DNA-protein crosslinks. Agents that produce interstrand crosslinks (ICLs) exist naturally and are widely used in chemotherapy. Therefore, it is important to understand how the lesions induced by these agents are repaired. In bacteria, the repair is mainly dependent on nucleotide excision repair (NER) together with homologous recombination (HR) or translesion synthesis (TLS). In human cells, it is not clear how these lesions are repaired, and it is believed to be a more complicated process in which NER does not play as important a role as in prokaryotes. Here, we investigated the repair mechanisms mainly after treatment with psoralen but also with acetaldehyde, cisplatin and mitomycin C in some studies. As expected from studies on plasmids and in bacteria, we used new techniques to confirm that various ICL-inducing agents block replication fork elongation in mammalian cells. We also found that the replication fork was unable to bypass these lesions. We confirmed that ERCC1/XPF and the HR proteins BRCA2 and XRCC2/3 are vital for protection against ICL treatments. These proteins were also found to be equally important for the repair of monoadducts. To better understand ICL repair in mammalian cells, we developed a method to study the induction and unhooking of ICL in human fibroblasts. We found that ICLs were repaired and that 50% of the induced ICLs were unhooked within 3 hours following exposure. Additionally, we determined that XPA, but not XPE, is involved in ICL unhooking, although not affecting lethality. A step in ICL repair is the formation of double-strand breaks (DSBs), and we identified a replication-dependent formation of DSBs following ICL treatment. Furthermore, ERCC1/XPF was not necessary for DSB formation. The repair of these DSBs was performed by HR and involved ERCC1/XPF. Additionally, we were able to quantify the ICL unhooking in human fibroblasts and found that they can unhook ~2500 ICL/h. We also determined that a dose of approximately 400 ICL/cell is lethal to 50% of the cells, indicating that ICL unhooking is not the most critical step during the repair process.

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  • 433.
    Vare, Daniel
    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.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    XPA has a key role in the unhooking step of ICL repair in intact mammalian cellsManuscript (preprint) (Other academic)
  • 434.
    Vare, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Groth, Petra
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Carlsson, Rickard
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cellsIn: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856Article in journal (Refereed)
  • 435.
    Vare, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Groth, Petra
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Carlsson, Rickard
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells2012In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 11, no 12, p. 976-985Article in journal (Refereed)
    Abstract [en]

    DNA interstrand crosslinks (ICLs) are highly toxic lesions that covalently link both strands of DNA and distort the DNA helix. Crosslinking agents have been shown to stall DNA replication and failure to repair ICL lesions before encountered by replication forks may induce severe DNA damage. Most knowledge of the ICL repair process has been revealed from studies in bacteria and cell extracts. However, for mammalian cells the process of ICL repair is still unclear and conflicting data exist. In this study we have explored the fate of psoralen-induced ICLs during replication, by employing intact mammalian cells and novel techniques. By comparative studies distinguishing between effects by monoadducts versus ICLs, we have been able to link the block of replication to the ICLs induction. We found that the replication fork was equally blocked by ICLs in wild-type cells as in cells deficient in ERCC1/XPF and XRCC3. The formation of ICL induced double strand breaks (DSBs), detected by formation of 53PB1 foci, was equally induced in the three cell lines suggesting that these proteins are involved at a later step of the repair process. Furthermore, we found that forks blocked by ICLs were neither bypassed, restarted nor restored for several hours. We propose that this process is different from that taking place following monoadduct induction by UV-light treatment where replication bypass is taking place as an early step. Altogether our findings suggest that restoration of an ICL blocked replication fork, likely initiated by a DSB occurs relatively rapidly at a stalled fork, is followed by restoration, which seems to be a rather slow process in intact mammalian cells.

  • 436.
    Vare, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Persson, Jan-Olov
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The quantification and repair of psoralen-induced interstrand crosslinks in human cellsManuscript (preprint) (Other academic)
  • 437.
    Vielfort, Katarina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Neisseria gonorrhoeae and Lactobacillus from initial adherence to effects on human cells2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The causative agent of gonorrhoea Neisseria gonorrhoeae (gonococcus) colonises the urogenital tract epithelia. The vaginal tract microbiota of healthy females is dominated by Lactobacillus species. In paper I, the ability of lactobacilli to protect cervical cells against gonococcal adherence was investigated. The number of adhered lactobacilli did not correlate to the level of protection against gonococci. Instead, the protection was dependent on specific Lactobacillus isolates. Gonococci able to outmanoeuvre the normal microbiota colonise and may elicit an influx of neutrophils. In paper II the initial interaction between pathogens and neutrophils was investigated. N. gonorrhoeae was found to bind to the non-phagocytic rear (uropod) of the neutrophils. Results suggest that uropod binding is a trait specific of Neisseria species. By binding to the uropod bacteria could avoid the phagocytic front part of the neutrophils whilst being transported across the epithelial cell layer to new sites. Since gonorrhoea has been associated with cancer in several studies, effects of gonococcal colonisation on eukaryote genome integrity was investigated in paper III.  N. gonorrhoeae caused DNA stand breaks in vaginal epithelial cells and decreased the level of tumor protein p53. Infected cells showed increase of cyclin-dependent kinase inhibitors p21 and p27 along with reduced proliferation. The impact of lactobacilli colonisation on cervical cell proliferation was investigated in paper IV. Three out of four Lactobacillus isolates tested reduced cell proliferation. Decreased pH due to lactic acid production was found to be a contributing factor. However, vaginal isolated L. gasseri required a direct bacteria-cell interaction to affect cell cycle progression. Additional unknown factors also contributed as in the case of saliva isolated L. reuteri. In summary, this thesis investigates N. gonorrhoeae pathogenesis and the impact of Lactobacillus species in protection and colonisation.

  • 438.
    Vielfort, Katarina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The interaction between Neisseria gonorrhoeae and Lactobacillus spp. and their effect on host cell cycle.2010Licentiate thesis, monograph (Other academic)
  • 439.
    Vielfort, Katarina
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Söderholm, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Weyler, Linda
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Vare, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Löfmark, Sonja
    Aro, Helena
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells2013In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 126, no 1, p. 339-347Article in journal (Refereed)
    Abstract [en]

    The constant shedding and renewal of epithelial cells maintain the protection of epithelial barriers. Interference with the processes of host cell-cycle regulation and barrier integrity permits the bacterial pathogen Neisseria gonorrhoeae to effectively colonize and invade epithelial cells. Here, we show that a gonococcal infection causes DNA damage in human non-tumor vaginal VK2/E6E7 cells with an increase of 700 DNA strand breaks per cell per hour as detected by an alkaline DNA unwinding assay. Infected cells exhibited elevated levels of DNA double-strand breaks, as indicated by a more than 50% increase in cells expressing DNA damage-response protein 53BP1-positive foci that co-localized with phosphorylated histone H2AX (gamma H2AX). Furthermore, infected cells abolished their expression of the tumor protein p53 and induced an increase in the expression of cyclin-dependent kinase inhibitors p21 and p27 to 2.6-fold and 4.2-fold of controls, respectively. As shown by live-cell microscopy, flow cytometry assays, and BrdU incorporation assays, gonococcal infection slowed the host cell-cycle progression mainly by impairing progression through the G2 phase. Our findings show new cellular players that are involved in the control of the human cell cycle during gonococcal infection and the potential of bacteria to cause cellular abnormalities.

  • 440. Vineis, Paolo
    et al.
    Anttila, Sisko
    Benhamou, Simone
    Spinola, Monica
    Hirvonen, Ari
    Kiyohara, Chikako
    Garte, Seymour J.
    Puntoni, Riccardo
    Rannug, Agneta
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Strange, Richard C.
    Taioli, Emanuela
    Evidence of gene-gene interactions in lung carcinogenesis in a large pooled analysis2007In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 28, no 9, p. 1902-1905Article in journal (Refereed)
    Abstract [en]

    To test the hypothesis of interaction among genetic variants in increasing the individual risk of cancer, we have studied the cumulative effect on lung cancer risk of variants in three metabolic genes, CYP1A1, GSTM1 and GSTT1, which are involved in the metabolism of the tobacco smoke constituents and environmental contaminants, polycyclic aromatic hydrocarbons and of other lung carcinogens. We have selected from the Genetic Susceptibility to Environmental Carcinogens pooled analysis all the studies on lung cancer conducted after 1991 in which all variants were available. The data set includes 611 cases and 870 controls. We found a cumulative effect of the combination of the a priori ' atrisk ' alleles for these genes (P for trend 0.004). The risk of lung cancer was increased with the combination of CYP1A1*2B or CYP1A1*4 alleles and the double deletion of both GSTM1 and GSTT1 up to an odds ratio (OR) of 8.25 (95% confidence interval 2.29-29.77) for the combination including CYP1A1*4; among never smokers, the latter combination was associated with an OR of 16.19 (1.90-137). Estimates did not change after adjustment by the number of cigarettes smoked and duration of smoking were consistent across ethnicities and were approximately the same for adenocarcinomas and squamous cell carcinomas. These observations from a large pooled analysis strongly suggest the existence of gene-gene interactions in lung carcinogenesis. People with rare combinations of common gene variants have a high risk of cancer and can be assimilated to subjects with highly penetrant mutations.

  • 441.
    von Stedingk, Hans
    et al.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Osterman-Golkar, Siv
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Törnqvist, Margareta
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Hemoglobin adducts2011In: Biomarkers and Human Biomonitoring. Vol. 2 : Selected biomarkers of current interest / [ed] Lisbet E. Knudsen and Domenico Franco Merlo, Cambridge: Royal Society of Chemistry , 2011, p. 1-22Chapter in book (Other academic)
  • 442.
    Waluk, Dominik P
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Studies on the human glycine N-acyltransferase-like 2 (GLYATL2) and its role in production of glycine-conjugated signaling molecules.2010Licentiate thesis, monograph (Other academic)
  • 443.
    Waluk, Dominik P.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hunt, Mary C.
    Identification of glycine N-acyltransferase-like 2 (GLYATL2) as a transferase that produces N-acyl glycines in humans.2010In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 24, no 8, p. 2795-2803Article in journal (Refereed)
    Abstract [en]

    The discovery of glycine conjugates of long-chain fatty acids (N-acyl glycines) in the brain and other non-neuronal tissues has led to the identification of an emerging class of bioactive lipids. The biological activities of N-acyl glycines include antinociceptive, anti-inflammatory and antiproliferative effects, and activation of G-protein-coupled receptors. However, despite the fact that N-acyl glycines are emerging as a distinct lipid signaling family, pathways for their production are not fully elucidated. Here we report on the characterization of human glycine N-acyltransferase-like 2 (hGLYATL2), a member of a gene family of 4 putative glycine conjugating enzymes, and show that it synthesizes various N-acyl glycines. Recombinantly expressed hGLYATL2 efficiently conjugated oleoyl-CoA, arachidonoyl-CoA, and other medium- and long-chain acyl-CoAs to glycine. The enzyme was specific for glycine as an acceptor molecule, and preferentially produced N-oleoyl glycine. The hGLYATL2 enzyme is localized to the endoplasmic reticulum, and the mRNA shows highest expression in salivary gland and trachea, but is also detected in spinal cord and skin fibroblasts. The expression pattern and the identification of high levels of N-acyl glycines in skin and lung may indicate a role for N-acyl glycines in barrier function/immune response and the potential role of hGLYATL2 in this regard is discussed.

  • 444.
    Waluk, Dominik P.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sucharski, Filip
    Sipos, Laszlo
    Silberring, Jerzy
    Hunt, Mary C.
    Reversible lysine acetylation regulates the activity of human glycine n-acyltransferase-like 2 (hGLYATL2): Implications for production of glycine-conjugated signalling molecules2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 20, p. 16158-16167Article in journal (Refereed)
    Abstract [en]

    Lysine acetylation is a major post-translational modification of proteins, and regulates many physiological processes such as metabolism, cell migration, ageing and inflammation. Proteomic studies have identified numerous lysine-acetylated proteins in human and mouse models (Kim et al, (2006) Mol. Cell. 23, 607-618). One family of proteins identified in this study was the murine glycine N-acyltransferase (GLYAT) enzymes, which are acetylated on lysine 19 (K19). Lysine 19 is a conserved residue in human glycine N-acyltransferase-like 2 (hGLYATL2) and in several other species, showing that this residue may be important for enzyme function. Mutation of lysine 19 (K19) in recombinant hGLYATL2 to glutamine (K19Q) and arginine (K19R) resulted in a 50-80% lower production of N-oleoyl glycine and N-arachidonoylglycine, indicating that lysine 19 is important for enzyme function. LC/MS/MS confirmed that K19 is not acetylated in wild-type hGLYATL2, indicating that K19 requires to be deacetylated for full activity. The hGLYATL2 enzyme conjugates medium- and long-chain saturated and unsaturated acyl-CoA esters to glycine, resulting in the production of N-oleoyl glycine and also N-arachidonoyl glycine. N-oleoyl glycine and N-arachidonoyl glycine are structurally and functionally related to endocannabinoids and have been identified as signalling molecules that regulate functions like the perception of pain, body temperature, and also have anti-inflammatory properties. In conclusion, acetylation of lysine(s) in hGLYATL2 regulate the enzyme activity, thus linking post-translational modification of proteins with the production of biological signalling molecules, the N-acyl glycines.

  • 445.
    Waluk, Dominik P.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Tillander, Veronika
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hunt, Mary C.
    Molecular characterization of two members of the glycine N-acyltransferase gene family in human: glycine N-acyl transferase-like 1 (GLYATL1) and glycine N-acyltransferase-like 3 (GLYATL3).Manuscript (preprint) (Other academic)
    Abstract [en]

    N-acyl amino acids are a group of endogenous lipid mediators that regulate a variety of cellular physiological functions. The discovery of N-acyl amino acids in many biological systems has allowed research to focus on their functions as well as pathways for production of these signalling lipids.

    We have previously identified that human glycine N-acyltransferase-like 2 (hGLYATL2) is involved in the enzymatic formation of N-acyl glycines. hGLYATL2 is localized in a gene cluster with other glycine N-acyltransferase genes. Here, we have characterized human glycine N-acyltransferase-like 1 (hGLYATL1) and human glycine N-acyltransferase-like 3 (hGLYATL3), which are members of this gene family. Our results show that hGLYATL1 is localized to the endoplasmic reticulum (ER) but the intracellular localization of hGLYATL3 remains to be determined. The hGLYATL1 mRNA shows highest expression in liver and kidney, whereas mRNA of hGLYATL3 is expressed in pancreas and liver. Using bioinformatics we determined the overall three-dimensional (3D) structures of hGLYATL1 and hGLYATL3 enzymes, with predicted binding site residues.

    In summary, we have characterized novel members of glycine N-acyltransferases that may be involved in the production of lipid signalling molecules, in particular N-acyl glycines.

  • 446.
    Waluk, Dominik P.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Vielfort, Katarina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Derakhshan, Sepide
    Aro, Helena
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hunt, Mary C
    N-acyl taurines trigger insulin secretion by increasing calcium flux in pancreatic b-cellsManuscript (preprint) (Other academic)
    Abstract [en]

    Pancreatic b-cells secrete insulin in response to various stimuli to control blood glucose levels. This insulin release is the result of a complex interplay between signalling, membrane potential and intracellular calcium levels. Various nutritional and hormonal factors are involved in regulating this process. N-acyl taurines are a group of fatty acids which are amidated (or conjugated) to taurine and little is known about their physiological functions. In this study, treatment of pancreatic b-cell lines (HIT-T15) and rat islet cell lines (INS-1) with N-acyl taurines (N-arachidonoyl taurine and N-oleoyl taurine), induced a high frequency of calcium oscillations in these cells. Treatment with N-arachidonoyl taurine and N-oleoyl taurine also resulted in a significant increase in insulin secretion from pancreatic b-cell lines as determined by insulin release assay and immunofluorescence (p<0.05). Our data also show that the transient receptor potential vanilloid 1 (TRPV1) channel is involved in insulin secretion in response to N-arachidonoyl taurine and N-oleoyl taurine treatment. However our data also suggest that receptors other than TRPV1 are involved in the insulin secretion response to treatment with N-oleoyl taurine.

  • 447.
    Waluk, Dominik P.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Vielfort, Katarina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Derakhshan, Sepide
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Aro, Helena
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hunt, Mary C.
    N-Acyl taurines trigger insulin secretion by increasing calcium flux in pancreatic beta-cells2013In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 430, no 1, p. 54-59Article in journal (Refereed)
    Abstract [en]

    Pancreatic beta-cells secrete insulin in response to various stimuli to control blood glucose levels. This insulin release is the result of a complex interplay between signaling, membrane potential and intracellular calcium levels. Various nutritional and hormonal factors are involved in regulating this process. N-Acyl taurines are a group of fatty acids which are amidated (or conjugated) to taurine and little is known about their physiological functions. In this study, treatment of pancreatic beta-cell lines (HIT-T15) and rat islet cell lines (INS-1) with N-acyl taurines (N-arachidonoyl taurine and N-oleoyl taurine), induced a high frequency of calcium oscillations in these cells. Treatment with N-arachidonoyl taurine and N-oleoyl taurine also resulted in a significant increase in insulin secretion from pancreatic beta-cell lines as determined by insulin release assay and immunofluorescence (p < 0.05). Our data also show that the transient receptor potential vanilloid 1 (TRPV1) channel is involved in insulin secretion in response to N-arachidonoyl taurine and N-oleoyl taurine treatment. However our data also suggest that receptors other than TRPV1 are involved in the insulin secretion response to treatment with N-oleoyl taurine.

  • 448.
    Waluk, Dominik Paweł
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Biosynthesis and physiological functions of N-acyl amino acids2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    N-acyl amino acids are lipid signalling molecules that have recently been identified in biological systems. These lipids are structurally related to the endocannabinoids, although they do not activate cannabinoid receptors. In 2001, N-arachidonoyl glycine was the first signalling lipid in this group to be identified in bovine and rat brain and since then, about 50 novel N-acyl amino acids have been identified in mammalian systems. These N-acyl amino acids are involved in regulating pain processes, are anti-inflammatory and regulate body temperature, but the metabolic pathways for production and metabolism remain poorly understood.

    This thesis focussed on the identification of pathways for production and regulation of N-acyl amino acids, in particular N-acyl glycines, and in identifying physiological functions for N-acyl amino acids (particularly N-acyl taurines). Our results identified an enzymatic pathway for production of N-acyl glycines in human and we identified that the human glycine N-acyltransferase-like 2 (hGLYATL2) conjugates (amidates) medium- and long-chain, saturated and unsaturated acyl-CoAs with glycine, to produce N-acyl glycines, with the preferential production of N-oleoyl glycine. Furthermore, we have characterized two other members of the gene family of glycine N-acyltransferases (GLYATs) in human, the hGLYATL1 and hGLYATL3 that may be involved in the production of N-acyl amino acids.

    As N-acyl glycines are bioactive signalling molecules, it is likely their production requires a rapid on/off switch. The post-translational modification of proteins can result in enzyme regulation, without the need for transcriptional regulation. We have identified that hGLYATL2 is regulated by acetylation/deacetylation on lysine 19, and using mutation analysis, we show that deacetylation of lysine 19 is important for full enzyme activity.

    The physiological functions of N-acyl amino acids are not well studied to date. In this thesis, we have identified that N-arachidonoyl taurine and N-oleoyl taurine trigger insulin secretion by increasing the calcium flux in pancreatic b-cells via the activation of transient receptor potential vanilloid subfamily 1 (TRPV1).

    This work on N-acyl amino acids has led us to identify new pathways and physiological functions for these lipid signalling molecules, which advances our knowledge of the importance of these lipids in mammalian systems.

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  • 449.
    Waluk, Dominik
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sucharski, Filip
    Sipos, Laszlo
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Derakhshan, Sepide
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sillbering, Jerzy
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hunt, Mary
    Reversible lysine acetylation regulates the activity of human glycine N-acyltransferase 2 (hGLYATL2)-implications for the production of glycine conjugated signalling lipids2011In: Chemistry and Physics of Lipids, ISSN 0009-3084, E-ISSN 1873-2941, Vol. 164, p. s35-S35Article in journal (Other academic)
  • 450.
    Wang, Zhi
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wilhelmsson, Christine
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Hyrsl, Pavel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Loof, Torsten G.
    Dobes, Pavel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Klupp, Martina
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Loseva, Olga
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Moergelin, Matthias
    Ikle, Jennifer
    Cripps, Richard M.
    Herwald, Heiko
    Theopold, Ulrich
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Pathogen Entrapment by Transglutaminase - A Conserved Early Innate Immune Mechanism2010In: PLOS pathogens, ISSN 1553-7366, Vol. 6, no 2, p. e1000763-Article in journal (Refereed)
    Abstract [en]

    Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or-as previously shown-the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions.

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